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).
Iwata
J
J
Department of Clinical Biochemistry, Tokyo College of Pharmacy, Japan.
Suga
T
T
D015068:17-Ketosteroids / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D003480:Cushing Syndrome / Q000652:urine; D003619:Dansyl Compounds; D005470:Fluorometry; D005965:Glucuronates / Q000652:urine*; D006801:Humans; D006834:Hydrazines; D006863:Hydrogen-Ion Concentration; D006929:Hyperaldosteronism / Q000652:urine; D008297:Male; D000644:Quaternary Ammonium Compounds; D012997:Solvents; D013431:Sulfates / Q000652:urine*
D015068:17-Ketosteroids; D003619:Dansyl Compounds; D005965:Glucuronates; D006834:Hydrazines; D000644:Quaternary Ammonium Compounds; D012997:Solvents; D013431:Sulfates; C027913:dansyl hydrazine; C027435:tetrapentylammonium