Amplifex keto reagent

Lipid extraction of powdered bone was performed by using 100% HPLC grade methanol from VWR BDH® Chemicals (Radnor, PA, USA). Isotopically labeled internal standards, d₄‐cortisol, ¹³C₃‐testosterone, ²H₉‐progesterone, and ²H₅‐estradiol, were obtained from Sigma‐Aldrich (St Louis, MO, USA). Non‐isotopically labeled hormones used to create calibration curves were also acquired: hydrocortisone, β‐estradiol, and testosterone from Sigma‐Aldrich and progesterone from Calibiochem (San Diego, CA, USA). HPLC grade methanol for LC/MS/MS analysis performed at Bindeley Science Center at Purdue University was supplied by Fisher Chemicals (Fair Lawn, NJ, USA). Dansyl chloride and acetone for the Dansyl chloride solution for the derivation of samples were purchased from Sigma‐Aldrich. Sodium carbonate added to samples with Dansyl chloride solution was procured from Sigma‐Aldrich. Formic acid and acetonitrile used as buffer solutions during LC/MS/MS analysis were from Sigma‐Aldrich and Fisher Chemicals, respectively. Keto derivatives were prepared using the Amplifex keto reagent (AB Sciex, Framingham, MA, USA).

Male mice were castrated or sham-operated at 14 weeks of age and, after a recovery period of 14 d, tarsal plates were isolated. Approximately 2–3 mg of the tarsal plates were homogenized in methanol/H₂O (3:1) and extracted into dichloromethane using ISOLUTE columns (Biotage). Following evaporation to dryness, this fraction was reconstituted in methanol containing 5% acetic acid and processed for derivatization using the Amplifex Keto Reagent (AB Sciex)^{42 (link)}. The concentration of the testosterone derivative (MRM transition, m/z 403 > 164) was measured using LC–MS/MS QTRAP 4500 system (AB Sciex) and normalized to the weight of the tissue analyzed.