Direct determination of four sulfates and seven glucuronides of 17-oxosteroids in urine by fluorescence "high-performance" liquid chromatography.

We describe a direct method for determining four sulfates and seven glucuronides of 17-oxosteroids (17OS) in urine without hydrolysis, by use of "high-performance" liquid chromatography (HPLC) with fluorometric detection. After pretreatment of urine samples with a Sep-Pak C18 cartridge, four 17OS sulfates and seven 17OS glucuronides in the pretreated urine samples were reacted with tetrapentylammonium ions to form ion pairs. Ion-paired 17OS sulfates were extracted with benzene. By adding sodium sulfate to the remaining sample, we could then extract ion-paired 17OS glucuronides with dichloromethane. Each extract was labeled with dansyl-hydrazine in an acetic acid-acetonitrile solution. The labeled steroids were separated by HPLC on a reversed-phase Capcell-Pak C8 (silicon-polymer-coated silica gel modified with octyl groups). We monitored each effluent with a fluorometric detector (330 nmexcitation, 535 nmemission).