Use of a direct high performance liquid chromatography method for multiple testosterone hydroxylations in studies of microsomal monooxygenase activities

A high performance liquid chromatography method is described for separation of the products of testosterone monooxygenation. The method involves direct injection onto a reverse phase octadecylsilane column of the supernatant from incubations containing as little as 25 μg microsomal protein. Isocratic elution with formate buffer/acetonitrile and detection at 254 nm permits the separation and simultaneous quantitation of multiple discrete testosterone-derived peaks on the chromatogram. The production of the eight major oxygenated testosterone metabolites in hamster liver microsomes was determined. This profile was altered uniquely after pretreatment with either phenobarbitone, β-naphthoflavone, rifampicin or pregnenolone 16α-carbonitrile. These changes reflected analogous dissimilar effects of the enzyme inducers on the metabolism of the exogenous cytochrome P-450 substrates aldrin, acetanilide, benzo[α]pyrene and ethylmorphine. Therefore, the testosterone assay provides a sensitive and effective method for separating and quantitating testosterone monooxygenation products and may offer a single alternative to the use of multiple exogenous substrates for categorizing and defining the metabolic activity of cytochromes P-450.