Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

Abstract

A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantitation of endogenous steroid sulfates has been developed to be able to evaluate these metabolites as biomarkers to detect the misuse of endogenous androgenic anabolic steroids in sports. For sample preparation, a mixed-mode solid-phase extraction was optimized to eliminate the glucuronide fraction in the washing step thus obtaining only the sulfate fraction. Chromatographic separation was optimized to achieve adequate resolution between isomers. The electrospray ionization and the product ion mass spectra of the sulfates were studied in order to obtain the most specific and selective transitions. The method was validated for quantitative purposes for 11 steroid sulfates obtaining satisfactory values for linearity, accuracy, and intra- and inter-day precision (relative standard deviation better than 16.2%). Limits of quantitation ranged between 0.5 and 2 ng/mL. Extraction recoveries for sulfate metabolites were between 90 and 94%. Matrix effect ranged from 90 to 110% showing the absence of significant ion suppression/enhancement. Samples were found to be stable after 2 freeze/thaw cycles. The applicability of the method was checked by the analysis of 75 urine samples from healthy volunteers (54 males, 37 Caucasian and 17 Asian, and 21 Caucasian females) to evaluate the concentration levels of endogenous sulfate metabolites in basal conditions.

Sulfate metabolites improve retrospectivity after oral testosterone administration

“…25 The calibration curves were based on the peak area ratios of the sulfate metabolites versus the corresponding deuterated internal standard. 25 The calibration curves were based on the peak area ratios of the sulfate metabolites versus the corresponding deuterated internal standard.…”

“…Three isomers of androstanediol sulfate (Diol-S1, Diol-S2, and Diol-S3), 25 which were not commercially available, were semi-quantified …”

“…25 Only the upper limit was used assuming that the administration of T will increase the concentration of the metabolites or ratios and, thus, the suspicious samples will be having a concentration greater than the RL. 25 Only the upper limit was used assuming that the administration of T will increase the concentration of the metabolites or ratios and, thus, the suspicious samples will be having a concentration greater than the RL.…”

“…As described in our previous work, 25 these compounds are androstanediol sulfates with different configuration or different conjugation position compared to Diol-S (5α-androstane-3β,17β-diol 17β-sulfate), available as reference standard. As described in our previous work, 25 these compounds are androstanediol sulfates with different configuration or different conjugation position compared to Diol-S (5α-androstane-3β,17β-diol 17β-sulfate), available as reference standard.…”

“…25 The aim of the present study was to evaluate the usefulness of these sulfate metabolites to detect oral T misuse. 25 The aim of the present study was to evaluate the usefulness of these sulfate metabolites to detect oral T misuse.…”