Chloride present in biological samples as a tool for enhancement of sensitivity in capillary zone electrophoretic analysis of anionic trace analytes

Abstract

Effects originating from the variability of the sample matrix can be efficiently eliminated when the separation conditions are selected so that compounds of like charge with high concentration referred to as macrocomponents are embodied into the system of transient isotachophoresis. For stacking and separation of anionic trace analytes in biological samples, the presence of chloride is shown to be important to balance out effects of other macrocomponents that act against isotachophoretic stacking. Having acetoacetate, malate, citrate, and some drug metabolites in untreated human serum samples, the stacking mechanism of these compounds in an electrolyte system comprising 5 mM mandelic acid and epsilon -aminocaproic acid, pH 3.8, is explained. Analytes are monitored by indirect UV-absorption detection. Attention is paid to the minimum chloride concentration required with respect to the concentration ratio of phosphate (stacker) and lactate (destacker) present in the sample so as to ensure both stacking and separation of trace analytes. Insight into the separation process is given both with computer simulations and experiments. For selected analytes, the effect of chloride concentration on quantitative evaluation, sensitivity and limit of detection is demonstrated as well. Moreover, the applicability of the mobility window between phosphate and lactate for an additional group of metabolites is sketched.

Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking

“…5A). Chloride and phosphate present in serum were previously identified as being beneficial for the stacking of analytes of like charge for which they act as transient stackers [33,34]. These two anions were also found to enhance the stacking of the two disaccharides.…”

“…Human serum contains a high concentration of salts and this precludes online preconcentration techniques based on conventional sample-stacking or field-enhanced sample injection which require low salt matrices for efficient analyte focusing. Transient ITP or sample self-stacking [29][30][31][32][33][34], formation of a transient moving chemical reaction boundary [35], sweeping [36][37][38], and use of a dynamic pH junction [39], however, are online concentration techniques that can be used to temporarily induce narrow analyte bands in the presence of large amounts of salts. In the present study, electrokinetic sample injection together with sample stacking was studied for high-sensitivity analysis of di-HA and di-CS in model mixtures and human serum digested by chondroitinase ABC using a borate buffer at alkaline pH, reversed electroosmosis (EO) provided by addition of CTAB, and direct UV detection at 232 nm.…”

Dynamic computer simulations of electrophoresis: A versatile research and teaching tool

“…For biological and environmental samples such as serum, urine and seawater, the presence of high concentrations of chloride is perfect to function as a suitable leading electrolyte for the ITP step, and all that is required to ensure that an appropriate terminator is used and that there is sufficient time to complete the ITP stage and then to destack and separate the components by ZE. Transient ITP (tITP) systems with samples naturally abundant in chloride have been the basis of several studies, the most significant belonging to Gebauer et al for the analysis of biological samples with tITP or originally referred to as sample self stacking [196][197][198][199]. Their first report [196] presented a Electrophoresis 2010, 31, 726-754 simple theory to provide explicit description of various zone parameters such as detection time, variance and resolution, which was confirmed by computer simulation using the GENTRANS software.…”

“…This work was later continued to look at the concentrations of components required to induce tITP stacking, with the concentration of the sample macrocomponent or stacking ion required to be above a critical concentration [197], and to study the effect of multiple macro-components upon stacking and conditions under which one of them would function as a destacker [198]. Using the concepts elucidated in this work, Křivánková et al [199] performed simulations to examine the conditions required to induce tITP stacking of minor components in serum (citrate, malate and acetoacetate) at various phosphate to lactate ratios (the three minor anions migrate between phosphate and lactate), with simulation results for different amounts of salt in the sample shown in Fig. 19.…”

“…The conditions for panels B and C represent those for analysis of citrate, malate and acetoacetate in a typical human serum; from Ref. [199].…”