High performance liquid chromatography of steroid metabolites in the pregnenolone and progesterone pathways

Rapid separation of gonadal steroids in the progesterone (delta 4) and pregnenolone pathway (delta 5) has been accomplished by the use of high performance liquid chromatography (HPLC). Two HPLC systems are utilized. THe first requires the use of two separate radial compression columns (C-18 and C-8), with steroids being eluted with a methanol-water gradient. The second employs a stainless steel C-18 (reversed phase) column with a 12% octadecylsilane coating. The latter system separates seven of the eight steroids in the delta 4 and delta 5 pathways in thirty-five minutes. For the quantitation of steroids directly, integration of the peak areas, using 254 nm absorption for the delta 4 pathway steroids (5 ng minimum limit), and 210 nm absorption for the delta 5 pathway steroids (25 ng minimum limit) is used. For the quantitation of radiolabeled metabolites resulting from incubation of gonadal tissue with radiolabeled steroid precursors, either one of two methods is used: (1) the eluent can be recovered from the HPLC using a fraction collector, and counted in liquid scintillation counter or (2) the entire eluent (or a portion of it) can be counted immediately by directing the flow through a radioactivity detector.