Separation of Free Steroid
Samples initially extracted with 10% methanol were reextracted using ethyl ether to obtain primarily unconjugated testosterone and nonpolar metabolites of testosterone. Extracts were concentrated into 2-ml vials, dried under nitrogen, and reconstituted in ethanol. HPLC separations were obtained using a Waters 5 X 100-mm Nova-Pac C18 column contained in an RCM Radial Compression Cartridge Holder (Waters, Milford, MA). A linear gradient of 20-75% acetonitrile (Fisher) in water at a 1 ml/min flow rate over 30 min, followed by 75% acetonitrile for 5 min, was used to separate testosterone metabolites. After separation on the C18 column, samples were passed through a UV detector to determine retention time of cold steroid samples or spiked 14C samples. HPLC elution fractions containing 3H-labeled and nonlabeled steroid standards were run to determine separation efficiency and retention times of the metabolite peaks. proventil inhaler
Samples were collected using a fraction collector. Half of each sample was measured on the LSC and the other half with the testosterone ELISA. Some samples were also measured using a Packard Flo-One Beta Series A-500 ra-dio-chromography in-line detector. The percentage unconjugated testosterone excreted in feces and the number of major metabolites were determined.