High Performance Liquid Chromatography Determination of Chlorpropham and Its Metabolites in Isolated Rat Hepatocyte Incubations

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Abstract
Free chlorpropham (CIPC) and its metabolites (4-hydroxy CIPC, 3-chloroani-line and 3-chloroacetanilide) were separated by reverse phase high performance liquid chromatography (H.P.L.C.) using an isocratic procedure. The separation was used to quantify the metabolism of CIPC in rat hepatocyte incubations. The free metabolites were diethylether-extracted at pH 7.4. The sulfate and glucuronide conjugates of 4(OH)CIPC were enzymatically split and the aglycone determined by HPLC. Beside the unmetabolized CIPC which accounts for 33% of the dose, 4(OH)CIPC as the sulfoconjugate and glucuroconjugate represents 35% and 20% respectively, while free 4(OH)CIPC accounts for only 3%. About 10% of the CIPC is hydrolytically split yielding 3-chloroaniline (2%) which further transformed into 3-chloroacetanilide (5%). In spite of the great sensitivity of the proposed method (0.2 μg/ml incubation medium) no other metabolites were recorded. This extraction and separation procedure could be applicable to future metabolic studies in cellular incubation systems.