Analysis of orotic acid in human urine by on-line combination of capillary isotachophoresis and zone electrophoresis

Abstract

The techniques of the on-line combination of capillary isotachophoresis with zone electrophoresis in two coupled capillaries (ITP–CZE) and a single capillary zone electrophoresis (CZE) were used for the sensitive determination of orotic acid (OA) in human urine. The simple CZE system was successfully applied for fast and reliable analyses of urine of healthy adult volunteers (the detection limit 1.7·10 −6 M OA, the total time of analysis 6 min). However, this method failed in analyses of OA in urine of ill children due to more complex matrix of the samples. Here, the ITP preconcentration and preseparation step coupled on-line with CZE proved to serve well with an electrolyte system developed and optimized for this purpose. The maximum selectivity and resolution of OA from other sample constituents in ITP–CZE was achieved by use of an electrolyte system of very low pH 2.15 both for ITP and CZE stage. The sensitivity of detection and simplicity of OA identification were enhanced by use of an external UV scanning detector. High sensitivity of ITP–CZE combination (limit of detection 3·10 −7 M OA), low sample consumption (1 μl), good reproducibility of migration times (inter-day RSD<1.86%) and acceptable reproducibility of the determination of OA in urine samples (average RSD=7.27%) make this technique suitable for routine determination of trace concentration of OA especially in urine of ill children under various pathological conditions and medication.