Development and Validation of a Fecal Testosterone Biomarker: MATERIALS AND METHODS(4)

MATERIALS AND METHODS(4)Extraction Efficiency

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.

This entry was posted in Testosterone and tagged Biomarker, Mus musculus, Peromyscus maniculatus, Testosterone.