Liquid chromatography determination of the anti-androgen vinclozolin and its metabolites in rat serum

The objective of this study was to develop a chromatographic method for the analysis of the anti-androgen vinclozolin (V) and its metabolites 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) and 3,5-dichloroaniline (M3) in rat serum. V, M1–M3 were resolved using an HPLC gradient program with a mobile phase consisting of 60–75% methanol:acetonitrile (70:30) and 0.05 M monobasic sodium phosphate buffer pH 3.3 at 1 ml/min, a C18 column, and monitored at 212 nm. Incubates of 0.01 M monobasic potassium phosphate buffer (PB) pH 7.4 and rat serum were spiked with V and its metabolites and processed by diluting samples (1:4) with 0.1 M PB pH 3.3, to limit methodological hydrolysis of analytes, followed by addition of acetonitrile. Recoveries of V, M1 and M2 ranged from 85 to 105%, whereas recovery of M3 was <25%. V was hydrolyzed to M1 and M2 after incubation in PB pH 7.4 and rat serum, with M1 the predominant metabolite. This method was successfully applied in the analysis of V and its metabolites in the serum of a male rat after oral administration of V (100 mg/kg).