A normal phase high-performance liquid chromatography system for steroid 17 alpha-hydroxylase/C17-20 lyase (cytochrome P-450 21scc) assays

A normal phase HPLC system has been developed which is applicable to all of the steroid separations involved in the assay of steroid 17 alpha-hydroxylase (EC 1.14.99.9) and C17-20 lyase activities, in both the delta 4 (progesterone) and delta 5 (pregnenolone) pathways. A hexane-tetrahydrofuran (THF) gradient system is used with silica stationary phase and a flow cell radioactivity detector having a high efficiency for tritium. Folch extraction produces uniform extraction of substrates and products from the microsomal incubates, and this uniformity is maintained through HPLC separation and measurement. The hexane-THF mobile phase is convenient for product isolation and substrate purification and should be adaptable to other steroid separations. The system is especially useful for steroid enzyme assays utilizing radiolabeled substrates, since internal standards are not required for measuring recoveries of substrates and products.