A TLC fluorescence derivatization assay for chlorpromazine and its non-conjugated hepatic microsomal metabolites

Procedures for the assay of chlorpromazine (CPZ) and its metabolites were compared. An improved assay, involving organic extraction, thin-layer chromatography and fluorescence derivatization, was developed. The compounds were extracted from microsomal protein suspensions into 15% n-propanol in dichloromethane by successive extractions at pH 2 and pH 12. CPZ and its mono- and di-desmethyl, 7-hydroxy, N-oxide, sulphoxide and N-oxide-sulphoxide metabolites were separated using TLC on silica gel developed with methanol-acetone-ammonia (50:50:1 v/v/v). The compounds were extracted from the TLC plate with chloroform-methanol (2:1 v/v) and subjected to oxidation with hydrogen peroxide. The highly fluorescent oxidized derivatives were identified as sulphoxides by their fluorescence characteristics. Amounts of derivatized CPZ and metabolites were measured using derivatized promazine as the internal standard.