Mass spectrometry in bioanalytical and
clinical applications
Habilitation thesis
by
Ondřej Peš
2022
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All likelihood-based models are Bayesian models in which the prior distribution is an appropriately selected uniform
prior, and as the size of the data gets large they are identical given any finite appropriate prior. So such empirical
researchers are really Bayesian; they just do not know it yet.
— Jeff Gill and Christopher Witko
This page is intentionally left blank
Originality and conflict of interest statement
The author performed the original research presented in the habilitation thesis in cooperation with his colleagues,
students, and members of the cooperating research groups; all of them are appropriately quoted as coauthors of the
manuscripts. Funding of the research was supported by the following projects:
• Specific University Research Grant of Masaryk University:
MUNI/A/1090/2021, MUNI/A/1266/2020, MUNI/A/1167/2019, MUNI/A/0976/2018, MUNI/A/0910/2017,
MUNI/A/1205/2016, MUNI/A/1056/2015, MUNI/A/1195/2014, MUNI/A/0992/2009
• Internal development projects:
MUNI/11/SUP/08/2022, ROZV/28/LF11/2020, ROZV/23/LF7/2019, FRVS/746/2008
• Czech Science Foundation:
GAP206/12/G014, GCP206/10/J012, GA203/09/1025
I hereby declare that I have no conflict of interest.
Preface
This cumulative habilitation thesis consists of 9 original and 2 review articles published in years 2008–2020 and
focuses on the development and applications of mass spectrometry techniques in bioanalytical, medicinal, and clinical
chemistry. The entire thesis has been thematically divided into three parts, as follows.
1. Mass spectrometry hyphenated via an offline interface
Many fields of chemistry, biology, or medicine usually require more than a simple approach in observing
critical processes and inferring specific conclusions. Usually, mass spectrometry on its own is not capable
of providing answers to complex questions. It is thus beneficial to employ different platform setups to
achieve a maximum gain of information entropy.
2. Mass spectrometry as a tool in identification and characterization
Unambiguous identification of a compound belongs to tasks, which have been haunting chemists for
centuries. Any chemical compound may be essentially classified as a known unknown (its structure has
already been de novo elucidated) or an unknown unknown (no records exist about the structure). Mass
spectrometry is a compelling and still readily available technique capable of identifying and characterizing
chemical substances, provided they might be ionized. Fragmentation techniques and different types of
detection may orthogonal information, leading to minimizing ambiguity in the compound’s identity.
3. Mass spectrometry as a sensitive and specific detector
Recent developments in mass spectrometry allow detection of very low amounts while keeping very high
specificity. Theoretical limits of common and abundant mass spectrometric detectors (under optimal
conditions) are as low as a few femtograms in a single injection. Additional coupling with a separation
technique may enhance the limits even further. Hence, it is reasonable to exploit such a feature in
quantitative research, especially when sensitivity and specificity of other methods applied so far may
have been reaching their limits.
Acknowledgments
I would like to thank all my colleagues, students, and others involved in my research career. The presented thesis
would never have existed without their support and encouragement. Mainly, I would like to express my gratitude to
the following people (in alphabetical order):
Mirka Bittová, Jiří Dostál, Pavla Foltynová, Jana Gregorová, Martina Hanousková, Jan Juřica, Viktor Kanický,
Katarína Kostolanská, Michaela Králíková, Pavel Krásenský, Lenka Kubinyiová, Josef Kučera, Adam Midlik, Miriam
Minaříková, Lenka Nerudová, Lucie Novotná, Hana Paulová, Jan Preisler, Jan Přikryl, Jiří Schlaghamerský, Kristýna
Šebrlová, Lucie Šimoníková, Jiří Slanina, Pavel Šmak, Jindra Smutná, Eva Táborská, Petr Táborský, Tomáš Vaculovič,
Tomáš Vašina, Michaela Vašinová-Galiová, Ondřej Zendulka
I would like to thank my family as well.
Contents
1 Introduction 1
2 Mass spectrometry hyphenated via an offline interface 3
2.1 Offline coupling of microcolumn separations to desorption mass spectrometry . . . . . . . . . . . . . 4
2.2 Off-Line Coupling of Capillary Electrophoresis to Substrate-Assisted Laser Desorption Inductively
Coupled Plasma Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3 Substrate-assisted laser desorption inductively-coupled plasma mass spectrometry for the determination
of copper in myeloid leukemia cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3 Mass spectrometry as a tool in identification and characterization 35
3.1 Identification of metabolites of selected benzophenanthridine alkaloids and their toxicity evaluation . 37
3.2 Sanguinarine is reduced by NADH through a covalent adduct . . . . . . . . . . . . . . . . . . . . . . 46
3.3 Seasonal variation in alkaloid composition and antiproliferative activity of Stylophorum lasiocarpum
(Oliv.) Fedde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.4 Heavy water enhancement of fluorescence signal in reversed-phase liquid chromatography . . . . . . 67
3.5 A study on bioluminescence and photoluminescence in the earthworm Eisenia lucens . . . . . . . . . 76
4 Mass spectrometry as a sensitive and selective detector 83
4.1 Determination of lansoprazole, 5-hydroxylansoprazole, and lansoprazole sulfone in human plasma for
CYP2C19 and CYP3A4 phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.2 Online solid-phase extraction liquid chromatography-mass spectrometry of hair cortisol using a
surrogate analyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.3 Therapeutic Drug Monitoring of Sunitinib in Gastrointestinal Stromal Tumors and Metastatic Renal
Cell Carcinoma in Adults-A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5 Conclusion 122
6 References 123
1 Introduction
Originally, mass spectrometry (MS) was developed to measure atom masses, and its first scientific contribution was
to demonstrate the existence of isotopes. At that time, MS was anticipated to fade from science when all isotopes
were discovered. Fortunately, petroleum chemists in the 1940s were using MS to measure small hydrocarbons, and in
the 1960s, the complex fragmentation processes were studied and a range of possible applications was apprehended.
Currently, mass spectrometry has been with scientists for more than a hundred years although it was doomed several
times since its inception.
The discoverer of MS, Sir Joseph John Thomson, made the discovery involuntarily while studying a phenomenon of
cathode rays. Over 50 years, the cathode rays were continuously debated whether they were made of particles or
waves. Those who believed in particles reasoned that their mass should be evidenced. With his laboratory assistant
Ebenezer Everett, Thomson first measured the charge-to-mass ratio (e/m) of the cathodic rays – electrons in 1897,
for which Thomson received the 1906 Nobel Prize.1
Ultimately, Thomson, with Francis Aston’s help, built the first
mass spectrometer to measure masses of charged particles by deflecting them using electric and magnetic fields onto
a photographic plate.2
The beginning of the 20th century might be viewed as a hunt to improve the resolving power
to refine the accuracy of measurements of the relative abundance and masses of the elements and their isotopes and
broaden the MS applications outside the realm of physics. The practicality of MS fully showed in the 1940s when
Alfred Nier built several MS instruments capable of preparing 13
C-enriched carbon.3
Enrico Fermi, John Dunning,
and Alfred Nier began the nuclear era when nanogram quantities of 235
U were prepared. Since 1940, MS has been
used quantitatively to control production processes although the mass spectrum-to-molecular structure relationship
was not known and thus extensively studied. The operating principle of the quadrupole mass spectrometer was
made in 1953 by Wolfgang Paul and Helmut Steinwedel,4
for which Paul received the 1989 Nobel Prize. However, it
wasn’t until the 1970s that the quadrupole found its true calling in computerized gas chromatography coupled to
MS in enviromental analysis.
In the 1960s, Fred McLafferty, Klaus Biemann, and Carl Djerassi were methodologically drawing out fragmentation
mechanisms allowing chemists to employ MS to determine structures of unknown molecules.5–7
Ultrahigh resolving
power instruments came around 1970 when Melvin Comisarow and Alan Marshall applied Fourier transform to ion
cyclotron resonance (ICR),8
developed in 1949 by John Hipple.9
The discovery of time-of-flight (TOF) MS in 1946
is given to William Stephens, who pioneered a conceptually simple mass analyzer with theoretically unlimited mass
range and the ability to capture the entire spectrum at once.10
Absence of inherently pulsed ionization techniques
left TOF MS inapplicable until its renaissance about thirty years later when laser desorption made its appearance.
By the 1980s, small organic molecules were routinely being analyzed by MS. The area of elemental analysis was
further strengthened by development of inductively coupled plasma (ICP) in 1980, which enables to measure nearly
all of the elements of the periodic table when interfaced to MS.11
However, macromolecules such as proteins, nucleic
acids, and complex carbohydrates were challenging as the macromolecules could not be ionized without being too
fragmented. Techniques like fast atom bombardment, plasma desorption, secondary ion MS or thermospray were
making little progress as they worked only with small proteins at high concentrations. In 1988, the discovery of
the electrospray ionization (ESI) interface by John Fenn revolutionized biological MS.12
Fenn was awarded the
1
2002 Nobel Prize together with Koichi Tanaka, who shared the prize for using a laser desorption technique in a
1988 analysis of proteins.13
Fenn gave a lot of credit to Malcolm Dole who demonstrated that nonvolatile solutes,
when dissolved in volatile solvents, produced highly charged droplets in which they stayed intact even if transferred
into the gaseous phase.14
On the other hand, the lifetime effort of Michael Karas and Franz Hillenkamp, who are
considered the inventors of matrix-assisted laser desorption ionization (MALDI), was not rewarded by any prize
whatsoever.15
The development of ESI and MALDI opened the MS field to new researchers pursuing protein and
peptide investigations. Analysis of neonatal dried blood spot samples for a range of inborn errors of metabolism
was a primary early application for liquid chromatography (LC)–ESI MS, which persevered to current times.16
Bacterial identification and diagnosis is currently driven by MALDI fingerprinting.17
ESI and MALDI are considered
competing and complementary at the same time.
The 21st century has brought significant innovations in the design of TOF MS and multiple collector ICP MS;
ESI techniques evolved into ambient ionization techniques – desorption electrospray ionization (DESI)18
and direct
analysis in real time (DART)19
, and the orbital ion trap (Orbitrap) analyzer was introduced. Orbitrap, which was
first given as a proof-of-principle in the 1990s by Alexander Makarov, was commercialized in 2005.20,21
The history
of ion mobility spectrometry (IMS) goes back to Thomson and Rutherford who conceptualized the technique in
1896.22
In the 1960s IMS was more commonly known as plasma chromatography and used to gas-phase separate a
wide variety of substances, e.g., many airport explosives detectors are currently based upon IMS. The introduction
of a routine IMS MS instrument in 2006 drove the technology from research to analytical laboratories.23
Since then
many manufactures have developed their own IMS MS platform based on unique mobility separations, thereby
adding the analytical power of IMS to MS for challenging systems in complex mixtures.24,25
The unique ability to separate the ionized molecules themselves according to m/z has driven advances across science
over a hundred years, and at the same time, it is the primary reason why mass spectrometry has become one of the
dominant analytical methodologies of the 21st century.
2
2 Mass spectrometry hyphenated via an offline interface
MS itself is a very powerful technique; however, complex mixtures can easily overcome the power in terms of
resolution, sensitivity, or ion suppression. Coupling MS to separation techniques, which may decrease the complexity
of a sample, is an additional and sometimes a mandatory step to increase the reliability and efficiency of qualitative
and quantitative analysis. Such hyphenation may be performed online, inline, or completely offline. While the
online coupling of chromatography to MS using ESI is the predominating technique it still possesses shortcomings
originating from the time constraints of MS measurements. Particularly, a short time frame limits the analysis
of all components in highly resolved peaks. In some cases, the entire analysis has to be repeated to complete
data acquisition on missed analytes. Or the duration of online analysis is given by the time in which the entire
separation is accomplished; however, in the offline arrangement, the separation, which may take hours, can be stored
and analyzed (repeatedly) in minutes. The offline coupling of MS to separation techniques presents a significant
advantage in actual decoupling the processes of separation and detection, which both may be performed under
their optimal conditions. Sometimes, an online interface is lacking, such as in 1D/2D gel electrophoresis (GE), or
thin-layer chromatography (TLC). MALDI is generally less responsive to ion suppression than ESI, and furthermore,
since singly-charged ions are typically formed in MALDI, simpler spectra are produced than those in ESI. Yet, the
offline coupling of chromatography to MS represents a minority of publications indexed by the Web of Science, as
seen in Figure 1.
0
25
50
75
100
125
2001 2006 2011 2016 2021
Year
Publicationcount
Figure 1: Publication results in 2021 on the Web of Science for a query: "(offline OR off-line) chromatography mass
spectrometry"
Microcolumn techniques such as capillary LC (cLC) or capillary electrophoresis (CE) may be easily recorded and
3
stored without fabricating complex and fragile online interfaces. The classification of junctions between the separation
column and the deposition probe and the deposition mechanisms are given in Figure 2. Generally, a sheath flow
from a T-junction is commonplace in LC; a liquid junction with a separation buffer addition is a viable alternative
in CE. When a separation technique is coupled offline, the sheath liquid is usually evaporated and is thus not part of
the sample.
Figure 2: Off-line coupling scheme. Junctions used for coupling of microcolumn techniques include (A) coaxial
sheath flow, (B) T-junction, (C) sheathless, (D) porous junction, (E) liquid junction (F) droplet electrocoupling.
Another great advantage lies in the availability to archive the separation, perform on target reactions, or employ
different detection techniques avoiding the throughput mismatch when the separation and detection techniques are
not compatible in their data acquisition rates. In other words, a detection technique working at a slow acquisition
rate, e.g., MS/MS acquisition or a high resolving power instrument, may be employed in an offline design without
compromised efficiency due to time constraints. The availability of commercial mass spectrometers equipped with
<1 kHz lasers capable of high data acquisition speeds in both MS and MS/MS modes pushes the advantages of
offline coupling even further.
2.1 Offline coupling of microcolumn separations to desorption mass spectrometry
Essentially, the simplest mode of coupling LC to desorption MS is offline, which could be achieved by manual
fractionation and collection into vials or tubes, followed by concentration and spotting for desorption analysis.
The majority of offline systems includes MALDI or LDI as an ionization source although other techniques like
4
desorption/ionization on silicon, material-enhanced laser desorption/ionization, laser ablation, surface-assisted laser
desorption were described. Direct fraction collection via automated robotic interfaces is far more reliable in terms
of labor and repeatability. The effluent is deposited by a needle mounted in a z-axis direction to the spotting
target. The fraction collection itself may be classified from various points of view. The deposition of the continuous
streak has proven to be beneficial only in fast separations. In other separations, the mass spectra are accumulated
along a finite trace segment which provides an average spectrum similar to that obtained from a discrete fraction.
A slight decrease in chromatographic resolution due to the segmented acquisition is usually negligible. However,
chromatographic resolution may deteriorate as a result of axial diffusion in the case of a relatively long evaporation
time of the streak. Hence, the discrete spot fractionation is the main mode of operation in commercial spotting
devices. The effluent deposition could be performed either contact or contactless. Many spotting devices rely on
contact deposition even as it suffers from significant memory effects as the advancing needle remains wet from the
previous fraction. Most of the time, addition of sheath liquid in microcolumn separations incurred a problem of
sensitivity loss due to significant dilution caused by a much larger volume of the supplemental liquid. A predesigned
sample support with hydrophobic surface could concentrate the analyte in the deposited sample. Contactless
deposition modes are scarcer and include spraying (ESI, nebulizer), dispensing (piezoelectric, impulse-driven),
heated droplet deposition or deposition assisted by electric field. Later, as ambient MS have become available, for
instance, DESI, desorption atmospheric pressure chemical ionization (DAPCI), desorption atmospheric pressure
photoionization (DAPPI), DART, paper spray ionization (PSI), or diode laser thermal vaporization (DLTV), they
helped in disease diagnosing and drug monitoring tasks26
. Their contribution can as well be traced to the analysis
of recorded separations.27–30
Several step-wise processes of transferring the solid analytes into the gaseous state of
charged particles have given a name to a range of techniques used in the analysis of solid samples across different
fields of science.31
Many commercially-available spotting devices have become available, and the authors made an
effort to review and summarize all of the manufacturers, interfaces, and their applications to date.
PES, Ondrej and Jan PREISLER. Off-line coupling of microcolumn separations to desorption mass spectrometry.
Journal of Chromatography A 2010, 1217(25), 3966–3977. ISSN 0021-9673. doi:10.1016/j.chroma.2010.02.058
Document Type: Review, IF = 4.194; JCR Category + Category Quartile: BIOCHEMICAL RESEARCH METHODS
Q1 + CHEMISTRY, ANALYTICAL Q1; AIS = 0.872
Author’s contribution: 60 %
5
Journal of Chromatography A, 1217 (2010) 3966–3977
Contents lists available at ScienceDirect
Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
Review
Off-line coupling of microcolumn separations to desorption mass spectrometry
Ondˇrej Peˇs, Jan Preisler∗
Department of Chemistry and Department of Experimental Biology, Faculty of Science, Masaryk University, Kotláˇrská 2, 611 37, Brno, Czech Republic
a r t i c l e i n f o
Article history:
Available online 3 March 2010
Keywords:
Off-line coupling
Microcolumn separation
Mass spectrometry
Spotter
Deposition interface
a b s t r a c t
The presented review provides comprehensive and detailed characteristics on microcolumn separation
techniques off-line coupled to mass spectrometry. Major attention is paid to the classification of junctions
between the separation column and the deposition needle and to the process by which the liquid is
transferred onto the target. Both contact and non-contact deposition techniques are covered. In order to
emphasize the significance of the topic of off-line separations, current commercially available devices
have been compared in terms of their potential utilization in analytical chemistry with a summarization
of applications used over the past few years.
© 2010 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3966
2. Off-line vs. on-line interfacing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3967
3. Effluent collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3967
4. Contact deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3968
4.1. Target electrocoupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3969
5. Non-contact deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3969
5.1. Electrospray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3969
5.2. Nebulizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3969
5.3. Piezoelectric dispensers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3970
5.4. Electric field-assisted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3970
5.5. Heated droplet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3970
5.6. Impulse driven. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3970
5.7. Chip-based systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3970
6. Commercial spotters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3972
7. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3972
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3974
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3975
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3975
1. Introduction
Mass spectrometry (MS) is a powerful technique capable of
obtaining essential data about a wide range of samples. However,
not even mass spectrometry is able to provide the required results
when applied to complex sample mixtures without prior separation.
To reduce sample complexity, coupling mass spectrometry to
one or more available separation techniques, in either an on-line
or off-line arrangement, is unavoidable.
∗ Corresponding author. Tel.: +420 549 496 629; fax: +420 549 492 494.
E-mail address: preisler@chemi.muni.cz (J. Preisler).
Microcolumn separation techniques, such as capillary liquid
chromatography (cLC, ␮LC) or capillary electrophoresis (CE), are
among the most important analytical tools in separation science.
The advantages of these techniques include high separation efficiency,
low limits of detection, minute sample size requirements
and economical operating costs. With MS detection, both LC and
CE belong to the most powerful solutions for the identification of
unknown substances in complex matrices.
Whilst the common on-line, in-line (at-line) approaches have
been comprehensively covered by many reviewers [1–6], challenges
resulting from time scales imposed by analyzing complex
mixtures on-line seem to be a major driving force for technological
advances in the field of off-line coupling. Basically, the separation
0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2010.02.058
O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977 3967
is recorded on a target plate in the form of a solid as either discrete
fractions or a continuous track. Such a separation record is predestined
for desorption techniques capable of effective introduction
of the sample into a mass spectrometer. The majority of offline
systems includes matrix-assisted laser desorption/ionization
(MALDI) [7] or laser desorption/ionization (LDI) as an ionization
source. The sample introduction techniques further include desorption/ionization
on silicon (DIOS) [8], material-enhanced laser
desorption/ionization (MELDI) [9], desorption electrospray ionization
(DESI) [10], laser ablation (LA) [11], or the recently presented
surface-assisted laser desorption inductively coupled plasma (SALD
ICP) [12]. As MALDI MS is amongst the leading ionization techniques
nowadays, most examples of off-line coupling discussed in
this paper pertain to the proteomic area. The principles and applications,
however, can be extended to the other desorption techniques
and analyte types.
Historically, many proteomic studies have relied on 2D gel electrophoresis
(GE) followed by excision of spots from the gel. The
proteins contained within the spot are then subjected to protease
digestion, and MS/MS is used to identify the peptides [3]. Although
2D gels have been widely used, they are limited by presence of
not always advantageous gel matrix [13]. In brief, proteins with
extreme values of molecular mass and/or pI tend to be underrepresented.
Variations in the protein charge and size is affected by
post-translational modifications which may result in several bands
of the same protein over a wide pH range. Protein bands may not
be detected due to a low expression of the protein, i.e. the dynamic
range of gels is limited. Last but not least, 2D gels can be time
consuming to prepare, run and analyze; these gels suffer from variable
reproducibility and a skilled operator is required to run them
successfully.
Alternative approaches may employ 1D GE followed by LC of
peptides from proteolytically digested bands on-line coupled to
tandem MS, also known as Gel-C-MS/MS [14], multidimensional
protein identification technology (MudPIT) [15,16] and column or
microcolumn separations. As these techniques are complementary
and do not suffer from the limitations of 2D gels, they could become
the method of choice in many proteomic applications.
Published reviews mostly cover only a part of the topic, such
as CE–MS [2,17], or quantitative proteomics [18]. The aim of this
review is to provide a comprehensive view on microcolumn separation
techniques coupled off-line to MS. It includes the classification
of junctions between the separation column and the deposition
probe, deposition mechanisms as well as characterization and
availability of commercial spotters. In the last part of the review, a
list of applications mapping the area of off-line coupling is given.
2. Off-line vs. on-line interfacing
Due to the inherent nature of liquid column separations, electrospray
ionization [19] (ESI) offers easier and more straightforward
on-line coupling, relative to MALDI. While being the predominating
technique, it still possesses shortcomings originating from the
time constraints of MS measurements. Particularly, there is a short
time available for the analysis of all components in highly resolved
peaks, i.e. by the time MS/MS of a selected compound has been carried
out, co-eluting or closely eluting compounds may have been
skipped. In some cases, the entire LC–MS analysis has to be repeated
in order to collect data on missed analytes.
Generally, the simplest mode of coupling LC to desorption MS is
off-line, which has the advantage of completely decoupling the two
techniques, enabling separate optimization of each. MALDI is, relative
to ESI, less responsive to traces of additives in the separation
medium, such as sodium dodecylsulphate, trifluoroacetic acid, or
salts. Furthermore, since singly charged ions are typically formed
in MALDI, it is known to produce simpler spectra than ESI. Compared
to on-line interfacing, papers dealing with off-line coupling of
microcolumn techniques are less frequent, presumably due to the
requirement of a relatively complex deposition interface and early
expansion of MS/MS-capable mass spectrometers equipped with
ESI. It is important to note that stable deposition depends on many
variables, such as effluent flow rate, temperature and viscosity, concentration
of organic solvents in effluent, substrate surface, surface
tension, or environment pressure. On the other hand, the key difference
between time-dependent and result-driven analyses [20] is
constantly being equalized by increasing the power of data acquisition
and processing hardware. Although on-line coupling prevails,
the off-line approach is capable of combining the advantages of
column (ruggedness) and gel (archiving) separations. The ability
to store the eluted fractions on a MALDI plate and expose them
to another spectrometric technique has appeared to be rewarding,
e.g. in metallomics, where both species, metal and bio-ligand, are
expected to be determined together [12,21].
In combination with multicolumn separation, the off-line
approach offers improved sample throughput and cost-effective
exploitation of a mass spectrometer. In the on-line approach,
the duration of MS analysis is given by the time in which the
entire liquid separation is accomplished. In the off-line arrangement
LC separations, which may take hours, can be stored on
a MALDI target and analyzed repeatedly, under various conditions,
in minutes. Alternatively, multiple samples, separated and
deposited on a target in parallel, can be analyzed by a single mass
spectrometer without compromising separation and mass spectrometry
performance as demonstrated by in-line capillary array
electrophoresis (CAE) MALDI MS of eight protein digests [22]. This
concept is valid also for off-line workflow [23–25]. Separation
records obtained from multiple columns can be analyzed with a single
high-throughput mass spectrometer which is usually the most
expensive part of the setup. The throughput of axial MALDI TOF MS
is, up to a certain limit (∼10 kHz), proportional to the rate at which
individual spectra can be acquired; this rate being primarily a function
of the laser repetition rate.[26] Reliable solid-state UV lasers
with fast repetition rates (0.1–5 kHz), power density and pulseto-pulse
stability adequate for MALDI applications have become
available, which may significantly reduce the time required for MS
analysis of large sample arrays [27]. Recently, the introduction of
commercial mass spectrometers equipped with a 1 kHz laser capable
of high data acquisition speeds in both MS and MS/MS modes
has been announced [28,29].
3. Effluent collection
Off-line LC–MS can simply be achieved by manual fractionation
and collection into microtubes, followed by concentration and
spotting for traditional MALDI analysis. This process is labor intensive
and can result in sample loss [30,31]. This review will focus
on direct fraction collection on a sample target. In addition, offline
coupling to CE requires an electrical contact to be maintained
at both ends of the capillary during the separation. Typically, the
capillary output end is located in an electrolyte reservoir acting as
a conductor and pH buffer. In order to prevent degradation of the
CE resolution, intake and outflow of the auxiliary buffer has to be
arranged in such a manner as to prevent the induction of laminar
flow in the separation capillary.
Naturally, in CE and after the introduction of nanoflow separation
techniques such as cLC, sample volumes from both systems are
directly compatible with spotting onto a MALDI target without any
preconcentration [3]. However, low flow rates may require supplementary
liquid for the cLC or CE effluent to be deposited with
it. Viable connections as well as available deposition interfaces are
3968 O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977
Fig. 1. Off-line coupling scheme showing supplementary liquid addition and deposition mechanisms. Junctions used for coupling of microcolumn techniques include (A)
coaxial sheath flow, (B) T-junction, (C) sheathless, (D) porous junction, (E) liquid junction (F) droplet electrocoupling. (A)–(C) are typical for cLC whereas (D)–(F) are common
in CE.
shown in Fig. 1. Generally, a sheath flow from a T-junction is preferred
in LC; a liquid junction with a separation buffer addition is a
viable alternative in CE. Alternatively, the voltage may be switched
off, the vial at the cathode end removed and the desired peak eluted
by applying pressure at the inlet end [32,33]. Because prior knowledge
of the analyte retention time is required, the pressure method
relies on an on-line detector for the separation to be stopped at the
appropriate point. Disadvantages include prolongation of the analysis
time and difficulty in handling small analyte volumes that are
produced at flow rates of a few nanoliters per second. The addition
of supplementary liquid at the capillary output has been used to
overcome this problem [34–36].
The fraction collection itself may be classified from various
points of view. As previously mentioned, effluent can be collected
in the form of discrete fractions or a continuous streak. The deposition
of the continuous streak has proven to be beneficial only in
fast separations where the peak widths are as low as 2 s [37,38].
In other separations, the mass spectra are accumulated along a
finite trace segment which provides an average spectrum similar
to that obtained from a discrete fraction. A slight decrease in chromatographic
resolution due to the segmented acquisition is usually
negligible. However, chromatographic resolution may deteriorate
as a result of axial diffusion in the case of a relatively long evaporation
time of the streak. Therefore, most spotters only aim at discrete
spot fractionation which fulfills most common needs. The spotting
devices have been sorted according to the principles of operation.
4. Contact deposition
The simplest way to transfer the liquid is assumed to be the
deposition needle or rather the droplet formed underneath, that
touches the target plate surface and most commercial devices work
on this principle. Although mechanically simple, the contact deposition
may suffer from significant memory effects as the advancing
needle remains wet from the previous fraction. Takigiku et al. [39]
presented the use of a previously described porous glass joint [40]
near the cathodic end of the CE column to collect fractions for
desorption MS; later it was used by Keough et al. [41] for the
determination of proteins. Next, off-line microcolumn separation
techniques coupled to MALDI MS involved elution onto the sample
target by the addition of a supplementary liquid. It included
cLC [42–45], CE [34–36,46–50], capillary isoelectric focusing (CIEF)
[51–55], capillary electrochromatography (CEC) [56] and 2D LC CE
[57]. Whilst being effective, a problem of sensitivity loss could
occur due to significant dilution caused by a much larger volume
of supplemental liquid from the junction. To address this issue,
a prestructured sample support [58] was used for fraction collection
[59]. In this design, dilution by the matrix/buffer solution
was counteracted by the concentrating effect of the hydrophobic
Teflon surface. In 1998, Stevenson et al. designed a device comprising
a modified pen plotter programmed to apply the HPLC
effluent directly to a membrane that was attached to the MALDI
sample target stage and used it for N-terminal sequencing of
peptides via automated microblotting onto membrane substrates
[60,61].
Rejtar et al. introduced continuous vacuum fraction collection
from CE coupled to MALDI MS [43], which arose from an on-line
vacuum deposition device introduced by the same group in 1998
[37]. They were able to produce ∼100 ␮m of wide streaks, only discrete
segments of which were consumed in MS. After data analysis
the rest of the sample, containing unidentified peptides as well as
several species chosen for sequence confirmation, was re-analyzed
in the MS/MS mode. An example of the 3D MS-chromatogram with
highly resolved peaks in both directions is shown in Fig. 2. Moreover,
a similar principle, sub-atmospheric deposition interface, was
used in 2008 for coupling of CE to ICP MS [12]. Although the vacuum
(sub-atmospheric) interface demands a large deposition chamber,
it allows for the generation of smaller spots or a narrower streak
due to the solvent evaporating more rapidly than in the case of
deposition at atmospheric pressure.
A device for contact deposition developed by Nagele and
Vollmer [62] read the actual flow rate of the mobile phase from the
pump’s flow sensor in order to adjust the draw speed of the deposition
probe. If the liquid volume per spot was greater than 500 nL,
O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977 3969
Fig. 2. 3D MS-chromatogram of an in-gel digest of an excised 40–50 kDa SDS-PAGE
band of a yeast lysate, analyzed by continuous deposition MALDI MS analysis. Reproduced
with permission from [38].
the capillary tip started to move continuously upwards, keeping
the droplet in contact with the surface.
In 2008, Hattan et al. deposited LC effluent onto both conventional
MALDI plates and plates with collimated-hole structures
(CHS) [63]. These plates contained monolithic chromatography
media filling the holes and provided additional capacity for capturing
and concentrating samples. Using these three-dimensional
targets, they achieved about a 30-fold enrichment of sample concentration
in the matrix crystals on the surface in comparison to
the conventional stainless steel MALDI targets.
An off-line interface incorporating sheathless flow and counterflow
balance was developed to couple CE to MALDI Fouriertransform
ion-cyclotron resonance (FT-ICR) MS for neuropeptide
analysis of complex tissue samples [64]. The interface integrated
a porous cellulose acetate membrane joint constructed near the
capillary outlet for the electrical circuit completion which simplified
the CE interface by eliminating a coaxial sheath-liquid. The
electro-osmotic flow (EOF) at reversed polarity (negative) CE mode
was balanced and reversed by a pressure-initiated capillary siphoning
(PICS), which generated a low flow (<100 nL/min) for fraction
collection. Thus, the sample deposition occurs at a speed appropriate
to chromatography giving enough time for FT-ICR MS to record
high-resolution spectra.
4.1. Target electrocoupling
A few off-line CE interfaces involving no supplemental sheath
flow have been introduced. The electrical circuit was maintained
by deposition onto a conductive target. Such fraction collection of
CE effluent was first performed by Zhang and Caprioli in 1996 [65].
A sample track was produced by linear movement of the target
during the electrophoretic separation and subsequently analyzed
by MALDI MS.
In 2001, the capillary was put into a droplet of buffer solution
or matrix solution in order to make an electrical connection and
to apply a high voltage between the metallic sample plate and the
counter pole of CE [66]. The applied high voltage was automatically
turned off during the transfer of the capillary outlet between
droplets. Separated components were collected and concentrated
in droplets on the plate.
Another example of droplet electrocoupling (DEC) for CE MALDI
MS with a structurally silicone coated target was presented by Jacksén
et al. [67] The migration times from a UV-electropherogram
were used to pinpoint the fractionation start time and the number
of fractions needed to cover all UV-detected peaks. With a transfer
time of ∼0.05 s between two fractions, evaporation during this
short transfer time resulted in a minute concentration of analytes
and the voltage was not turned off during the transfer. To avoid the
formation of a sparking spray-like sample transfer or air bubbles,
the capillary was positioned as close to the MALDI plate as possible
without actually touching it.
In 2006 an elastomeric device reversibly sealed on the MALDI
target, capable of concentrating and purifying samples directly on
the MALDI target, was described [68]. A poly(dimethyl siloxane)
(PDMS) sample concentrator with an array of wells was sealed on
the MALDI target; sample solutions were deposited in the wells
that match the spots on the MALDI target. The device was used for
the collection of sialylated glycopeptides separated by CE on the
MALDI target [69].
A special case of DEC was described by Chiu et al. [70]; the capillary
outlet was coated with conductive epoxy thus requiring no
capillary fracture, liquid junction, or sheath-liquid. Busnel et al.
[71] modified the setup by painting the outer outlet part of the
separation capillary with a silver conducting paste to adapt the
interface to neutrally coated capillaries producing no significant
electro-osmotic flow.
5. Non-contact deposition
5.1. Electrospray
A conductive spraying technique was used to spray the CE effluent
onto a matrix-coated MALDI target, which moved periodically
underneath the spraying tip [72–74]. A steel plate with a small
hole in it prevented the aerosol from overspreading. However, the
masked sample was lost and this contributed to the relatively low
sensitivity of the method, especially due to dilution effects and
the excessively wide sample distribution. Signal intensity and sample
homogeneity would have been enhanced if the entire sprayed
output had been collected into sample wells [75].
Off-line narrow-bore size exclusion chromatography (SEC)
MALDI MS analysis was performed by Lou and van Dongen [76]. The
SEC effluent, as well as a suitable matrix solution added coaxially
through a T-junction with no dead volume, was directly deposited
on the MALDI plate.
DeVault et al. [77] modified the conditions to produce a narrow
(20 ␮m) liquid filament (electrofilament), which was capable of
depositing spatially preserved bands with ∼100 ␮m track widths.
Even though it was not coupled to MS, it enabled recording CE
separations without decreasing its efficiency.
Electrospray deposition was also used to couple LC with DIOS
MS. A spatially preserved linear track of the separated sample was
generated on a specially designed DIOS chip [78].
Wei et al. [79] electrosprayed nanoliter volumes of
matrix/analyte mixture from a high voltage (1–2 kV) fused-silica
capillary onto a grounded MALDI plate mounted 100–500 ␮m from
the capillary outlet. Electrospray deposition with these conditions
produced sample spots 200–300 ␮m in diameter.
5.2. Nebulizers
Wall et al. presented a heated nebulizer interface, where LC
effluent was continuously collected as linear tracks on a MALDI
3970 O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977
target [80]. MALDI target plates were pre-coated with a matrix
prior to sample deposition and a fine mist of droplets that rapidly
evaporated was sprayed onto the target plate. The heated capillary
nebulizer provided rapid on-line desolvation of effluent from flow
rates of 200 nL/min up to 50 ␮L/min which permitted splitless, continuous
deposition of LC separations of peptide and protein digest
samples at nanoflow and capillary scales.
A simple interface based on an oscillating capillary nebulizer
(OCN) [81], was first used for deposition of a post-column mixture
of cLC effluent and MALDI matrix onto a MALDI target [82]. Later,
the effluent from a thermal field-flow fractionation system was premixed
with a matrix and deposited directly and continuously onto
a moving MALDI probe [83]. The result was a continuous sample
track representative of the separation process. Subsequent off-line
MALDI MS was performed in automated and manual modes.
5.3. Piezoelectric dispensers
In 1998, a piezoelectric flow-through microdispenser system
was introduced for the detection of attomole amounts of proteins
from a single droplet of analyte mixed with a matrix; the droplets,
ranging from 30 to 200 pL, were formed at a frequency 500 Hz
on a MALDI target plate [84]. In 2000, the piezoelectric microdispenser
was modified to interface capillary LC with MALDI for the
identification of biomolecules [85]. The MALDI target plate was
placed on a computer-controlled high-resolution x–y stage on to
which the column of effluent was deposited as discrete spots. The
entire target plate was sprayed with a homogeneous layer of ␣cyano-4-hydroxy-cinnamic
acid mixed with nitrocellulose using
an airbrush. Multiple (1800) droplets of 1 nM peptide mixture
deposited on the same target spot, corresponding to ∼100 amol,
could be detected. Application of the method was extended to the
identification of phosphopeptides generated from a trypsin digest
of recombinant autophosphorylated ZAP-70; chromatography and
on-line tandem mass spectrometry were combined to ascertain the
identity of peptides [86,87].
5.4. Electric field-assisted
An automated platform for high-throughput proteomics combining
LC and MALDI FT-ICR was developed in 2002 by introducing
a charge induction mechanism which transferred the column of
effluent to a MALDI plate by applying a negative potential to
the plate [23,88]. The effluent from the LC formed an electrically
grounded droplet. An electric field formed by the application of
a negative voltage (−2 kV) pulse to a MALDI target (positioned
2–5 mm from the outlet) polarized the droplet and pulled it onto the
target. This resulted in a series of droplets of precisely controlled
volume. Surface-modified targets were used to facilitate concentration
and precise positioning of samples. High ionic strength
solutions and pure organic solvents could be deposited, enabling
their use in various modes of chromatography as well as on-plate
automated recrystallizations. Moreover, the deposition platform
was capable of electrospraying the effluent as continual streams.
5.5. Heated droplet
LC–MS coupling using a heated droplet interface developed by
Zhang et al. [89] was based on the formation of hanging droplets
at the outlet of an LC effluent transfer tube. The transfer tube and
the MALDI plate were heated in a controlled manner in order to
evaporate the carrier solvent without spraying, ionizing, nebulizing,
splattering or uncontrolled boiling of the carrier solvent. Even
with liquid flow rates of up to 50 ␮L min−1, about 800 ␮m analyte
spots were formed on the MALDI plate.
Similar to the heated droplet interface, heated sheath gas and a
capillary nozzle were used to remove most of the mobile phase as
well as deposit the GPC-LC effluent onto the pre-coated matrix on
a moving MALDI plate [90].
5.6. Impulse driven
Another non-contact interface introduced by the same group
[91] involved a pulsed voltage operated solenoid, which generated
impulses that transported hanging droplets from the LC outlet
directly to a MALDI plate via a momentum transfer process. This
impulse-driven system is claimed to be compatible with solvents
of varying polarity and viscosity and hydrophobic and hydrophilic
MALDI matrices. The generated droplets with volumes ranging
from 20 nL to 2 ␮L make the interface useful for depositing the
effluent from a capillary LC column that typically operates at flow
rates below 2 ␮L min−1. In 2007, the interface was integrated with
the heated droplet interface resulting in an impulse driven heated
droplet system, which could accommodate a wide range of LC flow
rates giving the user greater flexibility in choosing the methodology
to suit the experimental requirements [92]. The maximum flow
rate of about 5 ␮L min−1 at room temperature could be extended
to a relatively high flow rate of up to 50 ␮L min−1 with heating.
Recently, a solenoid valve and a He-pressurized pump have been
used to dispense sample and matrix for coupling CE to MALDI MS
[93].
5.7. Chip-based systems
The concept of rapid open-access channel electrophoresis has
been introduced with sample mixtures being separated in open
microchannels cut into glass microchips [94]. With a matrix added
to the buffer solution prior to separation, the channel yielded a
complete MALDI sample and was ready for analysis at the end of
the separation after solvent evaporation. The laser was then used to
raster the channel providing mass spectra as a function of channel
position.
CIEF separation on three model proteins in a pseudo-closed
microchannel built on a polymethylmethacrylate (PMMA) chip was
performed in 2003 [95]. The chip consisted of a base with separation
channels and a cover. The cover minimized sample drying and
the effects of atmospheric carbon dioxide. Sample drying was also
reduced by cooling down the chip on a thermoelectric cooler. After
completion of CIEF, the cover was removed and the solvent evaporated.
The base was put onto a commercial MALDI target using
a home-made adapter; a MALDI matrix was deposited by syringe
pump on top of the focused sample in the channel, and a protein
signal was recorded along the channel.
Matrix-free infra-red (IR) LDI of polypeptides and proteins
directly from a polyacrylamide gel after electrophoretic separation
in a microfluidic chip fabricated from PMMA was performed by Xu
et al. [96]. A simple T-structure of gel was prepared on a PMMA chip
and covered with an annealed PDMS cover. After electrophoresis,
the gel could be analyzed when attached either to the PDMS cover
or to the PMMA chip.
MALDI MS in an open microchannel has advanced to off-line
hyphenation between CE and MALDI MS in the so-called closedopen-closed
microchannel system [97]. Two fused-silica capillaries
were connected via a silicon chip comprising an open microcanal,
which was utilized as a MALDI detection window within the CE capillary
system. With a MALDI target integrated in the electrophoresis
setup, electrocoupling was assured and the external sample transfer
from the CE capillary to the target was avoided.
It is worth noting a technique with the potential for coupling
a microseparation to MS—the microfluidic compact disk (CD)
described by Gustafsson et al. [98]. The device involved parallel
O.Peˇs,J.Preisler/J.Chromatogr.A1217(2010)3966–39773971
Table 1
Commercially available microfraction collectors—spotters.
Manufacturer Model Max. spotting
rate/Hz
Min. fraction
volume/nL
Plate capacitya Flow rate Matrix flow
rate
Positioning precision
(X/Y/Z axis/␮m)
Spotting needle T-junction
volume
Deposition Manufacturer’s
comments
Agilent 1200 Series
Micro
collector/spotter
0.33 100 4 MTP 100 ␮L/min 100 ␮L/min
total flow
Not specified Quartz, 360 ␮m
O.D.
29 nL Discrete Possibility of two
spotters connected via a
micro valve.
Applied
Biosystems
TempoLC MALDI
spotter
4 0.8 8 ABI large/16
ABI small
200 nL–3 ␮L/min 200 nL–20 ␮L/min 10 Teflon coated
fused silica,
360 ␮m O.D.,
50 ␮m I.D.
Less than 500 nL Discrete/
continuous
Tested with monolith
columns.
Bruker
Daltonics
Proteineer fcII 0.3 200 4 Bruker MTP 200 nL–50 ␮L/min 1000 nL–120␮L/min 100 Fused silica,
50 ␮m I.D.
Few nL Discrete Controlled by
HyStar-Software
Package.
CTC Analytics PAL MALDI
Option
0.33 5 23 shallow/11
deepwell MTP
20 nL–300 ␮L/min 20 nL–300 ␮L/min 100 Stainless steel
capillary, 50 ␮m
I.D.
10 nL–3 ␮L Discrete
Dionex ProBot 0.25 5 2 MTP/6 ABI
small
200 nL/min 248 nL–308 ␮l/min. 20 Fused silica,
30 ␮m or
100 ␮m I.D.
30 nL Discrete/
continuous
Eksigent Tech-
nologies
Ekspot 0.25 25 8 MTP/16 ABI
small/4
deepwell MTP
200 nL–4 ␮L/min 400 nL–50 ␮L/min 100 Fused silica 17 nL Discrete Possibility to collect into
MT plates at low flow
rates due to a sideway
capillary movement
once lowered into a well.
Gilson 223 fraction
collector
0.555 200 1 Bruker MTP 1000 nL–100 ␮L/min 5000 nL–50 ␮L/min 250 Spring loaded
probe, 1/32”
O.D., 200 ␮m I.D.
3 ␮L Discrete
Shimadzu AccuSpot 0.33 10 9 MTP/18 ABI
small
50 nL–10 ␮L/min 100 nL–10 ␮L/min 100/120/0b Fused sillica,
285 ␮m O.D.,
25 ␮m I.D.
On-target
matrix addition
Discrete A plate sensor
controlling the distance
of the tip to the metal
target.
SunChrom SunCollect
MALDI Spotter
1 2 4 MTP 1 nl–100 ␮L/min 1 nL–10 ␮L/min 30/125/39 Quartz, 360 ␮m
O.D.
100 nL Discrete/
continuous
Tissue spray option,
tested for MALDI
imaging, 260 ␮m O.D.
needle; micro fraction
collection into any MTP
of fractions from 2 nL up
to 1 mL.
a
MTP: microtitration well plate-sized target (any manufacturer), ABI small: dimensions 44 mm × 44 mm, ABI large: dimensions 123 mm × 81 mm.
b
With a plate sensor employed.
3972 O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977
sample preparation and MALDI MS of 96 samples with volumes
ranging from one to several microliters. Centrifugal force was
applied to move liquid through multiple microstructures, each
containing a 10-nL reversed phase chromatography column. The
peptides in the digests were concentrated, desalted, and subsequently
eluted from the columns directly into MALDI target areas
on the CD using a solvent containing the MALDI matrix. After crystallization,
the target areas of the CD were inserted into the MALDI
instrument for peptide mass fingerprinting and database identification.
The microfluidic disk was also used for the detection of
phosphorylated peptides with combined immobilized metal affinity
chromatography (IMAC) and enzymatic dephosphorylation [99].
Phosphoproteins were digested with trypsin, processed by nanoliter
scale IMAC on the CD with and without subsequent in situ
alkaline phosphatase treatment and detected in MALDI MS.
An automated proteolytic digestion bioreactor and droplet
deposition system was constructed with a PMMA microfluidic
device for off-line interfacing to MALDI MS [100]. The bioreactor
was mounted in the micro fraction collector for protein digestion
and deposition of the digests with a matrix solution on the MALDI
plate.
At-line coupling of LC to chip electrospray MS was performed
using a commercially available Advion TriVersa Nanomate® chip
electrospray system [101–105]. In the case of ultra-high pressure
LC (UPLC) coupled to FT-ICR MS [102], the LC gradient effluent was
collected in microliter fractions onto a 96-port plate which was
put into the autosampler of a chip-based nanoelectrospray ionization
system serving the FT-ICR MS. The approach maintained
the advantage of off-line coupling; i.e. the time difference between
the separation speed of UPLC and the data acquisition speed of the
FT-ICR MS buffered to preserve the high efficiency of both methods.
The fractions of target components were collected manually
for greater flexibility which is needed during gradient elution.
6. Commercial spotters
Automated robotic interfaces allow direct collection and transfer
onto MALDI plates. The analyte, with or without a matrix, is
deposited by a capillary needle which is mounted in a z-axis direction.
In most systems the needle is held stationary while the MALDI
target plate moves underneath. Generally, all commercially available
instruments are flexible in using various types of MALDI target
plates. The connection between separation output and deposition
input is usually provided by a T-junction. The matrix solution can
be instantly mixed with the separation effluent from a side input of
the T-junction, deposited onto a target before sample introduction
or added to fractions in a separate step. In the first approach, the
deposited mixture is ready for MALDI MS immediately after evaporation.
However, in CE, a matrix addition inside the T-junction can
result in a change of separation electrolyte composition inside the
separation capillary, thus affecting the migration times and peak
resolution.
Table 1 lists, to the best of our knowledge, all current commercially
available spotters and information collected from the
manufacturers. Although the commercial spotters are primarily
intended for interfacing LC to MALDI MS, a slight modification
of the LC–MS spotting device, involving sheath flow interface
grounding, allows off-line CE–MS to be performed. Nevertheless,
most of the spotters used for off-line CE–MS are laboratory-
built.
7. Applications
Previous reviews [106,107] covering off-line coupling of column
separations and MS were mostly focused on the development
Fig. 3. Comparison of the number of overlapping and unique protein assignments
obtained by LC/ESI/MS/MS and LC/MALDI/MS/MS analysis of the 39S
subunit of bovine mitochondrial ribosomes. Reproduced with permission
from [110].
of interfaces which were tested on model samples. As the field
matured and the number of commercial spotters in the separation
community grew, off-line systems were applied to solve many real
problems, especially in the proteomic area. In addition to papers
dealing with the interface development for off-line coupling, we
have listed the applications in Table 2. The table does not include
the off-line systems using columns with an I.D. larger than 300 ␮m
and applications in which fractions of microcolumn effluent are
collected into a relatively large volume (>5 ␮L) in microvials prior
to MS processing.
Utilization of microcolumn separation techniques off-line coupled
to MS is suitable in peptidomics, proteomics, metallomics, as
well as in low mass screening applications such as metabolomics,
pharmacokinetics, and drug development. Although the ESI MS
interface is unquestionably easier to implement [108], the offline
systems have spread rapidly in recent years. Although various
interfaces for on target liquid spotting have been developed, the
majority of applications are performed by cLC MALDI MS using a
T-junction and contact deposition. CE MALDI MS may be an attractive
choice for certain areas. For instance, Amon et al. have shown
that CE–MS can be considered as a competitive and complementary
method to capillary reversed phase HPLC MS for glycopeptide
mapping [109]. Detection limits for the glycopeptides were found
to be near 100 fmol for CE ESI MS and 50–300 fmol for CE MALDI
MS. In the latter case, it was strongly dependent on the size of the
glycopeptide.
Analytical schemes combining off- and on-line approaches have
been performed by many groups. For instance Bodnar et al. evaluated
the improvement in proteome coverage of complex protein
mixtures gained by analyzing samples using both LC ESI MS/MS
and LC MALDI MS/MS [110]. Parallel analyses of a single sample
were accomplished by interfacing a commercially available fractionation
system (Probot) with a nanoscale LC system. The Probot
was configured to perform a post-column split so that a fraction
(20%) of the column effluent was sent for on-line LC ESI MS/MS
data acquisition and the majority of the sample (80%) was mixed
with a matrix solution and deposited onto the MALDI target plate.
The split-flow approach takes advantage of the concentration sensitive
character of ESI and provides sufficient quantity of sample for
MALDI/MS/MS; the complementary nature of the ionization techniques
results in a significantly improved protein coverage, as seen
in Fig. 3.
O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977 3973
Table 2
Applications of microcolumn separations coupled to desorption MS.
Analyte Sample/objective Instrumentation Fractionation Year Reference
Protein digest Escherichia coli SCX cRPLC MALDI Probot 2009 [111]
Protein digest Corynebacterium glutamicum RP cRPLC MALDI Probot 2009 [108]
Protein digest Ventricular cerebrospinal fluid CE MALDI Probot 2009 [112]
Proteins digest Cervical-vaginal fluid cRPLC MALDI 2009 [113]
Protein digest Escherichia coli cRPLC MALDI Probot 2009 [114]
Protein, protein digest Follicular fluid IEF CE MALDI Probot 2009 [115]
Protein digest Bax HCT116 clones SCX cRPLC MALDI Impulse-driven heated
droplet
2009 [116]
Peptides, proteins Saliva cRPLC MALDI Probot 2009 [117]
Melanoma-specific
markers, protein
digest
Plasma SCX cRPLC MALDI Probot 2009 [118]
Protein digest Honeybee royal jelly cRPLC MALDI Laboratory-built
contact
2009 [119]
Protein digest Ribosomal subunit of Escherichia coli,
human protein mixture, HEK293 cells
cRPLC MALDI 1100 Series 2009 [120]
Protein digest Neuronal cells cSCX cRPLC MALDI Probot 2009 [121]
Protein digest Yeast mitochondria, bovine serum
albumin
cRPLC MALDI 2009 [122]
Protein digest Aqueous humor cRPLC MALDI 1100 Series 2009 [123]
Protein digest Mouse cortex cRPLC MALDI Probot 2009 [124]
Protein digest Bovine milk cRPLC MALDI Probot 2009 [125]
Protein digest Tumor-bearing kidneys cRPLC MALDI Probot 2009 [126]
Neuropeptides Rhodnius prolixus cRPLC MALDI Probot 2009 [127]
Small molecules Yeast CE MALDI Laboratory-built
contact
2009 [128]
Myoglobin digest Sphingomyelin-coated CE MALDI 1100 Series 2009 [129]
Neuropeptides Cancer borealis CE MALDI PICS 2009 [130]
Small molecules Sophora japonica CE MALDI Laboratory-built
non-contact
2009 [131]
15
N-labeled protein
digest
Methanococcus maripaludis cRPLC MALDI FT-ICR Probot 2009 [132]
Neuropeptides Cancer borealis CE MALDI FT-ICR PICS 2009 [133]
Hepatocyte proteins Human liver RP cRPLC MALDI Probot 2009 [134]
Chromium species CE SALD ICP Laboratory-built
sub-atmospheric
contact
2008 [12]
Oligosaccharides cNPLC MALDI Probot 2008 [135]
Peptides Blood cells cRPLC MALDI 2008 [136]
Glycosphingolipids Erythrocytes cRPLC MALDI Proteineer fc 2008 [137]
Laminin ␤1 digest cRPLC MALDI Proteineer 2008 [138]
Proteins Liver tissue SCX cRPLC MALDI Probot 2008 [139]
Phosphorylated
peptides, protein
digest
cRPLC MALDI Probot 2008 [140]
Proteins, peptides Urine cRPLC MALDI Probot 2008 [141]
Proteins cRPLC MALDI Tempo LC MALDI 2008 [142]
Protein digest Arabidopsis cRPLC MALDI Probot 2008 [143]
Protein digest Escherichia coli cRPLC MALDI Probot 2008 [144]
Protein digest Porphyromonas gingivalis cRPLC MALDI Proteineer fc 2008 [145]
Protein digest Cerebrospinal fluid cRPLC MALDI Probot 2008 [146]
Protein digest Bovine conceptus fluids cRPLC MALDI 1100 Series 2008 [147]
Man-6-P glycoprotein
digests and
glycopeptides
Mouse serum SCX cRPLC MALDI Probot 2008 [148]
Protein, protein digest Follicular fluid IEF cRPLC MALDI 1100 Series 2008 [149]
Fiber connexins Bovine lens cRPLC MALDI Laboratory-built
contact
2008 [150]
Arabinoxylans cNPLC MALDI Probot 2007 [151]
Protein digest White spot syndrom virus cRPLC MALDI Probot 2007 [152]
Glycated peptides Serum albumin cRPLC MALDI AccuSpot 2007 [153]
Peptides Serum cRPLC MELDI 1100 Agilent 2007 [154]
Peptides, protein digest cRPLC MALDI AccuSpot 2007 [155]
Protein digest D20 liver cancer tissue SCX cRPLC MALDI Probot 2007 [156]
Protein digest Apoptotic Jurkat T cells cRPLC MALDI Probot 2007 [157]
Protein digest Vitreous humor cRPLC MALDI Probot 2007 [158]
Protein digest Arabidopsis thaliana seed extracts cRPLC MALDI Probot 2007 [159]
Protein digest Breast-cancer cell lines MCF-7/WT cRPLC MALDI Micro-FC, G1364D 2007 [160]
Protein digest Red blood cell membrane proteins SCX cRPLC MALDI Probot 2007 [161]
Protein digest Sinorhizobium fredii cRPLC MALDI Probot 2007 [162]
Protein digest cRPLC MALDI 1100 Series 2007 [163]
Proteins Examination of DropStop foil CE MALDI Probot 2007 [164]
Peptide mixture Prediction of retention times cRPLC MALDI Laboratory-built
contact
2007 [165]
Glycoprotein,
proteinase P69B
Tomato cRPLC MALDI Laboratory-built
contact
2006 [166]
3974 O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977
Table 2 ( Continued ).
Analyte Sample/objective Instrumentation Fractionation Year Reference
Sialylated
glycopeptides
Bovine ␣1-acid glycoprotein CE MALDI Target electrocoupling 2006 [69]
Serum constituents Serum cRPLC MELDI Probot 2006 [167]
Protein digest Plasma and serum cRPLC MALDI Probot 2006 [168]
Proteins Esophageal adenocarcinoma
epithelium
cRPLC MALDI Laboratory-built
non-contact
piezoelectric
2006 [169]
Proteins, protein digest cRPLC MALDI Probot 2006 [170]
N-glycans Honey bee phospholipase A2 cNPLC MALDI Probot 2006 [171]
Peptide biomarkers Urine CE MALDI Probot 2006 [172]
Bovine serum albumin
digest
Microchip LC MALDI Laboratory-built
electric field-assisted
2006 [173]
Protein digest Singapore grouper iridovirus cRPLC MALDI Probot 2006 [174]
Glycopeptides Plasma CE MALDI Probot 2006 [109]
Proteins Rat liver tissue cRPLC CIEF MALDI Probot 2006 [175]
Protein digest HCT116 cell lysate cSCX cRPLC MALDI Laboratory-modified
FC204
2006 [176]
Glycated serum
albumin
cRPLC MALDI AccuSpot 2006 [177]
Protein digest Candida albicans cRPLC MALDI 2006 [178]
Protein digest Rat liver proteasome cRPLC MALDI 1100 Series 2006 [179]
Colicin E9 protein
digest
Escherichia coli cRPLC MALDI Probot 2006 [180]
Protein digest Multipotent adult progenitor cell line SCX RP cRPLC MALDI 1100 Series 2006 [181]
Proteins Rat liver SCX cRPLC MALDI Probot 2006 [182]
Neuropeptides Brain rat tissue cRPLC MALDI Laboratory-built
electric field-assisted
2006 [183]
Peptide mixtures Kinetics of deamidation cRPLC MALDI Laboratory-built
contact
2006 [184]
Peptide mixtures Prediction of retention times cRPLC MALDI Laboratory-built
contact
2006 [185]
Protein digest Escherichia coli Multi cRPLC MALDI Laboratory-built 2005 [24]
Protein digest Squamous carcinoma cells SCX cRPLC MALDI Heated droplet 2005 [186]
Protein digest Escherichia coli cRPLC MALDI Heated droplet 2005 [187]
Polydisperse
fluorosurfactant
FC134
CE MALDI Laboratory-built 2005 [188]
Protein digest Yeast SCX/SAX/cSCX cRPLC MALDI Probot 2005 [189]
Tumor peptides Renal cell carcinoma cRPLC MALDI 1100 series spotter and
Probot
2005 [190]
Proteins Blood serum CIEF MALDI Laboratory-built 2005 [54]
Protein digest Milk cRPLC MALDI Heated droplet 2005 [191]
Protein digest GST fusion proteins cRPLC MALDI Laboratory-built
contact
2005 [192]
Protein digest Human serum cRPLC MALDI Probot 2005 [193]
Protein digest teratocarcinoma cell lines cRPLC MALDI Probot 2005 [194]
Protein digest Escherichia coli cRPLC MALDI Laboratory-built
contact
2005 [195]
Membrane protein
digest
Staphylococcus aureus N315 cRPLC MALDI Laboratory-built 2005 [196]
Membrane protein
digest
HT29 cells cRPLC MALDI Heated droplet
interface
2004 [197]
Proteins Tear fluid CE MALDI Probot 2004 [46]
Penicillin-binding
proteins, digest
Escherichia coli SCX cRPLC MALDI Probot 2004 [198]
Glycopeptides Asialofetuin digest cRPLC MALDI Probot 2004 [199]
Lectin affinity purified
glycoprotein digest
K562 cells cRPLC MALDI Laboratory-built
contact
2004 [200]
Peptide mixtures Prediction of retention times cRPLC MALDI Laboratory-built
contact
2004 [201]
Peptide mixtures Site-specific glycosylation cRPLC MALDI Laboratory-built
contact
2004 [202]
Protein digest Murine brain prion protein, tracheal
epithelium gland secretion
SCX cRPLC MALDI Probot 2003 [203]
Protein digest Bovine mitochondrial ribosomes cRPLC MALDI Probot 2003 [110]
Nonionic emulsifiers Cremophor EL CE MALDI Probot 2002 [204]
8. Conclusions
Recent technological advancements, namely increased repetition
rate of desorption experiments and faster data acquisition and
processing promote the expansion of microcolumn technique offline
interfacing. The inability to adequately sample all proteins
in a complex matrix due to the high dynamic range of protein
concentrations and to identify the same proteins reproducibly in
different laboratories using different MS platforms re-enables the
off-line systems to enter the science foreground. Ultimately, a similar
degree of automation as with LC ESI MS may allow researchers to
take full advantage of the benefits offered by the off-line interface.
While ESI is still considered the foremost ionization technique in
MS coupling, off-line coupling has increased utilization in various
fields of chemistry, especially due to dissimilar ionization efficiencies
of ESI and MALDI and the decoupling of separation and MS.
O. Peˇs, J. Preisler / J. Chromatogr. A 1217 (2010) 3966–3977 3975
It includes avoiding a throughput mismatch where a too slow or
a too fast mass spectrometer is coupled on-line to LC. Since the
MS is performed separately, the use of high speed MS, such as
TOF, can significantly decrease the time needed for analysis and
re-analysis of the separation record. Moreover, the separation can
be archived and/or exposed to other analytical techniques, such as
laser ablation/desorption ICP MS. Alternatively, it can be subjected
to on-target chemical reactions prior to MS.
Acknowledgments
We gratefully acknowledge the financial support of the Czech
Science Foundation (Grants No. 203/09/1025 and P206/10/J012)
and the Ministry of Education, Youth and Sports of the Czech Republic
(MSM0021622415).
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2.2 Off-Line Coupling of Capillary Electrophoresis to Substrate-Assisted Laser Desorption
Inductively Coupled Plasma Mass Spectrometry
Interfacing different MS techniques to separation methods have always been challenging by means of online coupling.
Flow splitting or additional detectors connected in series may deteriorate the entire method’s sensitivity, robustness,
or applicability. A throughput mismatch may be an issue if too slow or too fast detection technique is used in an
online-hyphenated separation. Another option is recording the separation on a suitable target, which may serve for
archiving or reanalysis by a complementary technique. Analysis of metalloproteins is very challenging due to the
presence of a low molecular metal (or a metal-containing group) and a large molecule of protein – both requiring
a hard and soft ionization technique to be determined by MS. A sub-atmospheric chamber, originally capable of
depositing microcolumn separations and analyzing them by MALDI MS, was developed in 2000.32,33
Nowadays,
many commercial spotting devices capable of storing liquid separations for different analyses have been readily
available.34
A specific type of disposable, co-polymer target has been tested in assisting the laser ablation process in laser
ablation (LA) ICP MS. This surface-assisting process resulted in mere desorption of the deposited sample rather
than ablation of the bulk material. Since the laser energies may have been kept as low as tens of MW · cm−2
, the
limits of detection (LOD) reached approximately an order of magnitude lower than in the case of common LA
ICP MS involving high energies (units to tens of GW · cm−2
). Even at low laser energies, a reliable transfer of the
sample to an ICP cell was assured. This surface-assisted laser desorption (SALD) mechanism has been proposed,
investigated, and finally applied in a model speciation analysis to demonstrate the feasibility of a complementary
analysis of metalloproteins. The SALD technique was further applied in myeloid leukemia cells,35
improved by cold
plasma,36
employed in analysis of nanoparticles,37
or analysis of sterols.38
PES, Ondrej, Pavla JUNGOVA, Radek VYHNANEK, Tomas VACULOVIC, Viktor KANICKY and Jan PREISLER.
Off-Line Coupling of Capillary Electrophoresis to Substrate-Assisted Laser Desorption Inductively Coupled Plasma
Mass Spectrometry. Analytical Chemistry. 2008, 80(22), 8725–8732. ISSN 0003-2700. doi:10.1021/ac801036x
Document Type: Article, IF = 5.712; JCR Category + Category Quartile: CHEMISTRY, ANALYTICAL Q1; AIS
= 1.681
Author’s contribution: 45 %
18
Off-Line Coupling of Capillary Electrophoresis to
Substrate-Assisted Laser Desorption Inductively
Coupled Plasma Mass Spectrometry
Ondrˇej Pesˇ, Pavla Jungova´ , Radek Vyhna´ nek, Toma´ sˇ Vaculovicˇ , Viktor Kanicky´, and
Jan Preisler*
Department of Chemistry, Faculty of Science, Masaryk University, Kotla´rˇska´ 2, 611 37, Brno, Czech Republic
A novel off-line coupling of capillary electrophoresis (CE)
and inductively coupled plasma mass spectrometry
(ICPMS) is reported here. The coupling interface is based
on the connection of a separation capillary to a deposition
capillary via a liquid junction maintaining high separation
efficiency and sample utilization due to the self-focusing
effect and lack of pressure-induced flow in comparison
with nebulizer-like interfaces. The separation is recorded
in the form of droplets of CE effluent on a suitable
substrate s a poly(ethylene terephthalate) glycol (PETG)
sample plate placed inside a partially evacuated chamber.
Substrate-assisted laser desorption (SALD) is used to
vaporize the sample fractions and to enable further
transfer to the ICPMS. The mechanism of SALD is
examined using model samples deposited on a variety of
substrates. The highest response is obtained for a PETG
substrate; sample desorption due to ablation of PETG is
found to outweigh direct ablation of sample. Detection
limits are given for several metal elements. Finally, a rapid
(2.5-min), high-resolution separation of CrIII
/CrVI
species
injected in subpicomolar quantity is shown.
Microcolumn separation techniques, such as capillary electrophoresis
(CE) or microliquid chromatography (LC), continue to
mature as important analytical tools in separation science. Advantages
of the techniques include rapid analysis, minute sample
size requirements, high separation efficiency, low limits of detection,
and economical operating costs. With mass spectrometric
(MS) detection, both CE and LC belong to the most powerful
techniques for the identification of unknown substances in
complex matrixes.1
Most hyphenated techniques usually combine a separation
module with an online spectrometric detection technique, such
as electrospray ionization MS or inductively coupled plasma (ICP)
optical emission spectrometry (OES) or MS, to obtain structural
or quantitative information. For quantitative elemental analysis,
ICPMS is preferred to electrospray or ion spray mass spectrometry,
because the signal from these techniques does not necessarily
have to be related to the total elemental concentration of
the metal, even if a bare metal peak is observed.2
An increasing demand for information about element speciation
at trace levels leads to coupling microcolumn separation techniques
to ICP.3
The toxicity of most elements depends on their
chemical form; in some cases, one form of the metal can be toxic
whereas the same metal in a different form is nontoxic or even
essential for proper functioning of living organisms. The determination
of the concentration of specific chemical forms is vital
for the interpretation of their biochemical behavior or assessment
of the potential danger to organisms.
In the literature, all approaches considering online coupling
of LC4,5
or CE6-8
with ICPMS assume the efficient introduction
of analytes into the ICP. Furthermore, the run buffer, micellar
agent, salt concentration, pH, complexing agents, and analyte
should not interfere with the ICP measurement, and ICPMS
interface should not influence the performance of the separation.
In the case of CE, utilization of a proper enrichment preconcentration
technique (such as sample stacking, on-column transient
isotachophoresis, field amplitude sample injection, precolumn
extraction, or concentration) will overcome the low concentration
sensitivity and limited capacity of CE.9
There are three main requirements that a CE-ICP interface
design has to satisfy.10,11
First, the interface must provide an
electrical connection and a stable electrical current for reproducible
electrophoretic separations. Second, the interface must adapt
the flow rate of the electroosmotic flow inside the CE capillary to
the flow rate of the nebulizer, and finally, the interface should
prevent the occurrence of laminar flow inside the CE capillary
caused by the nebulizer.
* To whom correspondence should be addressed. E-mail:preisler@
chemi.muni.cz. Tel.:+420 549 496 629. Fax: +420 549 492 494.
(1) Lehmann, R.; Liebich, H. M.; Voelter, W. J. Capillary Electrophor. 1996,
3, 89–110.
(2) Olesik, J. W.; Thaxton, K. K.; Olesik, S. V. J. Anal. At. Spectrom. 1997,
12, 507–515.
(3) Michalke, B. Electrophoresis 2005, 26, 1584–1597.
(4) Sutton, K.; Sutton, R. M. C.; Caruso, J. A. J. Chromatogr., A 1997, 789,
85–126.
(5) Montes-Bayon, M.; DeNicola, K.; Caruso, J. A. J. Chromatogr., A 2003,
1000, 457–476.
(6) Kannamkumarath, S. S.; Wrobel, K.; B’Hymer, C.; Caruso, J. A. J. Chromatogr.,
A 2002, 975, 245–266.
(7) Alvarez-Llamas, G.; de laCampa, M. D.; Sanz-Medel, A. TrAC-Trends Anal.
Chem. 2005, 24, 28–36.
(8) Olesik, J. W.; Kinzer, J. A.; Olesik, S. V. Anal. Chem. 1995, 67, 1–12.
(9) Barnes, R. M. Fresenius J. Anal. Chem. 1998, 361, 246–251.
(10) Schaumloffel, D.; Prange, A. Fresenius J. Anal. Chem. 1999, 364, 452–
456.
(11) Prange, A.; Schaumloffel, D. J. Anal. At. Spectrom. 1999, 14, 1329–1332.
Anal. Chem. 2008, 80, 8725–8732
10.1021/ac801036x CCC: $40.75  2008 American Chemical Society 8725Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
Published on Web 10/15/2008
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Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.
Several interface constructions based on concentric,12-16
microconcentric, 10,11,17-22
cross-flow,14,17
or ultrasonic23
nebulizers
have been presented and widely used, but the pressureinduced
laminar flow inside the capillary still has a negative
influence on the overall performance of such interfaces, in terms
of separation efficiency and precision of the CE-ICPMS system.
The laminar flow is usually counterbalanced using a back pressure
of the makeup liquid or by applying negative pressure to the inlet
vial. The interface construction introduced by Schaumlo¨ffel and
Prange10,11
addresses the issue of detrimental suction effects via
optimization of the fluid mechanics in the interface. This leads to
negligible laminar flow inside the CE capillary, and the migration
times of the analytes are thus both stable and reproducible. This
interface has been commercialized (CEI-100, CETAC Technologies,
Omaha, NE). Finally, the electrophoretic resolution has
usually been traded off for analysis speed since ions with similar
mobilities do not have to be electrophoretically separated. A direct
injection nebulizer, introduced in 1995 by Avila and co-workers,24
is probably the most efficient liquid sample introduction method
for CE; however, the exact amount of the injected sample was
not reported.
In the case of off-line configuration, the analyte separated by
CE could be collected in vials, and elements determined from
separated fractions (after dilution to 100 µL) by electrothermal
vaporization ICPMS.25-27
Advantages include easier sample
handling than in an online approach, with no special interface like
a nebulizer required. Since the CE was run with online UV
detection independently on the ICPMS detector, migration times
and UV spectra were obtained prior to the element detection with
ICPMS.
In another area of chemistry, proteomics, approaches dealing
with fraction collection of CE effluent via a deposition interface
have been presented,28,29
and commercially available deposition
devices, spotters, have been introduced by virtually all the major
manufacturers and are widely used in LC-MS.30
Fractions can
be collected on a wide variety of substrates, such as matrix-assisted
laser desorption/ionization (MALDI) targets,31
poly(vinylidene
difluoride) membranes,32
or microchip structures. To enable offline
coupling to ICPMS, desorption of the spotted samples has to
be involved to enable vaporizing and transferring the sample spots
from the substrates to element detector.
In common laser ablation, introduced in 1985 by Gray et al.,33
a short-pulsed, high-power laser beam is focused onto a sample
surface. The laser beam converts a finite volume of the solid
sample instantaneously into its vapor-phase constituents. The
vapor is then analyzed by measuring atomic/ionic emissions in
the induced plasma by laser-induced breakdown spectroscopy or
by transporting the vapor to another measurement system, such
as ICPMS. With ICP as an atomization source, laser ablation (LA)
has proved to be one of the most powerful analytical tools that
can serve for nearly nondestructive determination of numbers of
elements with very low detection limits in solids. Applications of
LA ICPMS cover a great range of academic and industrial fields
that include environmental,34-36
geological,37-39
archeological,40,41
forensic,42
and semiconductor manufacturing sectors.43
However,
the primary role of LA is engaged in analysis of sample material
itself, and the possibility of laser desorption of deposited samples
has not been considered.
Off-line coupling of LA ICPMS has been reported for planar
separation techniques, such as gel electrophoresis44-46
and thinlayer
chromatography,47
where a laser was used to ablate bulk
separation medium with incorporated analytes. Recently, effluent
fractions from high-performance liquid chromatography were
pipetted in replicates on a polystyrene substrate, dried, manually
excised, and analyzed using LA ICPMS.48
In all these cases, laser
fluences reached units of GW·cm-2
In this paper, laser desorption is used to vaporize sample spots
or dried separation fractions that had been deposited on a
(12) Lu, Q. H.; Bird, S. M.; Barnes, R. M. Anal. Chem. 1995, 67, 2949–2956.
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Anal. At. Spectrom. 2000, 15, 635–642.
(17) Baker, S. A.; Miller-Ihli, N. J. Appl. Spectrosc. 1999, 53, 471–478.
(18) Polec, K.; Szpunar, J.; Palacios, O.; Gonzalez-Duarte, P.; Atrian, S.; Lobinski,
R. J. Anal. At. Spectrom. 2001, 16, 567–574.
(19) Prange, A.; Schaumloffel, D.; Bratter, P.; Richarz, A. N.; Wolf, C. Fresenius
J. Anal. Chem. 2001, 371, 764–774.
(20) Schaumloffel, D.; Prange, A.; Marx, G.; Heumann, K. G.; Bratter, P. Anal.
Bioanal. Chem. 2002, 372, 155–163.
(21) Wang, Z. X.; Prange, A. Anal. Chem. 2002, 74, 626–631.
(22) Polec-Pawlak, K.; Schaumloffel, D.; Szpunar, J.; Prange, A.; Lobinski, R. J.
Anal. At. Spectrom. 2002, 17, 908–912.
(23) Lu, Q. H.; Barnes, R. M. Microchem. J. 1996, 54, 129–143.
(24) Liu, Y.; Lopezavila, V.; Zhu, J. J.; Wiederin, D. R.; Beckert, W. F. Anal.
Chem. 1995, 67, 2020–2025.
(25) Michalke, B.; Schramel, P. J. Chromatogr., A 1996, 750, 51–62.
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(27) Michalke, B.; Lustig, S.; Schramel, P. Electrophoresis 1997, 18, 196–201.
(28) Foret, F.; Preisler, J. Proteomics 2002, 2, 360–372.
(29) Preisler, J.; Hu, P.; Rejtar, T.; Moskovets, E.; Karger, B. L. Anal. Chem.
2002, 74, 17–25.
(30) Zarei, M.; Kirsch, S.; Muthing, J.; Bindila, L.; Peter-Katalinic, J. Anal.
Bioanal. Chem. 2008, 391, 289–297.
(31) Yeung, K. K. C.; Kiceniuk, A. G.; Li, L. J. Chromatogr., A 2001, 931, 153–
162.
(32) Stevenson, T. I.; Loo, J. A.; Greis, K. D. Anal. Biochem. 1998, 262, 99–
109.
(33) Gray, A. L. Analyst 1985, 110, 551–556.
(34) GarbeSchonberg, C. D.; Reimann, C.; Pavlov, V. A. Environ. Geol. 1997,
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2000, 366, 167–170.
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J. Astrophys. J. 2000, 536, L49–L53.
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Spectrom. 1998, 13, 263–270.
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X. Z.; Hall, C. M.; Ballentine, C. J.; Pettke, T.; Stirling, C. Geochim.
Cosmochim. Acta 1998, 62, 919–940.
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1141.
(44) Schaumloffel, D. J. Trace Elem. Med. Biol. 2007, 21, 18–22.
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1–15.
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585–591.
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F. J. Anal. At. Spectrom. 2007, 22, 1158–1162.
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435.
8726 Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
substrate-sample plate and to enable further transfer to ICPMS.
The role of the substrate, absorbing additives, sample morphology,
reproducibility, and limits of detection for five metal elements will
be examined and discussed. Finally, automated off-line CE-ICPMS
of CrIII
/CrVI
speciation will be demonstrated. CrIII
is considered
an essential trace element for the maintenance of an effective
glucose, lipid, and protein metabolism in mammals.49
On the other
hand, CrVI
can be toxic for biological systems,50,51
and watersoluble
CrVI
is extremely irritating and toxic to human body tissue
owing to its oxidizing potential and easy permeation of biological
membranes.52,53
EXPERIMENTAL SECTION
Chemical Reagents. Solutions of NaOH (Merci), HCl (Lachema),
and HCOOH (Riedel-de Hae¨n) in water (Agilent, Waldbronn,
Germany) were used for separation capillary treatment. EDTA
(Lachema) was used as a complexing agent. Elements of Cr, Co,
Ni, Sn, Cu, Fe, and Zn defined as certified reference materials in
aqueous solutions (Analytika) were used as analytes for ICPMS.
All chemicals were analytical-reagent grade.
Capillary Electrophoresis with Online UV Detection.
Fused-silica capillaries (Polymicro Technologies) with an i.d. of
75 µm and an o.d. of 375 µm were washed with 10 mM solutions
of NaOH and HCl (10 min each) and then filled with 10 mM
solution of formic acid as the separation running buffer. The
sample was injected from unbuffered solution by a pressure of
0.2 kPa for 5 s. Electrophoresis was driven at –480 V·cm-1
by a
high-voltage power supply (model CZE1000R, Spellman, Hauppauge,
NY). Platinum wires served as the electrodes in the
electrolyte reservoirs. Electrophoretic current of 1.7 µA was
measured with an ammeter placed within the circuit. For UV
detection, the total length of the capillary was 26 cm and the
effective length 20 cm. Absorbance at 214 nm was measured with
a CE detector (model Spectra 100, Spectra Physics, Ontario,
Canada).
Capillary Electrophoresis with Off-Line ICP Detection. To
facilitate fraction collection, a liquid junction filled with 10 mM
formic acid was employed as the joining component between the
separation and the deposition capillaries. The same separation
capillary was used as in the above case except that the total and
effective length was 20 cm. The sample was injected again from
an unbuffered solution at 0.2 kPa for 5 s, and the applied voltage
was –480 V·cm-1
.
Subatmospheric Deposition Chamber. A laboratory-built
subatmospheric chamber28,29
served for deposition of precisely
defined sample volumes or collection of separation effluent
fractions similarly to commercial spotter devices (e.g., Probot,
Dionex, Sunnyvale, CA). The deposition of solutions onto a sample
plate (50 × 27 × 0.5 mm) fastened onto the XY-stage in the
subatmospheric chamber was performed via a 29.6-cm-long, 51µm-i.d.,
fused-silica capillary. The end of the deposition capillary
was silanized by immersing into tributylchlorosilane (SigmaAldrich)
for 1 min to minimize potential memory effects. Dissimilar
to commercial spotters, the flow in the deposition capillary was
induced by lower pressure inside the subatmospheric chamber
instead of pressurizing the separation capillary. For ∼20-kPa
pressure inside the cylindrical chamber (45-cm diameter, 19-cm
height) generated with a membrane pump (model N35AT18, KNF
Neuberger, Freiburg, Germany), the aqueous solutions flow rate
of ∼50 nL·s-1
was calculated from Poiseuille’s equation. The
capillary total volume was ∼610 nL and the dead time ∼12.1 s.
The XY-stage and the probe for vertical movement, both operated
by a laboratory-built controller running under a LabView (National
Instruments, Austin, TX) program, were responsible for sample
plate movement and droplet deposition. Four sample plates of
different materials were tested; aluminum and copper plate, glass
slide, and PETG sheet (Bayer sheet, Darmstadt, Germany).
Samples were spotted onto these plates as discrete fractions
in a serpentine-like trail with 1.5-mm spacing in both directions.
At the given flow rate, 2-s fractions of aqueous solutions corresponded
to ∼100 nL of total volume in each fraction. Afterward,
the sample plate was carried over to the ablation cell of the New
Wave system described below.
LA ICPMS. Instrumentation for LA ICPMS consisted of a laser
ablation system (model UP 213, New Wave, Fremont, CA) and
an ICPMS spectrometer (model Agilent 7500 CE). The commercial
Q-switched Nd:YAG laser ablation system works at the
fifth harmonic frequency, 213 nm. The ablation device was
equipped with programmable XYZ-stages to move the sample
along a programmed trajectory during ablation/desorption. Visual
target inspection, as well as photographic documentation was
accomplished by means of a built-in microscope/CCD camera
system. A sample was inserted in the SuperCell (New Wave) and
was scanned by the laser beam, which was focused onto the
sample surface through a quartz window. The ablation cell was
flushed with a carrier gas (helium), which transported the laserinduced
aerosol to the inductively coupled plasma, at a flow rate
1 L·min-1
. A sample gas flow of argon was admixed to the helium
carrier gas flow subsequent to the laser ablation cell. Therefore,
the total gas flow was 1.6 L·min-1
. To reduce possible polyatomic
interference, a helium collision cell was used (gas flow 3
mL·min-1
). Optimization of LA ICPMS conditions (gas flow rates,
sampling depth, electrostatic lens voltages of the MS) was
performed with the glass reference material NIST SRM 612
regarding the maximum signal-to-noise (S/N) ratio and minimum
oxide formation (ThO+
/Th+
counts ratio 0.2%, U+
/Th+
counts
ratio 1.1%). Other ICPMS parameters were adjusted in compliance
with the manufacturer’s recommendations.
If not otherwise stated, the laser fluence was 71 MW·cm-2
and the repetition rate 10 Hz. The ions were measured with an
integration time 0.1 s/isotope. The beam waist was set to a
maximum of 300 µm to establish a large effective area for sample
ablation/desorption. Dwell time 2 s, i.e., the time the sample was
irradiated by the laser beam; ablation cell flush time 3 s and laser
warm-up time 3 s were adjusted. With the 10-Hz laser repetition
rate, dwell time 2 s corresponded to 20 laser shots/sample. In
the case of laser raster scans, dwell times were adjusted according
to the scan duration. Dynamic reaction cell was turned on during
measurements.
(49) Barnowski, C.; Jakubowski, N.; Stuewer, D.; Broekaert, J. A. C. J. Anal. At.
Spectrom. 1997, 12, 1155–1161.
(50) Prasad, A. S.; Ortega, J.; Brewer, G. J.; Oberleas, D.; Schoomaker, E. B.
JAMA-J. Am. Med. Assoc. 1976, 235, 2396–2398.
(51) Gwizdala, A. B.; Johnson, S. K.; Mollah, S.; Houk, R. S. J. Anal. At. Spectrom.
1997, 12, 503–506.
(52) Andrle, C. M.; Jakubowski, N.; Broekaert, J. A. C. Spectrochim. Acta, Part
B: At. Spectrosc. 1997, 52, 189–200.
(53) Mugo, R. K.; Orians, K. J. Anal. Chim. Acta 1993, 271, 1–9.
8727Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
Pneumatic Nebulization ICPMS. The Babington nebulizer
with Scott double-pass spray chamber (Agilent) was used.
Optimization of ICPMS conditions was performed regarding the
maximum S/N ratio for 52
Cr and 53
Cr and in compliance with the
manufacturer’s recommendations. Other ICPMS parameters were
as follows: carrier gas (argon) flow, 0.90 L·min-1
; makeup gas
(argon) flow, 0.19 L·min-1
; nebulizer pump flow, 0.022 mL·s-1
,
and spray chamber temperature, 2 °C. Nebulization efficiency of,
at most 5% was determined for this setup.
RESULTS AND DISCUSSION
While common LA ICP measurements rely on ablation processes
upon the sample itself, in this case, the sample intended
for analysis is deposited on a sample plate and a laser pulse is
used to vaporize the sample. Principally, the transfer can be
performed in three manners. Laser ablation, respectively laser
desorption (LD) of sample itself, substrate-assisted laser desorption
(SALD), or matrix-assisted laser desorption (MALD). In the
two latter cases, a substrate or a supplementary additive with
absorption of light greater than sample are employed, in an
analogy to SELDI and MALDI processes. Initially, sample solutions
were deposited onto different substrates to investigate the
influence of experimental conditions, such as sample morphology,
the nature of laser ablation/desorption processes, and data
acquisition modes.
Model System Selection. For investigating the overall performance
of LA(LD) ICPMS, chromium as an example element
was selected. Chromium belongs to a group of elements, which
can, without difficulty, be determined by ICPMS; CrIII
and CrVI
differ in toxicity and CE may easily be used for Cr species
separation. Chromium speciation using separation techniques has
been addressed in the literature many times; therefore, the simple
system of CrIII
/CrVI
was selected to demonstrate the potential of
the new coupling. The ion signal of two chromium isotopes, 52
Cr
(83.8% natural abundance) and 53
Cr (9.5% natural abundance), was
monitored.
Sample Plate Material Selection. The materials considered
for sample plates were glass, PETG, copper, aluminum, and
aluminum alloys (Al, AlMg, AlNi). Common stainless steel was
regarded as an inappropriate plate material because of a perceptible
content of Cr, which would have presumably increased the
background level. First, LA ICPMS of each empty sample plate
was performed to observe the background intensity of 52
Cr and
53
Cr; ion signals were integrated 0.1 s/isotope. The progress of
the ion signals is shown for PETG and glass substrates on Figure
1A and B. The S/N ratio for 52
Cr and 53
Cr was obtained as the
sum of the average ion intensities recorded with the laser on
divided by the standard deviation obtained from 5 data points
before and 25 points after turning the laser on. Copper and
aluminum alloys were found to contain a significant level of
chromium and therefore were avoided in further experiments.
Mass of 52 amu in the PETG sheet showed major polyatomic
interference as a result of the formation of a polyatomic molecule
40
Ar12
C. This interference occurred even if with the dynamic
reaction cell switched on. Alternatively, 53
Cr could be monitored
as the interference of 40
Ar13
C is less severe. Additionally, the
transparency of glass and PETG was found to be beneficial as it
allowed easy visual inspection of deposited droplets in the ablation
cell. To avoid possible cross-contamination, a fresh PETG sample
plate was always used. The glass slide was reused after cleaning
in ethanol, deionized water, and acetone in an ultrasonic bath.
Sample Morphology. The sample size, shape, and appearance
depended on the solution constitution, hydrophobic/hydrophilic
character of the sample plate, sample plate batch in the case of
PETG, and shape and treatment of the deposition capillary tip.
This presumably influenced sample behavior during the deposition
process, and the acquisition mode (a point or raster acquisition).
As shown in Figure 2, the morphology of the same samples on
various substrates is unique. While evenly spaced 100-500-µmdiameter
dots were formed on the relatively hydrophobic surface
of the PETG sheet, the spots on the glass sheet were spaced more
randomly and spot diameter ranged from 500 to 1000 µm due to
the more hydrophilic nature of glass surface. Sample-to-sample
variations of spot diameter did not exceed 10% for given deposition
conditions. For influencing the sample spot size, acetonitrile
(ACN) was admixed to the Cr solution in 1:1 (v/v) ratio and the
Figure 1. Time record of the ICPMS signal (amu ) 52 and 53) of
(A) bare glass substrate (B), PETG, and (C) 10 µM CrIII
solution
deposited on a PETG plate (see text for details). The laser was turned
on for 2.0 s at t ) 1.0 s.
8728 Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
solution was deposited on glass and PETG plates. Nonetheless,
fast evaporation of the organic solvent caused 2-3 times larger
spots, and therefore, no ACN addition was further employed.
Laser Scan Pattern. A typical experiment performed on a
chromium sample spot is shown in Figure 1C. Signal from ICPMS
was continuously obtained at the noise level of ICP with the laser
turned off. After the laser was turned on, the sample was ablated/
desorbed, and the signal increased proportionally to the amount
of Cr in the sample with ∼1-s delay, in the same manner as in
the case of background measurements. After switching the laser
off 2 s later (the dwell time), the signal decreased and tailed due
to the delayed transfer from the ablation cell to ICP. The
experiment also included the laser warm-up time (3 s) and the
cell flush time (3 s). The reduction of the laser dwell time below
2 s was not imperative as it would not markedly decrease the
overall analysis time.
According to the sample spot size, two different laser scan
patterns were proposed. For sample spots with diameters of e300
µm (which was the maximum size of the adjustable laser beam
waist), a point acquisition was selected; i.e., the laser beam was
focused on the center of the sample spot and the ablation cell did
not move during laser ablation. The entire sample was irradiated
for a certain time (typically 2 s, i.e., 20 laser pulses) and the
desorbed aerosol continuously transferred to the ICP. For spot
diameters larger than 300 µm, the laser beam was moved in an
S-shaped raster (covering typically 700 µm × 700 µm area) to
ablate/desorb the entire sample from the given sample spot. Three
linear speeds of scan 50, 100, and 200 µm·s-1
were tested. The
speed of 100 µm·s-1
was selected because the scanning of one
spot ended in an acceptable time and transfer of the sample to
ICP was still complete (data not shown). Both point and raster
desorption yielded the same integrated ICPMS signal; i.e., the
laser scan pattern did not affect the sample recovery as long as
the entire sample was desorbed. Nevertheless, the raster acquisition
resulted in a relatively time-consuming procedure (10-25
s/spot), which would have considerably increased the overall
analysis time, compared to the point acquisition (2 s/spot). Hence,
PETG was preferred to glass as the substrate because the small
spot size allowed desorption from either a point or a raster of
smaller size.
SALD Mechanism. To characterize the nature of the processes
arising from the interaction of the laser radiation and the
deposited sample, an experiment comparing vaporization of
chromium from low-absorbing glass and highly absorbing PETG
was performed. An aqueous solution of 10 µM CrIII
was deposited
onto glass and PETG substrates in the form of 10-nL droplets.
The size of the created spots was ∼400 µm on PETG substrate
and ∼800 µm on the glass substrate. The samples were desorbed
from both substrates by employing raster scan pattern in three
replicates for each value of laser fluence in the range from 2.4 to
98 MW·cm-2
. As seen in Figure 3, the ICPMS signal reached a
plateau at laser fluence ∼10 MW·cm-2
in the case of PETG
substrate, while a gradual increase of the signal over the entire
range of laser fluence can be observed for the glass substrate.
Yet, the signal magnitude for the glass substrate obtained at the
highest laser fluence is about a third of the value characteristic
for the plateau in the case of the PETG substrate. Relative signal
variation reflects an incomplete desorption and, in the case of glass
substrate, less regular spot shapes. After the first scan was
performed, a second scan within the same raster coordinates was
performed to reveal any remaining analyte. With the exception
of vaporization at laser fluence of 2.4 MW·cm-2
, the integrated
ICPMS signal from the second scan did not exceed 3% of the signal
from the first scan on both substrates, which points to a nearly
complete removal of the sample. In the case of glass, no
destruction of the slide was observed within the applied laser
fluences, which means direct ablation of sample was responsible
for chromium signal. Virtually all sample was removed from the
glass slide even at low laser fluence. The increasing laser fluence
resulted in higher signal probably due to formation of finer
particles of sample. In the case of PETG, ablation of the substrate
plays the key role as it sputters the deposited sample into the
carrier gas. The SALD is the dominant mechanism, although the
role of sample ablation increases at higher laser fluences.
Consequently, laser fluence was set to ∼70 MW·cm-2
to ensure
complete sample desorption and low signal variation for experiments
on PETG. Under these conditions, more than 99% sample
was removed and the relative standard deviation was below 3%.
Another idea of enhancement via supplementary desorption of
another compound was tested with a glass slide as the target plate.
Figure 2. Photographs of 10 µM CrIII
solution deposited on (A) glass,
(B) PETG, and (C) on PETG after laser desorption. Laser desorption
of samples on glass is not shown due to a complete cleanup of
deposited material. Slight vertical cracks on the photographs B and
C were formed after unrolling PETG from a storage coil.
8729Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
The R-cyano-4-hydroxycinnamic acid, commonly used matrix in
MALDI, was prepared as a 1 mg·mL-1
solution in 50% acetonitrile
and added to the Cr sample. Desorption of such prepared samples
was performed. Because a signal increase due to the addition of
absorbing matrix was not reproducible, the use of the absorbing
PETG substrate was preferred for sample vaporization.
Limits of Detection (LODs). For future speciation and
determination of other metals by CE-SALD ICPMS, limits of
detection (3σ) of deposited solutions Cr, Ni, Sn, Cu, and Zn were
determined. Solutions containing all metals in concentrations of
1 and 0.1 µM were deposited onto a PETG plate, and 10 spots
were analyzed separately for each metal. Three signals were
acquired in ICPMS: signals for the two most abundant isotopes
and 13
C as background level. Integration time was 0.1 s/isotope.
Peak height and peak area (calculated as the sum of the rectangles
between neighboring data points) estimations of LODs are
summarized in Table 1. LODs were calculated from the 0.1 µM
solution. For the LODs calculated from the peak height, the
corresponding range of mass LODs is 20-650 fg, and the
concentration LOD ranges are 2-100 nM or 0.2-6.5 µg·L-1
. The
LODs expressed in amounts are 1 order of magnitude lower than
in the case of the desorption from polystyrene,54
probably due to
a more quantitative desorption, different sample morphology, or
ablation system and ICPMS parameters. This, together with a
smaller size of deposited droplets, facilitates coupling with a
microcolumn separation.
In comparison to ICPMS limits of detection obtained with
nebulizer and a quadrupole instrument,55
the concentration LODs
reported in this work are up to 2 orders of magnitude higher. The
reason for such a difference is generally due to the fact that the
nebulizer interface allows signal accumulation over time. It should
be mentioned at this point that, for the intended analyte determination
in CE fractions, low sample volume is also important in addition to
low concentration detection limit. For a more direct comparison of
detection limits, 2-s sample plugs of 0.1, 1.0, and 10 µg·L-1
chromium
nitrate solution in 2% HNO3 were delivered in five replicates into the
stream of 2% HNO3 at a flow rate 22 µL·s-1
. The length of the sample
plugs matched the laser dwell time during the point desorption. The
injected amounts of Cr were 0.084, 0.84, and 8.4 pmol. Based on the
slope determined for the three Cr amounts, the LODs (3σ) calculated
from peak area and height were 190 and 160 fmol for 52
Cr and 140
and 90 fmol for 53
Cr. In contrast to the concentration LODs, the LODs
expressed in amounts are ∼2 orders of magnitude higher for the
nebulizer. This can be attributed mostly to the nebulization efficiency
of the pneumatic nebulizer, which was at most 5% for this setup.
Solvent vaporization might also contribute to further reduction of the
overall ionization efficiency in the case of wet aerosol from the
nebulizer.
Speciation Conditions. The optimal conditions for chromium
speciation using CE-SALD ICPMS were selected according to the
above results concerning the reproducibility of signal during
ablation/desorption, spot size, and the laser energy. The PETG
plate, the laser fluence 71 MW·cm-2
, and the dwell time, 2 s,
were chosen as the optimized conditions for further CE-SALD
ICPMS speciation.
CE Separation. Capillary zone electrophoresis separates on
the basis of mobility in response to a voltage gradient along the
capillary. The mobility of species is related to their charge and
(54) Yang, L.; Sturgeon, R. E.; Mester, Z. Anal. Chem. 2005, 77, 2971–2977.
(55) Becker, J. S.; Dietze, H. J. Spectrochim. Acta, Part B: At. Spectrosc. 1998,
53, 1475–1506.
Figure 3. Dependence of integrated ICPMS signal (53 a.m.u.) on laser fluence for 10 µM CrIII
solution deposited on glass (2) and PETG (9)
plates.
Table 1. Limits of Detection of Sn, Cr, Cu, Ni, and Zn
Estimated from Depositing 0.1 µM Solution (Deposited
Volume ∼100 nL)
metal isotope LOD, peak area (fmol) LOD, peak height (fmol)
118
Sn 0.8 0.2
120
Sn 0.1 0.3
52
Cr 1.1 0.4
53
Cr 7.7 1.7
63
Cu 1.9 1.1
65
Cu 2.8 1.1
58
Ni 2.1 0.7
60
Ni 3.5 1.5
64
Zn 13 9.7
66
Zn 14 9.8
8730 Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
size. A simple electrophoresis of Cr3+
species in a formic acid
electrolyte, from chromium(VI) existing as Cr2O7
2,
CrO4
2-
,
HCrO4
,
or H2CrO4, cannot be accomplished. The mobility of
highly charged species caused the loss of the Cr3+
signal in the
electropherogram. Jung et al.56
used EDTA as a complexing agent
to create a negatively charged species CrIII
(EDTA),
which could
be well separated from CrVI
under the same conditions. CE with
UV detection was used to optimize the separation of Cr species
prior to deposition on a target. The 100 µM solution of chromium
species in water prepared from CrIII
(EDTA)and
CrVI
was used
for hydrodynamic injection; ∼200 fmol of each species was
injected. A record of 2.5-min separation consisting of 75 fractions
on PETG plate was desorbed in 12.5 min in the point acquisition
mode at laser fluence 71 MW·cm-2
.
The UV detection of CE separation is shown in Figure 4A;
ICPMS detection is in Figure 4B. The first peak in the UV
electropherogram probably corresponds to NO3
,
which is the
anion of the initial CrIII
salt before complexation. The peak areas
of the integrated ICPMS signal (53 a.m.u.), 11.7 × 106
and 13.2 ×
106
a.u. for CrVI
and CrIII
, respectively, are similar, as expected.
It can be noted from comparison of the CrVI
peak in both detection
modes that the off-line coupling preserves high separation efficiency.
When injecting sample, no sample stacking has been
employed in order to determine the actual amount of the injected
sample. With sample stacking, injection of similar amounts can
be achieved from more diluted solutions.
CONCLUSIONS
This paper has presented a new approach in coupling a
microcolumn separation technique, capillary electrophoresis, to(56) Jung, G. Y.; Kim, Y. S.; Lim, H. B. Anal. Sci. 1997, 13, 463–467.
Figure 4. CE separation of CrIII
/CrVI
species with (A) UV (214 nm) and (B) ICPMS (53 a.m.u.) detection. Injection from 100 µM CrIII
(EDTA)and
100 µM CrO4
2solution;
the injected amount was ∼200 fmol.
8731Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
ICPMS. The coupling is performed via a fraction collection in a
subatmospheric chamber, which has been used in previous
works28,29
to couple microcolumn separation techniques to MALDI
TOF MS; similar commercial fraction collectors are available
nowadays. The ICPMS analysis is enabled by laser ablation/
desorption of deposited samples on a substrate material; the
process can be characterized as substrate-assisted laser desorption
due to a significant contribution of the absorbing substrate. The
suitability of materials, which can be used as the substrate, has
been tested. The PETG plate showed to be the most appropriate
material for Cr speciation analysis because of its properties, such
as hydrophobicity, transparency, and commercial availability. The
sample morphology, detection limits, and spot-to-spot reproducibility
were examined. Due to submicroliter sample volumes used
for analysis in SALD, concentration LODs were up to 2 orders of
magnitude higher compared to conventional pneumatic nebulizer
ICPMS. On the other hand, LODs expressed in analyte amount
were ∼2 orders of magnitude lower than those obtained with the
pneumatic nebulizer as a result of enhanced overall sample
transport efficiency. For most examined metals, the LODs lie
around 1 fmol (∼100 fg), 1 order of magnitude lower than
previously published results,48,54
which facilitate coupling to
microcolumn separations. The spot-to-spot reproducibility was
below 3%, which is adequate for the analysis of deposited
separation fractions. The same solution is suitable for ICP OES
detection.
The simple model system of CrIII
/CrVI
has demonstrated the
prospects of coupling a microcolumn separation to ICPMS in offline
mode. The separation efficiency has been conserved while
the rapid analysis time has remained. The separation can be
further optimized, e.g., by modification of separation conditions
or increasing the capillary length. Alternatively, capillary liquid
chromatography can be employed. Contrary to the use of nebulizers,
low flow rate in the separation columns is advantageous as
it leads to small sample spots, high sensitivity, and short analysis
time. Furthermore, a single separation record on a sample plate
can be analyzed in different detection modes. Thus, we expect
the off-line coupling will be used e.g. for analysis of separated
metalloproteins with complementary ICPMS and MALDI MS
detection techniques to gain information on both metal content
and protein mass or identity.
ACKNOWLEDGMENT
We gratefully acknowledge the financial support of the Ministry
of Education, Youth and Sports of the Czech Republic MSM00216-
22411, MSM0021622412, MSM0021622415 and LC06035. We also
thankPavelKra´sensky´ forhishelpwithinstrumentationdevelopment.
Received for review May 21, 2008. Accepted September
12, 2008.
AC801036X
8732 Analytical Chemistry, Vol. 80, No. 22, November 15, 2008
2.3 Substrate-assisted laser desorption inductively-coupled plasma mass spectrometry
for the determination of copper in myeloid leukemia cells
SALD ICP MS has been characterized and utilized in model samples containing copper followed by an investigation
of intracellular copper levels in disulfiram-treated myeloid leukemia cells. Disulfiram (DSF), as other thiocarbamates,
is able to complex metals and permeate cell membranes while being more cytotoxic to leukemic cells than to cells of
solid tumors. Hence, it may induce growth arrest and apoptosis of various cell types by increasing the intracellular
level of copper up to several ng per 106
cells. Copper belongs to a group of essential trace metals and plays a key
role in various biological processes. To be effective, many critical enzymes and transcription factors require copper;
however, its amount in an organism is very tightly regulated. Analysis of Cu(II) model samples showed a constant
signal over a wide range of laser power density, independence of signal on desorption modes and a wide linear
dynamic range. Before treating the leukemic cells, several parameters were required to be optimized, e.g., droplet
deposition morphology, power density, cell lysis, or desorption raster as LOD and dynamic range depended upon
these. Copper determined in DMSO-treated and untreated cells corresponded to natural intracellular concentration
in leukemic cells. There was no statistically significant difference found in concentrations of naturally intracellular
copper in untreated, DMSO-treated low density (LD, 50 000 cells per mL) and high density (HD, 200 000 cells per
mL) cells. The intracellular concentration of copper in DSF-treated cells (both HD and LD) increased six times
when 25 ng·mL-1
DSF was used and seven times when 50 ng·mL-1
DSF was used. Accuracy of copper determination
by SALD ICP MS was verified by conventional Babington nebulizer ICP MS.
In conclusion, complete sample desorption could have been achieved using ten pulses with spot-to-spot reproducibility
below 6%. With a linear dynamic range over six orders of magnitude and LOD of about 26 fg for copper, SALD ICP
MS has proved to be a powerful tool for analyzing metals in real samples at micro-volume and trace concentrations,
as demonstrated on the determination of copper in leukemia cells.
JUNGOVA, Pavla, Jarmila NAVRATILOVA, Ondrej PES, Tomas VACULOVIC, Viktor KANICKY, Jan SMARDA a
Jan PREISLER. Substrate-assisted laser desorption inductively-coupled plasma mass spectrometry for determination
of copper in myeloid leukemia cells. Journal of Analytical Atomic Spectrometry. 2010, 25(5), 662–668. ISSN
0267-9477. doi:10.1039/b919811c Document Type: Article, IF = 4.372; JCR Category + Category Quartile:
CHEMISTRY, ANALYTICAL Q1 + SPECTROSCOPY Q1; AIS = 0.816
Author’s contribution: 15 %
27
Substrate-assisted laser desorption inductively-coupled plasma mass
spectrometry for determination of copper in myeloid leukemia cells
Pavla Jungova,a
Jarmila Navratilova,b
Ondrˇej Pes,a
Tomas Vaculovic,a
Viktor Kanicky,a
Jan Smardab
and Jan Preisler*ab
Received 25th September 2009, Accepted 8th January 2010
First published as an Advance Article on the web 28th January 2010
DOI: 10.1039/b919811c
This work presents a detailed characterization of substrate-assisted laser desorption inductively
coupled plasma mass spectrometry (SALD ICP-MS), a new approach in the determination of trace
elements in submicrolitre volumes. SALD ICP-MS allows quantitative analysis of metals in liquid
samples with defined volumes deposited onto an absorbing substrate due to complete sample
desorption at relatively low laser power density. Analysis of model samples of Cu(II) showed a constant
signal over a wide range of laser power density, an independence of signal on desorption modes and
a wide linear dynamic range. SALD ICP-MS with its limit of detection for copper below 26 fg in
submicrolitre samples was successfully applied to investigate cytotoxic effects of disulfiram in human
myeloid leukemia cells U937 depending on intracellular levels of copper.
Introduction
The development and application of laser ablation inductively
coupled plasma mass spectrometry (LA ICP-MS) has rapidly
advanced over the past years. The method is most frequently
applied for local microanalysis1,2
and for bulk analysis of steels,
alloys, glasses, ceramics and/or powdered materials.3
Additionally,
focusing a laser beam on a specific spot or sample movement
during ablation allows a local or spatial analysis of a sample
surface, which is necessary for elemental distribution mapping,
thin metal layer profiling,4
elemental mapping in biological tissue
sections5
and elemental imaging in metalloprotein gel bands after
electrophoretic separation.6
Key benefits of LA ICP-MS include
direct and fast characterization of solid or liquid materials with
no or minimal sample preparation, small sample size requirement
and nearly non-destructive analysis. The most common techniques
used for the selective detection of metals in liquid samples
include FAAS,7–9
GF-AAS,10
ETAAS,11
ICP-AES,12
ICP-MS,13–17
ESI-MS.18,19
However, conventional methods fail when
analyzing biological and clinical samples with low analyte
concentration, small sample volume or a complex matrix. The
use of direct liquid ablation as a calibration technique for solid
sample trace element determination has also been presented,20,21
and more recently, a method based on the complete ablation of
dried droplets22
has been used for the quantification of Ni, Cd
and Pb in drinking water and Se in yeast digest. In these studies,
samples were mixed with a sodium acetate matrix, deposited on
a polystyrene weighting boat and the maximum laser power
density was applied to ensure complete sample ablation/desorption.
Consequently, dried-droplet LA-ICP MS has been applied
to determine SeMet in yeast after separation and fractionation by
HPLC.23
Dried residues of picolitre droplets generated by an
inkjet printer were employed as calibration standards for LA
ICP-MS.24
In our previous study25
we presented a new substrate-assisted
laser desorption (SALD) ICP-MS approach, applicable for the
analysis of liquid samples deposited onto an absorbing substrate
followed by complete sample desorption using a relatively low
laser power density. Moreover, SALD ICP-MS has demonstrated
full potential for coupling with microcolumn separation
techniques, such as capillary electrophoresis (CE), and for the
determination of trace elements in biological samples at submicrolitre
level. Despite the fact that LA ICP-MS is currently one
of the most employed methods for quantitative imaging of trace
metals in biological samples,26–28
it has been limited to the
analysis of bulk samples where sample volume cannot be
precisely defined. Herein SALD ICP-MS is presented as being
able to analyze dried droplets with well-defined volumes from
absorbing substrates.
In this work, we offer a detailed characterization of SALD
ICP-MS on model samples (CuSO4) followed by an investigation
of intracellular copper levels in disulfiram-treated myeloid
leukemia cells. Copper belongs to a group of essential trace
metals and plays an important role in various biological
processes. To be effective, many critical enzymes and transcription
factors require copper;29
however, its amount in an organism
is very tightly regulated.30,31
Disulfiram (DSF), an irreversible
inhibitor of aldehyde dehydrogenase, is a drug approved for the
treatment of alcoholism. Several studies have shown that DSF in
safe concentrations possesses anticancer effects.32–34
As other
thiocarbamates, DSF is able to complex metals and permeate cell
membranes35
and is more cytotoxic to leukemic cells than to cells
of solid tumors.36
Next, DSF may induce growth arrest and
apoptosis of various cell types by increasing the intracellular level
of copper,37
which was verified in our studies on human myeloid
leukemia cells U937.38
The regular amount of copper in 106
U937
cells reaches several nanograms.39,40
A typical volume of such
a
Department of Chemistry, Faculty of Science, Masaryk University,
Kotlarˇska 2, 611 37 Brno, Czech Republic. E-mail: preisler@chemi.muni.
cz; Fax: +420 549 492 494; Tel: +420 549 496 629
b
Department of Experimental Biology, Faculty of Science, Masaryk
University, Kotlarˇska 2, 611 37 Brno, Czech Republic
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View Article Online / Journal Homepage / Table of Contents for this issue
a sample amount corresponds to less than 100 mL, which does
not allow dilution or using conventional techniques for copper
determination. SALD ICP-MS with its limit of detection for
copper $180 fg in submicrolitre sample volume25
will be
successfully applied for analysis of these samples.
Experimental
Chemicals
Aqueous solutions of CuSO4 by Lachema, Czech Republic, with
concentrations 1.15 Â 10À6
; 5.75 Â 10À6
; 1.15 Â 10À5
; 5.75 Â 10À5
and 1.15 Â 10À4
M were used as model samples for the characterization
of SALD ICP-MS. For visualization, samples might
have contained 10À5
M solution of rhodamine B by SigmaAldrich,
Germany. Water was redistilled in a quartz apparatus
from Heraeus (Hanau, Germany). All reagents were analyticalreagent
grade.
Cell cultivation
U937 (ATCCT, cat. number CRL-1593.2) were seeded at low
density (LD, 50 000 cells per mL) and high density (HD, 200 000
cells per mL) in Roswell Park Memorial Institute 1640 (RPMI
1640) medium purchased from Sigma-Aldrich, Czech Republic,
containing 10% fetal calf serum by Invitrogen, UK, L-Glutamine
in concentration 2 Â 10À3
M, penicillin in concentration
100 U/mL and streptomycin in concentration 100 mg mLÀ1
(all Sigma-Aldrich) and cultured in humidified 5% CO2 atmosphere
at 37 
C. The cells were treated with tetraethylthiuram
disulfide disulfiram (100 ng mLÀ1
, Sigma-Aldrich) dissolved in
dimethylsulfoxide (DMSO, 100 ng mLÀ1
, Sigma-Aldrich) or left
untreated for 5.5 h. As a control, the cells were treated with
DMSO. Pelleted cells were washed twice with phosphate buffered
saline (PBS), PBS was pumped out and the number of cells was
counted using hemocytometer. The volume of $106
cells was
below 10 mL.
Cells lysis
Washed cells were lysed in 50 mL of deionized water for 10 min in
an ultrasonic bath or in 100 mL of mixture of 3 M HCl (Lachema)
and 10% trichloroacetic acid (TCA, Sigma-Aldrich) at laboratory
temperature for 3 h followed by incubation at 70 
C for
5 h,37
or in 100 mL of nitric acid (Lachema) and incubated at
37 
C overnight. The lysate was centrifuged (600 g/5 min) to
remove cell debris.
Sample preparation
Supernatant or model CuSO4 solution was, for easy visualization,
mixed with an aqueous solution of rhodamine B. The total
concentration of rhodamine B in any solution was 10À5
M. The
addition of rhodamine B at applied concentration did not have
any effect on the spot size. Sample solutions were spotted onto
a polyethylene terephthalate glycol plate (PETG), brand name
Vivak, as 200-nL droplets by a micropipette or in a laboratorybuilt
sub-atmospheric chamber41,42
providing deposition of
precisely defined sample volumes. Lower pressure ($20 kPa)
inside the cylindrical chamber (45-cm diameter, 19-cm height),
generated with a membrane pump, model N35AT18 (KNF
Neuberger, Germany), ensured stable flow of a sample solution.
The deposited sample volume was then calculated from the flow
rate and the deposition time based on Poiseuille’s equation.
SALD ICP MS
Sample plates were inserted into the laser ablation system, model
UP 213 (New Wave, USA), with Q-switched Nd:YAG working
at a wavelength of 213 nm and having a movable ablation cell,
model SuperCell (New Wave), for sample movement during
desorption. A built-in microscope/CCD camera system provided
visual control of samples. Sample spots were scanned by a laser
beam and the laser beamwaist was adjusted to the maximum size
$300 mm. The ablation cell was flushed with a carrier gas
(helium) at flow rate 1.0 L minÀ1
, which transported the aerosol
to an ICP mass spectrometer, model 7500CE (Agilent, Japan). A
sample gas flow of argon was mixed with the helium carrier gas
flow subsequent to the laser ablation cell (0.6 L minÀ1
). Optimization
of LA ICP-MS conditions such as gas flow rates,
sampling depth, electrostatic lens voltage of the MS was performed
with the glass reference material NIST SRM
612 regarding the maximum signal-to-noise ratio of 7
Li+
, 89
Y+
and 238
U+
for low, middle and high masses and minimum oxide
formation (ThO+
/Th+
counts ratio 0.2%, U+
/Th+
counts ratio
1.1%). Other ICP MS parameters were adjusted in compliance
with the manufacturer’s recommendations. If not otherwise
stated, sample spots were desorbed at the laser power density
$70 MW cmÀ2
, at a frequency of 10 Hz and a scan rate of 100 mm
sÀ1
(raster desorption) or dwell time 2 s (point desorption). Size of
the zigzag-shaped raster with spacing 200 mm was selected
according to the spot diameter to cover the entire sample area.
The ions were measured with an integration time 0.1 s/isotope.
Cell flush time and laser warm-up time were set to 3 s. In the case
that the helium collision cell was used, gas flow was 2.5 mL
minÀ1
. The 13
C ion signal was monitored to indicate the ablation
of the sample plate and to set the interval for integration of the
copper ion signal accordingly. The ion signal of two copper
isotopes, 63
Cu and 65
Cu, was recorded and the 65
Cu/63
Cu isotope
ratio was observed to reveal possible polyatomic interferences
(40
Ar25
Mg+
, 40
Ar23
Na+
, etc). The signal of 65
Cu+
was used for all
data evaluations further in the text.
Pneumatic nebulization ICP MS
A Babington nebulizer with Scott double-pass spray chamber
(Agilent,Japan)wasused.OptimizationofICPMSconditionswas
performedregardingthemaximum S/N ratiofor63
Cuand65
Cuand
in compliance with the manufacturer’s recommendations. The
other ICP-MS parameters were: carrier gas (argon) flow, 0.90 L
minÀ1
; makeup gas (argon) flow, 0.19 L minÀ1
; nebulizer pump
flow, 0.022 mL sÀ1
and spray chamber temperature, 2 
C. A
nebulization efficiency of 5% was determined for this setup.
Results and discussion
Substrate selection
Commercially available plastics, polyester polyethylene terephthalate
glycol (PETG) brands Vivak and Axpet produced by
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BayerSheet, Germany, polypropylene Evacast, foamed polyvinylchloride
Vikupor by Rochling, Germany, and polyvinylchloride
Vikunyl by EVC, Great Britain, were selected as suitable
substrates concerning availability, ease of plate fabrication and
transparency. Initially, the presence of common metals in plastics,
the deposited sample morphology and ablation crater
formation were examined. The intensity of ICP MS signal of the
two most abundant isotopes of Cr, Cu, Hg, Zn, Fe, Sn, Ni, Mo,
Cd, Pt, Pb and one isotope of Al, Mn, Co were monitored with
an integration time 0.1 s/isotope by point ablation with a dwell
time 5 s to study which of the plastics is applicable to trace
analysis of these common metals. Spot-to-spot reproducibility
was given by a raster desorption of ten 100 nL spots of 10À4
M
CuSO4. Sample morphology and ablation crater formation was
observed by microscope/CCD-camera system.
Carbon, a major element in plastics, raised formation of
polyatomic molecules 40
Ar12
C, with the same nominal mass as the
major isotope of Cr. Other observed molecules 40
Ar16
O interfered
with mass of Fe. Interferences might have been eliminated by
switching on the dynamic reaction cell or other isotope monitoring.
Polyatomic interferences with either of the copper
isotopes were not observed. Polyvinylchloride Vikupor was
excluded from further tests since a significant content of Al, Zn
and Pb were discovered. Polypropylene Evacast was contaminated
by Zn and spot borders were not recognizable on rough
surface. Although polyvinylchloride Vikunyl contained none of
the monitored metals, ablation formed irregular craters along the
irradiated area of the polymer resulting in an incomplete sample
desorption. Both PETG brands were found to be contaminated
by cobalt. While poorly visible large sample spots ($600 mm) and
uneven size of spots on Axpet inflicted a low spot-to-spot
reproducibility (RSD $34%). Samples deposited on PETG
Vivak created uniform, clearly visible and bordered spots
significantly increasing the reproducibility (RSD $9%).
Desorption of both latter plastics from irradiated areas proceeded
evenly.
In summary, all plastics except polyvinylchloride Vikunyl,
which is not suitable due to forming irregular craters, were
contaminated by at least one monitoring metal, but deposited
samples were uniform and clearly bordered only on PETG
Vivak. PETG Vivak was selected as the most suitable substrate
among the tested plastics since it was not found to be contaminated
by common metals but by cobalt, and deposited sample
spots were identical and distinct. Moreover, the high absorption
coefficient of PETG at the laser wavelength used (213 nm)
extensively enhanced sample desorption (SALD mechanism)
along the entire irradiated area.25
PETG Vivak used for all
further experiments will be referred to as PETG in the following
text.
Sample morphology
A microscope/CCD-camera system allowing visual inspection
was used to study sample morphology. Typical sample spots of
CuSO4, formed after depositing different volumes of its aqueous
solution, are shown in Fig. 1. Visual observation predicated that
copper, when the solvent evaporated, was concentrated in crystals
at the spot border. Subsequently, this fact has been
confirmed by analyzing a 100 nL spot of 10À4
M CuSO4. Laser
beamwaist was adjusted to 30 mm and different sample spot sites
were examined by point desorption as follows: desorption from
three points in a spot border, where the highest concentration of
crystals was noticed; three points in a spot center with no visible
crystals; and three points in a number of visible crystals within
the sample spot diameter. In the places with clearly visible
Fig. 1 Photographs of a CuSO4 aqueous solution droplet deposited on
PETG. Droplet volume and CuSO4 concentration: A) 6 nL, 10À4
M; B)
100 nL, 10À4
M and C) 100 nL, 10À6
M with 10À5
M rhodamine B.
664 | J. Anal. At. Spectrom., 2010, 25, 662–668 This journal is ª The Royal Society of Chemistry 2010
Publishedon28January2010.DownloadedbyMasarykovaUniverzitaVBrneon8/23/20218:53:13AM. View Article Online
crystals the intensity of the 65
Cu+
signal was 15-fold to 50-fold
higher than the signal intensity in the bare PETG surface.
Effect of laser power density on ICP MS signal
To investigate the optimal laser power density, a series of 100-nL
droplets of 10À4
M CuSO4 aqueous solution was deposited on
PETG. Five spots were desorbed for each value of laser power
density ranging from 2 to 129 MW cmÀ2
. To reduce possible
polyatomic interference, a helium collision cell was used. While
all sample spots were at least partially desorbed, a laser power
density >10 MW cmÀ2
was sufficient to desorb more than 98% of
the sample as evidenced by subsequent analysis of the same spot
at 70 MW cmÀ2
. Moreover, further increase in laser power
density had no effect on the integrated signal. Unfortunately, the
stated laser power density values may be slightly inaccurate
because the laser ablation system was not designed to be
routinely used at these low values of laser power density. On the
other hand, the higher the laser power density, the larger the
polyatomic interferences were observed. As a compromise,
a laser power density $70 MW cmÀ2
was selected for further
experiments.
Raster desorption optimization
A series of 100-nL droplets of 10À4
M CuSO4 aqueous solution
was deposited onto PETG. Scan rates of 30; 70; 100; 200 and
500 mm sÀ1
and laser frequencies 5; 10 and 20 Hz were tested in
all combinations. Natural ratio of copper isotopes
(65
Cu/63
Cu ¼ 0.447) was observed for scan rates up to 200 mm sÀ1
for all tested laser frequencies. An isotope ratio decrease was
observed at higher scan rates because of a signal increase at mass
63. However, bare PETG surface ablation did not result in any
signal change at any combination of laser frequency and scan
rate. Presumably, the samples contained traces of sodium, which
lead to polyatomic interference of 40
Ar23
Na. In order to prove
this hypothesis, an experiment with a dynamic reaction cell was
carried out. The sample spots were desorbed by the same raster
pattern at a scan rate of 100; 200; 300; 500 and 1000 mm sÀ1
at
20 Hz frequency. With the helium collision cell switched on, the
65
Cu/63
Cu isotope ratio was restored to its natural value and the
integrated signal of both isotopes was consistent along all scan
rates. It should be noted that the signal increase at mass 63 was
not always observed at higher scan rates. Yet, to eliminate
possible interferences, the dynamic reaction cell had to be
employed or scan rates kept below 100 mm sÀ1
.
Point desorption optimization
Point desorption is particularly influenced by laser frequency,
dwell time, and/or number of pulses. The integrated signal from
desorption of 6-nL droplets of 10À4
M CuSO4 in five replicates
was monitored at 1, 2, 5, 10 and 20 Hz for 1 s, which corresponded
to 1, 2, 5, 10 and 20 pulses. Second point desorption at
20 Hz for 1 s within the same coordinates was performed to
reveal any remaining sample.
Usually one pulse was sufficient for desorption of more than
90% of the sample but the spot-to-spot reproducibility calculated
from integrated signals of five copper spots exceeded 20%.
To desorb more than 98% of a sample five pulses were needed
and the spot-to-spot reproducibility decreased to below 10%
when using five or more pulses. Therefore ten pulses desorption
was selected for further experiments. Variance of ten pulses was
tested as a combination of frequency and time, i.e. 10 Hz for 1 s,
5 Hz for 2 s, 2 Hz for 5 s and 1 Hz for 10 s. Frequency 20 Hz
was not included because the instrument setup allowed minimal
dwell time 1 s. In any case, desorption of ten pulses yielded in
identical ICP-MS signal; the RSD, however, increased from 6%
at 10 Hz (dwell time 1 s) to 29% at 1 Hz (dwell time 10 s). Thus
for further experiments, dwell time 1 s and laser frequency
10 Hz were set to ensure complete sample desorption and low
signal variation.
Raster and point desorption comparison
As the maximum adjustable laser beamwaist is 300 mm, the
desorption mode was selected according to the size of the drieddroplet
spot. In order to investigate possible differences between
point and raster desorption, a series of volumes of 3, 6, 10, 20 nL
of 10À4
M CuSO4 aqueous solution droplets were deposited on
PETG. A typical spot diameter for volumes below 20 nL did not
exceed 300 mm, and both desorption modes were applicable.
Deposition of larger droplets might result in sample spot diameters
exceeding 300 mm, which could have been entirely desorbed
only by raster desorption. Thus, spots with a diameter below
300 mm (sample volume 3–20 nL, 0.3–2 pmol CuSO4) were desorbed
in five replicates by raster and point desorption. The
helium collision cell was used. As seen in Fig. 2, integrated signals
of 65
Cu+
reached similar values for both point and raster
desorption, proportionally increasing with the deposited volume.
Coefficients of determination were 0.9972 for point and 0.9954
for raster desorption. Total acquisition time for one sample spot
(including laser warm-up and cell flush-out) were 7 s for point
and $21 s for raster desorption. Therefore point desorption
should be preferred in the case of sample spots with diameters
lower than 300 mm. The lower integrated signal in the case of
point desorption is probably related to incomplete desorption of
the sample on the spot edges. Linearity of signal passed the F-test
for both raster and point desorption.
Fig. 2 Dependence of integrated ICP-MS signal (a.m.u. ¼ 65) on
droplet volume of 10À4
M CuSO4 solution obtained by (–-–) point
desorption; (–,–) raster desorption.
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Dynamic range
One of the advantages of the ICP-MS instrument employed is its
wide, linear dynamic range. In order to investigate the dynamic
range of SALD ICP-MS, 100-nL droplets of aqueous mixture of
10À5
M rhodamine B and CuSO4 at concentrations 10À7
; 10À6
;
10À5
; 10À4
; 10À3
; 10À2
and 10À1
M were deposited onto PETG and
desorbed in five replicates for each concentration. The helium
collision cell was used. Dynamic range proved to be linear within
the examined scale and coefficient of determination was 0.9990.
Limit of detection (LOD)
In order to determine limit of detection (3s) of Cu, 100-nL
droplets of aqueous mixture of 10À7
M CuSO4 and 10À5
M
rhodamine B were deposited onto PETG and desorbed in five
replicates. The helium collision cell was used. LOD was estimated
from peak area (calculated as the sum of the rectangles
between neighboring data points) was 0.4 fmol, i.e. 26 fg of Cu.
SALD ICP-MS of lysed cells
When cultivated, U937 cells were counted in a hemocytometer
and PBS was pumped out. This left a certain amount of cells in
a pellet without knowing sample volume. Thus, an addition of
defined volume of lysis solution was required for direct quantitative
analysis of cells. Three lysis solutions to LD cells were
tested: water, HNO3 and HCl/TCA, as in literature.37
Natural
intracellular concentration of copper in U937 cells is expected to
be several nanograms per 106
cells.40,43
An aqueous mixture of
10À5
M rhodamine B with various concentration of CuSO4 was
analyzed by SALD ICP-MS to produce a calibration curve.
From the calibration curve, concentration of copper in the cells
was determined.
First, analysis of a 200-nL water supernatant from cells containing
10À5
M rhodamine B showed the amount of copper to be
below 1 ng per 106
cells. This suggested that copper was bound in
an insoluble cell membrane, and for correct determination of the
total copper concentration, the cell membrane required to be
suitably disintegrated and re-dissolved.
Thus, lysis in 1.6 M HNO3
43
was tested, but visual inspection
showed that the cells were not completely lysed. More concentrated
nitric acid; 3.7 M and 7.3 M, was prepared to ensure
complete cell lysis. However, 200-nL supernatant droplets
deposited on PETG caused etching of the PETG surface which
resulted in a reduction of the integrated signal of Cu+
, probably
because of the penetration of copper into the polymer grid. To
avoid the etching, a cell lysate in 3.7 M HNO3 was diluted with
rhodamine B aqueous solution in volume ratio 1 : 4 and 1 : 7 so
that the resulting concentration of HNO3 was 0.9 M and 0.4 M.
No etching was observed and the determined amount of copper
was 1.7 and 3.5 ng per 106
cells. However, use of the diluted
HNO3 solutions was accompanied by a significant increase of
relative standard deviation to 75%, respectively 88%.
Finally, a mixture of HCl/TCA was used as a lysis reagent.
Completely lysed cells (visually examined) were deposited as
200-nL fractions showing minor etching of PETG and forming
2–3 times larger spots due to fast evaporation of the organic
solvent. SALD ICP-MS analysis of 200-nL droplets (five replicates)
resulted in 1.1 Æ 0.3 ng of copper per 106
cells. To
completely eliminate the PETG etching and minimize the spot
size, the lysate was diluted with rhodamine B aqueous solution in
volume ratio 1 : 3 so that the resulting concentration of rhodamine
B was 10À6
M. The determined amount of copper was 2.6 Æ
0.1 ng per 106
cells. To verify the visually established complete
cell lysis, the cell debris from HCl/TCA lysate was deposited on
the PETG covering an area of $1 mm2
. As the intensity of the
ICP-MS signal from the debris and the blank PETG surface did
not differ significantly, absence of copper in the cell debris with
all the copper entirely in the cell lysate was confirmed.
As seen in Table 1, comparison of different sample preparation
methods for SALD ICP-MS showed that lysis in the HCl/TCA
mixture followed by dilution with rhodamine B was found to be
the most suitable method for U937 cell preparation. The surface
of the PETG was not etched, the sample spots size was $800 mm
and the relative standard deviation reached 5%.
The effect of DSF on Cu content in U937 cells
With an accurate method for trace analysis of copper in cells,
physiologically relevant levels of copper may be further studied.
The effects of DSF on intracellular concentration of copper were
determined for low (50 000 cell per mL) and high (200 000 cell
per mL) densities of U937 cells. The cells were treated with DSF,
DMSO as a control or left untreated for 5.5 h, lysed in a mixture
of 3M HCl/10% TCA and analyzed by SALD ICP-MS. The cells
were treated with DMSO to inspect possible effects of DMSO
dissolvent. The intracellular concentration of copper was determined
as ng per 106
cells. Data were obtained from three independent
experiments over three days and processed using the
Student’s test; no data were excluded.
Copper determined in DMSO-treated and untreated cells
corresponded to natural intracellular concentration in U937
cells. There was no statistically significant difference found in
concentrations of naturally intracellular copper in untreated,
DMSO-treated LD and HD cells, as seen in Table 2. The intracellular
concentration of copper in DSF-treated cells (both HD
and LD) increased six times when 25 ng mLÀ1
DSF was used, and
seven times when 50 ng mLÀ1
DSF was used. The possible
difference between LD and HD cells was examined by treating
the cells with 100 ng mLÀ1
DSF; intracellular concentration of
copper increased to 41.0 Æ 1.7 ng per 106
cells in the case of LD
Table 1 SALD ICP-MS analysis of copper in different lysates of cells
U937
Lysis solution
Volume ratio
with aqueous
solution of
rhodamine B
Amount of
copper
(ng per 106
cells)
RSD
(%)
10À5
M rhodamine B — 0.89 Æ 0.36 41
7.3 M HNO3 — 0.82 Æ 0.93 113
1.6 M HNO3
a
3.7 M HNO3 — 0.22 Æ 0.32 150
3.7 M HNO3 1 : 4 1.17 Æ 0.88 75
3.7 M HNO3 1 : 7 3.52 Æ 3.08 88
3M HCl/10% TCA — 1.09 Æ 0.32 30
3M HCl/10% TCA 1 : 3 2.61 Æ 0.12 5
a
Cells were not completely lysed.
666 | J. Anal. At. Spectrom., 2010, 25, 662–668 This journal is ª The Royal Society of Chemistry 2010
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cells, while in the case of HD cells it did not increase considerably
(to 23.8 Æ 1.8 ng per 106
cells) compared to the addition of 50 ng
mLÀ1
DSF.
These results confirmed that DSF causes accumulation of
copper in U937 cells and that the process is significantly more
efficient in LD U937 cells. The lower intracellular concentration
of copper in the DSF-treated HD U937 cells was probably
caused by a limited supply of copper in the cultivation medium.
To verify this hypothesis, HD U937 cells were cultivated in 5 mL
of media treated with DMSO (conditioned medium 1, CM1) and
with DSF in concentration 100 ng mLÀ1
(conditioned medium 2,
CM2). Concentrations of copper in the medium determined by
SALD ICP-MS were 9.78 Æ 0.04 ng mLÀ1
in CM1 and 5.06 Æ
0.04 ng mLÀ1
in CM2. As expected, addition of DSF results in
a decrease of copper in the medium due to copper transport into
the cells. Furthermore, this documents that DSF, even at
a concentration of 100 ng mLÀ1
, is not able to transport any more
copper into the cells from the CM2 medium although this was
not completely depleted from copper. The difference of amounts
of extracellular copper between CM1 and CM2 was 23.60 ng for
5 mL media, which corresponds to the increase of intracellular
copper in HD 100 ng mLÀ1
DSF-treated cells. Further information
about Cu analysis in U937 cells is published elsewhere.38
Accuracy of copper determination in cells by SALD ICP MS
In order to examine the possible effect of HCl/TCA mixture on
ICP MS response on copper, solutions of 1.15 Â 10À4
; 5.75 Â
10À5
; 1.15 Â 10À5
; 5.75 Â 10À6
and 1.15 Â 10À6
M CuSO4 in 3 M
HCl/10% TCA, and in water, containing 10À5
M rhodamine B
were prepared. A series of 200-nL droplets were deposited on
PETG and analyzed by SALD ICP-MS. The slopes for calibration
curves of CuSO4 in HCl/TCA and in water were 1.61 Â 108
with a coefficient of determination 0.998 and 1.57 Â 108
with
a coefficient of determination 0.997, respectively. Therefore it
can be concluded that the ICP-MS signal is not affected by the
lysis solution.
Furthermore, accuracy of copper determination by SALD
ICP-MS was verified by conventional Babington nebulizer
ICP-MS. Because of the larger consumption of sample, ten times
larger volumes of media had to be used for cell culture preparation.
HD cells treated with DMSO and DSF (100 ng mLÀ1
)
were divided into two aliquots, each analyzed by SALD ICP-MS
and nebulizer ICP MS. Both CM1 and CM2 media were
analyzed by nebulizer ICP MS and the results were compared
with the results obtained by the previous SALD ICP-MS
experiment. As can be seen in Fig. 3, the results from SALD
ICP-MS and nebulizer ICP-MS are comparable for all the cases,
i.e. intracellular copper and extracellular copper in both media.
Conclusions
Detailed investigation and comprehensive characterization of
a substrate-assisted laser desorption technique has been performed.
Suitable commercially available substrates have been
tested, and PETG has shown to be the most appropriate
substrate especially due to absence of metal contaminants and
transparency. SALD ICP-MS has shown large signal stability
over a wide range of laser power density values. Moreover, the
signal has not been affected when switching between the point
and raster desorption modes. In the case of 63
Cu isotope determination,
raster desorption is, however, limited to scan rates
below 100 mm sÀ1
, if lacking dynamic reaction cell. Complete
sample desorption can be achieved using ten pulses with spotto-spot
reproducibility below 6%. With linear dynamic range
over six orders of magnitude and detection limits about 26 fg for
copper, SALD ICP-MS has proved to be a powerful tool for the
analysis of metals in real samples at micro volume and trace
concentration, as demonstrated on the determination of copper
in U937 cells. Moreover, the protocol for biological sample
preparation has been optimized by the minimization of substrate
etching, which was found to decrease the integrated ICP-MS
signal.
Acknowledgements
We gratefully acknowledge the financial support of the Czech
Science Foundation (Grant No. 203/09/1025) and the Ministry of
Education, Youth and Sports of the Czech Republic
(MSM0021622411, MSM0021622412 and MSM0021622415).
We also would like to thank to Pavel Krasensky for mechanical
engineering.
Notes and references
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Table 2 SALD ICP-MS estimated copper amount in DSF-treated U937
cells
Cell density
DSF
concentration/ng
mLÀ1
Amount of
copper
(ng per 106
cells)
RSD
(%)
LD (50 000 cells per mL) 0 2.61 Æ 0.12 5
25 14.9 Æ 2.6 17
50 22.6 Æ 4.5 20
100 41.0 Æ 1.7 4
HD (200 000 cells per mL) 0 2.4 Æ 0.3 13
25 18.4 Æ 4.6 25
50 22.1 Æ 2.4 11
100 23.8 Æ 1.8 8
Fig. 3 Comparison of SALD ICP MS with conventional ICP MS using
a Babington nebulizer. See text for details.
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3 Mass spectrometry as a tool in identification and characterization
In order to unambiguously identify and/or characterize a compound, MS is usually not sufficient and orthogonal
information should be involved.39
It has been proved that MS can provide maximum information when analyzing
pure substances. Hence, reducing the sample mixture complexity is mandatory, whether through a separation
technique or multiple-stage MS. The hybrid mass spectrometers capable of performing more than just measuring
the mass-to-charge ratio of analytes are eligible. Furthermore, the utility of various other LC detectors and/or
computational MS may help confirm identification, especially since collision-induced dissociation is not as reproducible
as electron ionization fragmentation across different instruments or even instrument types.
When analyzing an unknown compound, one of the most basic — but fundamental — goals is assigning its molecular
peak (if it exists) and retrieving its molecular formula. A few general principles could help in the task.
• Isotopic pattern
The isotopic pattern of the target molecular formula should be similar to the simulated one according to the binomial
expansion.40
Given the instrument resolving power, isotopic patterns are usually calculated for the most abundant
elements (C, H, O, N, S, P). Other elements are usually included in specific cases of known heteroatoms, e.g., analysis
of metals, presence of M + n elements, isotope dilution methods, etc.
• Molecular or ionic adducts
Ions like Na+
, K+
, or Clare
commonplace and cannot be easily removed from the final mixture during the ionization
process. They may easily replace the proton in ionization techniques producing even-electron ions. Hence, it is better
to accommodate the adducts’ specific mass shifts when searching for a molecular peak. Some classes of compounds
are even better suited to be analyzed using artificially added ions such as Li+
or Ag+
.41
• Multiply-charged ions and n-mers
Multiply-charged ions result from having more than a single site in a molecule where the charge could be located.
They are typically seen in the ESI of biomolecules, and deconvolution of the charge state of two adjacent peaks in
the positive mode can be calculated using a simple formula:
Peakm/z = (Mw + z)/z
Adjacent Peakm/z = (Mw + z + 1)/(z + 1)
Provided that the MS analyzer resolving power is sufficient, the isotopes of a multiply-charged ion will appear at
fractional m/z values. Fragmentation of multiply-charged ions provide (typically) peaks of higher m/z than those of
the precursor. The n-mers ions are considered merely gaseous phase-associated molecules, which happen to reach
the detector with some probability. The intensity of n-mers is usually lower than that of the molecular peak and
depends on the analyte concentration in the sample. The n-mers could easily form ionic adducts or lose neutral
compounds and their fragmentation provides (typically) peaks of m-mers, where m < n.
• Loss of a neutral molecule
35
There is a plenty of so called good leaving groups, which could be cleaved off an analyte molecule during any stage
of mass spectrum acquisition. A loss of molecules such as H2O or CO2 is often observed at certain compounds
(aliphatic –OH or –COOH) even under soft ionization conditions. The specific shifts in m/z can help to determine
the molecular composition.
• Nitrogen rule
Nitrogen has an even nominal mass (14) and odd valency (3). Neutral compounds, with all of the atoms in the
molecule having a number of covalent bonds equal to their standard valency, have odd nominal mass if they contain
an odd number of nitrogen atoms. Depending on the nature of the ionization process, either odd-electron or
even-electron ions may be produced. The nitrogen rule is reversed in the case of even-electron ions, yet the principle
is the same.
• Hydrogen/Carbon ratio
Another constraint for restricting formulas to those likely existing is including element ratios, especially the
hydrogen/carbon ratio. In most cases, the hydrogen/carbon ratio does not exceed H/C > 3 and should not be less
than 0.125 with typical ratios being found between 2.0 > H/C > 0.5.
• Hydrogen/deuterium exchange
Hydrogen/deuterium exchange (HDX) MS has been employed widely to determine the presence, the number and
the position of exchangeable hydrogen atoms in small organic molecules, peptides, or proteins. The exchange of
labile hydrogen with deuterium could happen in solution or gas phase of compounds containing groups such as
–OH, –NH2, –SH, –NH–, or COOH. Traditionally, deuterated solvents are used as mobile phase in liquid separation
techniques; but, a cost-effective approach utilizes ND3, CH3OD or D2O as sheath liquid in ESI or as an ionization
gas in chemical ionization mode. HDX MS is typically employed in identification of isomeric compounds or in
understanding the nature of chemical reactions formed via unpredictable pathways.
• Mass defect
The difference between the exact mass of an element (or a compound) and its closest integer value is called a
mass defect. Chemical reactions will introduce a mass defect shift between the parent and its product molecule,
which could be viewed using MS manufacturers algorithms when accurate mass is used. Typically, extracted ion
chromatograms are generated in a fixed, narrow mass defect range, yielding peaks selectively for the parent-product
compounds excluding majority of matrix components.
• Chemical reactions
A specific approach to identify a compound can involve a series of (bio)chemical reactions, e.g., chemical reactions
using a hemoprotein superfamily of cytochrome P450, in an in vitro or in vivo experimental design. In vitro designs
are advantageous as the final mixture of metabolites and parent compounds contain merely phase I metabolites. In
silico prediction software tools may help design the experiment in order to detect the prepared metabolites.
Additional approaches that complement the above-mentioned ones rely on utilizing chemical derivatization, different
36
ionization, instruments with high resolving power, fragmentation and a multiple-stage MS, retention-based characteristic,
or orthogonal information from other detectors e.g. UV-Vis, fluorescence, electrochemical etc. These are,
however, very situational and also depend on the available laboratory equipment.42
3.1 Identification of metabolites of selected benzophenanthridine alkaloids and their
toxicity evaluation
Benzo[c]phenanthridine alkaloids (QBAs) belong to a group of isoquinoline alkaloids and were first discovered in
the plant Sanguinaria canadensis, used in traditional medicine by Native Americans.43
Sanguinarine (SA) was first
isolated from the rhizome of the plant and bears its name. The plant contains other QBAs, such as chelerythrine
(CHE), sanguilutine (SL), chelilutine (CL), sanguirubine (SR) and chelirubine (CR), see Figure 3. Hexasubstituted
alkaloid macarpine (MA) was first isolated from Macleaya microcarpa in 1955.44
Major QBAs, SA and CHE, have
been produced commercially and belong to the best-investigated QBAs.
Figure 3: Benzo[c]phenanthridine alkaloid structures.
Minor QBAs have never been produced in large amounts even though their recent research suggested they exhibit
exclusively different properties despite their similarity with SA and CHE.45
SL exhibited high anti-proliferation
activity against specific melanoma cell lines, mediated by autophagy and and necroptosis.46
QBAs cytotoxicity
is predetermined by methylenedioxy rings at carbon positions C2–C3 and C7–C8. The most cytotoxic QBAs are
CR, SA, and MA with IC50 in a range of 0.5–6 · 10-6
M for THP-1 cell lines.47,48
Synthetically modified QBAs or
37
de novo synthesized QBA analogs were prepared to increase the cytotoxic properties or alter the metabolism.49,50
Another attempt to produce, isolate, characterize and assess the cytotoxicity of these minor QBAs metabolites have
been made in the following article using rat liver microsomes (RLMs) and LC–MS/MS. Two major metabolites,
O-demethylated CHE and O-demethylated SL, were successfully prepared, identified, isolated, and tested for
cytotoxicity in comparison to the corresponding parent alkaloid. Biotransformation reactions varied significantly
across all alkaloids despite the remarkable structural similarity of the studied QBAs. A few alkaloids were unable to
undergo specific transformation reactions, which could potentially be associated with their elevated cytotoxicity. On
top of that, by employing biotransformation reactions, it was possible to identify one of the metabolites of CHE. Two
O-demethylation reactions in phase I metabolism may produce two isomers, which substantially differ in toxicity.
When the prepared metabolite was purified, isolated, and tested for its cytotoxicity, the alkaloid fagaridine was
ultimately unraveled as the CHE metabolite.
SANDOR, Roman, Adam MIDLIK, Kristyna SEBRLOVA, Gabriela DOVRTELOVA, Kristyna NOSKOVA, Jan
JURICA, Iva SLANINOVA, Eva TABORSKA and Ondrej PES (corresponding author). Identification of metabolites
of selected benzophenanthridine alkaloids and their toxicity evaluation. Journal of Pharmaceutical and Biomedical
Analysis 2016, 121, 174–180. ISSN 0731-7085. doi:10.1016/j.jpba.2016.01.024 Document Type: Article, IF = 3.255;
JCR Category + Category Quartile: CHEMISTRY, ANALYTICAL Q1 + PHARMACOLOGY & PHARMACY Q2;
AIS = 0.618
Author’s contribution: 40 %
38
Journal of Pharmaceutical and Biomedical Analysis 121 (2016) 174–180
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
Identification of metabolites of selected benzophenanthridine
alkaloids and their toxicity evaluation
Roman Sandora
, Adam Midlika,b
, Kristyna Sebrlovaa
, Gabriela Dovrtelovac
,
Kristyna Noskovac
, Jan Juricac,d
, Iva Slaninovae
, Eva Taborskaa
, Ondrej Pesa,∗
a
Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
b
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
c
Department of Pharmacology, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
d
Central European Institute of Technology CEITEC MU, Kamenice 5, 62500 Brno, Czech Republic
e
Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 27 October 2015
Received in revised form 11 January 2016
Accepted 12 January 2016
Available online 15 January 2016
Keywords:
Benzophenanthridine alkaloids
Metabolism
LC MS
Microsomes
Biotransformation
a b s t r a c t
Selected benzo[c]phenathridine alkaloids were biotransformed using rat liver microsomes and identified
by liquid chromatography and mass spectrometry. While the metabolites of commercially available sanguinarine
and chelerythrine have been studied in detail, data about the metabolism of the minor alkaloids
remained unknown. Reactions involved in transformation include single and/or double O-demethylation,
demethylenation, reduction, and hydroxylation. Two metabolites, when isolated, purified and tested for
toxicity, were found to be less toxic than the original compounds.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
Quaternary benzo[c]phenanthridine alkaloids (QBAs) belong to
a group of isoquinoline alkaloids widely distributed in the family
Papaveraceae and, to a limited extent, in some species of the
families Fumariaceae and Rutaceae. Sanguinarine (SA) and chelerythrine
(CHE) are the best-known QBAs. In some plants, SA and CHE
are accompanied by less-abundant pentasubstituted derivatives
sanguilutine (SL), chelilutine (CL), sanguirubine (SR), chelirubine
(CR), and hexasubstituted macarpine (MA) (Fig. 1).
The effects of QBAs on biological systems have been studied
extensively, especially for commercially available SA and CHE. The
results have been summarized in several reviews [1–4]. Research
has primarily focused on antitumor effects of both alkaloids, but
they also display antimicrobial, antifungal and anti-inflammatory
activities. They may affect many targets on the molecular level and
modify the activity and function of a wide variety of enzymes. The
fractions rich in QBAs from Sanguinaria canadensis or Macleaya cor∗
Corresponding author.
E-mail address: ondramayl@gmail.com (O. Pes).
data have been used in toothpastes and mouthwashes as antiplaque
agents [1,3].
While it seems that low concentrations of QBAs have no observable
toxic effects [5–7], epidemiological case studies have shown an
association between SA use and oral premalignant lesions [8,9] and
a study with higher doses of SA discovered that SA displayed signs
of significant liver damage in mice [10]. Further, it has been shown
that SA creates complexes with DNA which could potentially lead to
the changes in DNA structure and subsequently affect cell viability
[11]. From a medical standpoint, the most interesting is the ability
of QBAs to cause apoptosis. While SA causes apoptosis in cancer and
normal cells alike, a few studies have shown that cancer cells are
significantly more sensitive to the effects of SA than normal cells
[12,13]. In contrast, Debiton et al. did not find any difference in
effect of SA between cancer and normal cells [14]. Still, because of
their medicinal potential, both SA and CHE have been substantially
studied in this respect [15–18]. Nevertheless, the exact biological
paths by which both substances affect cells are still not entirely
known. Although the commercially available SA and CHE have been
extensively studied, data about the effects of other minor QBAs on
cells are still limited. For instance, it has been shown that MA and
SR show a considerable fluorescence shift when bound to DNA, a
property, which make them useful as fluorescent probes [19]. While
http://dx.doi.org/10.1016/j.jpba.2016.01.024
0731-7085/© 2016 Elsevier B.V. All rights reserved.
R. Sandor et al. / Journal of Pharmaceutical and Biomedical Analysis 121 (2016) 174–180 175
SL and CL exhibit antiproliferative and anti-microtubular activities
[20], SR, CR, and MA are antiproliferative and pro-apoptotic, which
has been well documented [21]. SL has been found to preferentially
induce necroptosis over apoptosis in melanoma cells [22]. Their
limited availability is the major challenge to be overcome when
assessing their full potential and/or risks.
One of the reasons for inconsistent results of various studies
could be the difference in metabolism of QBAs by different cells.
There are substantial differences in the metabolism and enzymes
present even between various healthy cell types and tissues, which
can lead to different metabolite profiles. This could explain why
some studies found a significant ROS production, leading to apoptosis
[23–25] while others found no increase in ROS production
when studying the pro-apoptic properties of QBAs [14,26]. The
rapid apoptotic response induced by a glutathione depletion effect
could be a part of a mechanism of SA cytotoxicity [14].
Plant cells producing QBAs are able to reabsorb SA and CHE
reducing it to dihydro derivatives, thereby protecting themselves
from their own product of synthesis [27]. It seems that also in
animals and humans the first step in biotransformation of CHE
and SA is their conversion to the respective dihydro derivatives.
Psotova et al. identified in experiments with rats dihydro SA
(DHSA) as the main metabolite in liver and plasma after the
oral administration of SA [28]. Kosina et al. showed that formation
of the dihydro-metabolites, which may be followed by
specific O-demethylenation/O-demethylation processes, is probably
the main route of biotransformation (detoxification) of the
benzo[c]phenanthridines in human hepatocytes [29]. Deroussent
et al. studied oxidative biotransformation of SA using eight human
recombinant CYPs; however, only CYP1A1 and CYP1A2 displayed
any metabolic activity. Up to six metabolites were detected. The
main metabolite M2 (m/z 320) resulted from a ring-cleavage of
SA followed by demethylation; 5,6-dihydrosanguinarine was the
prominent derivative formed from SA in cells expressing no CYP
[30].
The enzyme cofactors NADH and NADPH can participate in the
reduction of SA to DHSA in cells, and this conversion has been
demonstrated in vitro by several authors in the absence of any
enzyme [29,31,32]. Wu et al. demonstrated that the SA iminium
bond can be reduced by rat liver microsomes and cytosol in the
absence of NAD(P)H. The reductase activity of both fractions was
significantly enhanced in the presence of NADH. The amount of
DHSA formed in liver cytosol was 4.6-fold higher in the presence
of NADH. In this case, NADH was more effective than NADPH as
an electron donor for the SA iminium bond reduction [33]. These
observations indicated that cytosolic enzymes are highly likely to
be involved in SA reduction in the rat liver. DHSA and dihydrochelerythrine
(DHCHE) was proved in other animal cells, however;
details of such processes have not yet been clarified [28–30,32,34].
While the metabolic biotransformation of SA has been well studied
[35], the exact properties of many of its metabolites are not
known. Without the knowledge of the metabolic fate of QBAs and
properties of their metabolites, it is impossible to understand or
predict the potential therapeutic options and side effects. Vacek
et al. have performed a well-designed study where they have produced
both phase I and II metabolites of CHE and DHCHE in vitro
using human hepatocytes [36]. Authors tried testing cytotoxicity
of DHCHE and O-demethylated CHE (DMCHE); however, both were
prepared in vitro by reduction with NaBH4. As enzymatic, stereoselective
O-demethylation of CHE might provide two isomers, C8
DMCHE (fagaridine) and C7 DMCHE (NK 109 or isofagaridine), they
could correspondingly differ in toxicity [37]. Metabolic studies of
minor QBAs have not been yet performed and herein presented
approach comprising of drug biotransformation, isolation of its
metabolites, and (cyto-) toxicity assessment is required for their
comprehensive study.
2. Materials and methods
2.1. Reagents and materials
Methanol (MeOH, p.a.) was obtained from Penta (Czech Republic).
Phosphoric acid (p.a.), formic acid (LC MS), ammonium
hydroxide (28–30% NH3 in water), sodium formate (LC MS),
Tris, KCl, sucrose, EDTA, NADP, glucose-6-phosphate, MgCl2,
glucose-6-phosphate dehydrogenase, fetal bovine serum, glutamine,
penicillin, streptomycin, MeOH (LC MS), acetonitrile (ACN,
HPLC grade, LC MS) and dimethylsulfoxide (DMSO, p.a.) were purchased
from Sigma–Aldrich (Czech Republic). All water used was of
Fig. 1. Benzo[c]phenanthridine alkaloid structures.
176 R. Sandor et al. / Journal of Pharmaceutical and Biomedical Analysis 121 (2016) 174–180
an ultra-pure grade supplied by an in-house Milli-Q system (Millipore,
MA, USA).
2.2. Alkaloid isolation
The standards of the benzophenanthridine alkaloids originated
from isolations during the systematic studies in the laboratories of
the Department of Biochemistry from plant material of Macleaya
microcarpa (Maxim.) Fedde, Dicranostigma lactucoides Hook. F. et
Thomson, S. canadensis L., and Stylophorum lasiocarpum (Oliv.)
Fedde. Briefly, SA, CHE, CR, CL, SL, SR and MA were prepared by
extraction of dried, ground plant material with methanol in a
FexIKA® apparatus (IKA, Germany). The extract was purified, concentrated
and the last step of isolation was performed by means
of semi-preparative reversed-phase (RP) chromatography [38,39].
The identity of the alkaloids was verified by EI-MS, 1H NMR and
13C NMR and their purities were at least 98% according to an HPLC
analysis [40,41].
2.3. Isolation of rat liver microsomes
Male Wistar albino rats (280 ± 20 g), 8 weeks of age were used
for the isolation of liver microsomal fraction. Animals were housed
under standard laboratory conditions (22 ± 2 ◦C room temperature;
55 ± 5% room humidity; 12:12 h light/dark cycle) with free access
to water and food. After 5 days of acclimatization, animals were
sacrificed by decapitation and subjected to laparotomy.
A liver tissue from 10 animals (3 g) was homogenized, and
rat liver microsomes (RLM) were isolated by differential ultracentrifugation
(19 000 × g and 105 000 × g) in Tris/KCl buffer (pH 7.4;
20 mM) including washing with 0.15 M KCl and finally diluted in
Tris/sucrose buffer (pH 7.4; 0.25 M). The total protein content in
the microsomal preparations was assessed according to Lowry et al.
using bovine serum albumin as a standard [42]. Aliquots of 250 ␮L
were stored in −80 ◦C until needed. All experimental procedures
were approved by both Local Committee for Animal Welfare and
the Czech Central Committee for Animal Welfare, approval No.
MSMT-158/2013-310 valid till 10/2017.
2.4. Incubation, extraction and analysis of samples
Each alkaloid (50 ␮g) was in two replicates incubated in a
horizontal vortex mixer (120 min−1) with water bath (37 ◦C) for
120 min at 37 ◦C with RLM and a NADPH generating system according
to the previously described method of Wójcikowski et al.
with a slight modification [43]. The incubation mixture of final
volume of 1.0 mL contained phosphate buffer (pH 7.4; 50 mM),
EDTA (1.1 mM), NADP (1.2 mM), glucose-6-phosphate (4.4 mM),
MgCl2 (3.2 mM), glucose-6-phosphate dehydrogenase (1 U mL−1),
RLM (50 ␮L, 20 mg mL−1). The reaction was stopped by addition of
100 ␮L of ice-cold methanol and transferred to an ice bath.
Metabolites were purified by adding 1 mL of MeOH:ACN:formic
acid (50:50:0.02, v/v/v) into a 100 ␮L aliquot of the incubation mixture,
followed by vortex and centrifugation. A 50 ␮L portion of the
supernatant was five times diluted with water and injected (20 ␮L)
onto an LC column. Each alkaloid was designed for a set of incubations,
where one sample contained the alkaloid, RLM and a NADPH
generating system, the second one contained only RLM and the
NADPH generating system (blank), and the third one tested the
alkaloid in the NADPH generating system with no P450 enzymes.
For isolation of selected metabolites, 200 ␮g of the alkaloid in
two replicates was mixed with the same RLM system, incubated
overnight (16 h) and purified accordingly.
2.5. LC MS
The LC MS method was developed using a Dionex Ultimate
3000RS (Thermo Scientific, CA, USA) module. Compound separation
was achieved with a 3.0 mm × 150 mm, 5 ␮m Synergi RP-Max
C18 (Phenomenex, CA, USA) column, thermostated at (23 ± 0.1) ◦C,
and a flow-rate of 0.5 mL min−1. The binary mobile phase system
consisted of 0.1% (v/v) of formic acid and ACN. After a 20 ␮L injection,
the volume of ACN was linearly increased from 20% to 40%
over 10 min and then to 80% over the subsequent 10 min. ACN was
held at 80% for 10 min, followed by equilibration under the initial
conditions for 3.0 min. A complete HPLC run was 33 min. The
HPLC system was connected to a Bruker MicrOTOF-Q II (Germany)
mass spectrometer, operated in the positive electrospray ionization
mode. The ionization conditions were determined by the
software as follows: capillary voltage: 4500 V, end plate offset:
−500 V, source temperature: 220 ◦C, desolvation gas (nitrogen)
flow: 10 L min−1, nebulizer (nitrogen) pressure: 300 kPa and collision
cell voltage: 35 eV. The base-peak chromatogram (BPC) was
acquired in MS mode by monitoring the m/z range of 50–3000
with a spectra sample time of 1 s. The mass spectrometer was calibrated
using 10 mM sodium formate in 50% isopropyl alcohol on
a daily basis and in the beginning of each LC run with a 20 ␮L loop
flush. This step ensured the mass accuracy was below 5 ppm across
the entire chromatogram. High-resolution MS and MS/MS spectra
were first investigated to obtain the elemental formula of each
compound. The final identification of target compounds relied on
isotope pattern matching with a combination of MS/MS and retention
behavior.
2.6. Metabolite isolation
An Agilent 1200 Series LC system (Agilent Technologies, CA,
USA) equipped with a fraction collector employed in alkaloid isolation
was used for purification and isolation of selected metabolites
as well. It consisted of a ternary pump, a DAD, a syringe-loading
sample injector (ECOM, Czech Republic) with a 5 mL external sample
loop and a fraction collector. Alkaloids were separated on a C12
column (Synergi RP-Max, 4 ␮m, 150 mm × 10 mm ID, Phenomenex,
CA, USA).
The mobile phase contained ammonium formate (0.1 M HCOOH
titrated with NH4OH to pH 3.75) and ACN with a following elution
profile: ACN was linearly increased from 20% to 40% over 10 min and
then to 80% over the subsequent 10 min. ACN was held at 80% for
10 min, followed by equilibration under the initial conditions for
3.0 min. The purified mixture of alkaloid and its metabolites was
diluted five times with water and 5 mL of sample was injected onto
a semi-preparative column. The flow-rate was set at 6 mL min−1
and fractions were collected on a peak basis using a diode array
detector at 280 nm. Employment of ammonium formate provided
similar retention times as in the case of LC–MS and enabled simple
treatment of fractions, as it is readily volatile. Moreover, the
peak tailing resulting from electrostatic interactions of charged
benzophenanthridines with active silanol groups in the stationary
phase was minimized by competing ammonium ions.
Both identity and purity of the fractions were determined by
LC MS. The fractions from both replicates were joined and the solvent
volume was reduced to ∼500 ␮L. The final concentration of
the purified metabolite was calculated based on the amount of the
original alkaloid loaded into the RLM system and verified at 280 nm
according to external calibration of the precursor alkaloid.
2.7. Antiproliferative activity
The antiproliferative activities of metabolites prepared by
overnight incubation of CHE and SL were tested using the A-
R. Sandor et al. / Journal of Pharmaceutical and Biomedical Analysis 121 (2016) 174–180 177
5.Blank_bez_alkaloidu_RA5_01_1757.d
11.CHE_4h_RB3_01_1765.d
8.CHE+NADPH_4h_RA8_01_1762.d
13.CHE+NADPH+RLM_4h_RB5_01_1767.d
0.0
0.2
0.4
0.6
0.8
6x10
Intens.
0.0
0.2
0.4
0.6
0.8
6x10
Intens.
0.0
0.2
0.4
0.6
0.8
6x10
Intens.
0.0
0.2
0.4
0.6
0.8
6x10
Intens.
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Time [min]
1
1
1
2
2
3
4
5
A
B
C
D
Fig. 2. Base peak chromatograms of a 4-h incubation mixture consisting of (A) RLM, (B) CHE, (C) CHE and NADPH, (D) CHE, NADPH, and RLM. Peaks are labeled as follows (1)
CHE, (2) DHCHE, (3) O-demethyl CHE, (4) O-demethyl DHCHE, (5) demethylene O-demethyl CHE. For more details, see text.
375 human malignant melanoma cell line and a colorimetric MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay. The A-375 cell line was purchased from the European Collection
of Animal Culture (ECACC, Salisbury, UK). The MTT assay was
performed in a 96-well plate (Nunc A/S, Denmark). Individual wells
were seeded with 200 ␮L of cells (1 × 104 per mL) and incubated in
a RPMI 1640 medium supplemented with 10 mass% of fetal bovine
serum, 2 mM of glutamine, 100 IU mL of penicillin and 100 ␮g mL−1
of streptomycin at 37 ◦C in a humidified atmosphere (5 vol.% CO2).
After 24 h of incubation, the medium was replaced with an alkaloidcontaining
medium at concentrations ranging from 0.3 mg L−1 to
10 mg L−1. The MTT assay was performed 48 h after addition of the
alkaloid: 20 ␮L of MTT (5 mg L−1 in PBS) was added to each well
and incubated for 4 h under culture conditions. At the end of the
incubation period, the medium was removed, the formazan crystals
were dissolved in 200 ␮L of DMSO and the optical density was
measured at 570 nm using a DTX 880 Multimode Detector (Beckman
Coulter, CA, USA). The concentration of each compound was
examined in four replicates.
3. Results and discussion
3.1. Metabolite identification
Products of biotransformation were identified by LC MS as result
of a combination of MS, MS/MS, and retention behavior. Searching
for metabolites was supported by a differential comparison of a
series of chromatograms with and without RLM and in the NADPH
system only, as seen in Fig. 2. Such a comprehensive approach
with only protein precipitation as a purification step allowed for a
maximum number of products. Selection of potential metabolites
relied on accurate m/z and characteristic fragmentation patterns.
Benzophenanthridine alkaloids, unlike pavine or aporphine ones,
strictly keep their nitrogen inside the highly aromatic ring during
fragmentation even under conditions of high collision energy. If no
nitrogen is lost during fragmentation then any change in parity in
nominal mass resulted from a loss of a radical molecule. That is considered
characteristic for methyl groups substituted on the nitrogen
of the phenanthridine ring [30]. If a methoxy group was adjacent to
a hydroxyl or another methoxy group, a loss of methane (−16 Da)
followed by a loss of CO (−28 Da) was observed. Compounds in
the chromatogram that experienced such fragmentation behavior
were inspected as a possible product of enzymatic biotransformation.
The last step in confident identification consisted in a relevant
change in retention time of suggested structures. Generally, phase
I metabolism aims to produce more polar compounds to increase
their solubility and/or to make them available for conjugation reactions
occurring during the phase II. The change corresponded with
the chromatographic behavior with all identified compounds. The
reduced dihydroderivatives, lacking the quaternary nitrogen, are
known for their poor solubility in water, which was observable
by a large increase in retention time on an RP column. Moreover,
they can be distinguished in positive full scan MS, as they tend to
be easily oxidized in the ion source providing characteristic peaks
[M-2 + H]+. This was especially important when identifying doubly
O-demethylated or demethylenated dihydro derivatives, which
eluted from the column even earlier than the original quaternary
alkaloids. The dihydro derivatives are considered the major and
probably the initial metabolites that are produced from any QBAs.
It is reasonable to assume that the reductive transformation is the
fastest process as their formation was accomplished even when
they were subjected to a NADPH generating system with no RLM
in the reaction mixture. While their toxicity was found to be rather
low, they could serve as precursors for further metabolites whose
178 R. Sandor et al. / Journal of Pharmaceutical and Biomedical Analysis 121 (2016) 174–180
Table 1
Retention time, exact mass, elemental composition, a type of reaction, and metabolite content of QBAs after 120 min incubation with RLM.
Alkaloid Detected compounds tr (min) Ion type Observed m/z Sum formula [M] Nominal mass
shift (Da)
Reaction(s) involved a
Peak area
fraction (%)
SA 4.4 [M]+
332.0927 C20H14NO4
Demethylene SA 2.8 [M]+
320.0923 C19H14NO4 −12 Demethylenation 3.0
Demethylene DHSA 12.2 [M + H]+
322.1069 C19H15NO4 −10 Reduction, demethylenation 0.2
DHSA 22.1 [M + H]+
334.1069 C20H15NO4 +2 Reduction 96.8
CHE 5.7 [M]+
348.1241 C21H18NO4
Demethylene O-demethyl CHE 2.3 [M]+
322.1079 C19H16NO4 −26 O-demethylation,
demethylenation
5.6
Demethylene CHE 3.9 [M]+
336.1210 C20H18NO4 −12 Demethylenation 0.8
Hydroxy O-demethyl CHE 4.3 [M]+ 350.1028 C20H16NO5 +2 O-demethylation,
monooxygentaion
2.4
O-demethyl CHE 4.4 [M]+
334.1071 C20H16NO4 −14 O-demethylation 62.4
Demethylene O-demethyl DHCHE 5.8 [M + H]+
324.1236 C19H17NO4 −24 Reduction, O-demethylation,
demethylenation
0.2
Hydroxy DHCHE 15.6 [M + H]+
366.1341 C21H19NO5 +16 Reduction, monooxygenation 0.6
O-demethyl DHCHE 17.5 [M + H]+
336.1235 C20H17NO4 −12 Reduction, O-demethylation 15.4
DHCHE 21.5 [M + H]+
350.1391 C21H19NO4 +2 Reduction 12.6
CL 7.1 [M]+
378.1351 C22H20NO5
O-demethyl CL 5.7 [M]+
364.1180 C21H18NO5 −14 O-demethylation 22.1
Bis (O-demethyl) DHCL 9,9 [M + H]+
352.1176 C20H17NO5 −26 Reduction, O-demethylation
(2x)
2.0
O-demethyl DHCL 16.2 [M + H]+
366.1331 C21H19NO5 −12 Reduction, O-demethylation 22.2
DHCL 21.1 [M + H]+
380.1483 C22H21NO5 +2 Reduction 53.6
CR 6.1 [M]+
362.1033 C21H16NO5
O-demethyl CR 4.7 [M]+
348.0865 C20H14NO5 −14 O-demethylation 56.6
O-demethyl DHCR 17.8 [M + H]+
350.1027 C20H15NO5 −12 Reduction, O-demethylation 2.3
DHCR 21.9 [M + H]+
364.1168 C21H17NO5 +2 Reduction 41.1
SL 6.9 [M]+
394.1654 C23H24NO5
O-demethyl SL 5.6 [M]+
380.1498 C22H22NO5 −14 O-demethylation 2.7
Hydroxy SL 6.1 [M]+
410.1604 C23H24NO6 +16 Monooxygenation 1.5
Bis(O-demethyl) DHSL (A) 6.7 [M + H]+
368.1499 C21H21NO5 −26 Reduction, O-demethylation
(2×)
2.5
Bis(O-demethyl) DHSL (B) 8.5 [M + H]+
368.1487 C21H21NO5 −26 Reduction, O-demethylation
(2×)
0.9
O-demethyl DHSL (A) 12.5 [M + H]+
382.1668 C22H23NO5 −12 Reduction, O-demethylation 24.0
O-demethyl DHSL (B) 13.1 [M + H]+
382.1639 C22H23NO5 −12 Reduction, O-demethylation 0.4
O-demethyl DHSL (C) 14.1 [M + H]+
382.1639 C22H23NO5 −12 Reduction, O-demethylation 0.6
Hydroxy DHSL 17.0 [M + H]+
412.1759 C23H25NO6 +18 Reduction, monooxygenation 0.3
DHSL 18.5 [M + H]+
396.1853 C23H25NO5 +2 Reduction 67.0
SR 6.0 [M]+
378.1341 C22H20NO5
O-demethyl SR 4.6 [M]+
364.1185 C21H18NO5 −14 O-demethylation 22.8
O-demethyl DHSR 15.7 [M + H]+
366.1330 C21H19NO5 −12 Reduction, O-demethylation 1.1
DHSR 19.9 [M + H]+
380.1498 C22H21NO5 +2 Reduction 76.0
MA 7.8 [M]+
392.1169 C22H18NO6
Demethylene MA 5.1 [M]+
380.1122 C21H18NO6 −12 Demethylenation 9.9
Demethylene DHMA 11.2 [M + H]+
382.1283 C21H19NO6 −10 Reduction, demethylenation 1.5
DHMA 22.3 [M + H]+
394.1319 C22H19NO6 +2 Reduction 88.5
a
Peak area fraction roughly represents a portion of a metabolite from the sum of all metabolites.
presence, structure, and formation rate might be the determining
factors of the initial compound toxicity.
Biotransformation of seven QBAs with major and minor metabolites
is summarized in Table 1. Products of biotransformation that
contributed with their peak area to the mixture from more than
50% in the chromatogram (excluding the original alkaloid) were
assigned as major metabolites. It is important to note that the
purpose of representing metabolite distribution solely from the
peak area fraction was only to approximate the real situation, as
their true response might be different. The reason for such a semiquantitative
comparison is the fact that standards of metabolites
are not available.
3.2. Metabolite isolation
The purified mixture of alkaloids and their metabolites were
separated by a semi-preparative HPLC. The fractions containing
transformed dihydro compounds (e.g., O-demethylated DHCHE)
were kept for several days in dark and cold to let them re-oxidize to
the respective quaternary compound. The initial large difference in
retention times between oxidized, charged molecules and reduced,
less polar dihydro derivatives allowed for obtaining the purest quaternary
metabolites in the end. This was especially important when
assessing the toxicity, as even low amount of the original alkaloid
could have influenced the outcome of the tested metabolite. The
final concentrations of isolated compounds were 32 mg L−1 and
29 mg L−1 for O-demethylated CHE and O-demethylated SL, respectively.
None of the dihydro-metabolites was tested for toxicity, as it
was impossible to keep them in the reduced form without affecting
the toxicity itself, i.e., addition of NADH or NaBH4.
3.3. Metabolite structure to toxicity relationship
Despite the similar structures of studied QBAs, different QBAs
underwent slightly different reactions during metabolism and the
resulting distribution of metabolites for each alkaloid was unique.
R. Sandor et al. / Journal of Pharmaceutical and Biomedical Analysis 121 (2016) 174–180 179
Fig. 3. An MTT assay of CHE and O-demethyl CHE in a range of concentrations
0.1–10 mg/L.
Fig. 4. An MTT assay of SL and O-demethyl SL in a range of concentrations
0.1–10 mg/L.
The biosynthetic pathway and known data about toxicity seems
to be related to the metabolic fate of the compounds. In most
plants, the biosynthetic pathway ends with the creation of SA,
but a few plant species, such as M. microcarpa or S. lasiocarpum,
may induce two stepwise hydroxylation and methylation reactions
resulting in production of CR and then MA [44]. When SA, CR,
and MA were subjected to RLM the O-demethylation reaction was
observed in CR only. Sanguinarine obviously could have not undergone
O-demethylation, having no methoxy groups, and underwent
demethylenation instead; MA favored the same ring opening reaction
over O-demethylation, despite the additional methoxy group
when compared to CR. The rate of O-demethylation seems to be
associated with the cytotoxicity of each alkaloid, as SA and MA
have proved substantially more toxic than CR when assessed using
a standard MTT assay [45].
The major metabolite of CHE, O-demethylated CHE, was formed
by a stereospecific enzymatic reaction providing demethylation of
a methoxy group attached either to the carbon C8 (fagaridine) or C7
(NK-109) of the ring. The products substantially differ in toxicity;
fagaridine is non-toxic but NK-109 was found to be as toxic as SA
or nitidine [37]. The toxicity of the O-demethylated product was
lower when compared to the original CHE as seen in Fig. 3. This fact,
while strengthening the confidence in fagaridine formation, also
indicated that the relatively low toxicity of CHE might be caused
by its straightforward detoxification to a relatively high number of
metabolites through the P450 enzyme family.
CL with three and SL with five methoxy groups favored single
and double O-demethylation as a major type of reaction, respectively.
We did not observe a fully demethylated product from CL or
SL under any conditions, which could imply that the methoxy group
at C10 of the benzophenanthridine ring at these two alkaloids was
kept intact in all cases. However, this requires to be confirmed, e.g.,
by NMR spectrometry. The O-demethylated SL, when subjected to
an MTT assay, was found to be less toxic than the original SL as seen
in Fig. 4. It is worth noting that while this fact has been assumed
since the first oral administration of SA and/or CHE, experimental
data about the toxicity of their major metabolites have not yet been
shown.
4. Conclusions
Biotransformation of five minor QBAs was investigated using in
vitro incubations with RLM. The manner of biotransformation of
alkaloids varied significantly although the original alkaloids are of
a very similar structure and the incubation conditions were identical.
Typical transformation reactions included O-demethylation,
O-demethylenation, hydroxylation, and/or their combination. The
inability to undergo specific transformation reactions may explain
high retention of MA in the organism and thus high toxicity.
Toxicity of O-demethylated metabolites, which were successfully
isolated and their structure was confirmed by MS or MS/MS, was
in general lower in comparison to the original alkaloids.
Acknowledgments
The financial support for this work received from the Czech
Ministry of Education, Youth and Sports (LH12176-KONTAKT II)
and Specific University Research Grant (MUNI/A/1195/2014,
MUNI/A/1123/2014, and MUNI/A/1116/2014) is gratefully
acknowledged.
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3.2 Sanguinarine is reduced by NADH through a covalent adduct
QBAs have been known for several biological properties, which were empirically verified but not elucidated. The
ability of SA to deplete the reducing potential of a cell has been one of them. Elucidation of SA properties would
unlikely have started if another publication by Matkar et al. had not come first.51
The authors claimed to discover
a new, negatively-charged(!) species of SA, which they identified by electrophoresis. Since the authors provided
no sufficient reasoning about the proper existence of such a species, we decided to replicate the experiment, and
attempted to identify the species in a more sensible way. A careful investigation and examination of several TLC
bands revealed that the novel SA species is extremely polar and converts to SA after some time. This implied a
thought of a covalently bound adduct of SA and NADH, which could even be an intermediate product in a reaction
of SA with cell reducing equivalents NADH or NADPH. Both coenzymes have been well-known for the following
features:
1. High resistance to effective oxidation by air oxygen.
2. Net transfer of a hydride ion as the formal mechanism of their oxidation/reduction.
SA was able to covalently attach NADH and act as a mediator of NADH oxidation by air oxygen. In other words, SA
exhibited enzyme-like activity; it was not consumed during the reaction and accelerated the NAD(P)H reaction with
oxygen. From the other point of view, many researchers have been looking for a compound, which could elucidate
(reject) the theory of a net hydride transfer, and SA (and possibly other QBAs) could serve as candidate molecules.
If a hydride ion had really been formed inside the cell it would have been extremely unstable and immediately
reacted with protons in its surroundings, especially those originating from dissociation/ionization of water molecules.
The existence of the covalent adduct was identified using LC–MS and its reaction kinetics was characterized using
LC with UV-Vis detection. The presence of the adduct contributed to explaining the mechanism of depletion of
reducing equivalents in a cell while supporting an alternative theory of their electrochemical behavior beyond simple
hydride transfer.
SANDOR, Roman, Jiri SLANINA, Adam MIDLIK, Kristyna SEBRLOVA, Lucie NOVOTNA, Martina CARNECKA,
Iva SLANINOVA, Petr TABORSKY, Eva TABORSKA and Ondrej PES (corresponding author). Sanguinarine
is reduced by NADH through a covalent adduct. Phytochemistry. 2018, 145, 77–84. ISSN 0031-9422. doi:
10.1016/j.phytochem.2017.10.010 Document Type: Article, IF = 2.905; JCR Category + Category Quartile:
BIOCHEMISTRY & MOLECULAR BIOLOGY Q2 + PLANT SCIENCES Q1; AIS = 0.761
Author’s contribution: 50 %
46
Sanguinarine is reduced by NADH through a covalent adduct
Roman Sandor a
, Jiri Slanina a
, Adam Midlik b, c
, Kristyna Sebrlova a
, Lucie Novotna a
,
Martina Carnecka a
, Iva Slaninova d
, Petr Taborsky e
, Eva Taborska a
, Ondrej Pes a, *
a
Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
b
National Centre for Biomolecular Research, Faculty of Science, Masaryk University Brno, Kamenice 5, 62500 Brno, Czech Republic
c
Central European Institute of Technology CEITEC MU, Kamenice 5, 62500 Brno, Czech Republic
d
Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
e
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 21 April 2017
Received in revised form
26 October 2017
Accepted 27 October 2017
Available online 5 November 2017
Keywords:
Benzophenanthridine alkaloids
Ene adduct
Hydride transfer
LC-MS
NADH
NADH depletion
Redox cycling
Sanguinarine
a b s t r a c t
Sanguinarine is a benzo[c]phenanthridine alkaloid with interesting cytotoxic properties, such as induction
of oxidative DNA damage and very rapid apoptosis, which is not mediated by p53-dependent
signaling. It has been previously documented that sanguinarine is reduced with NADH even in
absence of any enzymes while being converted to its dihydro form. We found that the dark blue fluorescent
species, observed during sanguinarine reduction with NADH and misinterpreted by Matkar et al.
(Arch. Biochem. Biophys. 2008, 477, 43e52) as an anionic form of the alkaloid, is a covalent adduct
formed by the interaction of NADH and sanguinarine. The covalent adduct is then converted slowly to the
products, dihydrosanguinarine and NADþ
, in the second step of reduction. The product of the reduction,
dihydrosanguinarine, was continually re-oxidized by the atmospheric oxygen back to sanguinarine,
resulting in further reacting with NADH and eventually depleting all NADH molecules. The ability of
sanguinarine to diminish the pool of NADH and NADPH is further considered when explaining the
sanguinarine-induced apoptosis in living cells.
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
Sanguinarine (SA) belongs to a family of plant secondary metabolites
called quaternary benzo[c]phenanthridine alkaloids
(QBAs). These compounds have been extensively studied for their
numerous biological activities, such as antitumor, antimicrobial,
antifungal, and anti-inflammatory. While the results have been
summarized in several works, the greatest attention is given to the
anticancer activity of QBAs (Slaninova et al., 2014; Gaziano et al.,
2016). It has been reported that SA is in vitro cytotoxic preferentially
toward cancer cells than normal cells at concentrations that
are comparable to those of the current clinically used anticancer
agents (Ahmad et al., 2000). Under physiological conditions, a hydroxide
anion (OHÀ
) is reversibly attached to the iminium bond of
SA to give a 6-hydroxy product called an alkanolamine or a pseudobase
(Fig. 1). The alkanolamine form, which is a nonpolar uncharged
molecule, can easily enter a cell to establish a new, pHdependent
equilibrium between the iminium and alkanolamine
form inside the cell. The alkaloid toxicity depends on the ability of
the planar, charged quaternary form of SA to produce a stable
complex with DNA, which subsequently could affect the cell
viability (Slaninova et al., 2001; Vacek et al., 2011). Treatment of
cells with SA led to a rapid production of reactive oxygen species
(ROS) (Burgeiro et al., 2013), fast and severe glutathione depletion
(Debiton et al., 2003), oxidative DNA damage and very rapid
apoptosis that was not mediated by p53-dependent DNA damage
signaling (Matkar et al., 2008a; Hammerova et al., 2011). The first
step in the metabolism of SA in rat liver is the reduction of the
quaternary form to dihydrosanguinarine (DHSA) (Fig. 1). The conversion
might be mediated by several NAD(P)H dependent oxidoreductases
(Deroussent et al., 2010; Wu et al., 2013). In cell cultures
of Eschscholzia californica, SA is reabsorbed and reduced to DHSA by
sanguinarine reductase, which was isolated (Weiss et al., 2006) and
characterized (Vogel et al., 2010). Additionally, it has been observed
that SA underwent the conversion to its inactive reduced form even
when incubated with NADH in the absence of any enzyme (Kovar
et al., 1986; Matkar et al., 2008b), however; physiologically
important reducing agents, such as glutathione and L-ascorbic acid,
* Corresponding author.
E-mail address: ondramayl@gmail.com (O. Pes).
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
https://doi.org/10.1016/j.phytochem.2017.10.010
0031-9422/© 2017 Elsevier Ltd. All rights reserved.
Phytochemistry 145 (2018) 77e84
were unable to effectively reduce SA (Kosina et al., 2011). On top of
that, SA, as well as other QBAs, may undergo a complex series of
metabolic changes, some of which may contribute to their biological
effects (Sandor et al., 2016).
When studying a relationship between the cell death and the
ability of SA to deplete the cellular antioxidant capacity Matkar
et al. (2008b) observed a novel, dark blue fluorescent, anionic
form of SA formed by the reduction with NADH in the absence of
any enzyme. The authors detected a new form of SA by reversed
phase thin layer chromatography (RP-TLC) and determined the
negative charge of species by gel electrophoresis. While a new form
of SA would be somewhat fascinating, an alternative explanation of
these findings could be presented and interpreted.
In this study, we have employed chromatographic and spectrometric
techniques to give a novel interpretation of SA being
reduced with NADH to DHSA. We found that the dark blue fluorescent
anion produced during SA reduction with NADH, originally
observed by Matkar et al. (2008b), is a covalent adduct formed by
the reaction of NADH and SA. The adduct is then converted slowly
to products, DHSA and NADþ
, in the second step of reduction. DHSA
is continuously re-oxidized back to SA by the atmospheric oxygen
resulting in eventual NADH depletion.
2. Results
2.1. Reduction of SA with NADH forms an unexpected spot on thinlayer
chromatography
First, we have aimed to reproduce the RP-TLC performed by
Matkar et al. (2008b). Resulting chromatograms might be seen in
Fig. 2. The retention factor (Rf) for NADH was one as it traveled
along with the polar mobile phase with no retention on the nonpolar
solid phase. Visualization under UV light (340 nm) showed
a fluorescence band of NADH broadened over the entire chromatogram
concentrating at the head. The same band at the head of
the chromatogram was clearly visible in the line where NADH was
mixed with SA. This was in accordance with our expectations as the
NADH molecule was negatively charged and thus highly polar. Dark
blue fluorescence was clearly visible in the lines where SA was
mixed with NADH (Rf ~ 0.75) and with NaBH4 (Rf ~ 0.35). After four
hours in dark, both dark blue spots turned their fluorescence to the
same orange-yellow color as it has been observed in the sole SA line
(Fig. 2B). A lower Rf of the dark blue spot in the SA þ NaBH4 line
allowed for identification as DHSA as it was characterized by blue
fluorescence and a decrease in polarity, which had resulted from a
loss of the charged quaternary form. The same DHSA band would
have been observed in the SA þ NADH line if the mixture had been
applied onto a TLC plate after a longer time (data not shown). A spot
between SA and DHSA (Rf ~ 0.45) observed at lines of pure SA and
SA þ NADH probably represented an alkanolamine form of SA.
Another dark blue fluorescent spot in the SA þ NADH line with Rf
~0.75 could not have been attributed to any known species of SA
and was then studied comprehensively by liquid chromatography
with mass spectrometric detection (LC-MS).
2.2. Sanguinarine forms a covalent adduct with NADH
LC-MS was employed as a next step in revealing the origin of the
interaction between SA and the NADH molecule. A representative
chromatogram of an LC-MS method at very similar conditions to
those used for RP-TLC might be seen in Fig. 3. The product, formed
from the reaction between SA and NADH, was then expected to
elute around the tenth minute. Detection at 280 nm and by MS
showed a few distinct peaks around that retention time. In order to
be able to identify the product, positive mode MS and MS/MS
spectra were acquired (Fig. 4). Peaks observed in Fig. 4 might be
accordingly assigned to the pure reactants (SA at 332 Da and NADH
at 666 Da) as well as to the product of their reaction (997 Da for a
singly charged and 499 Da for a doubly charged ion). The MS/MS
spectrum of the doubly charged ion produces only SA as a major
fragment. This fragmentation might be explained by a favorable
formation of an aromatic quaternary form of SA and a neutral
residue of NADH. In other words, SA is a good leaving fragment
while retaining its charge as it contains quaternary nitrogen. In
negative mode, the adduct was observed at 995 Da (data not
shown) indicating that the mass of the neutral species was 996 Da.
Fig. 1. A molecule of sanguinarine in A) an iminium (a quaternary) form, B) an alkanolamine form, C) dihydrosanguinarine, and D) a hypothetic anionic form suggested by Matkar
et al. (2008b). The iminium form exhibits yellow fluorescence while the forms B and C glow dark blue under the UV light.
R. Sandor et al. / Phytochemistry 145 (2018) 77e8478
To confirm that the 997 Da ion is a covalent adduct and not just
an association of SA and NADH (332 Da þ 665 Da ¼ 997 Da) in a
liquid or gas phase, the diode-array spectra of the substrates and
products were evaluated, see Fig. 5. All chromatographic peaks of
the adduct absorbed at 280 and 340 nm which were also the absorption
maxima of NADH. However, the characteristic absorption
maximum of the SA quaternary form (470 nm) was not found in the
peaks of the adducts. Since the formation of the adduct led to
discoloration of SA, the 997 Da adduct should not be considered a
mass spectrometry gas phase species and a covalent bond was
likely to be formed. The existence of more than one peak around
the 10th minute evoked the product existed in several isomeric
forms; please see Discussion for more details.
The adduct is likely to be the same substance observed by
Matkar et al. (2008b) in their experiments due to the similar
properties (such as negative charge, polarity, and dark-blue fluorescence
changing with time). At neutral pH, the net charge of the
adduct is (À1), because the negative charge of phosphate groups
(À2) is only partially weakened by the positively charged nitrogen
atom of pyridine (þ1). The net negative charge of the product was
Fig. 2. RP-TLC of NADH, SA, an equimolar mixture SA þ NADH, and a mixture of SA with NaBH4 after A) 15 min, B) 240 min. SA-OH e an alkanolamine form of SA. For details, please
see text.
Fig. 3. Liquid chromatography of SA mixed with NADH after 2 h of reaction with detection A) MS of a base peak, B) MS of an extracted ion m/z 499.1089 ± 0.2 C) at 280 nm, D) at
340 nm, E) at 470 nm. Peaks are labeled as follows: 1) NADþ
, 2) NADH, 3) SA, 4) a group of adducts of SA with NADH, 5) DHSA. SA is the only colorful compound as it absorbs visible
light. For details, please see text. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
R. Sandor et al. / Phytochemistry 145 (2018) 77e84 79
originally incorrectly interpreted as a new form of SA, which was
not supported by any evidence (Matkar et al., 2008b). Furthermore,
if the anionic species had truly existed, it should have been a highly
unstable, as it would have immediately accepted a proton from
water (Fig. 1).
Fig. 4. A positive ESI A) MS spectrum of the SA þ NADH adduct from the 10th min (Fig. 3) and B) MS/MS spectrum of the doubly charged, monoisotopic adduct ion 499.1087 Da
(theoretical 499.1119 Da). Other monoisotopic signals belong to a singly charged adduct 997.1603 Da (theoretical 997.2165 Da), an ion of NADH 666.1166 Da (theoretical
666.1320 Da), and an ion of SA 332.0939 Da (theoretical 332.0917 Da).
Fig. 5. Diode-array spectrum of the NADH-SA adduct (solid), NADþ
/NADH (dash-dotted) SA (dotted), and DHSA (dashed). The spectra have been normalized to the adduct
maximum intensity at 280 nm.
R. Sandor et al. / Phytochemistry 145 (2018) 77e8480
2.3. The covalent adduct is an intermediate in the reduction of SA
with NADH
Kinetics of the reaction was measured to explore if the covalent
adduct was involved in the final reduction of SA to DHSA. It is worth
noting, that the same reduction reaction was described by Kovar
et al. (1986); however, the authors did not attempt to clarify the
mechanism of the reduction, although they suggested a rather
complex transition state, which must have been formed between
the target molecules. The formation kinetics at 280 nm might be
seen in Fig. 6, which clearly showed an increased concentration of
the adduct and DHSA over time. While the maximum concentration
of the adduct was observed in 120 min, DHSA reached its peak
concentration in about 480 min. In other words, the higher rate of
the adduct formation than the rate of its decomposition to DHSA
suggested that the adduct was truly necessary for the reaction to be
completed. The decline in SA concentration was biphasic with the
fast decrease occurring in the first 120 min followed by a slow
Fig. 6. NADH and SA were mixed as described in section 5.3, sealed under Ar and twelve consecutive LC-MS runs were performed. The initial rates expressed as a slope of a linear
regression from the first three points were À11.0, þ5.3, and þ2.8 peak area/minute for SA, the adduct, and DHSA respectively.
Fig. 7. Ten consecutive LC-MS runs from a vial with the adduct transferred from the fraction collected by semi-preparative chromatography. NADH peak area is kept at a constant
level while the peak area of DHSA increases.
Fig. 8. Representation of the NAD(P)H depletion effect caused by continual reoxidation
of DHSA. SAþ
- a quaternary form, SA-NAD(P)H - the adduct of SAþ
with
NADH.
R. Sandor et al. / Phytochemistry 145 (2018) 77e84 81
decrease. It corresponds to the rapid formation and a slow
decomposition of the adduct. The additional decrease in DHSA after
the 480th
minute is caused by re-oxidation of the entire mixture by
the atmospheric oxygen. If the vials had not been sealed under
argon atmosphere, the re-oxidation would have appeared around
the 400th
minute and the resulting DHSA would have been subsequently
converted to SA (data not shown).
In order to support our hypothesis that the adduct was a real
intermediate in the reduction of SA with NADH, the adduct has
been isolated by semi-preparative LC and its further conversion was
immediately analyzed by LC-UV-MS, as seen in Fig. 7. Initially, the
adduct was partially converted back to reactants, SA and NADH,
indicating that the formation of the covalent adduct was reversible.
The NADH concentration had peaked around 50 min and then
declined very slowly. This decrease in NADH concentration indicated
the presence of additional reaction. At the same time, the
concentration of the adduct and DHSA kept decreasing and slowly
increasing, respectively. This suggested that the additional reaction
was the conversion of the adduct to DHSA and that the adduct was
indeed a true intermediate of the reaction. The continuous increase
in the SA concentration could be explained as a sum of a velocity of
the initial reversed reaction and a velocity of DHSA oxidation
caused by the atmospheric oxygen.
It is worth to note that the equilibrium conditions in the vial
from the fraction collector were substantially different from those
when the mixture was fresh. When SA and NADH were freshly
mixed the excess of NADH made sure the reaction would progress,
up to some point, in a reducing environment. On the other hand,
when a fraction containing the adduct was removed from the collector
and transferred to the LC-MS vial, the equilibrium was shifted
to the left i.e., the adduct partially decomposed to reactants (NADH
and SA) and a part was converted to products (NADþ
and DHSA).
When the reaction was left overnight to proceed completely, SA
and NADþ
were the only compounds observed in the reaction
mixture. SA catalyzed the oxidation of NADH by the air oxygen, in
other words, sanguinarine showed an oxygen-NADH oxidoreductase-like
activity. This effect of SA is probably the reason why SA
and other QBAs could deplete NAD(P)H in the cells and induce rapid
cell death (Fig. 8).
3. Discussion
3.1. NADH is oxidized by SA in a two-step process involving the
covalent adduct formation
In our study, we observed a formation of a covalent adduct
during the reduction of SA with NADH. Although we have observed
the formation of several isomeric adducts between SA and NADH
with similar chromatographic properties, we expect solely a single
species was converted to the products e DHSA and NADþ
.
The existence of the covalent intermediate as a result of an
attack of NADH to the reactive iminium bond of SA is supported by
two facts:
Fig. 9. The proposed Ene mechanism for NADH dehydrogenases, the upper figure shows NADH oxidation, the lower figure shows NADþ
reduction, according to Hamilton (1971);
Libby and Mehl (2012).
Fig. 10. Reduction of SA with NADH based on the proposed Ene adduct mechanism.
R. Sandor et al. / Phytochemistry 145 (2018) 77e8482
1) The molecular mass of the adduct and its respective fragmentation
to SA and NADH.
2) Discoloration (a lack of 470 nm absorption band) and dark blue
fluorescence of the adduct.
The originally postulated one-step hydride transfer to NADþ
has
been still a prevailing mechanism of enzymatic NADþ
reduction
especially due to an inability to detect any reaction intermediates.
Here, we try to suggest another option to explain the net hydride
transfer.
Hamilton (1971) proposed the formation of the covalent intermediate
between an alcohol substrate and a pyridine coenzyme
during oxidation catalyzed by dehydrogenases (Fig. 9, the lower
panel). The first step was an electrocyclic Ene reaction, which was
facilitated by transition states with aromatic character. In the second
step, the adduct was decomposed to the products by an
elimination reaction triggered by a proton. The Ene mechanism for
hydride transfer did not receive much attention especially due to an
inability to detect any reaction intermediates. Libby and Mehl
(2012) found covalent Ene adduct intermediates in a dihydropyridine
model for NADH reduction reactions and Rosenthal et al.
(2014) detected a covalent Ene adduct intermediate in an enzymatic
hydride transfer from NADPH to the corresponding substrate.
The results suggested that Ene reaction mechanism could be shared
by more NAD(P)H dehydrogenases.
Since we have not characterized the exact structure of the
adduct, the mechanism of the SA reduction discussed here is only
tentative. One possibility suggests that the reduction of SA with
NADH should proceed via the Ene reaction mechanism, which is in
line with previous reports (Libby and Mehl, 2012; Rosenthal et al.,
2014), and the proposed reaction is given in Fig. 10. The net hydride
transfer then involves transition states with aromatic character,
as well as lowering activation energy.
The existence of several isomeric forms of the adduct observed
in the LC chromatogram around the 10th minute (Fig. 3B) might be
explained by a creation of two new adjacent stereogenic centers in
the molecule of the Ene adduct e the quaternary nitrogen of SA and
C-6 of the nicotinamide ring (Fig. 10). The Ene adduct then comprises
two pairs of enantiomers providing two peaks on the chromatogram
monitored at m/z 499. If we take into account the
possibility of using C-2 instead of C-6 for the formation of the Ene
adduct, we could obtain even four enantiomeric pairs. Such
explanation is in accordance with the previous findings (Libby and
Mehl, 2012), where the isomers of Ene adducts formed between a
model molecule of NADH and the substrates. Both SA and NADþ
in
the oxidized form have a pyridinium ring containing a reactive
iminium bond, which plays a key role in the covalent adduct formation
in the proposed Ene reaction. Considering a substrate
oxidation with NADþ
(Fig. 9, the lower panel), the iminium bond of
NADþ
is attacked by a nucleophilic oxygen of a hydroxylic group.
Correspondingly, during the SA reduction with NADH, the reactive
iminium bond of SA might have been attacked by any of the
nucleophilic atoms of NADH. This would also explain the reversible
formation of the isomeric adducts seen in LC-MS (Fig. 3).
4. Conclusions
In this work, we observed a novel species of a covalent adduct,
which was capable of acting as an intermediate in the facilitation of
the SA reduction by NADH. The idea of such an intermediate existence
was postulated by Kovar et al. (1986); nevertheless, there
have been no later attempts to characterize the intermediate. The
species is likely to be the same molecule Matkar et al. (2008b)
observed in their experiments; however, they misinterpreted the
intermediate as a negatively charged species of SA.
We have shown that the process of reduction went through a
stable covalent intermediate, which is contradictory to the
accepted model of a one-step hydride transfer. Our data supported
the covalent intermediate hypothesis, but we managed to form
such an intermediate with a native molecule of NADH, instead of a
hard-to-design synthetic model molecule.
The product of the reduction, DHSA, was continually re-oxidized
back to SA by the atmospheric oxygen to generate a new portion of
SA available for a further reaction with NADH. This fact guaranteed
that the concentration of the adduct remained constant and we
suppose this happens also in living cells, in which SA is quickly
reduced by NAD(P)H-dependent enzymes. This idea is supported
by other authors, who observed an induced apoptosis due to the
rapid depletion of a reducing potential of the cell (Debiton et al.,
2003; Hammerova et al., 2012; Pallichankandy et al., 2015). Inhibition
of NAD(P)H-dependent enzymes by SA previously observed
(Kalogris et al., 2014) could be caused by the covalent adduct due to
its structural similarity with NAD(P)H.
5. Experimental
5.1. Reagents and materials
Methanol (MeOH, p.a.) was obtained from Penta (Czech Republic).
Acetic acid (LC-MS), ammonium hydroxide (28e30% NH3 in
water), sodium formate (LC-MS), NADH disodium salt (Grade I),
MeOH (LC-MS), and acetonitrile (ACN, HPLC grade, LC-MS) were
purchased from SigmaeAldrich (Czech Republic). All water used
was of an ultra-pure grade supplied by an in-house Milli-Q system
(Millipore, MA, USA).
5.2. Alkaloid isolation
The standards of the benzophenanthridine alkaloids originated
from isolation during the systematic studies in the laboratories of
the Department of Biochemistry from plant material of Macleaya
microcarpa (Maxim.) Fedde, Dicranostigma lactucoides Hook. F. et
Thomson, Sanguinaria canadensis L., and Stylophorum lasiocarpum
(Oliv.) Fedde. Briefly, SA was prepared by extraction of dried,
ground plant material with methanol in a FexIKA®
apparatus (IKA,
Germany). The extract was purified, concentrated and the last step
of isolation was performed by means of semi-preparative reversedphase
(RP) chromatography. The identity of the alkaloid was verified
by the LC-MS analysis.
5.3. Reaction conditions
SA was incubated with NADH in an equimolar ratio at room
temperature under argon atmosphere in a sealed vial for 5 min, if
not stated otherwise. For an LC-MS analysis (Fig. 6), the mixture of
0.5 ml of 1 mmol/L of alkaloid and 0.5 ml 1 mmol/L NADH was
diluted ten times, sealed in an LC-MS vial under argon atmosphere,
and immediately injected onto the LC-MS system. For a semipreparative
analysis, the mixture of 0.5 ml of 1 mmol/L of alkaloid
and 0.5 ml of 1 mmol/L NADH was sealed under argon atmosphere,
left for 60 min, and then 1 ml was injected onto the semipreparative
column specified in the section 5.4.
5.4. Adduct isolation and purification
An Agilent 1200 Series LC system (Agilent Technologies, CA,
USA) equipped with a fraction collector was used for purification
and isolation of the adduct. It consisted of a ternary pump, a diode
array detector (DAD), a syringe loading sample injector (ECOM,
Czech Republic) with a 5 ml external sample loop and a fraction
R. Sandor et al. / Phytochemistry 145 (2018) 77e84 83
collector. The alkaloid-NADH complex was separated on a C12
column (Synergi RP-Max, 4 mm, 250 mm  10 mm ID, Phenomenex,
CA, USA). The mobile phase consisted of ammonium formate buffer
(0.05 M HCOOH titrated with NH3 to pH ¼ 4.50) and ACN with the
following elution profile: ACN was linearly increased from 25% to
27.5% over first 5 min, then to 41.5% over the subsequent 5 min, next
to 47% at the 15th minute and finally to 80% at 20 min, where it was
held for 10 min before equilibration under the initial conditions for
3.0 min. The flow rate was set at 4 mL minÀ1
and fractions were
collected on a peak basis using a DAD at 280 nm. Fraction identified
as the adduct was transferred into an LC-MS vial and ten LC-MS
runs (Fig. 7) were performed.
5.5. Liquid chromatography e mass spectrometry
The LC-MS method was developed using a Dionex Ultimate
3000RS (Thermo Scientific, CA, USA) module. Compound separation
was achieved with a 3.0 mm  150 mm, 5 mm Synergi RP-Max
C18 (Phenomenex, CA, USA) column, set at (23 ± 0.1) C, and a flowrate
of 0.5 mL minÀ1
. The binary mobile phase system consisted of
ammonium acetate buffer (10 mM, pH ¼ 4.4) and ACN. After a 10-mL
injection, the percentage of ACN was linearly increased from 20% to
40% over 10 min and then to 80% over the subsequent 1 min. ACN
was held at 80% for 5 min, followed by equilibration under the
initial conditions for another 5 min. A complete LC run was 21 min.
The LC system was equipped with a DAD detector and at the same
time, it was connected to a Bruker MicrOTOF-Q II (Germany) mass
spectrometer operated in the positive electrospray ionization
mode. The ionization conditions were determined by the software
as follows: capillary voltage: 4500 V, end plate offset: À500 V,
source temperature: 220 C, desolvation gas (nitrogen) flow:
8 L minÀ1
, nebulizer (nitrogen) pressure: 300 kPa, and collision cell
voltage: 6 eV. The base peak chromatogram (BPC) was acquired in
MS mode by monitoring the m/z range of 50e3000 with a spectra
sample time of 1 s and MS/MS spectra were collected in a datadependent
mode. The mass spectrometer was calibrated using
10 mM sodium formate in 50% isopropyl alcohol on a daily basis
and in the beginning of each LC run with a 20-mL loop flush. Highresolution
MS and MS/MS spectra were first investigated to obtain
the elemental formula of each compound. The final identification of
target compounds relied on isotope pattern matching with a
combination of MS/MS and retention behavior.
5.6. Thin-layer chromatography
Thin-layer chromatography was performed on aluminum plates
(6 Â 12 cm) covered by Silica gel 18 F254s (Merck, Germany) and a
mobile phase consisting of ammonium formate 0.1 M and MeOH
(92:8 v/v). The chamber with the mobile phase was left covered for
20 min to saturate before any separation. Samples were immediately
after mixing applied to the plate, which was after drying
(2e3 min) inserted into the chamber with the mobile phase. The
separation took approximately ten minutes. After the experiment,
the TLC plate was removed, allowed to dry for 15 min, visually
inspected and photographed under UV light at 340 nm.
Acknowledgement
This work was supported by funds from the Faculty of Medicine
MU to a junior researcher Ondrej Pes and by the Specific University
Research Grant (MUNI/A/1205/2016, MUNI/A/1237/2016, MUNI/A/
0810/2016) provided by the Ministry of Education, Youth and
Sports of the Czech Republic in the year 2016.
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3.3 Seasonal variation in alkaloid composition and antiproliferative activity of Stylophorum
lasiocarpum (Oliv.) Fedde
Since many QBAs cannot be efficiently synthesized, the major way for their obtaining is isolation and purification from
plant material. Minor QBAs, especially MA and CR, can be isolated from a plant Stylophorum lasiocarpum, which
is considered an essential source of minor QBAs. LC–MS was used to identify and characterize the alkaloids found in
roots and aerial parts of the plant. A combination of retention time, precise mass-to-charge ratio, isotopic patterns,
and fragmentation behavior allowed for identifying seventeen compounds produced in the benzo[c]phenanthridine
pathway starting from (S)-reticuline. In most plants, the end product is SA; however, S. lasiocarpum belongs to a
few plant species producing MA by involving two hydroxylation and methylation steps. The seasonal variation of
QBAs production was established to maximize the isolation efficiency for cultivation and harvesting of the plant
material.
SEBRLOVA, Kristyna, Ondrej PES, Iva SLANINOVA, Ondrej VYMAZAL, Jana KANTOROVA and Eva
TABORSKA. Seasonal variation in alkaloid composition and antiproliferative activity of Stylophorum lasiocarpum
(Oliv.) Fedde. Chemical Papers. 2015, 69(5), 698–708. ISSN 0366-6352. doi:10.1515/chempap-2015-0083 Document
Type: Article, IF = 1.326; JCR Category + Category Quartile: CHEMISTRY, MULTIDISCIPLINARY Q3; AIS =
0.235
Author’s contribution: 20 %
55
Chemical Papers 69 (5) 698–708 (2015)
DOI: 10.1515/chempap-2015-0083
ORIGINAL PAPER
Seasonal variation in alkaloid composition and antiproliferative
activity of ËØÝÐÓÔ ÓÖÙÑ Ð × Ó ÖÔÙÑ (Oliv.) Fedde
a
Kristýna Šebrlová*, a
Ondřej Peš, b
Iva Slaninová, b
Ondřej Vymazal,
a
Jana Kantorová, a
Eva Táborská
aDepartment of Biochemistry, bDepartment of Biology, Faculty of Medicine, Masaryk University,
Kamenice 5, 62500, Brno, Czech Republic
Received 20 August 2014; Revised 20 October 2014; Accepted 3 November 2014
Stylophorum lasiocarpum (Oliv.) Fedde (Papaveraceae) belongs to traditional Chinese medicine
herbs but there was minimal information on the content of alkaloids in this plant. Extracts from
the aerial part and roots were examined by liquid chromatography with UV and mass spectrometric
detection, with nineteen alkaloids identified. Changes in alkaloid content over the entire
vegetation period of a one- and two-year old plant were studied. The protoberberine alkaloids,
coptisine and stylopine, were found to be the main substances in extracts of the aerial part irrespective
of the plant’s age and time of harvest. Variable amounts of protopine, sanguinarine,
chelerythrine, chelirubine, macarpine, chelilutine and berberine were also recorded in the aerial
part. The roots contained significantly larger quantities of all alkaloids than the aerial part with the
levels of most alkaloids varying from May to October, peaking in the middle of the vegetation period.
Coptisine was the dominant alkaloid in all samples. The antiproliferative activities of the root
extract and of seven individual alkaloids were tested on A375 human malignant melanoma cells.
The significant dose-dependent toxicity of the root extract was attributed largely to the quaternary
benzo[c]phenanthridine alkaloids, macarpine and sanguinarine.
c 2014 Institute of Chemistry, Slovak Academy of Sciences
Keywords: Stylophorum lasiocarpum, traditional Chinese medicine, vegetation period, benzophenanthridine,
LC MS, cytotoxicity
Introduction
Stylophorum lasiocarpum (Oliv.) Fedde is a biennial
or short-lived perennial herb native to Central
and Eastern China. S. lasiocarpum is one of the 23
species of the tribe Chelidonieae (Papaveraceae family).
Due to its close similarity to the species Chelidonium
majus, the plant is also known as Chinese
celandine poppy or human bloodwort. Although the
dried whole herb of S. lasiocarpum is included among
traditional Chinese medicine (TCM) herbs, to the best
of our knowledge, information on its medicinal effects
is minimal (Lei et al., 2014). S. lasiocarpum is generally
applicable when treating injuries, controlling
bleeding, relieving pain or alleviating swelling (Feng
et al., 1985).
Stylophorum lasiocarpum is permeated by a welldeveloped
duct system of laticifers producing orangered
latex. The stems are erect, usually simple and
glabrous with basal leaves forming a rosette. Two or
three cauline leaves are borne from the upper stem, almost
opposite or whorled, similar to the lower leaves
but subpetiolate. Four to seven yellow flowers bloom
in an umbel-like cluster, opening in succession. The
fruits are narrow (approx. 60 mm × 5 mm) terete capsules
covered in short hairs; they bear numerous seeds
that are oval (approx. 1 mm), tessellate and cristately
carunculate (Zhang et al., 2008). The main secondary
metabolites are isoquinoline alkaloids (Table 1).
The alkaloids of the plant were first characterised
by Slavík et al. (1991). Stylopine (STY) and coptisine
(COP) were the principal alkaloids identified in the
*Corresponding author, e-mail: k.pencikova@mail.muni.cz
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Table 1. Stylophorum lasiocarpum alkaloids
Ring type Alkaloid R1 R2 R3 R3 R4 R5
Sanguinarine (SA) —CH2— —CH2— —H —H
Chelerythrine (CHE) —CH2— —CH3 —CH3 —H —H
Sanguilutine (SL) —CH3 —CH3 —CH3 —CH3 —H —H
Chelilutine (CL) —CH2— —CH3 —CH3 —OCH3 —H
Sanguirubine (SR) —CH3 —CH3 —CH2— —OCH3 —H
Chelirubine (CR) —CH2— —CH2— —OCH3 —H
Macarpine (MA) —CH2— —CH2— —OCH3 —OCH3
Berberine (BER) —CH3 —CH3 – – –
Coptisine (COP) —CH2— – – –
Canadine (CAN) —CH3 —CH3 – – –
Stylopine (STY) —CH2— – – –
Protopine (PRO) – – – –
Chelidonine – – – –
roots, while smaller quantities of sanguinarine (SA),
macarpine (MA), chelerythrine (CHE), chelirubine
(CR), chelilutine (CL), protopine (PRO), corysamine,
berberine (BER) and N-methylstylopinium hydroxide
were also isolated. Thin-layer chromatography (TLC)
detected trace amounts of chelidonine, allocryptopine,
cryptopine, scoulerine, isoboldine and magnoflorine.
The aerial part of the plant yielded STY, COP
and chelidonine as the dominant alkaloids and lower
amounts of PRO and N-methylstylopinium hydroxide.
Trace amounts of SA, CR, cryptopine, scoulerine,
isoboldine, corytuberine, corysamine, BER and magnoflorine
were detected by TLC in the aerial part of
the plant.
Suchomelová et al. (2007) subsequently studied the
roots of the plant species as a potential source of quaternary
benzo[c]phenanthridine alkaloids (QBAs) and
confirmed that the major alkaloids were COP and
STY and, in addition, found benzo[c]phenanthridine
(SA, CHE, CR, CL, MA) and protoberberine (BER)
alkaloids. Accordingly, S. lasiocarpum belongs to a
small group of plant species that, in addition to SA
and CHE, contain less common QBAs such as CR,
CL or MA. SA and CHE are present in relatively
high amounts in these plants, may be isolated, and
have been commercially available for years. They have
been the subjects of a number of biological studies
that have been summarised in recent reviews (Dvorák
et al., 2006; Slaninová et al., 2014). By contrast, the
content of other QBAs is significantly lower in these
plants and their availability is very limited, hence they
have long evaded attention. The first biological studies
recently described the effects of these alkaloids on
human cancer cells. Strong anti-proliferative and proapoptotic
properties were observed upon treatment
with MA, CHR, SR (Slaninová et al., 2007a), SL and
CHL (Slunská et al., 2010). The effects of SL and CHL
were independent of the activity of p53 (Hammerová
et al., 2011) and SL effectively killed cells overexpressing
the anti-apoptotic protein Bcl2 (Hammerová et al.,
2012). Despite the close similarity of the QBAs’ molecular
structures, the cell death mechanism was different
for each alkaloid (Hammerová et al., 2011). SL
induced the caspase-independent cell death, necropBrought
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700 K. Šebrlová et al./Chemical Papers 69 (5) 698–708 (2015)
tosis (Hammerová et al., 2012). These properties of
QBAs indicate their prospective applications in the
treatment of resistant tumours. In addition to these biological
effects on cells, QBAs in the quaternary form
have been reported as interacting with dsDNA and Gquadruplexes
(Rybáková et al., 2013). Currently, the
targeting of quadruplexes by small molecules seeks to
elucidate the biological effects and plays an interesting
role in the development of new potential anti-cancer
drugs (Galezowska et al., 2012).
Among the rare QBAs, MA is particularly distinguished
by its unusual properties, being reported as
a promising fluorescent probe for cell nuclei-labelling
as well as for cell cycle visualisation in fluorescence
microscopy and flow cytometry. Slaninová (2007b) described
the ability of MA to act as a supravital DNA
dye on various living human cells (HL60, HeLa and
LEP). On the addition of MA to cells, the instantly
appearing bright red nuclear fluorescence revealed the
nuclear architecture at various stages of the cell cycle
not only in living cells but also in yeasts and bacteria.
In addition, MA is highly antiproliferative in vitro,
predominantly by apoptosis, and is significantly more
cytotoxic than any other QBAs (Ishikawa et al., 1995;
Slaninová et al., 2007a).
The above information will undoubtedly advance
the research and application of minor QBAs in cancer
biology and therapy. However, an essential requirement
of any further research is to obtain the QBAs
in sufficient quantities. As they are not commercially
available and their synthesis is somewhat unsatisfactory
(Ishikawa et al., 1995), the best approach for
their large-scale production appears to be isolation
from the plant material. S. lasiocarpum is regarded
as a core source of minor QBAs, albeit that no comprehensive
data on the production, accumulation and
distribution of QBAs in this plant have ever been
published. Such data, as well as descriptions of the
antiproliferative activities of the plant extract and
its individual alkaloids, are provided in the present
study.
Experimental
Plant material
Stylophorum lasiocarpum Oliv. Fedde was cultivated
in the Medicinal Herbs Centre of the Faculty
of Medicine of Masaryk University (Brno, Czech Republic).
A voucher specimen of S. lasiocarpum (Oliv.)
Fedde (BRNU, ID number 620684) is deposited in
the herbarium of Masaryk University (Brno, Czech
Republic). Three individual plants of one- and twoyear
old cultures were randomly harvested during the
vegetation period from May to October. The plant
material was wiped, dried in the dark at ambient
temperature for 10 days and ground to a fine pow-
der.
Reagents and materials
Methanol (p.a.) was obtained from Penta (Czech
Republic), phosphoric acid (p.a.), formic acid (p.a.),
sodium 1-heptanesulphonate (p.a.), fetal bovine serum,
glutamine, penicillin, streptomycin, acetonitrile
(ACN) (HPLC grade, LC MS) and dimethylsulphoxide
(p.a.) (DMSO) were purchased from Sigma–
Aldrich (Czech Republic).
The standards of the alkaloids originated from isolations
during the systematic studies of Papaveraceae
in the laboratories of the Department of Biochemistry:
allocryptopine, berberine, canadine, coptisine,
chelerythrine, chelilutine chelirubine, macarpine, magnoflorine,
protopine, sanguinarine (Táborská et al.,
1978; Dostál et al., 1992), dihydrochelerythrine, dihydrochelilutine,
dihydrochelirubine, dihydrosanguinarine,
chelidonine, N-methylstylopinium, stylopine, norsanguinarine,
stylopine (Slavík & Slavíková, 1977).
The identity of the alkaloids was verified by EI-MS, 1
H
NMR and 13
C NMR and their purities were at least
98 % according to HPLC analysis (Marek et al., 1999;
Suchomelová et al., 2007).
Extraction and isolation
Dried, ground plant material (0.5 g) was extracted
with 100 mL of methanol in a FexIKA apparatus
(IKA, Germany), which is based on fluidised bed extraction.
The extraction protocol was optimised on the
basis of a comparison with Soxhlet extraction with
methanol (Urbanová et al., 2012). Extraction times of
4 × 15 min for the aerial part and 6 × 15 min for the
roots were used. The recoveries of both methods were
comparable. The solvent from the extract was then
evaporated to 25 mL. Prior to LC UV, the extract
was diluted 3 × and filtered through a 0.45- m nylon
syringe filter. For LC MS, the extract was diluted
20 × and filtered through a 0.22- m PTFE syringe
filter.
LC MS
The LC MS method was developed using a
Dionex Ultimate 3000RS (Thermo Scientific, CA,
USA) module. Compound separation was achieved
with a 3.0 mm × 150 mm, 5 m Synergi RP-Max
C18 (Phenomenex, CA, USA) column, thermostated
at (23 ± 0.1)◦
C, and a flow-rate of 0.5 mL min−1
. The
binary mobile phase system consisted of 0.1 mass %
of formic acid and ACN. After 20- L injection, the
volume of ACN was linearly increased from 20 % to
40 % over 10 min and then to 80 % over the subsequent
10 min. ACN was held at 80 % for 10 min, followed by
equilibration under the initial conditions for 3.0 min.
A complete HPLC run was 33 min. The HPLC system
was connected to a Bruker MicrOTOF-QII (Germany)
mass spectrometer, operated in the positive
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K. Šebrlová et al./Chemical Papers 69 (5) 698–708 (2015) 701
electrospray ionisation mode. The ionisation conditions
were determined by the software as follows: capillary
voltage: 4500 V, end plate offset: –0.5 V, source
temperature: 220◦
C, desolvation gas (nitrogen) flow:
10 L min−1
, nebuliser (nitrogen) pressure: 300 kPa
and collision cell voltage: 35 eV. The base-peak chromatogram
(BPC) was acquired in MS mode by monitoring
the m/z range of 50–3000 with a spectra sample
time of 1 s. The identification of target compounds relied
on isotope pattern matching with a combination
of MS/MS and retention behaviour. High-resolution
MS and MS/MS spectra were first investigated to obtain
the elemental formula of each compound. A compound
was identified unambiguously if the fragmentation
patterns of the unknown and the standard were
identical.
LC UV
The HPLC apparatus consisted of a LC-20 AD
high-pressure gradient pump LC-20 AD, an SPD
M20A diode-array detector (Shimadzu, Japan) and
a syringe-loading sample injector (ECOM, Czech Republic)
with a 20- L external sample loop. Alkaloids
were separated on a C12 column (Synergi RP-Max,
4 m, 150 mm × 4.60 mm ID, Phenomenex, CA,
USA).
The method used was detailed in our previous
study (Suchomelová et al., 2007). Briefly, the mobile
phase was prepared from a stock solution of 0.01 M
sodium 1-heptanesulphonate and 0.1 M triethylamine
in Milli-Q water (Millipore, MA, USA), adjusted to
pH 2.5 with phosphoric acid. Mobile phase A consisted
of 75 : 25 (vol.) stock solution : ACN. Mobile phase B
was 40 : 60 (vol.) stock solution : ACN.
The following elution profile was employed: 0–
1 min 100 % A; 1–10 min 0–20 % B; 10–22 min 20–
50 % B; 22–28 min 50–100 % B; 28–40 min 100 % B;
40–48 min 100 % A. The flow-rate was set at 0.5 mL
min−1
, the injection volume was 20 L and detection
was performed using a diode array detector at 280 nm.
The identification of the separated alkaloids was based
on comparisons of their retention times with those of
authentic standards. Quantitative analysis was performed
using external standards and was validated
previously (Suchomelová et al., 2007).
Antiproliferative activity
The antiproliferative activities of the whole extract
and of the individual alkaloids COP, MA, CR, SA,
CHE, BER and CL were tested using the A-375 human
malignant melanoma cell line and a colorimetric
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) assay. The extract was standardised
(n = 2) and contained: CL (0.76 ± 0.11) mg L−1
, BER:
(1.48 ± 0.22) mg L−1
, CHE (3.11 ± 0.58) mg L−1
, MA
(10.2 ± 1.1) mg L−1
, PRO (4.09 ± 0.38) mg L−1
, CR
(7.62 ± 0.85) mg L−1
, SA (6.04 ± 0.49) mg L−1
, STY
(10.6 ± 1.0) mg L−1
and COP (22.5 ± 1.9) mg L−1
.
The A-375 cell line was purchased from the European
Collection of Animal Culture (ECACC, Salisbury,
UK). The MTT assay was performed in a 96well
plate (Nunc A/S, Denmark). Individual wells
were seeded with 200 L of cells (1 × 104
per mL)
and incubated in a RPMI 1640 medium supplemented
with 10 mass % of fetal bovine serum, 2 mM of glutamine,
100 IU mL of penicillin and 100 g mL−1
of
streptomycin at 37◦
C in a humidified atmosphere (5
vol. % CO2). After 24 h of incubation, the medium
was replaced with an extract- or alkaloid-containing
medium at concentrations ranging from 0.3 mg L−1
to 10 mg L−1
. The MTT assay was performed 48 h
after addition of the extract: 20 L of MTT (5 mg L−1
in PBS) was added to each well and incubated for 4 h
under culture conditions. At the end of the incubation
period, the medium was removed, the formazan crystals
were dissolved in 200 L of DMSO and the optical
density was measured at 570 nm using a DTX 880 multimode
detector (Beckman Coulter, CA, USA). The
concentration of each compound was examined in four
replicates.
Results and discussion
Identification of S. lasiocarpum alkaloids
The extracts were first analysed by mass spectrometry
to confirm the identity of the alkaloids intended
for quantitation. The base peak chromatogram
of the root extract is shown in Fig. 1, and the retention
times, elemental composition and major fragments
of the root and aerial-part extracts are summarised
in Table 2. Seventeen compounds were identified
by comparing the fragmentation and retention
behaviours of the alkaloid peaks in the extracts obtained
from S. lasiocarpum roots with those of standard
samples. All, with the exception of COP, are
components of a benzo[c]phenanthridine pathway that
starts from (S)-reticuline and involves eleven highly
specific enzymes (Zenk, 1994). COP is regarded as a
side-product of the pathway because it is formed by
the oxidation of STY. Pathway intermediates, such as
N-methylstylopinium, PRO or dihydro-SA, were also
detected. In most plants, the biosynthetic pathway
ends with the creation of SA but a few plant species,
including S. lasiocarpum, may induce two subsequent
hydroxylation and methylation steps resulting in MA
production (Zenk, 1994). The presence of dihydro-CL,
dihydro-CHE, nor-SA, dihydro-CR, dihydro-SA and
canadine (CAN) was observed for the first time. The
detection of QBA dihydroderivatives is not surprising
as they are typically found in all plants producing
QBAs; however, they are often neglected due to their
non-polar character. The presence of chelidonine and
N-methylstylopinium in the extract of the aerial part
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702 K. Šebrlová et al./Chemical Papers 69 (5) 698–708 (2015)
Table 2. Retention time, exact mass of molecular ion or protonated ions and major fragment ions ([M]) of compounds found in
extracts of root (1–17) and aerial part (1–19) of S. lasiocarpum
Peak tR/min m/z Ion Observed Calculated Difference Formula Major fragment Compound
mass mass in ppm ions, m/z name
1 2.0 342.1694 [M]+ 342.1694 342.1705 –3.2 C20H24NO4
175.0763; 191.0931;
207.0828; 209.0953;
219.0795; 222.0675;
233.0606; 235.0762;
237.0899; 247.0749;
265.0854; 282.0895;
297.1126
Magnoflorine
2 3.3 354.1351 [M + H]+ 353.1278 353.1263 4.2 C20H19NO5
149.0597; 165.0546;
188.0703; 189.0779;
206.0799; 235.0762;
247.0762; 265.0856;
267.0658; 275.0711;
293.0801; 295.0968;
305.0909; 336.1232
Protopine
3 3.7 370.1648 [M + H]+ 369.1575 369.1576 –0.3 C21H23NO5
165.0903; 181.0865;
188.0725; 206.0808;
275.0702; 281.1112;
290.0935; 306.0895;
308.1057; 311.1266;
321.1191; 336.1234;
339.1238; 352.1535
Allocryptopine
4 3.8 320.0942 [M]+ 320.0942 320.0923 5.9 C19H14NO4
233.0824; 234.0918;
247.0646; 248.0711;
249.0786; 260.0696;
262.0864; 264.1018;
275.0601; 277.0736;
290.0820; 292.0980;
305.0685; 318.0763
Coptisine
5 4.1 324.1251 [M + H]+ 323.1178 323.1158 6.2 C19H17NO4
149.0596; 174.0556;
176.0706; 188.0711;
219.0797; 249.0888;
262.0824; 265.0887;
277.0820; 290.0815;
292.0963; 307.0976
Stylopine
6 4.4 340.1535 [M + H]+ 339.1462 339.1471 –2.7 C20H21NO4
149.0593; 165.0877;
174.0562; 176.0703;
324.1214
Canadine
7 4.6 332.0935 [M]+ 332.0935 332.0923 3.6 C20H14NO4
216.0808; 218.0960;
244.0757; 246.0921;
261.0752; 272.0697;
274.0871; 287.0576;
289.0737; 302.0810;
304.0961; 316.0611;
317.0686
Sanguinarine
8 5.0 336.1226 [M]+ 336.1226 336.1236 –3.0 C20H18NO4
246.0897; 263.0921;
274.0854; 275.0928;
278.0814; 291.0870;
292.0967; 302.0805;
304.0962; 306.0761;
317.0695; 320.0932
Berberine
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Table 2. (continued)
Peak tR/min m/z Ion Observed Calculated Difference Formula Major fragment Compound
mass mass in ppm ions, m/z name
9 5.8 348.1248 [M]+ 348.1248 348.1236 3.4 C21H18NO4
275.0895; 276.0919;
286.0881; 287.0928;
290.0809; 302.0775;
303.0886; 304.0961;
314.0803; 315.0881;
316.0956; 318.0765;
330.0742; 332.0939
Chelerythrine
10 6.2 362.1033 [M]+ 362.1033 362.1028 1.4 C21H16NO5
261.0793; 289.0745;
291.0869; 302.0490;
303.0825; 304.0753;
317.0683; 318.0771;
331.0829; 332.0563;
334.1058; 346.0700;
347.0782
Chelirubine
11 7.3 348.0861 [M]+ 378.1340 378.1341 –0.3 C22H20NO5
305.0702; 320.0906;
321.0938; 332.0910;
334.1101; 347.1166;
348.0860; 362.1014;
363.1090
Chelilutine
12 7.8 392.1145 [M]+ 392.1145 392.1134 2.8 C22H18NO6
291.0891; 319.0846;
330.0753; 331.0800;
334.0915; 346.0694;
347.0782; 348.0868;
360.0842; 361.0929;
362.0672; 364.1142;
376.0818; 377.0903
Macarpine
13 21.1 380.1495 [M + H]+ 379.1422 379.1420 0.5 C22H21NO5
319.0907; 320.0848;
332.0938; 334.0868;
335.0889; 348.1084;
350.1062; 363.1106;
364.1186; 378.1338
Dihydrochelilutine
14 21.6 350.1382 [M + H]+ 349.1309 349.1314 –1.4 C21H19NO4
288.0992; 289.0912;
290.0836; 302.0798;
303.0897; 304.0900;
315.0858; 316.0970;
317.1035; 318.0964;
332.0936; 334.1082;
348.1234
Didhydrochelerythrine
15 21.6 318.0752 [M + H]+ 317.0679 317.0688 –2.8 C19H11NO4
232.0756; 260.0704;
288.0643; 290.0807
Norsanguinarine
16 22.0 364.1185 [M + H]+ 363.1112 363.1107 1.4 C21H17NO5
290.0785; 291.0854;
304.0714; 305.0778;
306.0982; 317.0688;
318.0752; 319.0700;
332.0893; 334.0772;
346.0694; 347.0774;
348.0855; 349.0933;
362.1040
Dihydrochelirubine
17 22.2 334.1089 [M + H]+ 333.1016 333.1001 4.5 C20H15NO4
246.0882; 260.0752;
261.0772; 262.0828;
274.0817; 276.1009;
288.0747; 289.0750;
302.0791; 303.0835;
304.0927; 317.0717;
318.0779; 330.0748;
332.0926
Dihydrosanguinarine
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704 K. Šebrlová et al./Chemical Papers 69 (5) 698–708 (2015)
Table 2. (continued)
Peak tR/min m/z Ion Observed Calculated Difference Formula Major fragment Compound
mass mass in ppm ions, m/z name
18 3.8 354.1335 [M + H]+ 353.1258 353.1263 –1.4 C20H19NO5
163.0396; 188.0681;
217.0640; 235.0771;
237.0906; 245.0609;
247.0755; 265.0837;
275.0699; 293.0802;
295.0969; 305.0785;
323.0920; 336.1226
Chelidoninea
19 3.9 338.1404 [M]+ 338.1404 338.1392 3.5 C20H20NO4
149.0593; 176.0697;
188.0717; 190.0871;
207.0799; 219.0800;
235.0751; 237.0890;
247.0756; 249.0902;
265.0848; 277.0873;
295.0964; 307.1016
N-Methylstylopiniuma
a) Found only in the aerial part.
Fig. 1. LC MS base-peak chromatogram of 20 × diluted S. lasiocarpum root extract. Peaks labelled 1–17 were identified and are
summarised in Table 2. Unlabelled peaks contain compounds of no interest as their MS/MS provided different, non-alkaloid
structure patterns.
portions of S. lasiocarpum was observed in addition
to the alkaloids found in the root extract.
Seasonal analysis of S. lasiocarpum alkaloids
Aerial part
The QBA content in the aerial part of one- and
two-year plants was determined and is summarised in
Table 3. The protoberberine alkaloids COP and STY
were found to be the main substances in extracts of
the aerial part irrespective of plant age; SA and CR
were the major QBA portions. While the SA content
increased at the culmination of the first year of vegetation,
the CR content was relatively constant throughout
the first year of vegetation. In the second year,
both SA and CR displayed two maxima: in June and
at the end of the vegetation period. PRO exhibited exactly
the opposite trend, as it is a biosynthetic precursor
of SA and CR (Tanahashi & Zenk, 1990). Similar
behaviours were reported in the analysis of the aerial
parts of Macleaya microcarpa Fedde (Pěnčíková et al.,
2011).
Although the contents of CL and CHE were negligible
in both years, the production of MA was higher
toward the end of the second year. In the systemBrought
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Table3.ContentofisoquinolinealkaloidsfromaerialpartofS.lasiocarpumduringvegetationperiodsofone-andtwo-yearoldcultures;yearofharvest:2012;theamountofalkaloid
isofdrymass(mean±standarddeviation,n=2)
Amount/(mgg−1)
PlantMonth
age/yearCLBERCHEMAPROCRSASTYCOP
OneMay0.012±0.0030.040±0.0030.023±0.0080.093±0.0190.394±0.0590.188±0.0390.091±0.0201.61±0.263.92±0.64
June0.007±0.0020.018±0.0060.010±0.0040.037±0.0130.150±0.0270.199±0.0470.0640±0.00701.01±0.123.63±0.29
July0.002±0.0010.016±0.0020.026±0.0020.019±0.0050.215±0.0150.223±0.0190.304±0.0292.010±0.0674.47±0.24
August0.005±0.0010.021±0.0010.008±0.0020.005±0.0010.122±0.0170.262±0.0280.434±0.0551.985±0.0715.21±0.21
September0.006±0.0010.024±0.0040.039±0.0080.011±0.0040.104±0.0090.233±0.0101.30±0.271.787±0.0844.58±0.31
October0.023±0.0020.016±0.0020.018±0.0030.033±0.0090.152±0.0180.269±0.0161.70±0.172.475±0.0545.31±0.26
TwoMay0.011±0.0010.017±0.0030.013±0.0010.032±0.0070.516±0.0040.185±0.0250.081±0.0111.769±0.0673.88±0.22
June0.022±0.0090.020±0.0050.055±0.0030.125±0.0050.172±0.0281.332±0.0340.766±0.0301.826±0.0926.89±0.15
July0.023±0.0130.010±0.0010.018±0.0020.026±0.0050.501±0.0140.250±0.0310.204±0.0191.328±0.0773.82±0.24
August0.070±0.0110.021±0.0030.023±0.0040.114±0.0240.189±0.0210.67±0.201.00±0.142.01±0.173.93±0.19
September0.014±0.0030.021±0.0100.020±0.0080.054±0.0030.264±0.0210.392±0.0480.92±0.221.88±0.224.09±0.23
October0.008±0.0020.036±0.0060.030±0.0050.222±0.0230.144±0.0291.22±0.331.46±0.301.81±0.154.60±0.24
Table4.ContentofisoquinolinealkaloidsfromrootsofS.lasiocarpumduringvegetationperiodsofone-andtwo-yearoldcultures;yearofharvest:2012;theamountofalkaloidisof
drymass(mean±standarddeviation,n=2)
Amount/(mgg−1)
PlantMonth
age/yearCLBERCHEMAPROCRSASTYCOP
OneMay0.044±0.0070.92±0.220.54±0.112.45±0.211.55±0.201.552±0.0322.07±0.329.4±1.313.9±2.1
June0.183±0.0170.485±0.0550.930±0.0592.146±0.0400.1150±0.00501.588±0.0371.685±0.0813.77±0.209.50±0.41
July0.275±0.0210.585±0.0411.33±0.164.28±0.430.516±0.0982.76±0.274.07±0.627.16±0.679.6±1.1
August0.108±0.0120.746±0.0100.107±0.0121.79±0.250.354±0.0131.41±0.201.17±0.127.35±0.1314.96±0.25
September0.078±0.0101.95±0.100.126±0.0121.88±0.260.44±0.141.440±0.0761.735±0.0897.69±0.4018.32±0.96
October0.041±0.0040.857±0.0560.041±0.0111.21±0.190.063±0.0211.057±0.0330.872±0.0295.40±0.1813.15±0.70
TwoMay0.100±0.0080.585±0.0840.396±0.0423.92±0.480.151±0.0231.54±0.151.41±0.134.76±0.489.94±0.76
June0.148±0.0480.607±0.0790.68±0.157.05±0.680.939±0.0513.77±0.973.13±0.453.46±0.2213.91±0.57
July0.304±0.0160.649±0.0721.40±0.115.8±1.11.10±0.225.99±0.394.61±0.123.45±0.1911.05±0.63
August0.160±0.0261.159±0.0890.306±0.0522.96±0.140.214±0.0254.12±0.162.46±0.165.23±0.4013.06±0.67
September0.135±0.0450.695±0.0540.139±0.0312.37±0.220.548±0.0273.63±0.651.96±0.315.21±0.3814.2±1.2
October0.061±0.0080.738±0.0690.155±0.0262.02±0.380.238±0.0982.67±0.181.898±0.0525.00±0.3115.37±0.88
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706 K. Šebrlová et al./Chemical Papers 69 (5) 698–708 (2015)
Fig. 2. Mean (n = 4) percentage of viable cells after treatment with root extract or individual alkaloids (0.3–10 mg L−1). Established
by MTT assay. Legend: 0.3 mg mL−1 ( ), 1 mg mL−1 ( ), 3 mg mL−1 ( ), 10 mg mL−1 ( ).
atic analysis of QBA-containing plants, only S. lasiocarpum
included considerable amounts of MA and CR
in the aerial part (Pěnčíková et al., 2012).
Roots
Roots, in comparison with leaves, were found to
contain analogous alkaloids and the seasonal levels
of major alkaloids are summarised in Table 4. The
levels of most alkaloids varied from May to October,
peaking in the middle of the vegetation period. The
roots clearly contained larger quantities of all alkaloids,
whereas COP and STY were the major substances
in all samples. Although the levels of COP did
not differ in individual cultures, the concentrations of
STY were slightly lower in the two-year old plant.
Notably, the rarely occurring MA was found in
amounts readily comparable to SA, irrespective of the
age of the plant. An inter-species comparison shows
that the amount of MA in a one-year old plant was
equivalent to that found in the roots of M. microcarpa
13 years old (Pěnčíková et al., 2011). The levels of MA
in the roots of both cultures indicated a maximum
at the midpoint of the vegetation cycle, unlike in the
aerial part where few maxima were observed. Furthermore,
in the two-year old culture, the concentrations
of both MA and CR exceeded those of SA over the entire
vegetation period. The amount of CR in the roots
of a second-year S. lasiocarpum was also equivalent to
that in the roots of a 13-year old M. microcarpa, i.e.
approximately 5 mg g−1
of dry mass.
The changes in alkaloid content and their relative
proportion in the entire herb suggest that such seasonal
variations could be the result of long-distance
transport from leaves to roots and vice versa or local
de novo biosynthesis of the full range of alkaloids. The
localisation of isoquinoline alkaloid biosynthesis has
only been sporadically studied in a few species, suggesting
local de novo biosynthesis of isoquinoline alkaloids
in various organs (Weid et al., 2004; Samanani
et al., 2005, 2006; Khodorova et al., 2013).
Antiproliferative activity of standardised root
extract and pure alkaloids
The antiproliferative activities of the root extract
and of seven individual alkaloids were tested on A375
human malignant melanoma cells after 48-hours’ incubation
using a colorimetric MTT assay. Extracts at
concentrations of 1 mg L−1
, 3 mg L−1
and 10 mg L−1
and pure alkaloids over a concentration range of 0.3–
10 mg L−1
resulted in a dose-dependent decrease in
cell metabolic activity (Fig. 2). The QBAs generally
exhibited higher toxicity than other alkaloids, with
MA and SA being the most toxic. Several groups
have previously described the antiproliferative activity
of SA on various cancer cell types (Slaninová et al.,
2014). We previously described the strong antiproliferative
effects of MA and CR on several cancer cell lines
(Slaninová et al., 2001, 2007a), but this is the first
documentation of their effect on malignant melanoma
cells. The fact that these alkaloids exhibit a high antiproliferative
activity even against melanoma cells
suggests that they are strong candidates for further
cancer biology and therapy studies.
The toxicity of COP, the main alkaloid of S. lasiocarpum
extracts, on melanoma cells is much lower
than that of SA and MA. It was not possible to determine
the toxicity of PRO and STY due to their low solubility
in water. In an attempt to improve the solubility,
DMSO and/or hydrochloric acid were added, but
insoluble residues appeared each time. In respect of
the toxicity of the solvents (DMSO or HCl) at higher
concentrations, no definitive and accurate conclusion
was drawn (data not shown). The absence of derivatives
with higher solubility could be the reason for the
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K. Šebrlová et al./Chemical Papers 69 (5) 698–708 (2015) 707
ongoing lack of information regarding the cytotoxicity
of PRO and STY.
Conclusions
This study provides the first comprehensive qualitative
and quantitative set of data on alkaloids found
in S. lasiocarpum throughout the vegetation period of
a one- and two-year old plant. Although the content
of individual alkaloids varied during vegetation, the
major alkaloids were COP, STY, MA, CR and SA.
While the protoberberine alkaloid content was not
dependent on plant age, the amount of QBAs was
particularly high when isolated from the second-year
roots in July. Variations in the alkaloid content should
be taken into consideration when assessing the safety
and effectiveness of herbal remedies based on this
plant, especially due to the high cytotoxicity of QBAs.
S. lasiocarpum requires shade, abundant irrigation
and has relatively small roots; these factors might preclude
routine cultivation. However, the isolation of
MA and CR from plant material remains the best approach
for their large-scale production, with the older
roots being the prospective source.
Acknowledgements. The financial support for this work
received from the Czech Ministry of Education, Youth and
Sports (LH12176-KONTAKT II), the Czech Science Foundation
(GAP206/12/G014), and from the Science Foundation
of Masaryk University (MUNI/A/0954/2013 and MUNI/A/
0938/2013) is gratefully acknowledged.
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3.4 Heavy water enhancement of fluorescence signal in reversed-phase liquid chro-
matography
MS could prove essential even in fields and applications, which seem quite different such as fluorescence spectrometry.
It has been known that fluorescence intensity and lifetime heavily depend on the fluorophore’s surroundings.
Dynamic quenching is a process that enables dissipating the excitation energy in non-radiative pathways. In a water
environment, dynamic quenching is mediated mainly by –OH oscillators. To effectively decrease the quenching,
solutions with alkaline pH are used since many of those –OH groups become charged, and the quenching process
via non-radiative pathways is minimized. Sometimes however, the pH value cannot be efficiently increased, for
example, in chromatography using silica-based columns. Another approach to decrease dynamic quenching could be
the employment of deuterated solvents – specifically employing heavy water as a mobile phase in reversed-phase
chromatography. The labile (acidic) hydrogens in the fluorescent compound are dynamically exchanged for deuterium
atoms, which are heavier and oscillate with lower frequency. The overall fluorescence intensity could be thus increased
up to hundreds of percent. Although heavy water is relatively expensive to use as a common solvent in high flow-rate
chromatography, it may be easily included in microflow LC, cLC, or CE. Vitamin B, doxorubicin, and fluorescein
were selected, and the effect of fluorescence enhancement by substituting light water in the mobile phase with heavy
water was examined. The number of exchanged hydrogens was monitored by MS to provide a better understanding
of the structure-to-enhancement relationship. The overall process of fluorescence enhancement by deuterated solvents
is rather complex and was recently studied more comprehensively.52
TABORSKY, Petr, Josef KUCERA, Jan JURICA and Ondrej PES (corresponding author). Heavy water enhancement
of fluorescence signal in reversed-phase liquid chromatography. Journal of Chromatography B-Analytical Technologies
in the Biomedical and Life Sciences. 2018, 1092, 7–14. ISSN 1570-0232. doi:10.1016/j.jchromb.2018.05.037 Document
Type: Article, IF = 2.813; JCR Category + Category Quartile: BIOCHEMICAL RESEARCH METHODS Q2 +
CHEMISTRY, ANALYTICAL Q2; AIS = 0.557
Author’s contribution: 60 %
67
Contents lists available at ScienceDirect
Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb
Heavy water enhancement of fluorescence signal in reversed-phase liquid
chromatography
Petr Taborskya
, Josef Kuceraa
, Jan Juricab
, Ondrej Pesc,⁎
a
Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
b
Department of Pharmacology, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
c
Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
A R T I C L E I N F O
Keywords:
Liquid chromatography
Heavy water
Fluorescence
Deuterium
Mass spectrometry
Doxorubicin
Fluorescein
Vitamin B2
A B S T R A C T
Liquid chromatography with fluorescence detection has been used in analyses demanding high sensitivity and
selectivity. As majority systems rely on reversed-phase columns with water being the main component of the
mobile phase, fluorescent compounds with emission maxima higher than 500 nm might be dynamically quenched.
A simple replacement of H2O with D2O enhanced the sensitivity for selected compounds by 10–200%.
Affected compounds included an anti-cancer drug doxorubicin, a luminescent probe fluorescein, and naturally
occurring forms of vitamin B2. Similar levels of enhancement were obtained by fluorescence spectrometry. Such
simple yet effective approach may greatly improve HPLC analyses coupled to fluorescence detection.
1. Introduction
Fluorescence detection in liquid chromatography (LC) has been
widely spread due to a number of key features, such as high sensitivity,
high selectivity, and repeatability. Reversed-phase high-performance
LC (RP HPLC) with the C18 modified silica has become the most frequent
type of an HPLC column since the market introduction. There
might be some drawbacks when using light water in RP HPLC with
fluorescence detection as water molecules can cause numerous collisions
with the excited fluorophore molecules, which result in a nonradiative
relaxation. The process was described as luminescence dynamic
quenching and the energy is dissipated as heat via the eOH oscillation
[1]. Dynamic quenching is typical for luminophores with
emission maxima higher than 500 nm as it is accompanied by relatively
high absorbance of H2O above 500 nm [2].
When the water is replaced with another solvent the fluorescence
intensity, lifetime, as well as fluorescence quantum yield might change.
For example, luminescence lifetime of ethidium bromide was 1.8 ns in
H2O but increased to 5.0 ns in DMSO, 6.9 in ethanol, 5.9 ns in glycerol,
or to 6.3 ns in D2O [3]. The shorter fluorescence lifetime of ethidium
bromide in light water was attributed to the non-radiative deactivation
process described above. The quantum yield of the fluorescent dye
ATTO 655 (0.23 in H2O) was increased to 0.39 in methanol, 0.55 in
acetone, 0.56 in DMSO, and to 0.48 in D2O [1].
Dynamic fluorescence quenching induced by deuterated water was
found minimal in comparison to H2O. One of the explanations relies on low
absorbance of D2O in a region above 500 nm [2]. Hence, if H2O is replaced
by D2O in the mobile phase, the fluorescence signal-to-noise response may
increase without any further modification of the compound separation.
Although the enhancement induced by D2O replacement is neither uniform
nor known beforehand, an increase of fluorescence intensity and lifetime
between units and hundreds of per cent might be observed.
We have selected a couple of compounds to demonstrate the
aforementioned behavior of fluorophores surrounded by D2O instead of
H2O. In order to enlighten the relationship between fluorescence intensity
in light and heavy water, mass spectrometry was used as well.
Doxorubicin, which has been used as an anticancer agent, has been
analyzed with satisfactory sensitivity in various biological fluids by
chromatography [4–6] or targeted by fluorescence probes [7]. Fluorescein,
which is heavily employed as a fluorescence labeling agent [8],
has been used in imaging [9], chromatography [10,11], in cell targeting
[12], or sensing [13]. Trace amounts of vitamin B2 has been commonly
determined by fluorescence techniques [14–17]. The mentioned applications
may greatly benefit from the employment of heavy water into
the methodology even as the overall impact and utilization still remain
to be answered in future. In other words, we do not expect all researchers
immediately exchanging their mobile phases, however; it may
help in specialized types of applications or analyses where the limits of
detection need to be pushed even further. It includes micro- or nanoHPLC
to maintain the costs reasonable.
https://doi.org/10.1016/j.jchromb.2018.05.037
Received 26 April 2018; Received in revised form 25 May 2018; Accepted 26 May 2018
⁎
Corresponding author.
E-mail address: ondramayl@gmail.com (O. Pes).
Journal of Chromatography B 1092 (2018) 7–14
Available online 26 May 2018
1570-0232/ © 2018 Elsevier B.V. All rights reserved.
T
2. Experimental
2.1. Reagents and materials
Formic acid (LC MS), acetic acid (LC MS), ammonium hydroxide
(28–30% NH3 in water), sodium formate (LC-MS), acetonitrile (ACN,
LC-MS), heavy water, fluorescein, doxorubicin, riboflavin, flavin
mononucleotide (FMN), and flavin adenine dinucleotide (FAD) were
purchased from Sigma-Aldrich (Czech Republic). Light water was of an
ultra-pure grade supplied by an in-house Milli-Q system (Millipore, MA,
USA).
2.2. Fluorescence spectrometry
Stock solutions were prepared by dilution of solid chemicals in H2O.
The concentration of all stock solutions was 0.1 mM. Samples were
prepared by dilution of the stock solutions in D2O and H2O. Final
concentrations were 0.22 μM for fluorescein, 0.01 μM for doxorubicin,
and 1.4 μM for FAD, FMN, and riboflavin. The pH (pD) value of sample
solutions was checked to be 7 ± 0.05 and was not further adjusted.
A spectrofluorometer Aminco-Bowman Series 2 (Thermo Fisher
Scientific, MA, USA) equipped with a 150 W Xe lamp was used for
steady state measurements. Excitation and emission wavelengths were
set at 445 nm and 525 nm for riboflavin, FMN, and FAD; 490 nm and
590 nm for doxorubicin; and 480 nm and 515 nm for fluorescein. The
spectra were acquired with 1 nm resolution and a scanning speed of
1 nm/s.
Time-domain measurements were performed using TCSPC SPC-
130EM (Becker and Hickl GmbH, Germany) instrumentation equipped
with a 408 nm diode laser (picosecond resolution, repeating frequency
150 MHz). The emission monochromator was set at same values as in
steady-state measurements. The quartz cuvettes were used in all ex-
periments.
2.3. Liquid chromatography with fluorescence or mass spectrometric
detection
Samples were measured on an LC system Shimadzu LC-10 ADVp
(Shimadzu, Japan) consisting of a manual injector, low pressure gradient
ternary pump, and a fluorescence detector RF 10AXL and a
Dionex UltiMate 3000RS (Thermo Fisher Scientific, CA, USA) equipped
with a high-pressure gradient binary pump, an autosampler, and a
heated column compartment. The latter was connected to a Bruker
MicrOTOF-Q II (Germany) mass spectrometer.
Compound separation was achieved with a 2.1 mm × 150 mm, 5 μm
Acclaim 120 C18 (Thermo Fisher Scientific, CA, USA) column, and a
flow-rate of 0.4 mL min−1
. The binary mobile phase system consisted of
ACN and 0.1% formic acid for doxorubicin, ACN and 0.1% formic acid
with 0.1% ammonia for B2 vitamins, and pure H2O without any additives
for fluorescein analysis. H2O or D2O was used as a solvent for the
mobile phase preparation. After a 20-μL injection, the percentage of
ACN was linearly increased from 10% to 60% over 7 min where it was
held for 2 min, followed by equilibration under the initial conditions for
another 5 min. A complete LC run was 14 min.
Fluorescence detector was set at 480 nm excitation and 590 nm
emission wavelengths for doxorubicin, at 377 nm excitation and
530 nm emission wavelengths for B2 vitamins, and at 489 nm excitation
and 512 nm emission wavelengths for fluorescein analysis.
MS was operated in the positive electrospray ionization mode and
the ionization conditions were determined by the software as follows:
capillary voltage: 4500 V, end plate offset: −500 V, source temperature:
220 °C, desolvation gas (nitrogen) flow: 8 L min−1
, nebulizer (nitrogen)
pressure: 300 kPa, and collision cell voltage: 6 eV. The base
peak chromatogram (BPC) was acquired in MS mode by monitoring the
m/z range of 50–1500 with a spectra sample time of 1 s and MS/MS
spectra were collected in a data-dependent mode. The mass spectrometer
was calibrated using 10 mM sodium formate in 50% isopropyl
alcohol on a daily basis and at the beginning of each LC run with an
external 20-μL loop flush. High-resolution MS and MS/MS spectra were
Fig. 1. Liquid chromatography with fluorescence detection of A) doxorubicin (26 μM, λex = 480 nm, λem = 590 nm), B) vitamin B2 species (15 μM each,
(λex = 377 nm, λem = 530 nm), and C) fluorscein (542 nM, λex = 490 nm, λem = 518 nm) in H2O (solid line) and D2O (dashed line).
P. Taborsky et al. Journal of Chromatography B 1092 (2018) 7–14
8
first investigated to obtain the elemental formula of each compound.
The final identification of target compounds relied on isotope pattern
matching with a combination of MS/MS and retention behavior.
3. Results and discussion
Chromatograms of doxorubicin, B2 vitamin forms, and fluorescein
in H2O and D2O with fluorescence detection might be seen in Fig. 1A.
At doxorubicin, the fluorescence intensity is 2.98 times higher when
heavy water was used as a solvent in the mobile phase. Fluorescence
excitation and emission spectra of doxorubicin in H2O and D2O can be
seen in Fig. 2. Fluorescence spectrometry at pH = 7 showed an increase
in intensity (ID2O/IH2O = 2.87) as well as in lifetime (τD2O/τH2O = 4.04)
of doxorubicin (Table 1).
B2 vitamin forms provided 1.16 and 1.06 times higher fluorescence
intensity for FAD and FMN, respectively. Fluorescence intensity of riboflavin
in heavy water mobile phase was 0.95 times lower.
Fluorescence excitation and emission spectra of riboflavin, FMN, and
FAD in H2O and D2O can be seen in Fig. 3. Fluorescence spectrometry at
pH = 7 showed an average 10% increase in intensity as well as in the
lifetime of all vitamin B2 forms (Table 1).
Fluorescein is well known to greatly increase its fluorescence intensity
when ionized under high pH values (pH > 8) [18]. However,
the chromatography columns based on silica particles are prone to
degradation at high pH due to attack by excess hydroxide ions at the
silica surface [19]. Hence, only water without any additives was used in
a gradient elution of fluorescein from a column (Fig. 1C). The fluorescence
intensity increased 2.19 times when pure D2O was used in the
mobile phase. Fluorescence spectrometry in pure H2O or D2O showed
an increase in intensity (ID2O/IH2O = 2.22) as well as in lifetime
(τD2O/τH2O = 1.43) (Fig. 4, Table 1).
However, it has been reported that deuterated solvents might
modify the separation between deuterated and non-deuterated analytes
[20]. Even the relative response for unlabeled and deuterated forms of
analyte was found to vary significantly from one batch of matrix to the
other [21–23]. The exact cause of this deviation is unknown, but it may
be related to the phenomenon that the properties of deuterated compounds
somewhat differ from those of their unlabeled analogues.
When light water was used as a mobile phase solvent, all MS and MS
spectra were unambiguously attributed to doxorubicin, FAD, FMN, riboflavin,
and fluorescein. When heavy water was used, the MS and MS/
MS spectra were searched for peaks with a mass increase corresponding
to the number of hydrogens, which had been replaced by deuterium.
The exchangeable hydrogen atoms are usually bound to N, O, or S
atoms in functional groups such as eOH, eNHe, eNH2, and eCOOH.
Accurate measurements of exchangeable hydrogen atoms in a molecule
commonly assist in structural elucidation [24–30].
Consistently, all acidic hydrogens in the structure of doxorubicin,
riboflavin, FMN, and fluorescein were replaced with deuterium as seen
from Figs. 5, 6, 7, and 8. In the case of the FAD molecule, the number of
replaced hydrogen was determined from MS1
(data not shown) as no
MS/MS spectra were acquired due to poor ionization efficiency in the
positive mode. It is worth mentioning, that the consistency of the H/D
exchange might differ in gaseous and liquid phase and the conditions
for reliable quantitation should be confirmed e.g., by an overnight ex-
periment.
The question when the fluorescence intensity may increase due to
the heavy water employment might be answered by dynamic
quenching, which is caused by a difference in the eOH and eOD oscillator
ability to efficiently quench a fluorophore molecule. In other
words, when a fluorophore is heavily surrounded by eOH groups (either
intra- or intermolecularly) the effect of quenching was observed.
When the oscillator groups were replaced by eOD groups the
quenching effect was lower. However, the eOD groups had to be in a
direct contact with the fluorophore. Hence, doxorubicin and fluorescein
with the highest amount of eOH oscillators surrounding the fluorophore
showed the highest increase of fluorescence intensity when the
eOH groups were replaced by eOD (Table 1). The increase of fluorescence
intensity was higher than in the case of B2 vitamins even as the
number of totally exchanged atoms was lower since both deuterium
atoms have replaced the hydrogen atoms in the eOH oscillator directly
connected to a fluorophore part of the molecule. When fluorescein was
Fig. 2. Emission and excitation spectra of doxorubicin in H2O and D2O (λexc = 490 nm, λem = 590 nm, T = 293.15 K).
Table 1
Fluorescence properties of selected compounds in H2O and D2O. *Total number
of hydrogens replaced by deuterium atoms.
λex
(nm)
λem
(nm)
ID
(a.u.)
IH
(a.u.)
ID IH
τD
(ns)
τH
(ns)
τD τH
nD/H*
Doxorubicin 478 593 63.7 22.2 2.87 4.20 1.04 4.04 7
Fluorescein 489 512 58.9 26.6 2.22 4.85 3.40 1.43 2
FAD 370 525 45.8 41.5 1.10 3.66 3.19 1.15 10
FMN 370 525 75.1 64.9 1.16 5.11 4.58 1.11 6
Riboflavin 370 525 110 93.0 1.18 5.06 4.58 1.11 5
P. Taborsky et al. Journal of Chromatography B 1092 (2018) 7–14
9
put in the H2O/D2O adjusted to pH ≥ 10, the fluorescence intensity did
not differ as both eOH/eOD groups were ionized (data not shown).
In the case of the vitamin B2 species, the ability of heavy water to
enhance the fluorescence intensity and lifetime was observed at FAD
and FMN only, a riboflavin molecule showed no overall increase in
fluorescence intensity. Most likely, the size of the molecules needs to be
Fig. 3. Emission and excitation spectra of A) riboflavin, B) FMN, and C) FAD in H2O and D2O (λexc = 445 nm, λem = 525 nm, T = 293.15 K).
P. Taborsky et al. Journal of Chromatography B 1092 (2018) 7–14
10
taken into account, as well as the absence of eOH groups in a close
proximity to the fluorophore. The effect of the ]NHe to ]NDe replacement
may have contributed too. Nevertheless, the employment of
heavy water enhanced the fluorescence intensity of FMN and FAD,
when compared to doxorubicin, only by approximately 10%. Certainly,
other conditions, such as pH or ionic strength need to be taken into
account, as many molecules are known to be fluorescent in a specific,
usually alkaline range of pH; outside this range, they are heavily
quenched.
4. Conclusions
Replacement of light water in the mobile phase with heavy water
was found a simple yet effective approach to enhance the sensitivity of
selected fluorescent compounds. The increase of the fluorescence intensity,
as well as the lifetime was affected by the presence of eOH/
eOD groups in the nearest vicinity of the fluorophore. More molecules
have to be studied in order to clarify the structure-to-enhancement
relationship as there may be additional factors influencing the overall
change in fluorescence.
Fig. 4. Emission and excitation spectra of fluorescein in H2O and D2O (λexc = 480 nm, λem = 515 nm, T = 293.15 K).
269.0842
289.0780
1+
309.0670
1+
324.0922
1+
348.0557
1+
363.0846
1+
382.1072
Doxo 1uM_RA1_01_5747.d: +MS2(552.2214), 55.1eV, 4.3min #254
321.0745
1+
333.0769
1+
346.0451
1+
361.0666
1+
Doxo 1uM_RA1_01_5744.d: +MS2(544.1750), 54.8eV, 4.4min #262
0
1000
2000
3000
Intens.
0
500
1000
1500
2000
2500
250 300 350 400 450 500 550 m/z
Fig. 5. MS/MS of doxorubicin ions [M + H]+
(lower pane) and [M + D]+
(upper pane). The difference between M is 7 Da.
P. Taborsky et al. Journal of Chromatography B 1092 (2018) 7–14
11
246.1054
1+
383.1829
1+
Rfv, FAD, FMN _RC2_01_5791.d: +MS2(383.1827), 19.2eV, 3.5min #204
243.0866
1+
377.1458
1+
Rfv, FAD, FMN _RA2_01_5777.d: +MS2(377.1463), 18.9eV, 3.4min #200
0
1
2
3
4
5
4x10
Intens.
0.0
0.2
0.4
0.6
0.8
1.0
5x10
50 100 150 200 250 300 350 m/z
Fig. 6. MS/MS of riboflavin ions [M + H]+
(lower pane) and [M + D]+
(upper pane). The difference between M is 5 Da.
246.1024 344.1458
1+
363.1591
1+
383.1756
1+ 420.1460
1+
444.1300
1+
464.1535
1+
Rfv, FAD, FMN _RC2_01_5791.d: +MS2(464.1520), 22.1eV, 3.1min #181
243.0890
1+
341.1223
1+
359.1349
1+
414.1070
1+
439.1003
1+
457.1119
1+
Rfv, FAD, FMN _RA2_01_5777.d: +MS2(457.1113), 21.9eV, 2.9-3.1min #171-184
0.00
0.25
0.50
0.75
1.00
1.25
1.50
4x10
Intens.
0.0
0.5
1.0
1.5
2.0
4x10
150 200 250 300 350 400 450 m/z
Fig. 7. MS/MS of FMN ions [M + H]+
(lower pane) and [M + D]+
(upper pane). The difference between M is 6 Da.
P. Taborsky et al. Journal of Chromatography B 1092 (2018) 7–14
12
Acknowledgements
This work was supported by the Specific University Research Grant
(MUNI/A/0910/2017, MUNI/A/1132/2017) provided by the Ministry
of Education, Youth and Sports of the Czech Republic in the year 2017.
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233.0919
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273.0889
1+
289.0838
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317.0781
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Fluorescein in D2O .d: +MS2(336.0958), 50.0eV, 0.4min #24
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(lower pane) and [M + D]+
(upper pane). The difference between M is 2 Da.
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14
3.5 A study on bioluminescence and photoluminescence in the earthworm Eisenia
lucens
Bioluminescence is explained by a chemical reaction of two substances – luciferin and luciferase. The luciferin
molecule is typically a low-molecular weight substance, which is combined with a high-molecular weight compound
(typically a protein molecule), oxidized, and the light is emitted. Several well-known luciferin-luciferase systems have
been employed in chemiluminescence immunoassays.53
Bioluminescent segmented earthworms (Annelida) have been
reported predominantly from marine species; however, terrestrial bioluminescent species belonging to clitellates, e.g.,
Diplocardia longa, Microscolex phoshoreus, or Fridericia heliota have been studied as well. Eisenia lucens (also known
as Eisenia submontana) lives under wet bark in the central European region and its bioluminescent luciferin-luciferase
system has yet not been studied in detail. Fluorescence spectroscopy, fluorescence lifetime microscopy (FLIM), and
LC–MS with ESI and APCI were employed to uncover the culprit of the bioluminescence chemistry. Riboflavin was
assumed to play a key role as it was found to be in high concentrations in these earthworms and was fluorescent.
When the earthworm was irritated (mechanically, chemically, or electrically) it expelled bioluminescence coelomic
fluid from its dorsal pores. The emitted blue-green light faded away after a few seconds, and the composition of the
coelomic fluid was examined by several spectrometric techniques. An LC analysis with UV-Vis and MS detection
confirmed the presence of riboflavin in the coelomic fluid at a concentration of 23 µM. Photoluminescence of E.
lucens could be explained by the high content of riboflavin; however, bioluminescence exhibited a different emission
maximum. Since FLIM revealed no other emitting species being present inside the eleocytes, the bioluminescent
emitted light might have been mediated by riboflavin, but another luciferin molecule was likely involved. A few
luciferin molecules from other species, e.g., N-isovaleryl-3-aminopropanal, long-chain aldehydyc hydrogencarbons,
or their oxidized products, have been sought inside the coelomic fluid by ESI and APCI LC–MS. Nonetheless, no
particular evidence of a different luciferin molecule was found. Hence, the question about the common origin of the
earthworm and bacterial bioluminescence, which is based on luminescence of a riboflavin derivative molecule FMN,
was only suggested and left to be answered in future research.
PES, O., A. MIDLIK, J. SCHLAGHAMERSKY, M. ZITNAN a P. TABORSKY. A study on bioluminescence and
photoluminescence in the earthworm Eisenia lucens. Photochemical and Photobiological Sciences. 2016, 15(2),
175–180. doi:10.1039/c5pp00412h Document Type: Article, IF = 2.344; JCR Category + Category Quartile:
BIOCHEMISTRY & MOLECULAR BIOLOGY Q3 + BIOPHYSICS + CHEMISTRY, PHYSICAL Q3; AIS = 0.623
Author’s contribution: 40 %
76
Photochemical &
Photobiological Sciences
COMMUNICATION
Cite this: Photochem. Photobiol. Sci.,
2016, 15, 175
Received 4th November 2015,
Accepted 7th January 2016
DOI: 10.1039/c5pp00412h
www.rsc.org/pps
A study on bioluminescence and
photoluminescence in the earthworm
Eisenia lucens†
O. Pes,a
A. Midlik,a,b
J. Schlaghamersky,c
M. Zitnand
and P. Taborsky*b,e
Eisenia lucens is an earthworm living in the organic soil layer of
decomposing wood. When irritated, the worm expels coelomic
fluid through pores in its body wall, exhibiting blue-green bioluminescence.
The mechanism of the bioluminescence, which
seems to be different from other bioluminescence systems of terrestrial
animals, has been studied in this work. Many lines of evidence
indicate that riboflavin stored in coelomycetes plays an
important role in this glowing reaction.
Introduction
Bioluminescence has been reported from a considerable
number of species of segmented worms (Annelida). Many
belong to predominant marine polychaetes with several bioluminescent
representatives of the taxon Clitellata, specifically
of earthworms (Megadrili), from the families Lumbricidae and
Megascolecidae, and of the related but substantially smaller
potworms (Enchytraeidae).1
Except for a few species of the
megascolecid genus Pontodrilus,2,3
all bioluminescent clitellates
(at least 33 species) are terrestrial.4
Bioluminescence in
clitellates has been mostly studied in the earthworms Diplocardia
longa,5,6
Microscolex phosphoreus,4
and the enchytraeid Fridericia
heliota.7–11
Diplocardia longa, which has been studied
in detail by Wampler and colleagues,5,6,12,13
expels a bioluminescent
fluid from its pores with bioluminescence originating
from discrete subcellular loci within free chloragogen cells suspended
in the coelomic fluid.12
Wampler et al. proved that
N-isovaleryl-3-aminopropanal was the luciferin molecule.4,6
They also suggested that all bioluminescent earthworms used
the N-isovaleryl-3-aminopropanal/hydrogen peroxide reaction,
although they admitted that the colour of bioluminescence
varied among species (from 490 nm to over 600 nm).4
The
Siberian potworm Fridericia heliota (Enchytraeidae) exhibits
blue bioluminescence (maximum at 478 nm) with the light
being emitted from epidermal cells. The bioluminescent
system included a luciferin combined with CompX, lysine,
GABA, and oxalate. Apart from that, the reaction required
atmospheric oxygen, ATP, Mg2+
and Fridericia luciferase.7–11,14
Eisenia lucens (Waga, 1857) from Lumbricidae is an earthworm
found in Central and Eastern Europe. Its bioluminescence
has been firstly reported by the Polish scientist
Antoni S. Waga in 1854, who described it and named it as
Lumbricus lucens. Later, the species was re-discovered by
F. Vejdovský and described as Eisenia submontana, which is
now considered a junior synonym of E. lucens. The constitution
and shape of an Eisenia lucens body is similar to other
Eisenia species such as Eisenia fetida (erroneously given as
E. foetida) and Eisenia andrei. Adults have 60–130 segments
and reach 4.5–18 cm in length and 5–6.5 mm in width. The
body coloration is conspicuous: each segment has a transverse
band of red-brown to red-violet colour in its middle, whereas
the parts towards the intersegmental grooves are of lighter yellowish
to greyish colour (this colour also prevails ventrally).
The prostomium is epilobic, the clitellum being saddleshaped.
Typical montane forest habitats are in the Carpathian
arch but might also be found in other parts of Europe, west to
northern Spain; completely missing in Northern Europe. The
earthworm commonly lives under the bark and in wet decomposing
wood of downed tree trunks and stumps, but has been
reported in the organic soil, in particular under moist to wet
conditions. The bioluminescence reaction might be inspected
after a mechanical, chemical, or electrical irritation of the
earthworm when the coelomic fluid is expelled from its dorsal
pores. An attempt to describe the bioluminescence reaction of
E. lucens was made in the work of Bačkovský et al. from 1939;
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5pp00412h
a
Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice
753/5, Brno, CZ 62500, Czech Republic
b
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 753/5,
Brno, CZ 62500, Czech Republic. E-mail: taborak@email.cz
c
Department of Botany and Zoology, Faculty of Science, Masaryk University,
Kamenice 753/5, Brno, CZ 62500, Czech Republic
d
Material Research Centre, Faculty of Chemistry, Brno Technical University of
Technology, Purkyňova 464/118, Brno, CZ 61200, Czech Republic
e
Central European Institute of Technology (CEITEC), Masaryk University, Czech
Republic, Kamenice 753/5, Brno, CZ 62500, Czech Republic
This journal is © The Royal Society of Chemistry and Owner Societies 2016 Photochem. Photobiol. Sci., 2016, 15, 175–180 | 175
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however, the authors studied merely the photoluminescence
reaction by comparing the fluorescence spectra of riboflavin
and E. lucens extract. Although they reported an increased
amount of riboflavin in the earthworm, they did not examine
the chemical form of the emitting species (riboflavin, FAD,
FMN), and concluded that riboflavin acted as a luciferase.15
In this paper, we attempted to characterize the bioluminescence
system using a combination of techniques such as
fluorescence microscopy, fluorescence lifetime imaging (FLIM),
luminescence spectrometry, and mass spectrometry (LC MS).
Materials and methods
Riboflavin, FMN, FAD, palmitic acid, cis-11-hexadecenal, spectroscopy
ethanol, ammonium formate (LC MS), methanol
(MeOH) (LC MS) and acetonitrile (ACN) (LC MS) were purchased
from Sigma Aldrich (Czech Republic). Specimens of
Eisenia lucens were collected in two areas within the Czech
Republic: the Bohemian-Moravian Highlands (49°39′18.9″N
15°59′34.2″E) and the Moravian-Silesian Beskids (49°32′14.0″N
18°39′13.4″E). Earthworms were stored in plastic containers
filled with decaying wood and soil at a constant temperature of
20 °C. Earthworms were chemically stimulated by 50% ethanol.
The expelled fluid was collected, centrifuged, diluted 10 times
with water and filtered through a 0.22 μm PTFE syringe filter.
Luminescence
All spectra (photoluminescence and bioluminescence) were
measured on a spectroluminometer Aminco-Bowman Series 2
(Thermospectronic, USA), equipped with a Xe lamp (for excitation
between 200 and 900 nm). An Olympus BX60 fluorescence
microscope (blue light excitation) with a digital
image analysis system (Stream Motion 1.9.2) was employed in
the study of native photoluminescence. FLIM (Fluorescence
LifeTime Imaging) measurements were done using the confocal
microscope Leica TCS SP8 X, equipped with a tuneable
pulse white laser. A pulse laser of 405 nm was used as an excitation
source.
Mass spectrometry
The LC MS method was developed using a Dionex Ultimate
3000RS (Thermo Scientific, CA) module. Compound separation
was achieved with a 3.0 × 100 mm, 2.6 µm Kinetex C18 (Phenomenex,
CA) column equipped with a guard column at 23 °C,
and a flow rate of 0.3 mL min−1
. The binary mobile phase
system consisted of 0.1% ammonium formate (pH = 6.2) and
MeOH or ACN. The injection volume was 10 μL. For separation
of isoalloxazines (method A), MeOH was linearly increased
from 15% to 75% over 20 min, kept constant at 75% over the
next 2 min and followed by equilibration under the initial conditions
for 3.0 min. For separation of fatty compounds
(method B), ACN was held at initial 50% for 2 min and then
linearly increased to 95% over 10 min, kept constant at 95%
over the next 10 min and followed by equilibration under the
initial conditions for 3.0 min. A complete LC run in each
method took 25 min. The isoalloxazine ring was detected by
monitoring absorbance at 448 nm. The HPLC system was connected
to a MicrOTOF-QII (Bruker, Germany) mass spectrometer
operated in positive electrospray (ESI+) mode with
method A, negative electrospray (ESI−) mode and positive
atmospheric pressure chemical ionization (APCI+) mode with
method B. The ionization conditions were set by the software
according to the mode used; nebulizing and desolvation gas
was nitrogen. ESI+: capillary voltage +4500 V, end plate offset
−500 V, source temperature 250 °C, desolvation gas flow 5 L
min−1
, nebulizer pressure 2 bar, and collision cell voltage
35 eV. ESI–: capillary voltage −4500 V, end plate offset +500 V,
source temperature 250 °C, desolvation gas flow 5 L min−1
,
nebulizer pressure 2 bar, and collision cell voltage 35 eV.
APCI+: capillary voltage +4500 V, end plate offset −500 V,
corona needle 4000 nA, source temperature 210 °C, vaporizer
temperature 250 °C, desolvation gas flow 2 L min−1
, nebulizer
pressure 1 bar, and collision cell voltage 35 eV. A base-peak
chromatogram (BPC) was acquired in the MS mode by monitoring
in a range of 50 to 2000 m/z with a spectra sample time
of 1 s. Identification of target compounds relied on a combination
of accurate m/z, isotopic pattern, MS/MS and retention
behaviour. High-resolution MS and MS/MS spectra were first
investigated to obtain the elemental formula of each compound.
A compound was unambiguously identified if the fragmentation
patterns of the unknown and the standard
compound were identical. Due to the lack of fragmentation
spectra of non-polar compounds obtained by method B, compounds
were identified by comparing accurate m/z and retention
time with those of a standard compound.
Results and discussion
After irradiation by violet or blue light, several parts of the
earthworm bodies showed bright green photoluminescence
(occasionally reported as autofluorescence). The main centres
of photoluminescence emission were rows of bristles protruding
through the body wall and granules inside eleocytes
(Fig. 1). Eleocytes, a type of coelomocytes (cells floating in the
coelomic fluid), are detached chloragocytes, i.e. cells that originally
covered the outer wall of the intestine forming the chloragogen
tissue. Similar fluorescent coelomocytes were observed
also in other, non-bioluminescent species of earthworms e.g.,
E. fetida.16–18
To obtain coelomic fluid a few earthworms were irritated
with a 50% solution of ethanol on a glass plate. The expelled
fluid, when collected and diluted by re-distilled water, showed
two bands (370 and 440 nm) in the luminescence excitation
and absorption spectra, which were typical for tricyclic isoalloxazine
rings. If excited at 370 nm the sample emitted green
fluorescence with a maximum at 523 nm. By comparing such
spectra with those of biogenic isoalloxazines, riboflavin and its
derivatives flavinadenindinucleotide (FAD) and flavinmononucleotide
(FMN) were found as the best match. In order to
identify which of the three compounds is the emitting species
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in the earthworm, an LC analysis with UV-Vis and MS detection
was performed (Fig. 2). By comparing chromatograms of a
standard mixture of riboflavin, FMN, and FAD with a coelomic
fluid sample, we noticed that a single compound of riboflavin
appears in the system. To confirm this, a peak (m/z 377.1612)
at 9.4 min was subjected to MS/MS analysis and fragmentation
to lumichrome (m/z 243.0977), as in pure riboflavin, was
observed. This unambiguously confirmed the presence of riboflavin
in the earthworm, however; there were no signs of FMN
or FAD. Other peaks in the base peak chromatogram (BPC)
were not characterized in detail, nevertheless; they might be
attributable to phospholipids, peptides and other low-molecular
compounds commonly found in living organisms.
UV-Vis detection with external standard calibration provided
the content of riboflavin in the expelled coelomic fluid to
equal a concentration of 23 × 10−6
mol l−1
.
Photoluminescence of Eisenia lucens can be assigned to the
presence of such a high quantity of riboflavin. This essential
vitamin plays a key role in the immune potency of animals
including earthworms and its presence at high concentrations
was described for other earthworms as well e.g., the nonluminescent
E. andrei and E. fetida.19,20
While the expelled coelomic fluid containing coelomocytes
was of yellow colour in daylight, blue-green bioluminescence
was observed when earthworms were irritated in the dark (see
Fig. 1 and a video in the ESI†). When a freshly expelled portion
of the coelomic fluid was subsequently placed into a cuvette
and bioluminescent emission spectra were recorded, the emission
maximum observed immediately after irritation was
493 nm, and the dependence of intensity at 493 nm on time
was found to be exponential with a bioluminescence half-life
of about 6 s (Fig. 3). The measured bioluminescence emission
spectra of Eisenia lucens are clearly different from those
obtained when excited by light. On the other hand, they are in
a close match with the bioluminescence of bacteria from the
genera Photobacterium, Beneckea, Vibrio, and Xenorhabdus.21,22
Bacterial luminescence is generally based on a high molecular
weight flavoprotein (luciferase) catalyzing the oxidation of
FMNH2 by oxygen in the presence of a long-chain aldehyde.23
Emitted light maximum is ∼495 nm, which indicates the FMN
to be the emitting species. LC MS analysis confirmed that
although the expelled coelomic fluid of Eisenia lucens did not
contain any FMN/FAD, a precursor of FMN – riboflavin was
present in great excess. This may indicate that the bioluminescence
mechanisms of bacteria and E. lucens are
similar (Fig. 3) except for riboflavin being the emitting species.
We suggest that riboflavin is in vivo converted to the reduced,
Fig. 1 Visualisation of the earthworm Eisenia lucens by photography –
under daylight (upper-left) and in the dark after irritation (lower-left),
and by fluorescence microscopy – native green photoluminescence of
bristles (lower-right) and detached chloragocytes (upper-right). The red
fluorescence originates inside the intestinal tract.
Fig. 2 LC with detection at 448 nm of (A) a model solution containing
(1) FAD (6.6 min), (2) FMN (7.3 min), and (3) riboflavin (9.4 min), and the
expelled coelomic fluid of Eisenia lucens detected (B) at 448 nm and (C)
by MS of a base peak (BPC) in positive ESI mode. The injection volume
was 10 μL and the concentration of each analyte was 3 μg mL−1
except
for riboflavin (10 μg mL−1
). The retention time of riboflavin in BPC is
slightly increased (∼3.6 s) due to the sequential design of used
detectors.
Fig. 3 A suggested mechanism of Eisenia lucens bioluminescence,
maximum emission (493 nm) and bioluminescence half-life (6.2 s).
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highly-energetic, non-aromatic form by NAD(P)H as in the case
of FAD to FADH2 conversion in higher organisms. Such oxidation
of flavines (including the oxidation of the reduced riboflavin),
was simulated in vitro by several groups. Zeng et al.
prepared an experiment where riboflavin was converted to the
reduced form by Fe2+
ions in a pyrophosphate buffer.24
Chemiluminescence
with emission maximum at ∼490 nm was
induced by an addition of hydrogen peroxide. Similarly,
Towner et al. demonstrated in their report (Towner, Technical
manuscript, 1969) another experiment. Os(III) was used instead
of Fe(II) to reduce riboflavin in a phosphate or borate buffer.
All these experiments showed that the riboflavin chemiluminescence/bioluminescence
reaction is plausible.
When the coelomic fluid was studied by fluorescence
microscopy, it contained glowing granules inside the eleocytes,
which were identified as the apparent source of bioluminescence
(Fig. 4). FLIM revealed a uniform distribution of
fluorescence lifetimes (∼2 ns) along the entire cell strengthening
the fact that riboflavin is the only emitting species in the
earthworm. As a small organic molecule, its lifetime is
accepted to be in units of nanoseconds. Prior to any bioluminescence,
an oxidation step is required. Generally, two
oxidation agents – oxygen and hydrogen peroxide are associated
with a glowing reaction. A simple experiment confirmed
that bioluminescence of Eisenia lucens is caused by atmospheric
oxygen: a specimen, irritated under a N2 atmosphere,
was expelling coelomic fluid in the flask. The expelled fluid
was not bioluminescent until the flask was opened and aerial
oxygen was allowed for the reaction. By combining this observation
with microscopy, we suggest that riboflavin molecules
from the expelled eleocytes are oxidized by aerial oxygen.
The question whether any luciferin molecule was involved
in the reaction mechanism was again pursued using LC MS. It
is worth noting that the term “luciferin” has never been strictly
defined. Originally, it meant an essential ingredient needed
for emission of bioluminescence. Now it seems appropriate to
define it as the general term of an organic compound that
exists in a luminous organism and provides the energy for
light emission by being oxidized, normally in the presence of a
specific luciferase.21
We have included already known luciferin
molecules, such as N-isovaleryl-3-aminopropanal suggested by
Wampler in Diplocardia longa or long chain aldehydes (or
related products of oxidation carboxylic acids) typical for luminous
bacteria, in the search. Under conditions of analysis of
isoalloxazines, there were no signs of N-isovaleryl-3-aminopropanal
or fatty compounds being present. In order to bring
attention towards more non-polar compounds, the elution
strength of the mobile phase was increased and the MS was
operated in negative ESI and positive APCI modes. This
allowed for the detection of fatty acids or fatty aldehydes,
respectively, and palmitic and palmitoleic acids as well as
three isomers of hexadecenal were found. However, no structural
data on the location or conformation of the double bond
were acquired. While all these 16-C compounds may naturally
be present in living organisms and are required for specific
purposes (fuel, signalling, building blocks, etc.), they might
also be involved in the E. lucens bioluminescence reaction as a
potential luciferin molecule. Bioluminescence reactions are
also catalysed by compounds called luciferase enzymes. Identification
of the luciferase proteins is considered in further
research.
The use and adaptive value of bioluminescence in earthworms
are still not well understood. It is often mentioned that
it might deter predators when earthworms are attacked.25
Bioluminescence
is possibly a part of a complex defence strategy
of earthworms. We observed that after irritation, the earthworms
attempted to change their position quickly by convulsive
movements, leaving a glowing spot in the place of their
previous position, which should keep the predator confused
about the worm’s true location. Similar strategies are used by
some marine luminous organisms such as squids. Another
approach to deflect predators is used by a few species of terrestrial
earthworms e.g., Megascolides australis that expel nonluminescent
coelomic fluid, which is believed to be toxic or
repellent.25
The obvious resemblance of the bioluminescence
mechanism of luminous bacteria and earthworms could indicate
a similar origin of this ability. However, the adaptive value
of bacterial bioluminescence has not yet been uncovered.
Conclusions
The present work reports about novel findings in photoluminescence
and bioluminescence of the earthworm Eisenia
lucens. As its body contains a vast amount of riboflavin, there
is a high probability that among other functions in the organism,
riboflavin stored in the eleocytes plays a key role in the
bioluminescence of this species. We propose that the mechanism
of bioluminescence in E. lucens is analogous to the mechanism
described in bioluminescent bacteria except for the
emitting species to be riboflavin molecules oxidized by atmosFig.
4 Eisenia lucens coelomocytes (eleocytes) subjected to fluorescence
microscopy (left) and FLIM (right) – green colour here represents
a lifetime of about 2 ns.
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pheric oxygen. The relationship is deepened by the existence
of possible luciferins/oxoluciferins as derivatives of the 16-C
aldehyde/carboxylic acid redox pairs.
Acknowledgements
The authors would like to kindly acknowledge Dr Bozena
Koubkova and Dr Sarka Masova (Parasitology Research Group,
Dept. of Botany and Zoology, Faculty of Science, Masaryk University
Brno) for assisting in fluorescence microscopy; Jiří Procházka
(Terrestrial Invertebrates Research Group, the same
department as above), Ema Taborska, Lucie Taborska, Marek
Svoboda and Pavla Foltynova for collecting the earthworm
specimens in the Moravian-Silesian Beskids and BohemianMoravian
Highlands. This research has been financially supported
by the Ministry of Education, Youth and Sports of the
Czech Republic under the project CEITEC 2020 (LQ1601).
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4 Mass spectrometry as a sensitive and selective detector
Although triple quadrupole (QqQ) mass analyzers may sometimes be called “just” detectors, they are very specific
and sensitive at the same time. The ability to tune several compound-specific fragmentation patterns, accounting for
a constant difference in mass (x-axis) and, at the same time, a constant intensity ratio (y-axis) of target fragments,
results in invaluable hyphenation options with separation techniques. Manufactured QqQ mass analyzers share the
vast majority of the worldwide market in analysis of low molecular (< 3000 Da) compounds. In an overstating
way, it may be said that QqQ mass analyzers are rarely used in other modes of operation beyond selective reaction
monitoring (SRM). As any compound could be detected in concentrations as low as the signal-to-noise ratio allows,
the option to selectively decrease the noise is entirely valid and perfectly approachable by including two ion filtration
steps in the first and the third quadrupole. Quantitation guidelines using SRM recommend including one transition
(usually the most intensive) for quantitation and an additional one for confirmation of the target compound’s identity.
If the intensity ratio of the chosen transitions does not equal that of the authentic standards, the identity of the
target compound cannot be assured. Approximately 3,000 to 4,000 QqQ instruments per year are installed worldwide,
making them readily available and reducing the cost per sample. There are many clinical applications of LC–MS,
and the reasons for choosing LC–MS over LC with conventional detectors are high specificity, high sensitivity and
the ability to employ isotopically labeled internal standards.54,55
It is worth noting that other types of analytical
instruments that rely on chemical or spectrometric detection cannot profit from isotopically labeled compounds.
Quantitative determination in MS is usually assessed by one of the following calibration models:
• An authentic analyte without a sample matrix
It is a classical example of an external calibration curve using an analyte in calibration solutions differing in
composition from the authentic matrix used in samples. Sometimes it may provide reliable results in terms of
validation, e.g., for urine or saliva specimen.
• An authentic analyte in a surrogate matrix
The surrogate matrix is intentionally selected to mimic the authentic matrix yet containing no analyte. Such design
restricts the model for determining exogenous compounds not found in the sample matrix. The calibration samples
could be prepared in an analyte-free matrix such as animal, or human blank matrices. This approach is commonplace
across a majority of LC–MS analyses nowadays.
• A surrogate analyte in an authentic matrix
The analyte requires to be distinguishable in a different domain than chemistry. Stable isotopes are chemically
non-distinguishable but differ in physical properties such as mass. Generally, isotopic analogs involving stable
isotopes but deuterium are preferable as the extreme ratio of size to mass in hydrogen/deuterium negatively impacts
their chemical properties, such as retention times.56
• An authentic analyte in an authentic matrix
The most demanding and practically inconvenient approach restricts the calibration model to the addition of the
83
standard prior to or during the analysis. It immensely increases the number of analyses required to be performed to
obtain a reliable result from each sample.
Absolute MS responses are generally not used as they are subject to significant day-to-day variation because of a lot
of difficult-to-control factors influencing the absolute MS response, e.g., the cleanliness of ion source and ion optics,
collision cell pressure, the MS vacuum, or ion suppression. Ion suppression may be defined as a simultaneous presence
of more than one component in the ion source resulting in compounds competing during the ionization process
for a charge. The charge is always finite and redistributed to the competing species based on their concentrations
and their tendencies to ionize in the gaseous phase.57
Buffers and other mobile phase additives are also sources
of ion suppression (matrix effects). Although matrix effects cannot be avoided entirely, they can be minimized or
compensated for by optimizing sample preparation procedures, manipulating LC and MS conditions, and using
internal standards. One of the requirements for internal standard calibration is that the internal standard and
the analyte behave similarly during the entire analytical process including sample extraction and cleanup steps.
Isotopically labeled analogs of the analyte behave in all ways identically to the analyte except for the molecular
weight being shifted to higher mass-to-charge ratios. Synthesizing isotopically labeled chemicals can be expensive
and time-consuming; however, employing such internal standards circumvents many problems, and testing is more
rapid with significantly lower costs per test and fewer reanalyses. To avoid the natural occurrence of analyte isotopes
interfering with the labeled internal standard, the mass difference between the analyte and the corresponding
isotopically labeled analog should be at least 3 Da.58
Under certain circumstances, deuterated analogs do not
co-elute with their native analytes and cause inaccuracies in quantification. The deuterium isotope effect induces
this phenomenon due to a slight change in lipophilicity, and other, more expensive 13
C-, 15
N- or 17
O-labeled analogs
are considered more appropriate. Any validation procedure should assess the matrix effects by either postextraction
or postcolumn infusion strategies.59–61
Should the slopes of calibration curves obtained with and without matrix
differ, the matrix effects are present.
The process of quantifying and data collection inevitably leads to classification, prediction, and attempting to quantify
and understand the uncertainty inherent in phenomena underlying data – statistical modeling. Even calculation of a
mean value can be considered a statistical model. Such a model should optimally enable distinguishing between
noise and a true effect for generalization from the sample to the population. However, for more than a decade,
academic research has been mired in a “replication crisis,” in which the findings of thousands of published studies
have proven to be difficult if not impossible to reproduce. One of the reasons is definitely the tendency of journals to
prioritize papers that have surprising results, or that upset conventional wisdom. In other words, the publication bias
could prompt even well-intentioned researchers to engage in parsing data until statistically significant (and therefore
publishable) results come up. A term of p-hacking encompasses factors influencing the statistical significance by
manipulating p values (intentionally or not) e.g., by altering the sample size, removing outlying results, or multiple
testing without any corrections. Because p values are a frequency probability, if enough p values are calculated, then
one is bound to be statistically significant.62
And all statistically significant findings tend to overestimate the size of
effects.63
Null hypothesis significance testing (NHST) in its hybrid form, considered the golden standard in biological, medical,
84
social, or economical science, can be viewed as a collective work of Ronald Fisher, Jerzy Neyman, Egon Pearson,
and Everet Franklin Lindquist.64–66
Basically, there are two major flaws in NHST.
• Null hypothesis
Null hypotheses rarely can be true. Just about every treatment one might consider will have some effect, and no
comparisons or regression coefficient of interest will be exactly zero.67
It is then meaningless to formulate and test
null hypotheses that we know ahead of time cannot be true. With sufficient sample size, any hypothesis can be
rejected, and there is no real point in gathering a mountain of data just to reject a hypothesis that we did not
believe in the first place.63
Next, the research questions used to construct null and alternative hypotheses require to
be falsifiable while the null hypothesis is only the complement of the alternative hypothesis and contradicts the
researchers’ expectations. In other words, as NHST dichotomizes any question to reject/fail to reject, null hypotheses
are formulated only to be disproved.
• The p value
If the null hypothesis is true in the population, what is the probability of encountering a space of outcomes that are as
extreme as (or more extreme than) the actually observed outcome? Such probability is called a p value, and could be
viewed as a conditional probability of observing the current data given a true null hypothesis: Pr(Data|H0 = True).
The space of all possible outcomes under the true null hypothesis is however based on the intentions how the data will
be collected.68
Although p values are helpful in assessing how incompatible the data are with a specified statistical
model, contextual factors must also be considered, such as the design of a study, the quality of the measurements,
the external evidence for the phenomenon under study, and the validity of assumptions that underlie the data
analysis. The p values do not say anything about the effect size, the probability of replicating the result, or if the
result is of major theoretical, clinical or practical importance. Thus not all p values are equal and equal p values
may be interpreted very differently. For instance, a p value of 0.01 from a small underpowered study is more likely
to be a false positive that the same p value from a large, well-powered study.
The p value is often misunderstood as being the probability that the null hypothesis is true given the observed data:
Pr(H0 = True|Data).69
To turn p values into such probabilities requires Bayes’ theorem and is therefore not defined
in classical (frequentist) statistics. In other words, the probability that the null hypothesis is true given the data
depends indeed on the p value, but also on the power of the study to detect an effect and the prior probability that
the null hypothesis is true. For instance, the performance of a diagnostic test depends on its sensitivity, specificity,
disease prevalence, and positive predictive value. Test sensitivity is equivalent to statistical power. Test specificity is
equivalent to the p value. Disease prevalence in the population being tested is equivalent to the prior that the null
hypothesis is false. What the clinician wants to know is the probability that the subject has the disease given the
test is positive. This is the positive predictive value and, in hypothesis testing, is equivalent to the probability that
the alternative hypothesis is true given the data.70
If a p value is determining whether a particular null value of a
parameter can be rejected, the confidence interval (CI) provides a range of parameter values not being rejected.
Particularly, the 95% CI consists of all parameter values that would not be rejected by a two-tailed significance
85
test that allows 5% false alarms (type I errors). Since CIs are defined by p values, for every misconception of p
values, there can be a corresponding misconception of CIs.68
Such a common misconception is that CI represents
some sort of probability distribution over the parameter values. No, the 95% CI consists of merely two end points
covering (approximately) two standard errors (SE) from both sides of the parameter mean value. For a treatment
effect to be statistically significant, the CI cannot include zero. A CI that contains zero is uncertain whether there is
a treatment effect. In noisy studies when SE is large, a high effect requires to be observed for achieving the desirable
level of significance. Unrealistically high effects are then published and the same inherent problem of attempting to
do inference based only on sample probability for the data commits Bernoulli’s Fallacy.71
The way out of this trap
could be more and more accumulated evidence within subjective confines of the assumptions we make about the
world, i.e., criteria for publication should be determined not on the statistical significance of the findings but on the
extent to which those findings can move existing beliefs and shape new policies – the culprit of Bayesian statistics.
Bayesian statistics is named after Thomas Bayes (1702–1761), a British Presbyterian minister and amateur
mathematician who was interested in the notion of inverse probability, now referred to as posterior probability.72,73
Bayes’ rule was independently discovered by Pierre-Simon Laplace in 1774 who actually used the rule in practice.74
Over the next forty years he developed it into the form we use today.75
Bayesian inference could be seen as a
principled incremental process of scientific discovery encoded in the generic expression of Bayes theorem formally
given as
Pr(A|B) =
Pr(B|A) · Pr(A)
Pr(B)
where A, B are the specified events represented by conditional Pr(A|B), Pr(B|A) and unconditional Pr(A), Pr(B)
probabilities.76
In contrast to frequentist statistics, where parameters are treated as unknown constants, Bayesian
statistics is distinguished by a broad view of probability, i.e., parameters are treated as random variables with
specified prior distributions that reflect prior knowledge about the parameters before the observation of data. Bayes’
theorem can then be reformulated using a single parameter model consisting of a continuous response y, and a
parameter θ.
p(θ|y) =
p(y|θ) · p(θ)
p(y|θ) · p(θ) dθ
The denominator term is called the total or marginal probability of data and acts as a normalizing constant for the
posterior probability to lie within the unit interval. As statistical models grow parametrically larger the analytical
solution for the denominator begins to be impossible to compute, and it needs to be estimated numerically – usually
by some form of Markov chain Monte Carlo (MCMC) simulation. Hence, drawing inferences from data is represented
by the posterior distribution p(θ|y), which is proportional to the product of the likelihood p(y|θ) of data and the
prior p(θ) information.
p(θ|y) ∝ p(y|θ) · p(θ)
86
Such diachronic inference can generally be described by the following procedure:
1. Encode the initial beliefs (expert knowledge, previous results, similar studies performed, base rate, etc.) in the
form of a prior distribution.
2. Collect data via experimentation, observation, querying, etc.
3. Update the beliefs using Bayes’ theorem to the posterior distribution, which can be seen as a weighted
compromise between the prior and the observed data.
4. Repeat the entire process as more data become available.
Statistical modeling based on Bayesian inference has the ability to propagate uncertainty through the joint posterior
distribution of parameters conditioned on the data. A Bayesian credible interval says where the true parameter value
likely is (a frequentist confidence interval says where the true parameter value likely is not). A diachronic nature of
Bayesian updating enables to change hypotheses over time as new data become available – today’s posterior can be
used as tomorrow’s prior. Robust models originating from relaxing the assumptions are straightforwardly specified
using full Bayesian generative approach, e.g., instead of testing, transforming, or removing outlying data points,
it is easier to keep them all but use for example Student’s t-distribution with finite variance (degrees of freedom
ν > 2) to increase the likelihood that central tendency estimators are compatible with the observed data including
outliers. Any model may be easily generalized, any assumptions might be relaxed (heterogeneity, overdispersion,
inflation, normality, etc.), and any of the shape or scale distribution parameters might be conditioned on data in the
same fashion as the central tendency parameters. Bayesian models are able to test multiple hypotheses without
increasing the false alarm rate, or can even answer questions that cannot be answered using NHST, e.g., what is the
probability that the coefficient is zero under the null hypothesis? The process of learning inside a human brain is
based on Bayesian updating – credibility is reallocated across plausible events in everyday reasoning.77
The former diagnostic test could now be formulated more intuitively using Bayes’ theorem.
Pr(Disease|Test+
) =
Pr(Test+
|Disease) · Pr(Disease)
Pr(Test+)
where Pr(Test+
) is the average probability of a positive test result given as
Pr(Test+
) = Pr(Test+
|Disease) · Pr(Disease) + Pr(Test+
|Healthy) · (1 − Pr(Disease))
The important message here is the counterintuitive result obtained in rare events, e.g., in diseases with low prevalence.
If the prevalence (prior probability) is extremely low (< 1%) then even very high diagnostic sensitivity and specificity
(> 99%) will not dramatically raise the conditional probability of having the disease given being tested positive
(positive predicted value) because a great number of subjects would have been tested as false positives. Based on
that, any scientific inference could be framed in the following terms: (1) A hypothesis is either true or false; (2) a
statistical procedure is used to get an imperfect cue of the hypothesis’ falsity; (3) Bayes’ theorem should be used to
logically deduce the impact of the cue on the status of the hypothesis. The third term is hardly ever done.78
Suppose
the probability of positive finding when a hypothesis is true, is Pr(Significant | True) = 0.95. This is sensitivity
87
or statistical power of the test. Suppose that the probability of a positive finding, when a hypothesis is false, is
Pr(Significant | False) = 0.05. That is the false positive rate or 1 - Specificity, and it represents the 5% level (p value
= 0.05) of conventional significance testing. Finally, the base rate (the prior probability) at which hypotheses are
true could be given as 1 in every 100 hypotheses turns out to be true (Pr(True) = 0.01). The real value is unknown,
but the history of science suggests it is small. To compute the posterior, the appropriate values are plugged in Bayes’
theorem:
Pr(True | Significant) =
0.95 · 0.01
0.95 · 0.01 + 0.05 · 0.99
Pr(True | Significant) ≈ 0.16
So a significant finding corresponds to a 16% chance that the hypothesis is true. Even if the false positive rate
goes down to 1% (Specificity = 99%) the posterior probability (positive predicted value) reaches ~ 0.5, which is as
good as a coin toss. The most important thing is to improve the base rate (the prior probability Pr(True)), and
that requires thinking, not testing.79
For example, if the same hypothesis was independently studied with the same
statistical power (95%) and a false alarm rate (5%), the base rate (the prior probability) could be specified as the
former posterior probability Pr(True) ~ 0.16. In that case, the second significant finding would result in the new
~ 78% chance that the hypothesis was true, which is in concordance with the diachronic inference i.e., the initial
beliefs are being updated by incremental knowledge.
4.1 Determination of lansoprazole, 5-hydroxylansoprazole, and lansoprazole sulfone
in human plasma for CYP2C19 and CYP3A4 phenotyping
LC–MS is an ideal tool for metabolite prediction, detection, identification, and quantitation in phenotyping studies.
Typically, the administered parent compound is metabolized to major and minor metabolites at a given rate, which is
essentially dependent on the activity of the cytochrome P450 (CYP) system – the subject’s phenotype. It has shown
advantageous to determine the CYP activity before drug administration to adjust the pharmacotherapy accordingly.
The CYP metabolic activity is assessed by a specific substrate selectively biotransformed by distinct CYP isoenzymes.
The reaction rate or the metabolic ratio could then determine the metabolic phenotype. Lansoprazole (LAN) is used
as a proton pump inhibitor in duodenal ulcers, reflux esophagitis, Zollinger-Ellison syndrome, H. pylori eradication,
and gastroesophageal reflux disease in patients requiring therapy. Metabolites of LAN, 5-hydroxylansoprazole
(OHL) and lansoprazole sulfone (LS) were determined by LC–UV and LC–MS to assess the CYP2C19 and CYP3A4
metabolic activity. Although LC–MS provided low LOD, the linear dynamic range was insufficient to quantify the
parent compound (LAN) for the metabolic ratio assessment. Combining both detection techniques can provide
overlap in linear dynamic ranges of both methods and thus increase the reliability of the entire determination. In
conclusion, CYP2C19 and CYP3A4 phenotyping using combined LC–UV and LC–MS allowed reliable identification
of poor metabolizers of either CYP isoform.
KOSTOLANSKA, Katarina, Ondrej PES, Ondrej ZENDULKA, Jan MACHAL and Jan JURICA. Determination
88
of lansoprazole, 5-hydroxylansoprazole, and lansoprazole sulfone in human plasma for CYP2C19 and CYP3A4
phenotyping. Chemical Papers. 2019, 73(12), 2955–2963. ISSN 2585-7290. doi:10.1007/s11696-019-00682-4
Document Type: Article, IF = 1.680; JCR Category + Category Quartile: CHEMISTRY, MULTIDISCIPLINARY
Q3; AIS = 0.222
Author’s contribution: 20 %
89
Vol.:(0123456789)1 3
Chemical Papers (2019) 73:2955–2963
https://doi.org/10.1007/s11696-019-00682-4
ORIGINAL PAPER
Determination of lansoprazole, 5‑hydroxylansoprazole,
and lansoprazole sulfone in human plasma for CYP2C19 and CYP3A4
phenotyping
Katarína Kostolanská1
 · Ondřej Peš1
 · Ondřej Zendulka2
 · Jan Máchal3
 · Jan Juřica2,4,5
 
Received: 11 September 2018 / Accepted: 4 January 2019 / Published online: 18 January 2019
© Institute of Chemistry, Slovak Academy of Sciences 2019
Abstract
A simple and sensitive liquid chromatography method with absorbance or mass spectrometry detection was developed and
validated for the determination of lansoprazole, lansoprazole sulfone, and 5-hydroxylansorpazole in human serum with omeprazole
as an internal standard. Analytes were, after an extraction step with a mixture of isopropyl alcohol: dichloromethane
(2:8, v/v), separated in a gradient elution (acetonitrile: 20 mM ammonium formate) and detected at 280 and 300 nm or by
monitoring selective reactions in a triple quadrupole mass analyzer. The limits of detection were, for all analytes, below 2 ng/
mL and 3 pg/mL in the LC UV and LC MS method, respectively. The combined linear dynamic range exceeded 5 orders
of magnitude. The method was successfully applied in the assessment of CYP2C19 and CYP3A4 metabolic phenotypes in
ST-elevated myocardial infarction patients receiving novel anticoagulant treatment.
Keywords  Cytochrome P450 · Phenotype · Lansoprazole · 5-Hydroxylansoprazole · Lansoprazole sulfone · Liquid
chromatography · Mass spectrometry · Metabolites
Introduction
Cytochromes P450 (CYP) plays a pivot role in the metabolism
of most clinically used drugs and affects effective
drug concentrations in plasma. In some drugs, it is useful
to assess the CYP metabolic activity prior to or during the
pharmacotherapy to adjust the individual dosage according
to the patient’s phenotype. To determine the CYP metabolic
activity, a specific substrate, also known as a marker
of metabolic activity, which is metabolized selectively by a
distinct CYP isoenzyme, is used. The velocity of the reaction
or the ratio of the substrate to its metabolite concentrations
in plasma (metabolic ratio, MR) is then defining the metabolic
phenotype. (Fuhr and Rost 1994; Turjap et al. 2016).
With regard to the phenotype (or MR), metabolizers can be
classified as up to 4 different classes—poor metabolizers
(PM), intermediate metabolizers (IM), extensive metabolizers
(EM) and ultrarapid metabolizers (Zanger et al. 2004).
Poor metabolizers are prone to drug overdose, toxicity and
adverse drug reactions of CYP substrates and thus, knowledge
of metabolic phenotype is useful for the drugs with
low therapeutic index. Lansoprazole (LAN) belongs to
proton pump inhibitors used in the treatment of duodenal
ulcers, reflux esophagitis, Zollinger–Ellison syndrome, H.
pylori eradication, gastroesophageal reflux disease, or non-
steroidal antiinflammatory drugs-associated benign ulcers
in patients requiring therapy (MHRA 2017). It is administered
at the dose of 15 or 30 mg orally as a racemic mixture
of R- and S-isomers. LAN is rapidly absorbed from
This work was presented at the 16th Czech—Slovak Spectroscopic
Conference held in Luhačovice, Czech Republic on May 27–31,
2018.
*	 Jan Juřica
	jurica@med.muni.cz
1
	 Department of Biochemistry, Faculty of Medicine, Masaryk
University, Kamenice 5, 62500 Brno, Czech Republic
2
	 Department of Pharmacology, Faculty of Medicine, Masaryk
University, Kamenice 5, 62500 Brno, Czech Republic
3
	 Department of Pathological Physiology, Faculty
of Medicine, Masaryk University, Kamenice 5, 62500 Brno,
Czech Republic
4
	 Department of Human Pharmacology and Toxicology,
University of Veterinary and Pharmaceutical Sciences Brno,
Palackeho tr. 1946/1, 61242 Brno, Czech Republic
5
	 Department of Biochemistry, Faculty of Science, Masaryk
University, Kamenice 5, 62500 Brno, Czech Republic
2956	 Chemical Papers (2019) 73:2955–2963
1 3
the gastrointestinal tract after oral administration, reaching
maximal plasma concentrations in 1.7 h. The therapeutic
effect is observed within 1–3 h after oral dosing with the bioavailability
of about 80%. LAN is extensively (97%) bound
on plasma proteins (Dynamed American Society of Health
System Pharmacists 2018). Due to the irreversible inhibition
of ­H+
/K+
ATPase in the parietal cells, the effect persists
for 2–4 days. Lansoprazole is metabolized via CYP3A4
and CYP2C19 enzymes. CYP2C19 is responsible for lowcapacity
but highly selective hydroxylation and CYP3A4 for
high-capacity sulfoxidation. The velocity of both reactions is
greater from its S-enantiomer than from R-enantiomer (Kim
et al. 2003). There have been some mentions to use LAN as a
measure of CYP2C19 (Gumus et al. 2012; Sohn et al. 1997;
Xu et al. 2010; Zhang et al. 2011) and CYP3A4 (Yanagida
et al. 2009) metabolic activity, although no clear MR cutoff
value for distinguishing extensive and poor metabolizers
of CYP2C19 or CYP3A4 was established. However, poor
metabolizers had significantly higher molar ratio of LAN to
5-hydroxylansoprazole (OHL) than extensive metabolizers
(Gumus et al. 2012) and, based on in vitro data, LAN was
also suggested as a probe substrate for CYP3A4 phenotyping.(Yanagida
et al. 2009).
Essentially, LAN may be administered for determination
of both CYP2C19 and CYP3A4 metabolic activity in a single,
low dose. To distinguish between a poor and extensive
metabolizer, a visual inspection of MR histograms, probit
plot analysis, or receiver operator characteristics analysis is
performed (Jackson et al. 1989; Swar et al. 2016). Omeprazole
(OME) is used as CYP-specific probe for CYP2C19
phenotyping as a single probe substrate as well as a part
of several “cocktail” approaches for phenotyping CYP
enzymes. For this purpose, several different cocktails of
markers have been used (Asimus et al. 2007; Dierks et al.
2001; Sharma et al. 2004). Due to a variety of clinical reasons,
LAN is preferred over OME in many countries and
therefore, LAN may be a useful alternative for CYP phenotyping
without the necessity to use another drug probe
substrate.
The aim of this work was to develop a simple and sensitive
method for the determination of LAN, lansoprazole sulfone
(LS) and OHL using absorbance or mass spectrometry
(MS) detection. The validated method was then applied to
real plasma samples of ST-elevated myocardial infarction
(STEMI) patients receiving medication metabolized via
CYP2C19 or CYP3A4.
LAN can be determined by a variety of analytical methods
including chromatography, capillary electrophoresis, or
polarography (Dogrukol-Ak et al. 2001; Elkady et al. 2018;
Chung et al. 2017; Nassar et al. 2017; Uno et al. 2005b;
Yeniceli et al. 2004). Most of the methods deal with the
determination of LAN or impurities in pharmacy products,
such as capsules and tablets (Belal et al. 2004; Dogrukol-Ak
et al. 2001; Chung et al. 2017; Nassar et al. 2017; Yeniceli
et al. 2004). MS detection has been used to report LAN in
biological fluids (Bharathi et al. 2009; Elkady et al. 2018;
Ganesh et al. 2011; Luo et al. 2018; Oliveira et al. 2003;
Song et al. 2009; Sun et al. 2015; Wang et al. 2015; Wu
et al. 2008; Zhang et al. 2014). Some LC–UV methods for
the determination of solely LAN were also published (Yang
et al. 2011). (Bharathi et al. 2009). However, a few publications
are detecting LAN and its CYP-specific metabolites
simultaneously (Aoki et al. 1991; Gumus et al. 2012; Song
et al. 2008; Uno et al. 2005b).
While LC UV methods have a limit of quantification
(LOQ) in the range of 2–10 ng/mL for all analytes (Aoki
et al. 1991; Gumus et al. 2012; Landes et al. 1992; Uno et al.
2005a; Yanagida et al. 2009), which does not have to be sufficient
to evaluate a poor metabolizer profile, high-sensitive
LC MS methods usually suffer from a shifted working range,
which does not have to cover the levels of the drug and its
metabolites in a single, preferably linear, calibration curve.
A simple LC method employing either an absorbance or
MS detector might be used to provide reliable levels of LAN
and its metabolites over a broad dynamic range originating
from the overlap of dynamic ranges of both detection techniques.
It is a common situation in clinical practice when
a drug administered at rather high doses (~ 1–100 mg) is
metabolized to rather low-concentration products demanding
more sensitive methods to quantify them reliably. However,
the plasma level of the drug itself is usually extrapolated,
estimated, or even ignored, as it was found outside the calibration
range, or suffered from the detector saturation. The
aim of the presented approach is to simultaneously determine
plasma concentrations of LAN, LS, and OHL in a very
broad range of analyte concentrations. Another step towards
convenient determination of CYP2C19 and CYP3A4 metabolic
activity from a single dose of LAN would be to arrange
both detectors in a series.
Experimental
Chemicals
LAN, OHL, LS and internal standard OME were purchased
from Santa Cruz Biotechnology (Dallas, Texas, USA). Acetonitrile
(ACN), methanol (MeOH), acetic acid, sodium
hydroxide, and ammonium hydroxide were purchased from
Sigma-Aldrich (Prague, Czech Republic). Isopropanol and
dichloromethane were purchased from Merck (Darmstadt,
Germany). All reagents used were of an analytical grade
except for methanol and acetonitrile, which were of an LC
MS grade. All water used was of an ultra-pure grade supplied
by an in-house Milli-Q system (Millipore, MA, USA).
2957Chemical Papers (2019) 73:2955–2963	
1 3
Standard stock solutions, calibration standards,
and quality controls
LAN, LS, OHL, and OME were dissolved in ACN to prepare
stock solutions (2.20, 1.30, 1.20, and 2.20 mg/mL, respectively).
The stock solutions were stored at − 80 °C. Working
standard solutions were made by diluting the stock solutions
with water–acetonitrile (1:1, v/v). A working internal
standard solution was prepared at a final concentration of
3.0 µg/mL for LC UV and 150 ng/mL for LC MS method.
The calibration standards (Table 1) were prepared by spiking
pooled blank human plasma obtained from 8 healthy
volunteers. The serum was verified to be free of LAN, LS,
and OHL by using LC MS.
Quality control (QC) samples for both LC UV and LC
MS were prepared in three levels (low, medium, and high)
and are given in Table 1. The final concentration of OME
was 300 ng/mL in the LC UV method and 15.0 ng/mL in
the LC MS method.
LC UV
The liquid chromatographic system Dionex UltiMate
3000RS (Thermo Fisher Scientific, CA, USA) equipped
with a binary high-pressure gradient pump, an autosampler,
a heated column compartment, and a DAD detector
was employed in the LC–UV method. Data from the detector
were collected and analyzed using Chromeleon™ 6.8
software (Thermo Fisher Scientific, CA, USA). An Ascentis
Express C18 reversed-phase chromatographic column
(100 × 3.0 mm I.D.; 2.7 µm) with ­Ascentis®
Express C18
(5 mm × 3 mm; 2.7 µm) security guard (both Sigma-Aldrich,
Czech Republic) were used for the gradient separation. The
mobile phase consisted of (A) 20 mM acetic acid adjusted
to pH 4.5 with 25% ammonium hydroxide solution and (B)
acetonitrile. The LC UV initial composition of 75% A and
25% B was linearly increased to 60% A and 40% B over the
next 6 min. It was held for 1.5 min, followed by equilibration
under the initial conditions for another 2.5 min. The
flow rate was 0.6 mL/min and the column was operated at
40 ± 0.1 °C. The absorbance of the eluent was monitored at
300 nm (OHL, OME) and 280 nm (LAN, LS).
LC MS
The LC MS method was developed using a Dionex Ultimate
3000RS (ThermoScientific, CA, USA) module, equipped
with a binary high-pressure gradient pump, an autosampler,
and a column oven. The same column, pre-column, and
mobile phase were used as in the LC UV method.
The LC MS initial composition of 80% A and 20% B was
linearly increased to 80% A and 20% B over the next 6 min.
It was held for 0.5 min, followed by equilibration under the
initial conditions for another 1.5 min. The flow rate was
0.6 mL/min and the column was operated at 23 ± 0.1 °C.
The parameters were slightly modified to lower the retention
times as the MS detection usually does not demand chromatographically
resolved peaks all the time.
A Bruker EVOQ Qube triple quadrupole mass spectrometer
(Germany) operated in the positive heated electrospray
ionization mode was used in LC MS. The parameters
of operation were set as the following: Spray voltage
+ 4000 V, cone temperature 350 °C, cone gas flow 20 psi,
heated probe temperature 300 °C, probe gas flow 40 psi
(nitrogen), and nebulizer gas flow 45 psi (nitrogen). Argon
was used as collision gas. All other parameters were set
at default values provided by the manufacturer. The flow
from LC was diverted to waste in the range of 0–2 min and
5.5–8 min. Target compounds were scanned in the selected
reaction monitoring mode (SRM) as fragments of [M + H]+
ions: LAN 370.1–252.0 (8 eV) and 370.1–119.1 (13 eV);
LS 386.1–119.1 (26 eV) and 386.1–322.1 (18 eV); OHL
386.1–251.9 (10  eV) and 386.1–135.0 (26  eV); OME
346.0–198.0 (8 eV) and 346.0–151.1 (19 eV). Dwell time
for each transition was 50 ms.
Patients’samples
Real plasma samples were obtained from 101 STEMI
patients treated by percutaneous coronary intervention
and subsequently with novel anticoagulants prasugrel or
ticagrelor; both have been ascertained as CYP2C19 and/
or CYP3A4 substrates. The patients received the dose of
30 mg of lansoprazole and the blood samples were drawn
3 h after the administration. The study was approved by the
Table 1  Concentration levels of
the calibration standards for the
LC UV and LC MS method
Level 1 2 3 4 5 6
QC Low Medium High
LC UV (ng/mL) OHL 34.4 68.8 138 275 550 1100
LAN 68.8 138 275 550 1100 2200
LS 40.6 81.3 163 325 650 1300
LC MS (pg/mL) OHL 22.0 55.0 550 1100 2200 11,000
LAN 22.0 55.0 550 1100 2200 11,000
LS 26.0 65.0 650 1300 2600 13,000
2958	 Chemical Papers (2019) 73:2955–2963
1 3
local Ethical committee (an Ethical committee of St. Anne’s
faculty hospital).
Sample preparation
Aliquots (250 μL) of human blank plasma sample or
spiked human plasma were transferred into a screwcapped
glass tubes. 25 μL of the internal standard solution,
50 μL of 0.1 M NaOH and 5 mL of extraction mixture
(isopropanol:dichloromethane 2:8, v/v) were added and the
tubes were vortexed 10 min at 2000 rpm at the horizontal
vortex and centrifuged for 5 min at 4000 g at room temperature.
4 mL of organic layer was decanted into conical
glass centrifuge tubes and the aqueous phase discarded. The
organic phase in the conical tube was evaporated at 36 °C
under a gentle stream of nitrogen. The residues, after evaporation,
were redissolved in either 250 μL (LC UV) or 1000
µL (LC MS) of 20% ACN and 20 μL (LC UV) or 10 µL (LC
MS) was injected onto an analytical column.
Method validation
The method was validated for its selectivity, linearity, limit
of detection, limit of quantitation, accuracy, precision,
matrix effects, extraction recovery, and stability according
to the principles of the relevant Food and Drug Administration
guidance (FDA 2018).
LOD, LOQ, and linearity
Calibration curves were constructed using six non-zero levels.
Calibration standards were prepared by spiking 250 μL
blank plasma with 12.5–25 μL of standard stock solutions
to prepare calibration standards as indicated in Table 1. The
analyte to internal standard peak area ratios was plotted
against the corresponding concentrations. The calibration
curve was estimated by the least-squares regression procedure.
LOD and LOQ were determined for each analyte
by analyzing the lowest calibration point of calibration and
calculating a theoretical signal–noise ratio of 3:1 for LOD
and 10:1 for LOQ. The values were verified by measuring
samples containing each analyte using the calculated
concentrations.
Accuracy and precision
To determine the intra- and inter-day precision and accuracy
of the assay, five replicates of each analyte in blank plasma
at three concentration levels (low, medium, high) within the
calibration range were analyzed for five consecutive days.
Precision was calculated as intra- and inter-day relative
standard deviation (RSD) values and accuracy was expressed
as a mean relative error [(measured concentration − nominal
concentration) × 100/(nominal concentration)].
Matrix effect and extraction recovery
Matrix effect was measured by comparing the calculated
concentrations of LAN, LS, and OHL in spiked plasma samples
after extraction (A) to the spiked solvent at the same
concentration (B) (Matrix effect = A/B × 100). Recovery
was measured by comparing the calculated concentrations
of LAN, LS, and OHL from spiked plasma samples before
extraction (C) to concentrations obtained when spiked after
extraction (D) (Recovery = C/D × 100). Five replicates at
low, medium, and high concentration levels were used for
both parameters.
Stability of extracted samples
The stability of the samples extracted at low, medium, and
high concentration levels samples reconstituted in 20% ACN
was evaluated by LC UV as a percentage of the zero-day
concentration after 7 days of storing at 4 °C in the dark. The
stability of the dried extracts was evaluated by preparation
of seven replicates at the same concentration levels, which
were reconstituted directly before an LC UV analysis.
Results and discussion
LC UV
A representative LC UV chromatogram can be seen in
Fig. 1. Peaks were chromatographically resolved (R > 2)
with retention times under 7 min. Base peak widths were
about 0.2 min. The LC UV method, despite its simplicity,
can be straightforwardly applied to determine a ratio of a
concentration of LAN metabolites over that of LAN. When
the concentration of metabolites was below of LOQ, the LC
MS method was employed.
LC MS
Representative LC MS chromatograms are shown in Fig. 2.
Generally, the LC MS method was used in cases when the
OHL and LS concentrations were not approachable by LC
UV. On the other hand, LC UV was used to determine the
LAN concentration in most cases as the LC MS dynamic
range could not have covered the levels in the vast majority
of real samples. As both methods were spatially and temporally
uncoupled, slightly different conditions could have
been applied. Since MS usually does not require the peaks
to be chromatographically resolved, a modification of the
2959Chemical Papers (2019) 73:2955–2963	
1 3
separation gradient and temperature was done to favor faster
retention times with base peak widths of around 0.1 min.
LOD, LOQ, and linearity
The calibration curve in the LC UV method was linear in
the employed calibration range of each analyte. The coefficient
of determination (R2
) was higher than 0.999 for all
three analytes.
The LC MS method operated primarily in a range of
sub-ng/mL concentrations and was linear in the range of
22–11,000 pg/mL with R2
 > 0.989. The LOD and LOQ
obtained by the LC UV method were 0.85 and 2.8 ng/mL
for OHL, 1.5 and 5.0 ng/mL for LAN, and 1.7 and 5.8 ng/mL
for LS, respectively. These were comparable with formerly
published LC UV methods. The LC MS provided LOD and
LOQ to be 1.2 and 4.1 pg/mL for OHL, 0.73 and 2.5 pg/mL
for LAN, and 2.2 and 7.1 pg/ml for LS, respectively, which
allowed to phenotype of poor metabolizers. To the best of
our knowledge, no sub-ng/mL detection limits have been
published to date for any of the compounds.
Accuracy and precision
The intra- and inter-day variations for the measured analytes
in the LC UV method were lower than 4.23% and 14.6%,
respectively. The LC MS method provided intra- and interday
variations lower than 7.82% and 14.2%, respectively
(Tables 2, 3). A series of five replicates with concentrations
overlapping dynamic ranges of both methods was calculated
using both calibration curves to verify the unbiased accuracy
(data not shown).
Matrix effect and extraction recovery
Extraction recovery was calculated using the LC UV method
only and the results for all analytes are summarized in
Table 4. The matrix effect was calculated only by the LC MS
method and was not observed (≥ 75% and ≤ 125%; Table 4).
Stability of extracted samples
A substantial change (> 20%) in the concentration of all analytes
was verified by LC UV three days after extraction and
reconstitution when stored at 4 °C in dark (Table 5). Since
all compounds showed poor stability, it was not continued
to obtain a 7-day profile. Instead, the dried sample extracts,
when stored at 4 °C in dark and reconstituted prior to the
N-th day analysis, were evaluated in the same manner. No
significant change (< 15%) in the concentration of the analytes
was observed during the consecutive 7 days (Table 5).
Although it involved a combined error originating from the
preparation of replicates, it allowed for bulk processing of
a great number of patient samples without the necessity to
analyze them immediately after extraction.
Experimental data obtained from the experimental
samples
The LC UV and LC MS methods were successfully
applied in the evaluation of CYP2C19 and CYP3A4 metabolic
phenotype of 101 ST-elevated myocardial infarction
patients receiving novel anticoagulant treatment (Table 6).
The patients were phenotyped with the use of a single dose
lansoprazole (30 mg) and 3-h-postdose levels of LAN,
Fig. 1  LC UV chromatograms
of a blank plasma sample spiked
with OHL (2.9 min; 68.8 ng/
mL), LAN (5.1 min; 138 ng/
mL), LS (5.9 min; 81.3 ng/mL),
and IS OME (3.4 min; 150 ng/
mL). Detection at 302 nm
(OHL, OME—solid line) and
282 nm (LAN, LS—dashed
line) were used in calculations
2960	 Chemical Papers (2019) 73:2955–2963
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Fig. 2  LC MS chromatograms of a a blank plasma sample, b a blank
plasma sample spiked with OHL (22 pg/mL), LAN (22 pg/mL), and
LS (26 pg/mL), and c a real plasma sample from a poor metabolizer.
Two SRM transitions for OHL (tR = 2.6 min), LAN (tR = 3.4 min), LS
(tR = 3.8 min), and IS OME (tR = 2.7 min) are represented by the top
to bottom frames, respectively
2961Chemical Papers (2019) 73:2955–2963	
1 3
OHL and LS were determined. 12 out of 101 patients had
levels of LS lower than 3 ng/mL, which are below the
LOQ of the LC UV method and thus, these samples were
subjected to the LC MS analysis. On the other hand, 76 out
of 101 patients had levels of LAN greater than 100 ng/mL,
which are greater than the highest point of calibration in
the LC–MS method. Thus, poor metabolizers need to be
phenotypized with the use of LC–MS method and majority
of the samples should be measured with LC–UV method,
so as to avoid imprecision at higher concentrations of LAN
by LC–MS method. The combination of methods is useful
for the whole spectrum of metabolizers.
In the real patient samples, a clear cut-off in the histogram
distribution of the LAN/LS molar concentration ratio
was observed (a gap in the continuous distribution between
20 and 23). Ten subjects had LAN/LS greater than 23 and
were, thus, suspected as poor metabolizers of CYP3A4.
Accordingly, another two patients were suspected as poor
metabolizers of CYP2C19 due to a clear cut-off observed
in the histogram of LAN/OHL molar concentration ratio; a
Table 2  Intra- and inter-day
accuracy and precision of the
LC UV method
Nominal concentration
(ng/mL)
Intra-day (N = 5) Inter-day (N = 5)
Accuracy (%) Precision RSD (%) Accuracy (%) Precision
RSD (%)
OHL 1100 0.89 0.102 − 5.08 14.6
138 3.40 0.821 − 5.44 6.71
34.4 13.8 3.22 3.35 7.92
LAN 2200 1.50 0.121 − 7.73 7.37
275 6.17 1.18 − 5.94 10.9
68.8 23.1 3.40 16.2 5.19
LS 1300 0.587 0.0989 2.82 6.93
163 3.86 1.22 6.37 7.40
40.6 8.35 4.23 − 0.831 9.00
Table 3  Intra- and inter-day
precision and accuracy of the
LC MS method
Nominal concentration
(pg/mL)
Intra-day (N = 5) Inter-day (N = 5)
Accuracy (%) Precision RSD
(%)
Accuracy (%) Precision
RSD (%)
OHL 11,000 1.21 5.43 − 3.71 8.43
550 − 2.31 5.30 7.13 8.30
5.50 4.30 2.83 − 12.7 11.8
LAN 11,000 − 2.50 2.33 − 8.45 8.88
550 4.34 3.17 7.59 11.0
5.50 − 6.24 6.56 − 5.98 14.2
LS 13,000 1.78 4.55 4.65 10.9
650 11.9 7.82 − 6.37 9.50
6.50 − 13.5 6.88 15.7 10.1
Table 4  Extraction recovery
and matrix effect (N = 5)
OHL LAN LS OHL LAN LS
Recovery (%) RSD (%)
 Low 95.6 97.6 99.9  Low 13.7 11.7 10.9
 Medium 88.3 89.8 92.1  Medium 11.8 9.93 10.3
 High 82.2 87.0 88.5  High 9.02 12.8 9.65
Matrix effect (%) RSD (%)
 Low 115 104 118  Low 9.89 4.27 12.3
 Medium 111 96.7 98.2  Medium 11.2 10.3 7.65
 High 104 95.9 117  High 14.0 9.81 8.88
2962	 Chemical Papers (2019) 73:2955–2963
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gap started with 52 and the two subjects had MR of 85.8 and
90.5. Hence, both LC UV and LC MS methods were necessary
to reliably identify non-compliant patients (N = 4), who
could not have been identified without using the combined
dynamic range of both methods. It is worth noting that the
poor metabolizers often suffer from adverse drug reactions
and their phenotype should ideally be known beforehand.
Conclusion
To determine the CYP2C19 and CYP3A4 metabolic phenotype,
a simple and rapid method relying on either UV or
MS detection was developed and validated. The idea of an
involvement of two detectors allowed for an increased linear
dynamic range, which has proved essential for accurate
quantitation of the ratio of a low metabolite (LS, OHL) over
a high LAN concentration. The LOD and LOQ obtained by
LC MS considerably (two orders of magnitude) exceeded
the ones published so far. Utilization of both methods provided
a reliable identification of non-compliant patients and
poor metabolizers of either CYP isoform. A single system
equipped with both detectors could be employed as an ultimate
platform in routine CYP2C19 and CYP3A4 metabolic
phenotype determination from a single dose of LAN.
Acknowledgements  This work was supported by funds from the
Faculty of Medicine MU to a junior researcher Ondřej Peš and by
the Specific University Research Grant (MUNI/A/1132/2017,
MUNI/A/0910/2017) provided by the Ministry of Education, Youth
and Sports of the Czech Republic and by the project no. LQ1605 from
the National Program of Sustainability II (MEYS CR).
Compliance with ethical standards 
Conflict of interest  Authors declare there are no potential conflicts of
interest to disclose.
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LAN 94.4 (106) 81.6 (86.4) 20.5 (114) ND (94.6) NA (111) NA (102) NA (103)
LS 99.1 (102) 167 (89.1) 176 (94.1) 202 (114) NA (105) NA (102) NA (98.2)
High OHL 99.2 (101) 73.1 (105) 48.0 (92.0) 34.3 (104) NA (98.9) NA (96.7) NA (98.8)
LAN 98.5 (113) 63.7 (93.1) 33.1 (91.4) 15.5 (103) NA (101) NA (105) NA (100)
LS 99.5 (107) 93.3 (90.8) 115 (89.2) 144 (115) NA (103) NA (104) NA (96.5)
Table 6  Distribution of measured concentration levels of OHL, LAN,
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OHL LAN LS
c < 3 ng/mL 6 4 12
c > 100 ng/mL 1 76 19
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4.2 Online solid-phase extraction liquid chromatography-mass spectrometry of hair
cortisol using a surrogate analyte
Hair cortisol has been widely analyzed since its introduction in 2000.80,81
Commonly used methods include
immunoassays and LC–MS. The specificity of immunoassays is somewhat questionable,82
and the LC–MS approaches
asses the baseline levels by the most distal hair segment pretending it contains no endogenous cortisol. Binz et al.
first proved that the most distal segment may contain as much cortisol as anywhere along the hair strand,83
and
suggested a more reliable approach of a surrogate analyte in the authentic matrix. We decided to follow the same
approach to cope with the bias at baseline levels of blank samples. Even though hair cortisol analysis has been
performed for twenty years, the physiological ranges were scarcely been agreed on. The lower limit (~ 1 pg/mg) of
the reference interval was established consistently; however, the upper limit dramatically differed due to extreme
intraindividual variability. We have found out that the hair cortisol concentration (HCC) thresholds were not sharp;
quite oppositely, HCC might be classified along with the following guidelines:
1. Healthy individuals > 1 pg/mg.
2. Cortisol metabolism impairment > 100 pg/mg.
3. Topically applied corticosteroids > 1000 pg/mg.
Unsharp borders exist due to stress contributions, the onset of impairment, and other factors that could not have
been addressed solely by HCC determination.
Stress, which many scientists try to objectively quantify using HCC, could be viewed as a confounder (or a latent
variable), which could not be directly conditioned on. In other words, it is impossible to divide subjects into stressed
and non-stressed groups. Thus, the contribution of physical stress to HCC could be relatively high per hair segment.
On the other hand, hair cortisol analysis could be especially helpful in corticosteroid substitution therapy monitoring,
establishing ectopic ACTH production, diagnosing a cyclic version of Cushing’s syndrome, or psychiatric disorders.
The longitudinal measurement using hair segmentation could provide HCC values unaffected by diurnal rhythms
and momentary cortisol levels in the organism. Topically applied corticosteroids were easily distinguishable as HCC
reached values over 1000 pg/mg, which could have not originated from steroid therapy or impairment. In a linkup
article, plasma urine and hair cortisol levels of 22 patients subjected to substitution therapies were longitudinally
measured between Q2/2018 and Q4/2019. Hierarchical Bayesian modeling was applied to compare the effectiveness
of the therapy. Individual hair cortisol levels better reflected the optimal therapeutic state than routinely measured
plasma and urine cortisol. The discontinuation of the substitution therapy e.g., after adrenalectomy, could have
been suggested by HCC monitored retroactively via a segmentation process.84
KOSTOLANSKA, Katarina, Lucie NOVOTNA, Eva TABORSKA a Ondrej PES (corresponding author). Online
solid-phase extraction liquid chromatography-mass spectrometry of hair cortisol using a surrogate analyte. Chemical
Papers. 2019, 73(1), 151–158. ISSN 2585-7290. doi:10.1007/s11696-018-0560-1 Document Type: Article, IF = 1.680;
JCR Category + Category Quartile: CHEMISTRY, MULTIDISCIPLINARY Q3; AIS = 0.222
Author’s contribution: 50 %
99
Vol.:(0123456789)1 3
Chemical Papers (2019) 73:151–158
https://doi.org/10.1007/s11696-018-0560-1
ORIGINAL PAPER
Online solid‑phase extraction liquid chromatography–mass
spectrometry of hair cortisol using a surrogate analyte
Katarina Kostolanska1
 · Lucie Novotna1
 · Eva Taborska1
 · Ondrej Pes1
 
Received: 17 April 2018 / Accepted: 21 July 2018 / Published online: 25 July 2018
© Institute of Chemistry, Slovak Academy of Sciences 2018
Abstract
Online SPE LC–MS with 13
C3-cortisol as a surrogate analyte was developed, validated and applied for obtaining hair cortisol
levels of 114 healthy volunteers. The first 3 cm from a posterior vertex position were analyzed as three individual segments
resulting in 310 hair cortisol levels, which were used, after log transformation, to predict a range of values observed in future.
The median value of hair cortisol was 4.76 pg/mg. The employed method allowed simple processing, high throughput and
may be readily expanded to analyze additional steroid compounds in hair.
Keywords  Endogenous cortisol · Long-term hair concentration · Surrogate analyte · LC–MS · Online SPE
Introduction
Cortisol is a glucocorticoid hormone with major effect
upon a range of physiological homeostatic mechanisms. It
plays an important role in stressful situations when stressors
activate the hypothalamic–pituitary–adrenocortical (HPA)
system.
Cortisol has been routinely analyzed in the laboratory
from saliva, serum/plasma, and/or urine. However, the
HPA axis represents a highly dynamic system influenced by
several factors and expose circadian rhythmicity. The most
widely analyzed saliva or plasma reflect only acutely circulating
cortisol levels, urine provides usually 24-h assessments
of levels through the day. Measurements in the abovementioned
body fluids are limited in the temporal range of
assessment. Cirimele et al. were first who detected cortisol
and other glucocorticoids in hair (Cirimele et al. 2000). Cortisol
is being constantly incorporated into a growing hair
shaft and, accordingly, can allow the unique possibility to
assess the long-term systemic levels and obtain a retrospective
index of long-term hormone secretion. Based on an
average hair growth rate of 1 cm per month (Wennig 2000),
it is possible to distribute the hair along the length of individual
segments and determine the concentration of cortisol
reflecting secretion in the monitored period. But due to a
rapid decrease of cortisol in hair, the measure may be limited
to the past 6 months (Kirschbaum et al. 2009).
Concerning the diagnostic value, differences in cortisol
levels can be used to evaluate disturbances of the HPA axis.
It may serve in the diagnosis of Cushing’s syndrome disease
but it may also be useful as a marker of successful therapy.
Changes in cortisol levels have been observed in mental
diseases, e.g., depression, general anxiety disorder or posttraumatic
stress disorder as well as physical diseases where
it could serve as a marker for diagnosis or risk stratification
(Russell et al. 2012).
Commonly used methods for a hair steroid analysis are
immunoassays; principally enzyme-linked immunosorbent
assays (ELISA). The major disadvantage of immunoassays
is low specificity. Given to high structural similarity
of steroid compounds, there is a risk of cross-reactions
inside the immuno-complex that may overestimate the target
concentration. In this context, cortisone especially has
been observed as a major source of interference in immunoassays
research of salivary cortisol (Miller et al. 2013).
These approaches are often unsuitable for clinical applications
that require a low detection limit (Taieb et al. 2002).
Much higher specificity is provided by liquid chromatography–mass
spectrometry (LC–MS). Compared to immunoassays
with specificity to only a single analyte, LC–MS
proves to measure a panel of multiple steroid hormones
simultaneously, currently 28 steroids (Dong et al. 2017).
LC–MS offers other advantages, such as high sensitivity,
*	 Ondrej Pes
	ondramayl@gmail.com
1
	 Department of Biochemistry, Faculty of Medicine, Masaryk
University, Kamenice 5, 62500 Brno, Czech Republic
152	 Chemical Papers (2019) 73:151–158
1 3
wide dynamic range, high throughput, and an option for
automated sample preparation (Gao et al. 2016) Current
and potential future applications of hair cortisol analysis
have been summarized in a review by Wester and Rossum
(Wester and van Rossum 2015), however; the exact physiological
range of hair cortisol concentration has not yet
been established.
In general, cortisol content in hair is very low. Raul et al.
reported cortisol concentration ranged from 5.2 to 91 pg/
mg with a mean value of 19 pg/mg. LC–MS/MS with electrospray
ionization (ESI) in the positive mode was used
for quantification; limit of detection (LOD) and limit of
quantitation (LOQ) was 1 and 5 pg/mg, respectively (Raul
et al. 2004). Another study reported a median cortisol level
of 8.8 pg/mg for elementary school girls with a range of
5–1330.5 pg/mg (Vanaelst et al. 2013). Though, only 39 of
168 samples were higher than LOQ (5 pg/mg). Hair cortisol
levels 12.7 ± 14 pg/mg in hair samples of healthy volunteers
with an age range of 4–63 years were obtained by LC–MS/
MS/MS (Quinete et al. 2015). The broad range of physiological
values is likely affected by the fact that the mechanism
of cortisol incorporation into the hair shaft has yet not
been fully revealed.
As cortisol is normally present in hair, it is difficult to
achieve a high throughput methodology using an authentic
matrix calibration curve. A method of the standard addition
should be used instead (Tsakelidou et al. 2017). Some
authors have considered a distal segment of hair as an analyte-free
matrix even that ascertained cortisol concentration
(1.2 pg/mg) was found in the 4 cm segment of the distal end
of 20 cm long human hair (Binz et al. 2016). In such cases,
the matrix varies from sample to sample and a lack of a reference
material made all the validation studies ambiguous.
Cortisol added to the solution of “blank hair” might behave
differently and may significantly contribute to the high variation
of hair cortisol, especially at low concentration levels.
Hence, spiking with known amounts of reference standards
should be avoided if the method of standard addition is not
used for each sample. Alternatively, the results should be
associated with the synthetic matrix only (Doroschuk et al.
2016).
The only approach using an authentic matrix and a surrogate
analyte was demonstrated by Binz et al. (Binz et al.
2016) even as using a surrogate analyte might prove very
advantageous in the analysis of endogenous compounds in
challenging matrices and allows for facile method development
when there is no analyte-free matrix available. The
response of the isotopically labeled 13
C3-cortisol as a surrogate
analyte was consistent and nearly identical with that
of cortisol. Moreover, a method of a calibration curve in
the authentic matrix could have been employed without the
risk of deteriorating the results. The authors compared both
approaches and validated the surrogate analyte method to
unambiguously determine the lowest cortisol levels in all
hair samples.
In the present study, we aimed to assess a prediction interval
based on data of hair cortisol in the local adult population
using an approach based on online SPE LC–MS/MS
with 13
C-labeled cortisol as a surrogate analyte.
Results and discussion
Online SPE LC–MS
An automated on-line SPE LC–MS provided high throughput
of samples and minimized the retention time fluctuation
as the autosampler loop (void volume > 100 µl) was excluded
from the separation pathway. Moreover, we have, to our best
knowledge, presented an inexpensive approach of utilizing
a guard column cartridge (2 pcs) as a trapping column for
the SPE purification/enrichment for the first time. The first 2
min of a chromatogram served for trapping and washing of
the sample, which was done by a continuous flow of 2 mL
of 10% MeOH from the secondary pump. When detection at
260 nm was used instead of MS the compounds originating
from the washing step of SPE were eluted during the first
milliliter of 10% MeOH (data not shown).
Representative LC/MS chromatograms are shown
in Fig. 1. Figure 1a shows a hair sample from the hair pool;
Fig. 1b, c show a hair pool sample spiked with 13
C3-cortisol
and ­D8-cortisone, and Fig. 1d shows a representative hair
sample of a participant with spiked ­D8-cortisone. In most
cases, the endogenous cortisol levels in hair pool were below
limits of detection, however; as the sample matrix was not
homogenous there were cases when the quantitation would
have been ruined if the surrogate cortisol calibration had
not been used. The response factor for 13
C3-cortisol/cortisol
was 0.94. Peaks of cortisol and cortisone were chromatographically
resolved to eliminate errors resulting from very
similar mass-to-charge ratios of target compounds. This
was achieved by employing > 50% MeOH in the mobile
phase during a single gradient run. On the other hand, the
system backpressure was relatively high (~ 300 bar), which
may shorten the analytical column lifetime. The separation
conditions were chosen to allow the simple introduction of
additional steroid compounds into the method in future. Due
to the high MeOH content in the mobile phase, it might be
advantageous to employ a heating gradient inside the ESI
chamber (Şener et al. 2017).
153Chemical Papers (2019) 73:151–158	
1 3
Linearity, LOD, and LOQ
Calibration was carried out using a 13
C3-labeled cortisol.
The calibration curve showed linearity in the entire range
1–100 pg/mg. The square of the correlation coefficient
(R2
) of the calibration curve was 0.9995. The intercept
was tested by regression analysis and found insignificant
(p value 0.076, α = 0.05). The LOD and LOQ were found
0.3 and 0.8 pg/mg for 13
C3-cortisol with a signal–noise
ratio of 3:1 (LOD) and 10:1 (LOQ), respectively. The LOD
and LOQ were in a similar range than previously published
methods with LC–MS. A calibration curve from
1 to 100 pg/mg was used to determine cortisol levels in
authentic hair samples as the cortisol concentrations in
hair normally exceed 1 pg/mg.
Recovery and matrix effect
The data for recovery and matrix effects are summarized in
Table 1. Recovery was 91.4, 100.2 and 90.1% for the low,
medium and high concentration, respectively, with the relative
standard deviation (RSD) below 12%. Matrix effects
were not observed for 13
C3-cortisol (≥ 75 and ≤ 125%).
Precision and accuracy
The results for accuracy and precision for 13
C3-cortisol as an
analyte are summarized in Table 2. In this case, all the data
met the required criteria (RSD < 15%).
A B DC
corƟsol
corƟsol
corƟsol
corƟsol
13
C3
-corƟsol
13
C3
-corƟsol
D8
-corƟsone D8
-corƟsone D8
-corƟsone D8
-corƟsone
Fig. 1  Online SPE LC–MS chromatograms of a a hair pool sample,
b a hair pool sample spiked with 13
C3-labeled cortisol (1.25 pg/mg),
c a hair pool sample spiked with 13
C3-labeled cortisol (12.5 pg/mg),
and d a real hair sample (8 mg). Two SRM transitions for 13
C3-cortisol
(tR = 6.3  min), cortisol (tR = 6.3  min), and IS ­D8-cortisone
(tR = 5.8 min) are represented by the upper, middle, and lower frames,
respectively. Please notice, that the endogenous cortisol was found in
the hair pool sample at rather low, however, detectable levels
Table 1  Recovery and matrix effect, (N = 5)
13
C3-cortisol
Low (5 pg/mg) Medium
(20 pg/mg)
High
(100 pg/
mg)
Recovery (%) 91.4 100 90.1
RSD (%) 11 8.8 9.0
Matrix effect (%) 116 97.4 106
RSD (%) 6.2 9.6 5.8
154	 Chemical Papers (2019) 73:151–158
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Clinical application
To indicate a range of hair cortisol values, which might be
applicable as a reference range, a series of 114 volunteers
(40 men, 74 women, age 19–27 years) has been analyzed
for hair cortisol levels, see Table 3. Such effort was already
made by other groups, however; a range of expected values
should be determined among the population it is going to
be applied to. As we plan to correlate hair cortisol levels of
people with disorders associated with high levels of cortisol
in the organism, we had to compare the range obtained
with local population with that assessed by other groups.
Although we attempted to minimize within individual variation
in hair growth by taking hair from the vertex posterior
region of the head, hair growth rates may vary among individuals.
Furthermore, the participants in this cohort at this
phase have a mean age of 20.5 years; therefore, it may not
be possible to generalize the findings of this study to older
adults, even as the age-related variability appears mostly in
the range 0.5–18 years (Binz et al. 2018).
The original data were log-transformed to obtain normal
distribution, which was used to obtain re-transformed
mean (4.76 pg/mg), standard deviation (1.88 pg/mg), and a
prediction interval (α = 0.05; 1.22–18.7 pg/mg) in the first
three centimeters of hair (Table 3). The prediction interval
could be defined as an estimate of an interval in which future
observations will fall, with a certain probability and it was
very similar to the 2.5–97.5 quantile interval.
To reveal the relationship of the hair cortisol concentration
with the physical state of the organism, we asked the
participant with the value of hair cortisol, which was marked
as an outlier, to be sampled again after a 3-month period. In
this case, the hair sample was obtained from three posterior
vertex positions approximately 2 cm from each other (left,
middle, and right) in two replicates; each of the hair strands
was cut into six 1 cm segments. Essentially, it could have
represented hair cortisol concentrations approximately over
the past 6 months (Table 4). Despite the high variability
between individual sampling locations, it may be clearly
stated that hair cortisol had been increased in the time of
the first sampling (4–6 cm) and was probably lowered by a
washing-out effect. The values from the 1–3 cm may suggest
that the overall cortisol balance in the organism has been
settled and that the former values had been momentarily
increased. However, the participant was not able to provide
any additional information on the cause of the elevated values.
Careful interpretation has to be made as, according to
another study, the participant could have been identified as
a person with Cushing’s syndrome (cut-off = 31.1 pg/mg)
(Wester et al. 2017).
Materials and methods
Chemicals and calibration standards
Reagents, stock solutions of 13
C3-cortisol (2,3,4-13
C3)
(100 μg/mL), deuterated ­D8-cortisone (2,2,4,6,6,9,12,12D8),
formic acid (HCOOH), and methanol (MeOH, LC–MS)
were obtained from Sigma Aldrich (Switzerland). All water
used was of an ultra-pure grade supplied by an in-house
Milli-Q system (Millipore, MA, USA).
A stock solution of deuterated internal standard (IS)
­D8-cortisone was prepared at the final concentration of
100 ng/mL and was also used as a working solution. A
stock solution of a stable isotope-labeled form of the analyte,
13
C3-cortisol, was prepared in methanol to a concentration
of 50 ng/mL. Working standard solutions were
prepared by diluting the stock solution 13
C3-cortisol to a
Table 2  Accuracy and precision, on 5 consecutive days
13
C3-cortisol Low (5 pg/mg) Medium
(20 pg/mg)
High
(100 pg/
mg)
Characteristics (N = 25)
 Mean (pg/mg) 5.09 20.2 102
 SD (pg/mg) 0.307 1.46 6.01
Accuracy
 Variance (pg/mg) 0.090 0.20 2.0
 Bias (%) 1.7 1.0 2.0
Intraday precision (N = 5)
 RSD (%) 3.5 6.2 4.9
Interday precision (N = 25)
 RSD (%) 6.0 7.2 5.9
Table 3  Hair cortisol concentration (pg/mg) of the study participants (N = 114) containing mean (X), median (M), re-transformed mean (XR), retransformed
standard deviation ­(SDR), prediction interval (PI), and a 2.5–97.5 quantile interval (Q95)
Hair segment N % X M XR SDR PI Q95
1st cm 114 100 6.02 4.86 4.85 1.84 1.44–16.3 1.45–16.5
2nd cm 101 88.6 7.13 4.90 5.00 2.03 1.22–20.6 1.35–17.8
3rd cm 95 83.3 6.63 4.20 4.59 2.09 1.05–20.0 1.13–23.1
3 cm length 310 – 6.57 4.76 4.76 1.88 1.22–18.7 1.32–19.4
155Chemical Papers (2019) 73:151–158	
1 3
concentration of 0.08, 0.4, 0.8, 1.6, and 8 ng/mL resulting
in the final concentration levels in the calibration curve of
1–100 pg/mg.
All standard and stock solutions were individually
diluted with methanol and stored at − 20 °C until use.
Hair sample collection and preparation
The hair samples were taken from healthy volunteers
following the basic criteria for collection defined by the
Society of Hair Testing (Cooper et al. 2012). The protocol
was approved by the Masaryk University Ethical Committee,
and all participants gave written informed consent.
The hair strands with a thickness of several millimeters
in diameter were cut with fine scissors from a posterior
vertex position as closely as possible to the scalp. Subsequently,
they were tagged on the proximal side and stored
individually in a folded piece of paper in labeled foiled
bags. Until the day of analysis, the hair strands were stored
at room temperature in a dry environment away from direct
sunlight. For the analysis, the first 3 cm of the proximal
segment was cut into 1 cm segments. A minimum of 8 mg
of each segment was weighted to evaluate and determine
the level of cortisol.
Four centimeters of the distal end of 20 cm long human
hair was used as authentic matrix. The hair was cut into
snippets, homogenized and used as a hair pool during the
method development.
Hair sample extraction
Prior to analysis, the hair samples were manually washed in
vials by shaking them two times with 2 ml of isopropanol for
1 min at room temperature. Subsequently, they were dried on
a piece of filter paper and uniformly mixed with a laboratory
tweezer to ensure maximum homogeneity. Approximately
8 mg of washed and dry hair was carefully transferred into a
glass tube. Sequentially, 1800 µl of methanol and 10 μl of IS
from the working solution (100 ng/mL) were added.
Calibration curve standards were prepared following
the same procedure using the hair pool matrix. The 8-mg
hair sample was spiked with 13
C3-cortisol (100 µL from the
working standard solutions resulting in final concentrations
of 1–100 pg/mg) and 10 μl of IS from the working solution
(100 ng/mL) was added prior to incubation. The hair was
incubated for 18 h at room temperature, in darkness and
without shaking. Afterward, 1600 µl of the extract was transferred
into a new tube and fully evaporated at 40 °C under a
constant stream of nitrogen. The residue was reconstituted
in 500 µl of 20% MeOH, vortexed for 1 min, and transferred
into the LC–MS vials. A volume of 50 µl was directly
injected into the online SPE LC–MS/MS system for analysis.
Online SPE LC–MS
The online SPE LC–MS method for analysis of cortisol
was developed using a Dionex Ultimate 3000RS (ThermoScientific,
CA, USA) module, equipped with a binary
Table 4  Hair cortisol
concentrations (pg/mg) of
a single sample cut into 6
segments in two replicates
Values of the mean (X), standard deviation (SD), and relative standard deviation (RSD) were calculated
Position Hair segment
1 cm 2 cm 3 cm 4 cm 5 cm 6 cm
Left
 A 7.82 8.45 8.13 13.7 27.6 35.2
 B 7.58 10.2 9.62 13.1 20.2 22.7
 X 7.70 9.33 8.87 13.4 23.9 29.0
 SD 0.168 1.24 1.05 0.44 5.23 8.82
 RSD (%) 2.18 13.3 11.8 3.26 21.9 30.5
Middle
 A 8.82 10.2 16.0 41.0 60.2 52.4
 B 11.0 28.2 23.5 51.8 73.1 61.1
 X 9.90 19.2 19.7 46.4 66.7 56.8
 SD 1.53 12.7 5.29 7.63 9.13 6.11
 RSD (%) 15.4 66.4 26.8 16.4 13.7 10.8
Right
 A 11.4 11.1 11.4 12.6 18.9 19.0
 B 10.7 12.3 13.5 14.5 21.3 19.9
 X 11.1 11.7 12.5 13.6 20.1 19.5
 SD 0.506 0.810 1.50 1.37 1.63 0.616
 RSD (%) 4.57 6.92 12.0 10.1 8.13 3.17
156	 Chemical Papers (2019) 73:151–158
1 3
high-pressure gradient pump (HPG), a quaternary lowpressure
gradient pump (LPG) connected to an auto-sampler
(AS), and a column oven with a 6-port, two-way valve. The
trapping column (SPE column) consisted of a mounted guard
column holder (Phenomenex, CA, USA) with two pieces of
a ­SecurityGuardTM
cartridge (2 × 4 mm, C18, Phenomenex,
CA, USA). With the switching valve in the sample loading
position (A), the LPG pump was used to load within 2 min
50 µl of the samples on the SPE column operating at room
temperature using 10% MeOH in water at 1 ml/min. From
2 to 2.5 min (Fig. 2), the valve automatically switched to its
alternate position (B) and then back to the loading position
(A), allowing the analytes to be transferred (in a backflush
mode) from the SPE column onto the analytical column
(2.1 mm × 100 mm, 2.7 μm C18 Ascentis, Phenomenex,
CA, USA), shielded by a guard column (C18, 2 × 2 mm,
Phenomenex, CA, USA), both operated at 20 ± 0.1 °C. The
HPG binary mobile phase system consisted of water with
0.1% formic acid (v/v) and MeOH. The flow-rate was set
to 0.2 ml/min and the following 12-min gradient program
was employed. The initial 50% of MeOH was kept constant
for 2 min and then linearly increased from 50 to 85% over
the next 7.5 min. It was held at 85% for 1 min, followed by
equilibration under the initial conditions for another 1.5 min.
From 4.0 to 8.5 min, the SPE column was regenerated using
100% MeOH and then re-equilibrated using 10% MeOH.
The outlet of the analytical column was connected to a
Bruker EVOQ Qube triple quadrupole mass spectrometer
(Germany) operated in the negative heated-electrospray
ionization mode with the following parameters: Spray voltage
3500 V, cone temperature 350 °C, cone gas flow 20
psi, heated probe temperature 350 °C, probe gas flow 40 psi
(nitrogen), and nebulizer gas flow 50 psi (nitrogen). Argon
was used as collision gas. All other parameters were set at
default values provided by the manufacturer. The flow from
LC was diverted to waste in the range of 0–5 and 8.5–12 min.
Target compounds were scanned in the selected reaction
monitoring mode (SRM) as fragments of ionic adducts with
Fig. 2  Switching valves positions. a Loading and washing. b Sample transfer. The table specifies individual times of events during a single sample
run
157Chemical Papers (2019) 73:151–158	
1 3
formic acid [M+HCOO]−1
: cortisol 407.1–331.1 (12 eV)
and 407.1–297.1 (29 eV); cortisol 13
C3 410.1–334.1 (12 eV)
and 410.1–300.1 (29 eV); ­D8-cortisone 413.3–337.1 (10 eV)
and 413.1–139.1 (36 eV). Dwell time for each transition
was 50 ms.
Method validation
As the validation for using the 13
C3-cortisol calibration
instead of endogenous cortisol calibration was done by Binz
et al. 2016, we have validated only the parameters related to
the 13
C3-cortisol calibration. Statistica (v13, TIBCO Software
Inc.) was used for data processing.
LOD and LOQ
Five calibration levels (1–100 pg/mg) were used. Three
replicates for each calibration level (1, 5, 10, 20, 100 pg/
mg) were analyzed. The calibration curve was estimated by
the least-squares regression procedure. LOD and LOQ were
determined for 13
C3-cortisol by analyzing the lowest calibration
point of calibration and calculating a theoretical signalto-noise
ratio of 3:1 for LOD and 10:1 for LOQ. The values
were verified by measuring 13
C3-cortisol samples using the
calculated concentrations.
Accuracy, precision, matrix effects, and recovery
Accuracy and precision of the method were evaluated by
measuring five replicates of a QC sample at three different
concentration levels (low 5 pg/mg, medium 20 pg/
mg, high 100 pg/mg) on 5 consecutive days. Matrix effect
was measured by comparing the calculated concentration
of 13
C3-cortisol spiked hair after extraction (A) to
the spiked solvent at the same concentration (B) (Matrix
effect = A/B × 100). Recovery was measured by comparing
the calculated concentration of 13
C3-cortisol from spiked
hair before extraction (C) to spiked hair after extraction (D)
(recovery = C/D × 100). Five replicates at low (5 pg/mg),
medium (20 pg/mg) and high (100 pg/mg) concentration
levels were used for both parameters.
Conclusions
An online SPE LC–MS with a surrogate analyte was developed
and validated to analyze the hair cortisol concentration.
A range and median concentration levels of physiological
values correspond to those previously published in the literature.
Cortisol in hair represents a long-term value of cortisol
in the organism even that the incorporation mechanism has
not been fully uncovered yet. We have obtained a prediction
interval for hair cortisol to help differentiate between
short-term physical stress-related issues and long-term
non-physiological conditions, such as Cushing’s syndrome.
Hence, when hair cortisol values are being determined we
recommend analyzing a longer segment of a hair strand to
obtain a decline curve, which might negate the enormous
individual and sampling variability. Careful interpretation
of hair cortisol levels within the prediction interval should
be considered as well.
Acknowledgements  This work was supported by the Specific University
Research Grant (MUNI/A/0910/2017) provided by the Ministry of
Education, Youth and Sports of the Czech Republic in the year 2017.
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4.3 Therapeutic Drug Monitoring of Sunitinib in Gastrointestinal Stromal Tumors
and Metastatic Renal Cell Carcinoma in Adults-A Review
Sunitinib (SUN) and its major metabolite N-desethyl sunitinib (DES), belonging to multitargeted tyrosine kinase
inhibitors, exhibit a non-uniform therapeutic outcome in adult patients. In 2006, SUN was launched as a standard
care treatment for metastatic cell carcinoma in adults, gastrointestinal stromal tumors, and in 2011 for pancreatic
neuroendocrine tumors. Severe toxicity and adverse effects, frequently observed in clinical practice but not in type III
clinical trials, could be explained by poor adherence, CYP polymorphisms, and drug-drug interactions. Therapeutic
drug monitoring (TDM) could target the variable outcomes and decrease adverse effects’ toxicity and/or frequency.
Reviewing 73 articles related to a SUN or DES concentration-to-toxicity relationship concluded that the therapeutic
trough levels should be in a range of 50–100 ng/ml for a total concentration SUN + DES. LC–MS is the method of
choice for TDM as it provides unmatched sensitivity and specificity at the same time. SUN and DES may undergo
Z/E-photoisomerization, forcing many laboratories and hospitals to work in the dark. Nevertheless, the light-induced
isomerization is reversible, and the active (E-)isomer could be quantitatively reverted by heating the samples in the
dark.85
Quantitative determination was performed using isotopically labeled or chemically similar internal standards.
As many worldwide LC–MS instruments have become available, the cost per sample could be kept relatively low. On
the other hand, the high cost of therapeutic agents and molecular pathology-based indication rules should guarantee
an optimal dose for each patient.
DEMLOVA, Regina, Miroslav TURJAP, Ondrej PES, Katarina KOSTOLANSKA a Jan JURICA. Therapeutic Drug
Monitoring of Sunitinib in Gastrointestinal Stromal Tumors and Metastatic Renal Cell Carcinoma in Adults-A Review.
Therapeutic Drug Monitoring. 2020, 42(1), 20–32. ISSN 0163-4356. doi:10.1097/FTD.0000000000000663 Document
Type: Review, IF = 3.681; JCR Category + Category Quartile: MEDICAL LABORATORY TECHNOLOGY Q2 +
PHARMACOLOGY & PHARMACY Q2 + TOXICOLOGY Q2; AIS = 0.719
Author’s contribution: 20 %
108
REVIEW ARTICLE
Therapeutic Drug Monitoring of Sunitinib in
Gastrointestinal Stromal Tumors and Metastatic Renal Cell
Carcinoma in Adults—A Review
Regina Demlová, MD, PhD,* Miroslav Turjap, PharmDr, PhD,† Ondrej Pes, PhD,‡
Katarína Kostolanská, PharmDr,‡ and Jan Jurica, PharmDr, PhD§¶
Background: Sunitinib is an inhibitor of multiple receptor tyrosine
kinases and is a standard-of-care treatment for advanced and
metastatic renal cell carcinoma and a second-line treatment in locally
advanced inoperable and metastatic gastrointestinal stromal tumors.
A fixed dose of the drug, however, does not produce a uniform
therapeutic outcome in all patients, and many face adverse effects
and/or toxicity. One of the possible causes of the interindividual
variability in the efficacy and toxicity response is the highly variable
systemic exposure to sunitinib and its active metabolite. This review
aims to summarize all available clinical evidence of the treatment of
adult patients using sunitinib in approved indications, addressing the
necessity to introduce proper and robust therapeutic drug monitoring
(TDM) of sunitinib and its major metabolite, N-desethylsunitinib.
Methods: The authors performed a systematic search of the
available scientific literature using the PubMed online database.
The search terms were “sunitinib” AND “therapeutic drug monitoring”
OR “TDM” OR “plasma levels” OR “concentration” OR
“exposure.” The search yielded 520 journal articles. In total, 447
publications were excluded because they lacked sufficient relevance
to the reviewed topic. The remaining 73 articles were, together with
currently valid guidelines, thoroughly reviewed.
Results: There is sufficient evidence confirming the concentration–
efficacy and concentration–toxicity relationship in the indications of
gastrointestinal stromal tumors and metastatic renal clear-cell carcinoma.
For optimal therapeutic response, total (sunitinib + N-desethylsunitinib)
trough levels of 50–100 ng/mL serve as a reasonable
target therapeutic range. To avoid toxicity, the total trough levels
should not exceed 100 ng/mL.
Conclusions: According to the current evidence presented in this
review, a TDM-guided dose modification of sunitinib in selected
groups of patients could provide a better treatment outcome while
simultaneously preventing sunitinib toxicity.
Key Words: sunitinib, N-desethylsunitinib, mRCC, GIST, therapeutic
drug monitoring, toxicity-adjusted dosing
(Ther Drug Monit 2019;00:1–13)
INTRODUCTION
Sunitinib is a multitargeted receptor tyrosine kinase
inhibitor (TKi), acting on signaling cascades involved in
proliferation and tumor progression. It was launched in 2006
and became a standard-of-care treatment for advanced/
metastatic renal cell carcinoma (mRCC) in adults and in
unresectable and/or metastatic malignant gastrointestinal stromal
tumor (GIST) in adults after the failure of imatinib
treatment due to resistance or intolerance.1,2 In 2011, sunitinib
was additionally approved for the treatment of unresectable or
metastatic, well-differentiated pancreatic neuroendocrine tumors
(pNET) with disease progression in adults.3
Similar to other TKi, it seems that a fixed dose of
sunitinib does not produce a uniform therapeutic outcome in
patients. Furthermore, the frequency of adverse effects and
toxicity also fluctuates.4,5 One of the possible causes may be
high interindividual variability in the systemic exposure to TKi
due to the high variability in pharmacokinetics (PK) and
genetic polymorphisms of specific efflux transporters.6 In addition,
a relationship has been found between plasma levels of
several TKi and the treatment outcome and toxicity.5,7–10
In clinical practice, it has been documented that severe
toxicity and adverse events occur more frequently in “real-life”
patients than in phase III of clinical studies.11 The causes of
this discrepancy may be patients’ nonadherence to the treatment,
genetic polymorphisms of the cytochrome P450 (CYP)
enzymes and transporters, or drug–drug interactions. The question
of whether therapeutic drug monitoring (TDM) with an
individualized dose adjustment may improve the therapeutic
Received for publication January 11, 2019; accepted May 8, 2019.
From the *Department of Pharmacology, Medical Faculty, Masaryk University,
Brno; †Department of Clinical Pharmacy, University Hospital Ostrava,
Ostrava; ‡Department of Biochemistry, Medical Faculty, Masaryk University;
§Department of Pharmacology, Medical Faculty, Masaryk University,
Masaryk Memorial Cancer Institute; and ¶Department of Human Pharmacology
and Toxicology, University of Veterinary and Pharmaceutical Sciences,
Brno, Czech Republic.
Supported by the State budget through MEYS, Large Infrastructure Project
CZECRIN (No. LM2015090) , by funds from the Faculty of Medicine
MU to junior researcher Ondrej Pes and by the Specific University
Research Grant (MUNI/A/0976/2018, MUNI/A/1550/2018) provided by
the Ministry of Education, Youth and Sports of the Czech Republic in the
year 2018.
R. Demlová is currently receiving a grant CZECRIN_4PATIENTS—innovative
advanced therapies (CZ.02.1.01/0.0/0.0/16_013/0001826) and participates in
the grant projects H2020:TRANSVAC2 (NCT 730964_TRANSVAC 2) and
H2020: TENSION—Efficacy and safety of thrombectomy in stroke with
extended lesion and extended time window: a randomized, controlled trial
(NCT 03094715). J. Jurica has received honoraria from Angelini Pharma,
LERAM pharmaceuticals and ProMed.CS. The remaining authors declare no
conflict of interest.
Correspondence: Jan Jurica, PharmDr, PhD, Department of Pharmacology,
Medical Faculty, Masaryk University, Kamenice 753/5, 625 00 Brno,
A19, Czech Republic (e-mail: jurica@med.muni.cz).
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
Ther Drug Monit  Volume 00, Number 00, Month 2019 1
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outcome of sunitinib or at least improve its tolerability is yet to
be answered. Hence, the main aim of this review lies in gathering
maximum clinical evidence for the justification of TDM
of sunitinib and its major metabolite, N-desethylsunitinib, in
adult patients with approved indications in relation to an
exposure/efficacy and exposure/toxicity relationship.
Pharmacodynamics of Sunitinib
Sunitinib exerts an inhibitory action on several receptor
tyrosine kinase families, including receptors for PDGFR-a,
PDGFR-b, VEGFR1-3, and c-KIT. Furthermore, it also inhibits
FMS-like tyrosine kinase 3 (Tyrosine kinase involved in the
regulation of the family of genes originally isolated from the
Susan McDonough strain of feline sarcoma virus, FLT), a macrophage
colony-stimulating factor receptor (CSF-1R), and
a glial cell-line derived neurotrophic factor receptor (RET =
rearranged during transfection; protooncogene).12–14 Sunitinib,
compared with interferon-a, placebo, or other targeted agents,
showed improved progression-free survival (PFS) in randomized
phase III trials.15 Together with pazopanib and a combination
of nivolumab and ipilimumab, sunitinib is currently the
most preferred and used TKi in the treatment of mRCC2,16 and
according to the European Society for Medical Oncology, British
Sarcoma Group, and National Comprehensive Cancer Network
guidelines, also the second-line treatment of metastatic
GIST.1,16,17 Moreover, sunitinib is currently being investigated
in 102 clinical trials, including for type B3 thymoma and thymic
carcinoma (NCT03449173), glioblastoma
(NCT03025893), genitourinary cancer (NCT02060370), urinary
tract urothelial carcinoma (NCT00794950), brain metastases
(NCT00981890), endometrial adenocarcinoma
(NCT00478426), soft-tissue sarcoma, bone sarcoma
(NCT03277924), ciliary body and choroid melanoma
(NCT02068586), ovarian clear-cell adenocarcinoma
(NCT00979992), prostate cancer (NCT00329043), thyroid
gland carcinoma (NCT00381641), pheochromocytoma and
paraganglioma (PPGL) (NCT01371201), small-cell lung carcinoma
(NCT00453154), breast cancer (NCT00513695), lymphoma,
non-Hodgkin lymphoma, and multiple myeloma
(NCT02693535).
PK Properties
Sunitinib is absorbed slowly and reaches a maximal
plasma concentration (Cmax) within 6–12 hours (h) after oral
administration.3,14 Although the absolute bioavailability has
not been assessed in humans, the preclinical experiments
approximated it to reach ;50% owing to the extensive
first-pass effect.18–20 The type of meal does not seem to affect
the bioavailability.21 PK is linear in the range of doses from
50 to 350 mg/d3,19 The steady-state concentration (css), owing
to the long elimination half-life, is reached after 10–14 days
of regular administration.22 However, a simulation made from
1205 samples taken from 70 patients determined the time to
reach 90% of the steady state was 6 days for sunitinib and 8
days for N-desethylsunitinib.23 Sunitinib and N-desethylsunitinib
are bound extensively to plasmatic proteins (95% and
90%, respectively), and both have a large volume of distribution
(Vd) of more than 2300 and 3000 L, respectively.3,24
In the preclinical experiments, protein binding profiles seem
to be linear; hence, hypoalbuminemia may lead to decreased
total concentrations of sunitinib and N-desethylsunitinib and
an increased volume of distribution.25
On the other hand, sunitinib intracellular penetration is
not influenced by active transport; nevertheless, it may be
limited by the activity of the efflux transporters, ABCB1 and
ABCG2.19,26–30 The transporters also prevent the passage of
sunitinib through the blood–brain barrier.31 Sunitinib is subject
to microsomal oxidative dealkylation through CYP3A4 to
N-desethylsunitinib, which shows a similar inhibitory action on
tyrosine kinases as the parent drug. It has been estimated that
N-desethylsunitinib adds approximately 30% to the overall
sunitinib + N-desethylsunitinib exposure.3 N-desethylsunitinib
is further metabolized through CYP3A4, and its elimination
half-life is even longer, up to 80–100 hours, while its Vd is
similar or greater than that of sunitinib.19 More than 60% of the
administered dose is excreted in feces, and only a small percentage
appears in the urine.32 No accumulation of sunitinib or
N-desethylsunitinib was observed within 13 cycles (28 days of
treatment) in patients with GIST dosed with 37.5 mg/d.33
Biotransformation of sunitinib is influenced by sex—
female patients have up to 30% lower apparent clearance (Cl/
F), but even this difference does not imply different dosing
for women.3,34 Only minor differences in sunitinib plasma
concentrations within various age groups in the outpatient
population have been reported.35 Patients with mRCC older
than 70 years received significantly lower doses compared
with those received by younger individuals (median dose
37.5, and 50 mg/d, respectively).36
Mild to moderate hepatic impairment (Child–Pugh
scores A and B) has no significant effect on sunitinib exposure37;
the influences of more severe hepatic impairment have
not yet been studied. In the patients with renal impairment
(ClCr #30 mL/min), the sunitinib systemic exposure was
similar to that in patients with normal renal functions. In the
hemodialyzed patients, the exposure decreased to 47% of the
exposure observed in patients with normal renal functions.3,14
The influence of hemodialysis on the PK of sunitinib is
somewhat inconclusive.38 Terada et al39 concluded that sunitinib
could be used in hemodialyzed patients without any
special dose adjustments since its PK, owing to its large
volume of distribution, is affected by high plasma protein
binding and rather extensive liver metabolism.
The effect of biorhythms was also studied, with
sunitinib reported to attenuate circadian blood pressure
rhythmicity.40 Neither morning nor evening administration
had any apparent effect on the therapeutic outcome or
PK41; however, the severity of the adverse events was slightly
lower in patients receiving sunitinib in the morning compared
with the evening dosing.42
Adverse Effects and Toxicity
Sunitinib is, in the context of other targeted therapy
agents for mRCC and GIST, well tolerated at recommended
dosing, and most adverse effects observed were mild to
moderate. Reported adverse effects (occurring in $20% patients)
included anemia, skin discoloration, fatigue, gastrointestinal
disorders (diarrhea, nausea, and vomiting), stomatitis,
decreased appetite, taste disturbance, hypertension, hand-foot
Demlová et al Ther Drug Monit  Volume 00, Number 00, Month 2019
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syndrome (HFS), hypothyroidism, and mucosal inflammation.3,43
Hematological disorders such as neutropenia, thrombocytopenia,
and anemia are among the most common
adverse effects.44 A decline in the left ventricular ejection
fraction also appeared quite often.43 Severe but rare adverse
effects of sunitinib include renal or heart failure, pulmonary
embolism, gastrointestinal perforation, and hemorrhages.3,45
In addition to the adverse effects mentioned in the summary
of the product characteristics, hepatotoxicity (an increase in
liver transaminases), increased creatinine, anorexia, dysgeusia,
mucositis, and epistaxis have been observed in real-life
patients.46,47 Some of the aforementioned adverse effects may
diminish with treatment continuation.
Dosing Schedules
In mRCC and GIST indications, sunitinib recommended
dosing consists of 50 mg/d over 4 consecutive weeks
followed by a 2-week rest period (4/2). An alternative dosing
schedule of 50 mg/d for 2 weeks with a single-week rest
period (2/1) should help in reducing the sunitinib toxicity in
mRCC patients without compromising the efficacy.48,49 In
GIST, an alternative of 37.5 mg continuously has also been
tested and proved to be a possible dosing strategy.33 Recently,
in a small Japanese study, an alternative to 4/2 dosing was
evaluated. Administration of sunitinib in 23 patients every
other day with a standard dose (50 or 37.5 mg) resulted in
a better time to progression (TTP), overall survival (OS) outcomes,
and tolerability (lower frequency of HFS, thrombocytopenia,
and leucopenia). The total trough levels (TTLs) were
significantly lower (64.83 versus 135.8 ng/mL, P , 0.001).50
The approved dose of sunitinib in the pNET indication
is 37.5 mg/d administered continuously. Dose modification
based on individual safety and tolerability is possible in 12.5
mg steps; however, the daily dose should be kept in a specific
range (25–75 mg/d for mRCC and GIST, while the maximal
studied daily dose for pNET was 50 mg/d).3
In special situations, such as coadministration of
inducer or inhibitor of sunitinib metabolism, a dose modification
has been advised. When coadministration of a CYP3A4
inducer cannot be avoided, a dose modification up to 87.5 or
62.5 mg/d is recommended for mRCC/GIST or pNET,
respectively.3
Drug–Drug Interactions
PK interactions generally include (1) drugs influencing
the main biotransformation pathway (CYP3A4) of sunitinib
or (2) sunitinib theoretically influencing the effects of other
drugs. Inducers of CYP3A4 enzymes, such as anticonvulsants
(carbamazepine, phenobarbital, or phenytoin), rifampin, mitotane,
dexamethasone, or St. John’s wort might decrease
plasma levels or systemic exposure of sunitinib.14,51 On the
other hand, coadministration of sunitinib with CYP3A4
enzyme inhibitors might result in supratherapeutic trough levels
and increased incidence or a higher grade of toxicity. Such
drugs include macrolide antibiotics (clarithromycin), azole
antifungals (ketoconazole, itraconazole, voriconazole, or posaconazole),
HIV protease inhibitors (indinavir, ritonavir, nelfinavir,
or saquinavir), aprepitant, as well as food supplements
based on citrus juice.3,14 A few interactions were documented
in clinical studies, for example, in highly active antiretroviral
therapy, where ritonavir and efavirenz increased or decreased
the exposure of N-desethylsunitinib, respectively.52 It was
also reported that coadministration of sunitinib with protonpump
inhibitors is associated with higher plasma concentrations
of sunitinib (+53%).35 Sunitinib is a moderate inhibitor
of CYP3A4 and, therefore, might increase plasma concentrations
of drugs metabolized by this enzyme.53,54 If concurrent
administration with an enzyme inhibitor or inducer is necessary,
a dose modification with TDM should be considered.3
Sunitinib is a PgP inhibitor and, thus, may increase the toxicity
of PgP substrates including colchicine.55
The displacement of sunitinib/N-desethylsunitinib from
plasma proteins may also be theoretically relevant when administering
with a drug that has a high affinity to albumin. Such
interaction has yet not been reported; however, several antiretroviral
treatment schedules did not show a significant change in
the unbound fraction of sunitinib and/or N-desethylsunitinib.52
Pharmacodynamic (PD) interactions are expectable in
a combination of sunitinib with drugs affecting heart rhythmicity,
immune response, liver functions, and, most notably,
bisphosphonates, which may increase the risk of osteonecrosis
of the jaw.14 Because of these effects, sunitinib should be used
with caution in a combination with antiarrhythmics or drugs
suspected of prolonging the QT interval (eg, several antipsychotics,
antidepressants, 5-HT3 antagonists, prokinetics, quinolones,
fingolimod, vemurafenib, or nilotinib).56 Such an
interaction is dangerous and possibly the most common PD
interaction.57 Meanwhile, sunitinib should be used carefully
with drugs that are hepatotoxic or may increase levels of alanine
aminotransferase or aspartate aminotransferase (eg, leflunomide,
acetaminophen, lomitapide, or nimesulide) since
sunitinib was mentioned in the “black box warnings” by the
Food and Drug Administration.58 Since sunitinib is also thyrotoxic,
particular caution should be taken when sunitinib is coadministered
with amiodarone (thyrotoxic + proarrhythmic).47
Finally, all immunosuppressants might increase the risk of
opportunistic infections.14 Interactions with immunostimulant
food supplements such as Echinacea extracts or beta-glucans
should be considered as well.59
METHODS
The literature search was conducted in December 2018
using the PubMed online database. Search terms used were
“sunitinib” AND “therapeutic drug monitoring” OR “TDM”
OR “plasma levels” OR “concentration” OR “exposure” in
“all fields.” Searches were limited to human studies and publications
in English or German languages. The initial search
provided 520 journal articles. A single author (J.J.) read the
abstracts of these articles. In total, 447 publications were
excluded because they lacked relevance to the reviewed topic
or were focused on TDM of sunitinib in indications other than
mRCC and GIST. The relevant 73 articles were revised as
they contained prospective, retrospective, and cross-sectional
observational studies, interventional randomized controlled
trials, literature reviews, letters to editors, and case reports.
Currently valid guidelines were also included in the
review.1,2,16,60
Ther Drug Monit  Volume 00, Number 00, Month 2019 A Review of TDM of Sunitinib in GIST and mRCC
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RESULTS
Benefit of TDM of Sunitinib in the Treatment
of mRCC and GIST
The most significant benefits from TDM are observed
in drugs when (1) no clear or safe marker of the clinical effect
may be readily measured; (2) toxicity or inefficacy may harm
the patient; (3) the clinically relevant concentration–response
(effect/toxicity) relationship has already been established; (4)
a narrow therapeutic range exists; (5) no or poor correlation of
a dose–response relationship exists, or (6) a high degree of
interindividual variability of plasma concentrations exists.
Other circumstances such as the presence of an active
metabolite in clinically relevant concentrations, great interaction
potential of the drug, or nonlinear PK might be considered
as well.
In the case of sunitinib and N-desethylsunitinib, most of
the criteria are perspicuous (1–4), while Lankheet et al35 did
not find any relationship between the dose and the plasma
concentration (5). Therefore, as in the case of imatinib, it may
prove beneficial to monitor plasma concentrations of sunitinib
to avoid toxicity and/or to improve the therapeutic outcome.
A lack of correlation between the dose and the drug
exposure [area under the time–concentration curve (AUC)] or
predose concentration (Ctrough) also justifies the use of
TDM.35,61 As the recommended dose of fixed 50 mg/d (dose
reduction is considered only in extreme adverse effects) has
been used in all patients regardless of their weight, sex, or
age, the interindividual PK variability is very high (for AUC
and Ctrough up to 41% and 54%, respectively).62 Other authors
have reported the interpatient variation coefficient (CV) of
Cmax, AUC, and Ctrough to be in the range of 20%–
60%.33,35,63–65 Faivre et al63 have observed that differences in
Ctrough between individuals on the same dose might reach 1
order of magnitude. Similar intrapatient variability determined
as CV of Ctrough has also been reported.65,66 On the
other hand, a strong correlation between sunitinib, N-desethylsunitinib,
and its combined Ctrough (TTL) and AUC
(combined AUC0–24h) has been observed.67
Concentrations Achieved at Standard Dosing
Schedules
Demetri et al68 conducted a study with 79 patients with
GIST; 79% of the patients on the 50 mg/d dose had TTL over
50 ng/mL by the 14th day of treatment. Lower TTL in the 2/2
schedule (32–47 ng/mL) was observed than in the 4/2 (38–66
ng/mL) or 2/1 (48–63 ng/mL) schedules. Lankheet et al4 published
a study where 42 patients with advanced solid tumors
(6 mRCC, 8 neuroendocrine carcinomas, 8 colorectal carcinomas,
3 adenocarcinomas of an unknown origin, 3 uveal
carcinomas, and 14 miscellaneous) had the median TTL value
of 49.5 ng/mL by the 15th day of treatment when dosed with
37.5 mg continuously.
Interestingly, in toxicity-adjusted dosing (TAD), the
median TTL in 45 patients with mRCC was 67.4 ng/mL, and
the interindividual variability (represented by SD) was 25.8 ng/
mL.66 In the study with Chinese patients with mRCC who were
administered 37.5 mg/d in the 4/2 schedule, 84%–92% of patients
had Ctrough greater than 50 ng/mL in the cycles 1–3.69 The
latter finding could be explained by ethnic differences in the
occurrence of some polymorphisms (ABCG2).29,70,71 More frequent
adverse events in the Asian population are also in line with
these findings.72
Determination of Sunitinib in Human Plasma
for TDM Purposes
Since sunitinib and its salts are of intense orange-yellow
color, their concentrations are determined by liquid chromatography
(LC) UV–Vis methods with satisfactory results.73,74
However, TTLs are expected to range below the detection limit
for most LC UV–Vis methods with some exceptions.75,76 This
makes liquid chromatography–mass spectrometry (LC-MS/
MS), frequently based on triple quadrupole analyzers, the ideal
choice for sunitinib TDM. Sunitinib is very light sensitive, and
all samples should be prepared and analyzed under strict lightprotected
conditions,77 determined as a sum of both isomers, or
converted back to the (E)-isomer.78
To determine sunitinib alone or sunitinib + its metabolite,
most authors use stable isotopically labeled internal
standards35,79–91 because they are generally recommended
for use in quantitative MS.92,93 However, some methods were
successfully validated with the use of a chemically similar
internal standard such as dasatinib,77,94 buspirone,95 clozapine,96
imatinib,97,98 sorafenib,99 or testosterone.100 In 1 case,
an external standard calibration was used.101
TDM generally requires very sensitive methods to
establish TTL. Methods based on LC-MS/MS provide a rapid
and reliable option to monitor low concentrations of sunitinib
and N-desethylsunitinib in patient plasma. Even as the
number of LC/MS instruments worldwide has increased, it
still demands a high initial cost and a skilled operator. Such
facts may significantly contribute to whether the TDM can be
successfully performed at a specific institution.
Exposure–Efficacy Relationship and Therapeutic
Target Range
The primary focus of the preclinical in vitro/in vivo
studies performed by Mendel et al102 was to develop a quantitative
understanding of the plasma levels of sunitinib exposure
required to achieve target inhibition (for each of its
primary targets). It might be defined as the extent to which
the targets need to be inhibited to have some biologic activity
and the fraction of the dosing interval the targets need to be
inhibited to achieve optimal antitumor activity. In the preclinical
experiments, sunitinib selectively inhibited VEGFR2
and PDGFRb phosphorylation in a time- and dose-dependent
manner, achieving strong inhibition of both targets when
plasma concentrations reached or exceeded 50–100 ng/
mL.3,79,102,103 Moreover, constant inhibition of VEGFR2
and PDGFRb phosphorylation was not required for the efficacy;
at highly efficacious doses, the inhibition was sustained
for 12 hours of a 24-hour dosing interval.102,104 Hence, even
complete in vitro inhibition of phosphorylation was not reflected
in clinical outcomes.
Objective measures of treatment outcomes, such as
TTP, OS, PFS, time to treatment failure, time to next
treatment, or frequencies and severity of adverse effects, are
Demlová et al Ther Drug Monit  Volume 00, Number 00, Month 2019
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recorded in clinical studies to demonstrate the real benefit of
TDM. These are often recorded simultaneously, especially
when there are some drawbacks to distinct measures (eg, time
to treatment failure does not sufficiently reflect toxicity) or
because they require large population samples (eg, OS).
Such preclinical PK–PD relationship has been tested
and confirmed in early clinical trials. As an example, a phase
I dose-escalation study by Faivre et al63 was undertaken in
patients with advanced solid malignancies. PK data from the
study indicated that doses of 50 mg/d led to TTL ranging
from 50 to 100 ng/mL. The clinical activity was observed
once the patients achieved daily doses of sunitinib that resulted
in steady-state TTL of 50–100 ng/mL. The most convincing
evidence for a correlation between exposure and the
treatment response in humans came from a PK–PD analysis
performed by Houk et al.61 The authors investigated the relationship
between clinical end points and sunitinib exposure in
patients with advanced solid tumors. PD data were available
for 639 patients of whom 434 had PK data (146 with mRCC,
219 with GIST, and 69 with solid tumors). The results indicated
that the increased exposure to sunitinib was associated
with an improved clinical outcome (longer TTP, longer OS,
and higher chance of antitumor response), as well as with an
increased risk of adverse effects in patients with mRCC and
GIST.61 Low sunitinib exposure was associated with
a reduced response rate, TTP, and OS in both patients with
mRCC and GIST. The exposure–response relationship in
sunitinib has been reported in mRCC, GIST, and in other
solid tumors in the 4/2 sunitinib dosing schedule. In particular,
sunitinib AUC0–24 greater than 800 and 600 ng/mL$h
were associated with higher rates of OS, TTP, and overall
response rate in patients with mRCC and GIST.61,65 When
these AUCs were extrapolated to TTL, the levels of 36.4,
24.6, and 30.5 ng/mL were obtained, which were lower than
the recommended target TTL (50–100 ng/mL).
When sunitinib was dosed continuously at 37.5 mg/d,
a lower Ctrough of 37.5 ng/mL is considered a reasonable
target value in patients with pNET.65 Correspondingly, it
has been shown that exposure correlated with the treatment
outcome; the patients with combined AUC0–24 above the
median had greater OS (35.2 months) than patients with combined
AUC0–24 below the median (16.7 months).105 Yu
et al106 proposed practical guidelines for TKi TDM in GIST
with a continuous dosing schedule of 37.5 mg/d with TTL
37.5–50 ng/mL. Analogously, the authors proposed a dosing
schedule 4/2 of 50 mg/d, with TTL being higher than 50 ng/
mL in RCC.
It has been recommended that dose increments should
be undertaken in patients with TTL below 50 ng/
mL.4,5,35,106,107 In recent clinical studies, TTLs were reached
in up to 50% of the patients at a fixed dose.4,35 Similar percentages
of patients who did not reach TTL were also estimated
by a PK-based simulation.107
In a prospective study by Narjoz et al,105 55 patients
with mRCC were assessed for an exposure-survival analysis.
Patients with high combined AUC had a longer survival than
patients with low AUC. In a retrospective observational clinical
study in Japanese patients with RCC (n = 21), Noda
et al38 reported worse clinical outcomes in patients with
TTL $ 100 ng/mL. Most of these patients experienced unacceptable
toxicity. A dose reduction might be needed in such
cases to avoid unnecessary early discontinuation of the treatment
due to toxicity.38 The exposure–efficacy relationship
studies with sunitinib are summarized in Table 1.
The phase II TAD study of Uemura et al108 aimed to
assess the efficacy and safety of sunitinib in Japanese patients
with mRCC. Sunitinib was self-administered at a starting
dose of 50 mg daily (mRCC, 4/2, n = 51). Doses were
reduced to 37.5 mg/d in cases of treatment-related grade
$3 adverse events and by additional 12.5 mg/d to a minimum
dose of 25 mg/d if toxicity persisted. All 51 patients experienced
treatment-related adverse events, the majority of which
were grade 1 or 2 in severity. At final dose levels, median
TTL reached therapeutic levels (.50 ng/mL) on day 14 of the
first cycle. Nine patients experienced treatment-related
adverse events that led to discontinuation.108 In the TAD
study by Sabanathan et al66 with patients with mRCC (n =
45), the sunitinib dose was increased/decreased to ensure only
grade 1 or 2 toxicity on 10–20 days of each 42-day cycle.
Eventually, 91% of the patients achieved the target toxicity
with a final sunitinib dose of 25 mg (18%), 37.5 mg (27%),
50 mg (50%), and 62.5 or 75 mg (7%). At TAD, the mean
TTL of the study group was 72.0 ng/mL (median 67.4 ng/mL;
SD 25.8 ng/mL). Using TAD, the majority of patients (85%)
achieved TTL above the proposed effective range of .50 ng/
mL. Fifteen percent of patients had TTL . 100 ng/mL. Patients
requiring the dose reduction owing to increased toxicity
during the dose manipulation period had TTL of ,50 ng/mL
(2 patients), 50–100 ng/mL (5 patients), and .100 ng/mL (8
patients).66
In the observational study with an unselected cohort of
outpatients with cancer in routine clinical practice (patients on
sunitinib n = 31) by Lankheet et al,35 the sunitinib Ctrough
ranged from 14.7 to 93.7 ng/mL; however, the predefined
target concentration of $50 ng/mL was reached in only
51.4% of the patients.
The feasibility of PK-guided dosing was investigated in
the following studies. A prospective study by Lankheet et al4
was conducted to determine the safety and feasibility of sunitinib
dosing based on TTL in patients with advanced solid
tumors (n = 42). Patients were given sunitinib at 37.5 mg/
d continuously. On days 15 and 29 of the treatment, TTL was
measured. If TTL was ,50 ng/mL and the patient did not
show any grade $3 toxicity, the daily sunitinib dose was
increased by 12.5 mg. If the patient suffered from grade $3
toxicity, the sunitinib dose was lowered by 12.5 mg. The
assessment of 29 out of 42 patients revealed that grade $3
adverse events were experienced in 7 patients (24%) at the
starting dose and in 9 patients (31%) after the dose escalation.
TTL was below the proposed target in 15 patients (52%) at
the starting dose. Of these, 5 patients (17%) reached target
TTL after the dose escalation without any additional toxicity.
The aim of a retrospective cohort study by Lankheet
et al5 was to monitor how many patients reached the target
exposure after a PK-based dose optimization in daily practice.
Twenty-seven patients (23 with mRCC and 4 with GIST)
were treated with sunitinib and provided 75 samples. In the
study, the target range was 50–87.5 ng/mL for mRCC
Ther Drug Monit  Volume 00, Number 00, Month 2019 A Review of TDM of Sunitinib in GIST and mRCC
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intermittent dosing (starting dose 50 mg/d) and 37.5–75 ng/
mL for mRCC and GIST continuous dosing (starting dose
37.5 mg/d). For patients with TTL within the target range,
the advice was to maintain the same dose level if the toxicity
was manageable. For patients with TTL below the target
range, it was advised to increase the dose to reach the target
TTL. If the TTL was above the target range and signs of
toxicity were present, the dose was decreased to reach the
target TTL range. After the start of treatment, the first mean
TTL was 35.8 ng/mL (ranging from 23.9 to 53.5 ng/mL) with
14 (51.9%) patients being below and 6 patients (22.2%)
above the proposed TTL target range. At the last
TABLE 1. The Exposure–Efficacy Relationship of Sunitinib in mRCC and GIST
Tumor Type n PK Parameter Outcome Correlation Significance Reference
Advanced solid
(preclinical)
TTL Target inhibition A Ctrough of 50–100 ng/mL is the
minimum plasma
concentration required to
inhibit Flk-1/KDR (VEGFR2)
and PDGFRb
N/A 102
mRCC 146 Sunitinib AUC0–24;
combined AUC0–24
TTP TTP increased with increasing
AUC0–24
P = 0.001 61
OS OS increased with increasing
AUC0–24
P = 0.010
Sunitinib AUC0–24 PR or CR PR and CR increased with
increasing AUC0–24
P = 0.00001
SD SD increased with increasing
AUC0–24
P = 0.002
mRCC 21 TTL , versus $ 100 ng/mL TTF Median TTF, 590 versus 71 d P = 0.04 38
PFS Median PFS, 748 versus 238 d P = 0.02
mRCC 20 TTL , versus $ 50 ng/mL TTF Median TTF, 743 versus 56 d P , 0.001 109
PFS Median PFS, 731 versus 95 d P , 0.001
mRCC 55 Combined AUC . versus ,
1973 ng/mL$h at day 21
OS Patients with high combined
AUC had a longer survival
than patients with low
combined AUC
P = 0.0051 105
GIST 225 AUCss PR and CR Probability of PR or CR
correlates with exposure
P = 0.06 61
SD Probability of SD correlates with
exposure
P = 3 · 1029
TTP and OS Above median AUCss displayed N/A
Longer TTP and OS
GIST 278 AUC0–24 . versus , 600
ng/mL$h
TTP, OS, and SD TTP, OS, and SD increased with
AUC0–24
P = 0.001 65
ORR TTP, OS, and SD increased with
AUC0–24
P = 0.06
mRCC 33 Combined AUC0–24 Disease
progression
In patients with disease
progression, combined AUCss
tends to be lower than in
patients without disease
progression during the first
cycle (1678 versus 2004
ng/mL$h, respectively)
P = 0.072 11
mRCC 26 Sunitinib Ctrough PFS No association of TTL with PFS
or OS
P = 0.269 67
OS P = 0.681
TTL PFS No association of TTL with PFS
or OS
P = 0.425
(PFS)
OS P = 0.505
(OS)
N-desethylsunitinib Ctrough PFS Ctrough .15 ng/mL associated
with increased PFS (61 wks
versus 12 wks)
P = 0.004
OS Ctrough .15 ng/mL associated
with increased OS (36 mo
versus 8 mo)
P = 0.040
CR, complete response; ORR, overall response rate; PR, partial response; SD, stable disease; TTF, time to treatment failure.
Demlová et al Ther Drug Monit  Volume 00, Number 00, Month 2019
6 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
measurement, the mean TTL was 50.4 ng/mL (ranging from
44.2 to 57.6 ng/mL) with 5 (18.5%) and 2 (7.4%) patients
being below and above the proposed TTL target range,
respectively. Thus, the proportion of the patients within the
target range increased from 26% to 74%. The unusually low
proportion of patients in the target range at the start of the
study was probably caused by the selection bias. First, only
the minimal effective target levels were known from the previous
studies, and at the beginning of each study period,
TDM was not part of the routine care but was reserved for
the cases of suspected toxicity or suboptimal drug response.5
On the other hand, in a recent small study of Takasaki et al109
performed on patients with mRCC, better clinical outcomes
(TTP and PFS) were observed in the group with TTL ,50 ng/
mL than in the group with TTL .50 ng/mL.
Summary
Most studies with patients with mRCC and GIST
confirmed better efficacy of sunitinib when TTLs were greater
than 50 ng/mL. There were also reported better outcomes in
combined AUC0–24h greater than 600 and 800 ng/mL$h for
patients with mRCC and GIST.
Exposure–Toxicity Relationship
Sunitinib exposure (AUC) or TTL has been associated
with various kinds of toxicity. In one of the first studies with
sunitinib, most patients with dose-limiting toxicity had Ctrough
greater than 100 ng/mL. Furthermore, a correlation between
an advanced age and sunitinib exposure with grade $3 toxicity
and N-desethylsunitinib exposure with grade $2 thrombocytopenia
has been reported.105
In a phase I dose-escalation study conducted by Faivre
et al,63 the occurrence of dose-limiting toxicities was associated
with sunitinib Ctrough .100 ng/mL. Patients with TTL $100
ng/mL had a higher incidence of grade $3 toxicity in a retrospective
observational clinical study by Noda et al.38 Significantly
higher incidence of grade $2 thrombocytopenia and
grade $2 hypertension in patients with TTL $90 ng/mL than
those with TTL ,90 ng/mL was identified in a study by Mizuno
et al.29 In this study, the authors reported a hazard ratio of
3.0 for high versus low AUC with a threshold of 2600 ng/
mL$h for a shorter treatment period without discontinuation or
dose reduction. Similarly, a PK simulation in a study conducted
by Nagata et al110 indicated that the sunitinib Ctrough
should be ,100 ng/mL to avoid severe thrombocytopenia.
In the study by Teo et al,69 patients who manifested
grade $2 mucositis and altered taste had higher TTLs than
those who had grade 1 or no toxicity. In the prospective study,
Bello et al111 reported an association between sunitinib exposure
and prolongation of the QT interval. Sunitinib Ctrough was
positively correlated with the occurrence of fatigue.30,61 Furthermore,
the increased sunitinib exposure was negatively correlated
with changes in the absolute neutrophil count and
positively with changes in diastolic blood pressure. However,
at a dose of 50 mg/d (4/2), these effects were generally mild to
moderate and easy to manage.61 In a prospective PK study by
Kloth et al,30 patients with grade $3 toxicity had a significantly
lower clearance of sunitinib than patients without grade $3
toxicities. In the review of de Wit et al,65 there was a positive
correlation between Ctrough and increase in diastolic blood pressure,
AUC0–24, and fatigue, and a negative correlation between
cumulative (per cycle) AUC and grade $2 absolute neutrophil
count. Grade $2 thrombocytopenia was also significantly more
frequently observed in patients with sunitinib Ctrough .90 ng/
mL than in the patients with sunitinib Ctrough ,90 ng/mL
(100% versus 53%). In line with these findings, a combined
AUC0–24 value was independently associated with any grade
$3 toxicity. Receiver operating characteristics revealed a combined
AUC0–24 threshold of 2150 ng/mL$h for grade 3 toxicity.11
However, in the toxicity-based dosing study of Lankheet
et al,4 the initial proportion (before any dose modification) of
patients with toxicity was higher in the group with TTL ,50
ng/mL than in the group with TTL .50 ng/mL. The exposure–
toxicity relationship studies are summarized in Table 2.
Practical guidelines for TDM of TKi by Yu et al106
proposed a dosing schedule of 50 mg/d (4/2) with TTL
.50 ng/mL and 37.5–50 ng/mL as a PK target in RCC and
GIST, respectively. Recent practical recommendations for
TDM of kinase inhibitors by Verheijen et al112 proposed
a similar target TTL ($50 ng/mL for intermittent dosing
and $37.5 ng/mL for continuous dosing). Based on the toxicity
studies, a value of 100 ng/mL should be considered as
upper TTL.
Sunitinib SmPC,3 European Society for Medical Oncology
Clinical Practice Guidelines,2 National Comprehensive
Cancer Network guidelines,16 or European Association of
Urology guidelines60 do not mention the determination of
sunitinib plasma levels as a possible step in a dose individualization
strategy.
The therapeutic goal in the treatment of mRCC is the
prolongation of survival. Thus, it is critical to maximize the
chance for response by consecutively reaching and maintaining
adequate drug exposure and keeping treatment-related
toxicities at an acceptable level while increasing the drug
adherence and overall time on treatment. TDM may include
prevention of treatment failure or severe toxicity as a consequence
of inadequate drug exposure, avoidance of low
exposure in renal or hepatic impairment (including patients
in continuous renal replacement therapy), and management of
PK variability caused, for example, by drug–drug interactions
or suspected genetic polymorphism and concerns over adherence
to treatment. TDM could also be helpful in unique populations,
such as in age or weight extremes (children, elderly,
patients with limb amputation, or obese patients), or rare
ethnic/genetic groups of patients.
Summary
The promising benefit/risk ratio occurs when TTL are
below 100 ng/mL or combined AUC0–24h below 2150 ng/
mL$h.
Population PK–PD Modeling for Prediction of
Sunitinib Exposure
Several population-based PK models for individual
prediction of plasmatic concentrations of sunitinib have been
developed. They involve either a 1- or 2-compartment model
for sunitinib or a 2-compartment model for N-desethylsunitinib.23,61,107,113
Some of these models also include various
Ther Drug Monit  Volume 00, Number 00, Month 2019 A Review of TDM of Sunitinib in GIST and mRCC
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TABLE 2. An Overview of Exposure–Toxicity Relationship Studies
Tumor Type n PK Parameter Outcome Correlation Significance Reference
Solid 28 TTL .100 ng/mL Dose-limiting toxicity (DLT) Most patients with DLT had
TTL .100 ng/mL
N/A 63
mRCC 19 TTL . or #90 ng/mL Grade $2 thrombocytopenia 100% versus 55.6% P = 0.033 29
Grade $2 hypertension 90% versus 22.2% P = 0.0055
Solid, GIST, and
mRCC
443 Combined AUC0–24 Fatigue A positive correlation between
AUC0–24 and the incidence
of fatigue
N/A 61
Combined AUCcum28 ANC ANC decreased linearly with
increasing AUCcum28
N/A
TTL dBP dBP increased linearly with
increasing TTL
N/A
Solid 24 TTL $ versus , 180 ng/
mL
QTc prolongation 15.4 versus 9.6 milliseconds N/A 111
pNET, GIST, and
mRCC
52 CL/F Grade 3 toxicity 34.4 versus 41.4 L/h for
patients with versus without
grade 3 toxicity
P = 0.025 30
TTL Fatigue A positive correlation between
Ctrough and the occurrence of
fatigue
P = 0.007
mRCC 36 TTL Grade $2 versus grade #1
mucositis
126.46 versus 84.81 ng/mL for
patients with grade $2
versus grade #1 toxicity
P = 0.04 69
Altered taste 159.91 versus 105.22 ng/mL
for patients with grade $2
versus grade #1 toxicity
P = 0.05
RCC 21 TTL . or ,100 ng/mL Grade $3 toxicities Patients with .100 ng/mL TTL
(n = 8), compared with
patients with ,100 ng/mL (n
= 13), had a greater
incidence of grade $3
toxicities (6 patients, ie,
75.0% versus 3 patients, ie,
23.1%).
N/A 38
RCC 6 TTL Grade $2 thrombocytopenia PK/PD simulations indicate
that TTL should be ,100 ng/
mL to avoid severe
thrombocytopenia
N/A 110
Advanced 29 TTL Grade $3 toxicity (CTCAE
4.02)
TTL ,50 ng/mL N/A 4
17.2% no severe toxicity,
34.5% severe toxicity
TTL $50 ng/mL 28.6% no
severe toxicity
20.7% severe toxicity
mRCC, GIST 29 TTL Serum AST and ALT activity Higher plasma concentration
associated with higher AST
and ALT activity
N/A 35
Advanced solid 105 Combined AUC0–24 Any grade $3 toxicity Combined AUC0–24 associated
with grade 3–4 toxicity
P , 0.0001 11
Advanced/metastatic
solid
92 Sunitinib AUC Any grade $3 toxicity Sunitinib AUC independently
associated with any grade
$3 toxicity [OR = 1.16
(1.05–1.28)]
P = 0.005 105
N-desethylsunitinib
AUC
Grade $2 thrombocytopenia N-desethylsunitinib AUC
independently associated
with any grade $3 toxicity
[OR = 1.27 (1.03–1.57)]
P = 0.028
mRCC 26 Sunitinib Ctrough Grade $1 HFS Higher sunitinib Ctrough
associated with HFS (89.5
versus 48.3 ng/mL)
P , 0.01 67
Demlová et al Ther Drug Monit  Volume 00, Number 00, Month 2019
8 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
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covariates, which help in the explanation of interindividual
variability in plasmatic concentrations, for example, gene
polymorphism of ABCG2 and lean body mass.29,105 The univariate
analysis showed that single nucleotide polymorphisms
of CYP3A4, CYP3A5, and ABCB1 affected sunitinib and
N-desethylsunitinib clearance, but the effect was lower than
the interindividual variability.6 A study of Theo et al114 confirmed
that CYP3A5 did not play a significant role in sunitinib
exposure. This was not surprising since CYP3A5 barely
contributes to the overall sunitinib clearance.53 PK–PD models
were also developed to predict the clinical benefit of sunitinib
TDM.
Goulooze et al107 performed Monte Carlo simulations
with modified Weibull models to generate an individual survival
function (TTP) in virtual patients receiving 37.5 mg/d of
sunitinib continuously. The simulation revealed that about
half of the patients on a daily continuous dose of 37.5 mg
had Ctrough below the target values and decreased to 16% after
a 2-step dose increase based on TDM. The authors also
included covariates of the dose-limiting toxicity and the dropout
rate in the model. These virtual patients were not meant to
be eligible for the TDM-guided dose increase despite the
predicted Ctrough below the target 50 ng/mL value. The authors
finally concluded that TDM might increase the TTP by
approximately 1–2 months in eligible patients and estimated
that the number of patients required in the clinical study to
prove their results using real data was approximately 1600.
These simulations were partially confirmed by a small
study with advanced solid tumor patients (n = 43, PK data
evaluated in 29 subjects), of which, 15 (52%) had TTL below
the target 50 ng/mL.4 Target TTL without any additional
toxicity was reached in 5 patients (17%).
The models mentioned above, provided they are
implemented in related software tools (eg, MWPharm,
BestDose, TDMx, DoseMe, NextDose, and TUCUXI), may
significantly help with dose individualization according to the
observed plasma concentration levels (model-based dosing).
None of the studies included in this review used such tools to
individualize sunitinib dosing before the measurement of real
plasma levels. Unfortunately, because of the occasional
reporting of high intraindividual variabilities in Ctrough,35 dose
modification or routine TDM based on a single-point concentration
remains challenging. However, as sunitinib exposure
strongly correlates with the midazolam exposure,54 midazolam
might be used as a phenotypic probe to predict the metabolic
clearance of sunitinib. Nevertheless, such a prediction
remains a rough estimate of real sunitinib and N-desethylsunitinib
exposure.
Summary
Population models may become a valuable tool in the
prediction and dose adjustment based on TDM. However,
currently, there is no valid PK model for sunitinib implemented
as a software tool.
CONCLUSIONS
The widely accepted definition for personalized pharmacotherapy
emphasizes treating the right patient with the
right drug in the right time using the right dose. In a clinically
apparent malignant disease, we need to identify the disease/
tumor-dependent variables as well as the host dependent
variables, as each plays a key role during the clinical course
of the disease. Both variables are essential, and the concept of
personalized medicine is, therefore, needed to be perceived as
the “all-in-one” concept. A definition of personalized medicine
either as “targeted therapy” with tumor-dependent variables
or “targeted dosing” based solely on pharmacogenomics
would be an oversimplification.115
In real clinical practice, the statement to administer “the
right drug to the right patient” refers mainly to the tumordependent
variables, which nowadays are one of the most
studied pathological parameters and treatment predictive biomarkers.
With current technologies enabling an examination
of the cancer genome and interpatient variation in germline
DNA, we are in a much better position than ever in terms of
selecting patients for targeted therapies.116
Not only “the right drug to the right patient” but also
tailoring drug dosages and schedules to optimize the PK profiles
is equally important. This part of the treatment individualization
is assessed mainly in the early phase of the drug
development but is usually missing in clinical practice. In the
review of de Wit et al,65 the authors concluded that sunitinib
would, after imatinib, be another great candidate, and with the
most evidence, for dose individualization based on TDM.
Despite the evidence-based clinical efficacy of sunitinib, its
TABLE 2. (Continued) An Overview of Exposure–Toxicity Relationship Studies
Tumor Type n PK Parameter Outcome Correlation Significance Reference
Sunitinib Ctrough Grade $1 thrombocytopenia Higher sunitinib Ctrough
associated with
thrombocytopenia (89.5
versus 50.8 ng/mL)
P = 0.02
TTL Grade $1 HFS Higher TTL associated with
HFS (75.2 versus 64.2 ng/
mL)
P , 0.01
TTL Grade $1 thrombocytopenia Higher TTL associated with
thrombocytopenia (64.4
versus 75.2 ng/mL)
P = 0.01
ANC, absolute neutrophil count; ALT, alanine aminotransferase activity; AST, aspartate aminotransferase activity; AUCcum28, cumulative 0–28 day AUC of sunitinib + Ndesethylsunitinib;
dBP, diastolic blood pressure; OR, odds ratio.
Ther Drug Monit  Volume 00, Number 00, Month 2019 A Review of TDM of Sunitinib in GIST and mRCC
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severe toxicity in some patients has become an essential issue
in clinical decisions regarding proper treatment. High exposure
to sunitinib might be one of the reasons for the severe
toxicity of sunitinib. Hence, TDM should/could be undertaken
in all patients or at least in a subset of patients subjected
to dose extremes and/or in patients with an “unmatched”
toxicity (high dose-low toxicity or low dose-high toxicity).66
Another proposed method for dose individualization
using toxicity as a replacement for systemic exposure is
TAD.117,118 Such a method has been suggested for axitinib
using hypertension as the target toxicity, and for sunitinib using
a broader range of toxicities.119,120 A similar approach was used
in the study of Maráz et al,121 in which the dose escalation was
conducted in patients with a slight disease progression. The
clinical outcome (PFS and OS) was better in the group undergoing
the dose escalation strategy than in the control group (P =
0.009 and 0.016 for PFS and OS hazard ratio, respectively).
Similar results were also obtained in the study of
Raphael et al,122 where an additional benefit was observed after
the dose escalation in patients who had previously progressed
from the disease. The occurrence of targeted toxicity from
sunitinib (HFS, neutropenia, hypertension, or thyroid dysfunction)
was associated with greater OS. Thus, toxicity may serve
as a surrogate marker of the treatment outcome, and TAD may
thus be an alternative to TDM-guided dosing.47,119,123
In our opinion, the approach of TAD would not be the
best option in the light of the currently known therapeutic
reference range in the treatment of GIST and mRCC, at least
for patients who do not perceive any toxicity. Another group
which could also benefit from TDM, instead of TAD, includes
patients with severe toxicity ($3), where the TDM-guided
dose de-escalation could be achieved more precisely and preserve
therapeutically relevant plasma concentrations.
In our opinion, and in agreement with other authors,35
the greatest benefits of TDM could be achieved in patients
above the target Ctrough, with or without toxicity, and in patients
below the target Ctrough experiencing no toxicity. These
groups are perfectly eligible for TDM-guided dose modification.
The question is whether the patients with plasma levels
above the target Ctrough range and no toxicity would benefit
from a TDM-guided dose decrease or whether TAD would be
a sufficient alternative.
There is probably no benefit of TDM in patients with or
without toxicity while within the target Ctrough. In the group
of patients experiencing toxicity while below the target
Ctrough, dose modification is inadequate and alternative drug
treatment would be optional. The groups of the patients which
could benefit from TDM of sunitinib are summarized in the
Table 3. Sunitinib TDM should/could be performed if drug–
drug interactions were present.
Despite drug exposure definitely being one of the key
factors influencing treatment outcomes, many other factors,
such as time from diagnosis to treatment, genetic polymorphisms
(both tumor and “host-related”), pretreatment levels of
circulating ligands of regulating receptors, some clinical factors
(activity of the lactate dehydragenase, hemoglobin concentration,
and performance status), or complex clinical score
MSKCC (“Motzer score” defined by Motzer et al124 at the
Memorial Sloan Kettering Cancer Center) may play a role.
The high cost of targeted agents and the increasing use of
indications based on tumor molecular pathology rather than its
location provide another reason to ensure an optimal dose for
each patient. There has been sufficient evidence that TDMguided
dose modifications of sunitinib could provide considerably
better treatment outcomes while simultaneously preventing
its toxicities. TTLs should be used as a response for
sunitinib exposure, with the target range being 50–100 ng/mL.
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5 Conclusion
Mass spectrometry has been part of modern chemistry regardless of the focus field. Its capability to identify,
characterize, quantify, and hyphenate is unparalleled across other methods. Although no mass spectrometer can
provide maximum performance in all tasks simultaneously, a suitable combination of a ionization technique and a
mass analyzer helps overcome the significant challenges of current science. It is worth noting that possession of the
instrument itself is not sufficient; a skillful operator capable of acquiring, interpreting, and maintaining the entire
workflow is mandatory.
All the data gathered by applying mass spectrometry in this work represent a collection of individual inferences
and conclusions, which would have taken much longer if mass spectrometry had not been accessible. The first part
dealt with the hyphenation capabilities of MS and demonstrated that even in the world where majority of LC is
online-coupled to ESI MS, the offline coupling has its place, and it is irreplaceable in specific types of applications.
The second part employed MS in the molecular composition and structure investigation to uncover either new
molecules or novel processes associated with them. MS is perfectly suited for identification of known unknowns with
the help of computational in silico database matching and prediction. The third part utilized MS as a sensitive and
a specific detector, which could substitute several different detection techniques simultaneously. Mass spectrometry
helped to reject the long-term downtrend that if a detection technique is universal (such as refractometric or
conductometric detection) it is associated with low sensitivity and specificity. Nevertheless, quantitation studies
using any method provides inferences in the small world only; navigation to the large world via generalization
remains the central challenge of statistical modeling.86
122
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