Analysis of the nutritional supplement 1AD, its metabolites, and related endogenous hormones in biological matrices using liquid chromatography-tandem mass spectrometry.

1,5alpha-Androsten-3beta,17beta-diol and/or 1,5alpha-androsten-3,17-dione (1AD) is an over-the-counter pro-hormone nutritional supplement designed to enhance strength and performance in athletes. 1AD purportedly mimics the pharmacological activity of testosterone through activation of the pro-hormones to their active form 1,5alpha-androsten-17beta-ol-3-one or Delta(1)-testosterone. This testosterone analogue ostensibly possesses strong androgenic potency without the adverse effects associated with aromatization of testosterone to estrogens. We have developed a highly sensitive and selective liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of 1AD, its analogues, and several structurally related endogenous hormones in plasma and urine. The limits of quantitation for the analytes ranged from 0.25 to 0.5 ng/mL. The accuracy of the assay was 92-113% with a precision of 2-12% relative standard deviation (RSD) for all analytes at 1.0, 5.0, and 15.0 ng/mL, respectively. The interassay precision was 6-16% RSD, and the accuracy was 90-105%. We have used this assay to determine the unconjugated and total (conjugated and unconjugated) concentrations of 1AD, its analogues, androstenediol, androstenedione, testosterone, dihydrotestosterone, and estradiol, in plasma and urine, as well as to investigate the metabolic fate of the three 1AD analogues (diol, dione, and active forms) when incubated with rat liver microsomes or rat testicular homogenates. Concentrations of both unconjugated and total testosterone in plasma were approximately 1.5 ng/mL and ranged from undetectable to 4.1 ng/mL in urine. 1AD and its analogues were not detected in plasma or urine. In vitro metabolism experiments using rat liver microsomes and testicular homogenates provided evidence for the interconversion of the three 1AD analogues, biosynthesis, and decomposition of several endogenous hormones, as well as evidence for 1AD analogue-induced changes in the typical profiles of testosterone and androstenedione in testicular tissue.