Optimization of capillary electrophoretic separation electrolytes for simultaneous C^4D and LIF
detection of biologically important ions.

Júlia Lačná^1,4, Norio Teshima^3, Hiroya Murakami^3, František Foret^1,2, Jan Přikryl^2 and Petr
Kubáň^1,2


^1Bioanalytical Instrumentation, CEITEC Masaryk University, Veveří 97, 602 00, Brno, Czech Republic

^2Department of Bioanalytical Instrumentation, Institute of Analytical Chemistry of the Czech
Academy of Sciences, Veveří 97, 602 00, Brno, Czech Republic

^3Aichi Institute of Technology, Department of Applied Chemistry, Toyota 4700392, Japan

^4Department of Chemistry, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

Contact information: Júlia Lačná, juliahod@gmail.com


Capillary electrophoresis (CE) offers a unique separation platform for the analysis of biological
fluids due to its high separation efficiency, short analysis times and low sample volume
requirement. The complexity of biological samples (such as exhaled breath condensate, saliva),
however does not allow complete analysis of compounds of interest, for instance markers of
nitrosative and oxidative stress, using a single detection mode. Nitrite and nitrate, present in
biological fluids as metabolites of nitrosative stress promoter NO[x], are typically at micromolar
concentrations, while glutathione (GSH) is present at low nM concentrations. Simultaneous analysis
of complete biomarker pattern is therefore not possible using single detection approach.

In this work we present an attempt on developing an optimized background electrolyte for
simultaneous separation and detection of small ionic species including nitrosative stress markers
nitrite and nitrate, inorganic cationic species and GSH as an oxidative stress marker using dual -
capacitively-coupled contactless conductivity detector (C^4D) and laser induced fluorescence (LIF)
- detection mode. By applying the double opposite end injection, we achieve efficient separation of
small anionic and cationic analyte groups, including the fluorescently labelled GSH with low limit
of detections. Optimized background electrolyte (BGE) containing 30mM HEPES, 15mM TRIS, 2 mM
18-crown-6 offered an efficient separation of complete biomarker pattern in a single run. Using
only one CE system decreases the time for analysis, consumption of samples and chemicals and the
instrumental complexity. This work offers new insights into selection and design of BGEs for
simultaneous separations by CE, especially in cases when C^4D is combined with LIF.