The solvent with the highest recovery, as determined in the experiments outlined above, was used for comparison with a 10% methanol solubilization method. Three groups (n = 6) of pooled samples were weighed (25- to 30-mg portions) in 13 X 100-mm borosilicate tubes. For extraction of the first set, 1 ml of 10% methanol was added to feces to create a slurry. The samples were capped and shaken at room temperature (22°C to 25°C) for 16-24 h, then centrifuged for 30 min at 2000 X g The supernatant was decanted and saved. For extraction of the second set, 50 ^l 10% methanol was added to feces as a wetting agent. The samples were capped and set at room temperature for 16-24 h, then extracted two times with 4 ml ether and reconstituted in buffer (as described for the previous experiment). Samples in the last set were extracted two times with 4 ml ether and reconstituted in buffer A 20-^l aliquot of each reconstituted extract was measured with the ELISA. An additional 60-min extraction was performed on all of the samples with 4 ml ether, and these extracts were reconstituted in buffer A 20-^l aliquot of each of these reconstituted extracts was measured with the ELISA. ampicillin antibiotic
To determine the amount of enzyme-hydrolyzable testosterone conjugates, a subsample was buffered with 0.2 M acetate and then subjected to enzyme hydrolysis using (3-glucuronidase (Sigma). A combination of 250 units glucuronidase containing 13 units sulfatase was added to 20 mg fecal samples, incubated at 37°C overnight, extracted with ethyl ether as above, and then measured with the ELISA.