Direct assay of uroporphyrin and coproporphyrin in human urine by reverse-mode field amplified sample injection-sweeping and micellar electrokinetic chromatography

Abstract

In this study, an on-line stacking capillary electrophoresis (CE) method, reverse-mode field amplified sample injection equipped with sweeping micellar electrokinetic chromatography (RMFASI-sweeping MEKC), was established for direct determination of uroporphyrin and coproporphyrin in human urine. Porphyrins playing a very important role in the biosynthesis of heme, chlorophyll and other important enzymes are a series of important molecules in organism. Therefore, determination of porphyrin metabolites, uroporphyrin and coproporphyrin, was very important for clinical survey of some diseases. In this study, the urine sample after simple dilution could be directly analyzed by this on-line stacking CE method. The optimal CE separation buffer was 70mM phosphate buffer at pH 3. Before sample injection, a water plug was introduced (2.5psi for 10s), and then the samples were loaded by electrokinetic injection (−10kV, 200s). Finally, the phosphate buffer (70mM, pH 3) containing 100mM SDS was served as the sweeping buffer to stack and separate the analytes at −20kV. The calibration curves were linear over a range of 15–200ng/ml for uroporphyrin, and 300–1000ng/ml for coproporphyrin. The coefficient of correlation (r) in intra-batch (n=5) and inter-batch (n=5) analysis was above 0.983. The LODs (S/N=3) were 5 ng/ml for uroporphyrin, and 100ng/ml for coproporphyrin. The absolute values of relative standard deviation (RSD) and relative error (RE) in intra-batch (n=5) and inter-batch (n=5) assays were less than 8.6% showing the good precision and accuracy. The stacking method was successfully applied in real urine sample and feasible for serving as a tool for detection of uroporphyrin and coproporphyrin in clinical.