Two solvent extractions were compared to determine the optimum method for extracting testosterone from mouse feces. The first was 80% pentane/20% ethyl acetate, previously used by our laboratory for serum testosterone extraction. The second was ethyl ether, a solvent commonly used for steroid extraction. Aliquots of 100 ^l [3H]testosterone (VWR Scientific) in ethanol were added to pooled fecal samples (n = 5) and empty borosilicate tubes (n = 3) as controls, then dried overnight. Each tube had 3.8 ml solvent added before vortexing for 60 min on a Thermolym Maxi-Mix III (Dubuque, IA) at medium speed (1100 rpm). After vortexing, 250 ^l water was added and the tubes were placed in a dry ice and methanol bath. antibiotics levaquin
The organic layer was decanted from the frozen aqueous phase and placed in a second borosilicate tube. The extraction was then repeated for an additional 30 min. The solvent extract was evaporated in a bath at 37°C. After the second extraction, a 4-mm glass bead (Fisher) and 2 ml buffer were added to each extract tube and vortexed for 60 min at medium speed. An aliquot from each tube was transferred to a scintillation vial (Packard, Downers Grove, IL), and after addition of 4 ml triton-toluene scintillation fluid, radioactivity was counted for 10 min using a Packard Liquid Scintillation Counter (LSC) (Tri Carb 1600 CA). The counts were compared to dpm added to determine percentage recovery.