Steroid hormone quantification protocol

Reagents were of ACS grade or higher (e.g. HPLC and Optima LC/MS) and were purchased from Thermo Fisher Scientific and used without further purification. Testosterone, epiTestosterone (epi-T), DHEA, DHEA-S sodium salt, DHEA glucuronide (DHEA-G), AST, epi-AST, DHT, 5-adiol, 3α-adiol and 3β-adiol were purchased from Steraloids (Wilton, NH, USA). [2,3,4-¹³C₃]-T ([¹³C₃]-T) and [2,3,4-¹³C₃]-DHT ([¹³C₃]-DHT) were from C/D/N Isotopes (Point-Claire, Quebec, Canada) and Cambridge Isotopes (Andover, MA, USA), respectively. [2,3,4-¹³C₃]-3α-adiol ([¹³C₃]-3α-adiol) and [2,3,4-¹³C₃]-3β-adiol ([¹³C₃]-3β-adiol) were synthesized by an enzymatic method according to our published procedure (Zang et al. 2017 (link)). 4-Dimethylaminopyridine (DAP) 2-methyl-6-nitrobenzoic anhydride (MNBAn), picolinic acid (PA), triethylamine (TEA), anhydrous tetrahydrofuran (THF), β-glucuronidase from E. coli and sulfatase from Abalone entrails were from Sigma-Aldrich. Charcoal dextran stripped fetal bovine serum (CD-FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA).