Capillary zone electrophoresis of orotic acid in urine with on-line isotachophoresis sample pretreatment and diode array detection

Potentialities of capillary zone electrophoresis with on-line isotachophoresis sample pretreatment and diode array detection (ITP–CZE–DAD) to the separation, detection and identification of trace analytes present in biological matrices were investigated. Urine represented a multicomponent, variable and high ionic strength matrix while orotic acid was chosen as a model analyte of a practical clinical relevance in this investigation. Using the ITP–CZE combination in the column-coupling configuration of the separation system ITP provided an enhanced sample load capacity to the separation system (a 30 μl sample injection volume), concentrated the analyte and served as an on-line sample clean up technique. On the other hand, CZE performed a final separation of the analyte from matrix constituents present in the ITP pretreated sample and provided favorable conditions for its detection and identification by DAD. Using current correction and smoothing procedures analytically relevant DAD spectra of orotic acid could be obtained also in instances when this was injected in a model sample at a 2·10−7 mol/l concentration (an estimated limit of determination of orotic acid at a 218 nm detection wavelength). ITP–CZE separations of urine samples (based on differences in acid–base properties and host–guest complexations of the analyte and matrix anionic constituents) led to significant sample clean ups. Consequently, DAD spectra of orotic acid matching its reference spectrum, could be acquired also in instances when the acid was present in urine matrices (loaded in 30 μl injection volumes of 20-fold diluted urine samples) at 4–6·10−7 mol/l concentrations. Here, residual trace matrix interferents prevented a closer approach to the above value attainable for model samples. Although this work was focused only on one analyte and urine matrix it implies very promising potentialities of the ITP–CZE–DAD combination in the identification and quantitation of trace analytes present in biological matrices, in general.