Determination of orotic acid in urine by capillary zone electrophoresis in tandem-coupled columns with diode array detection

Analytical potentialities of capillary zone electrophoresis in the separation system with tandem-coupled columns to the spectral identification and determination of orotic acid (OA) in urine by diode array detection (DAD), coupled to the separation system via optical fibers, were investigated. A very significant “in-column” clean-up of OA from urine matrix was reached in the separation stage of the tandem by combining a low pH (2.8) with complexing effects of electroneutral agents [α- and β-cyclodextrins, poly(vinylpyrrolidone) and 3-(N,N-dimethyldodecylammonio)propanesulfonate]. Due to this, its DAD spectral data could be acquired in the detection stage of the tandem with almost no disturbances by matrix co-migrants. The concentration limits of detection obtained under such working conditions for a 200-nl sample load of OA and 320 μm I.D. capillary tubes were 3.5 μmol/l (218 nm) and 0.4 μmol/l (280 nm). Using chemometry procedures (target transformation factor analysis, fixed size moving window-evolving factor analysis, orthogonal projection approach and fixed size moving window–target transformation factor analysis) in processing of the acquired spectral data, the presence of OA in the loaded urine matrix could be confirmed with confidence when its concentration was 10 μmol/l or slightly less.