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

Abstract

Simple high performance liquid chromatography (HPLC) methods for the analysis of 4-chlorophenyl methyl sulphide (CPMS), diphenyl sulphide (DPS) and their corresponding sulphoxide and sulphone metabolites in rat liver microsomes are described. The assay methods are based on a reversed phase HPLC column (Spherisorb(R) 5 ODS, 15 x 0.46 cm) using a mixture of water and tetrahydrofuran (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 recoveries were more than 80%. The calibration curves for determining the sulphoxide and sulphone of CPMS or DPS were linear (r > or =0.995) in the range of 0-50 microg/ml and the assays were reproducible with low inter- and intra-assay variation of less than 13.5%. The lower limit of quantitation (LOQ) was 0.1 microg/ml for CPMSO and 0.025 microg/ml for CPMSO(2), diphenyl sulphoxide (DPSO) and diphenyl sulphone (DPSO(2)). The HPLC methods were successfully applied to measure enzymically formed CPMSO, CPMSO(2), 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 sulphoxides. About 20% of the initial CPMS (0.5 mM) concentration in the incubation was converted to the sulphoxide although the sulphone was not detected 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 DPSO(2). Eadie-Hofstee plot of CPMS sulphoxidation was biphasic. This suggests that the sulphoxidation of CPMS is a consequence of at least two enzyme systems, one characterized by low affinity and high capacity (K(m)=0.1 mM; V(max)=2.1 nmoles/mg protein/min) and the other by high affinity and low 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, respectively.