Extraction of Steroids

Before processing samples, internal standards (Sigma Aldrich) for testosterone (T) (-23,4-¹³C₃), E1 (-23,4-¹³C₃), and E2 (D₅) were added to each tissue and plasma sample (E1 and E2). Calibration curves using T (Sigma Aldrich, catalog No. T037), E1 (Sigma Aldrich, catalog No. E-075), and E2 (Sigma Aldrich, catalog No. E-060) were used to determine the quantity of each steroid. Steroids were extracted from approximately 150 to 200 μL of plasma via vortex using 500 mL of MTBE (×2). The resulting upper phase was removed and placed into a glass vial where it was dried down using N₂. The dried-down steroids were derivatized as previously described using 1-methylimidazole-2-sulfonyl chloride (24). For tissue samples, approximately 150 mg of skeletal muscle (1.5 gastrocnemii) was cut into sections with a razor blade, weighed, and homogenized in 1 mL of acetonitrile for 3 minutes using a bead beater (Biospec Products). The samples were centrifuged for 5 minutes × 12 000g and the resulting supernatant was removed and placed into glass vials. Subsequently, the tissue samples were resuspended in 1 mL of acetonitrile, homogenized, centrifuged, and the supernatant collected for a second time. The supernatants were dried down using N₂ and were resuspended in 200 mM sodium acetate. MTBE was added to the sample for liquid-liquid extraction of the steroids (×2). The MTBE layer was removed and dried under nitrogen gas before derivatization as previously described (24). After derivatization, the samples were placed in 0.2 poly(vinylidene fluoride) micro spin filters (Fisher Scientific) and centrifuged to remove any insoluble material before mass spectrometry analysis. Analyte recovery for plasma and tissue was greater than 80%.