HPLC analysis of 4-chlorophenyl methyl sulphide and diphenyl sulphide and their corresponding sulphoxides and sulphones in rat liver microsomes

Simple high performance liquid chromatography (HPLC) methods for the analys is of 4-chlorophenyl methyl sulphide (CPMS), diphenyl sulphide (DPS) and th eir corresponding sulphoxide and sulphone metabolites in rat liver microsom es are described. The assay methods are based on a reversed phase HPLC colu mn (Spherisorb(R) 5 ODS, 15 x 0.46 cm) using a mixture of water and tetrahy drofuran (THF) as mobile phase at a flow rate of 0.5 ml/min and ultraviolet detection at 260 nm. The compounds were extracted into diethyl ether (2 x 5 ml) from rat liver microsomal incubation mixture (2 ml) and the recoverie s were more than 80%. The calibration curves for determining the sulphoxide and sulphone of CPMS or DPS were linear (r greater than or equal to 0.995) in the range of 0-50 mug/ml and the assays were reproducible with low inte r- and intra-assay variation of less than 13.5%. The lower limit of quantit ation (LOQ) was 0.1 mug/ml for CPMSO and 0.025 mug/ml for CPMSO2, diphenyl sulphoxide (DPSO) and diphenyl sulphone (DPSO2). The HPLC methods were succ essfully applied to measure enzymically formed CPMSO, CPMSO2, DPSO and DPSO 2 in rat liver microsomes and to characterise the Michaelis-Menten kinetics associated with the metabolism of CPMS and DPS and their corresponding sul phoxides. About 20% of the initial CPMS (0.5 mM) concentration in the incub ation was converted to the sulphoxide although the sulphone was not detecte d under these optimum-incubation conditions. Similarly, about 15-20% of DPS was converted to the sulphoxide while less than 0.1% of DPS was converted to DPSO2. Eadie-Hofstee plot of CPMS sulphoxidation was biphasic. This sugg ests that the sulphoxidation of CPMS is a consequence of at least two enzym e systems, one characterized by low affinity and high capacity (K-m = 0.1 m M; V-max = 2.1 nmoles/mg protein/min) and the other by high affinity and lo w capacity (K-m = 0.05 mM; V-max = 1.5 nmoles/mg protein/min). On the other hand, the Eadie-Hofstee plot of DPS sulphoxidation was monophasic with an apparent V-max and K-m of 1.8 nmoles/mg protein/min and 0.036 mM, respectiv ely. (C) 2002 Published by Elsevier Science B.V.