13c3 testosterone

OEA, PEA, the deuterated standards D₉-progesterone and D₇-cortisone, and ¹³C-labeled standards ¹³C₃-androstenedione, ¹³C₃-testosterone, and ¹³C₃-progesterone were purchased from Sigma-Aldrich (Buchs, Switzerland). ¹³C₃-cortisol and ¹³C₃-cortisone were purchased from Isoscience (Ambler, USA). AEA, 2-AG, and the deuterated eCBs (D₄-AEA, D₅-2-AG, D₄-OEA, D₄-PEA, and D₁₁-AEA) were purchased from Cayman Chemicals (Ann Arbor, USA). Water and methanol (MeOH) were of LC–MS grade (Chromasolv®) and purchased from Sigma-Aldrich (Buchs SG, Switzerland). Acetone, ethyl acetate, and ammonium fluoride were purchased from Merck (Darmstadt, Germany). Reconstitution solution consisted of 0.2 mM NH₄F in water/methanol 97/3 v/v, respectively. Isolute® SLE + columns were purchased from Biotage® (Uppsala, Sweden). All chemicals were of highest analytical grade.

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).

Plasma androgens (testosterone and androstenedione) were analyzed by adapting a previously described method (17 (link)). Briefly, steroids were extracted from plasma (500 μL) by solid-phase extraction on an HLB Oasis (60-mg, 3-cc columns; Waters UK, Elstree, UK) with 10 ng ¹³C₃-testosterone and ¹³C₃-androstenedione (Sigma Aldrich, Dorset, UK) as internal standards. Extracted steroids were separated using liquid chromatography on a UPLC column (2.1 mm × 50 mm, 1.7 μm; Acquity UPLC BEH C18; Waters, MA). All steroids were analyzed in positive ion mode using electrospray ionization on an AB Sciex QTrap 5500 operating in triple quadrupole mode for testosterone (m/z 289 → 97, 25 V) and androstenedione (m/z 287 → 97, 25 V). Linear regression analysis of calibration standards, calculated using peak area ratios of analytes to internal standard, was used to determine the concentration of the analytes in the samples.