Determination of p-chloronitrobenzene and its metabolites in urine by reversed-phase high-performance liquid chromatography.

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method (HPLC) using ultraviolet detection was developed for the determination of p-chloronitrobenzene (p-CNB) and seven of its metabolites in rat urine. Analysis was performed before and after hydrolysis of the urine samples with acid to determine both free and conjugate forms of the metabolites. An equal volume of methanol was added to the urine sample and after centrifugation the mixed solution was injected into a high-performance liquid chromatograph. A column packed with 5-microns octadecylsilane (ODS) spherical particles was used at 30 degrees C. The metabolites were divided into three groups, and each group was subjected to three different mobile phase and detection wavelength conditions as follows: water-methanol (60:40, v/v) and 250 nm for p-CNB and 2,4-dichloroaniline; 0.005 M phosphate buffer (pH 3.6)-methanol (76:24, v/v) containing 1.2 mM sodium 1-octanesulphonate and 240 nm for p-chloroaniline, 2-chloro-5-nitrophenol, 2-amino-5-chlorophenol, p-chloroacetanilide and 4-chloro-2-hydroxyacetanilide; and 0.005 M phosphate buffer (pH 6.0)-methanol (80:20, v/v) and 340 nm for N-acetyl-S-(4-nitrophenyl)-L-cysteine. The response was linear at concentrations less than 200.0 micrograms/ml (r = 0.9998) for all metabolites, and the detection limits of each metabolite were between 0.05 and 0.2 micrograms/ml in non-hydrolysed urine. Analysis of the spiked samples demonstrated good accuracy and precision of the method in both intra- and inter-day assays. Storage stabilities of p-CNB and its metabolites at -20 degrees C, 4 degrees C and room temperature were examined for both neutral and acidic urine samples. This method was also shown to be applicable to toxicokinetic study of p-CNB following administration to rats.