Use of a direct high performance liquid chromatography method for multiple testosterone hydroxylations in studies of...

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 micrograms 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, beta-naphthoflavone, rifampicin or pregnenolone 16 alpha-carbonitrile. These changes reflected analogous dissimilar effects of the enzyme inducers on the metabolism of the exogenous cytochrome P-450 substrates aldrin, acetanilide, benzo[alpha]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.