Signal amplification using deuterated water-based buffers in capillary
electrophoresis coupled to laser induced fluorescence detection
Josef Kučera1
, Štěpán Káňa1
, Andrea Celá2
, Ondřej Peš3
, Ivona Voráčová4
, Petr Táborský1
1Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
2
Department of Biochemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
3
Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
4
Department of Bioanalytical Instrumentation, Institute of Analytical Chemistry, Czech Academy of Sciences,
Veveří 967/97, 60200 Brno, Czech Republic
Many fluorescent molecules can be quenched by OH oscillators present in water. The Increase
of fluorescence intensity or lifetime can be obtained by replacing the light water for the deuterium oxide.
We can take advantage of this phenomena in some analytical techniques coupled to the fluorescence
detection (e.g. capillary electrophoresis, high performance liquid chromatography, flow cytometry,
fluorescence microscopy, etc.).
The capillary electrophoresis coupled with laser-induced fluorescence detection can be used
for the determination of anthracyclines and related compounds which are widely used drugs, especially
in cancer treatment. The steady-state and time-resolved fluorescence measurements of chosen
anthracyclines in light and heavy water were carried out on the spectrofluorometer. Those experiments
have proved that the emission signal and lifetimes of anthracyclines can be increased up to 4.5 times
using deuterium water as a solvent. Therefore the deuterated water-based buffers can be used
for the detection of anthracyclines by the capillary electrophoresis with LIF.
The CE-LIF separation of chosen molecules was successfully done in borate buffers prepared
in light and heavy water. The fluorescence signal in D2O based buffer increased up to 4.4 times for rhein.
Around 2 times higher fluorescence emission signal was recorded also for other studied anthracyclines
(doxorubicin, daunorubicin, epirubicin). The calculated limit of detection for rhein was 5.1ꞏ10-8
mol.l-1
and 5.0ꞏ10-9
mol.l-1
in light and heavy water buffer respectively. Over one order better limit of detection
is not caused only by reducing of the dynamic quenching but also lower noise level achieved
in the analysis in D2O buffer. The main advantage of capillary electrophoresis is the high sensitivity
combined with the lower consumption of sample and background electrolyte (about hundreds
of microliters) which means that only small volume of heavy water is needed for the analysis.