Carbon isotope ratios of endogenous steroids found in human serum\u2014method development, validation, and reference population-derived thresholds

Eberhard Nieschlag,Thomas Piper,Lia Bally,Mario Thevis,Hans Geyer

doi:10.1007/s00216-021-03439-9

Abstract

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)–based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated.Graphical abstract

Highlights

Since decades the detection of testosterone (T) or testosterone prohormone misuse in sports drug testing relies on the Published in the topical collection Recent Trends in (Bio)Analytical Chemistry with guest editors Antje J
The limitations of the urinary steroid profile were demonstrated by data obtained on two female athletes, who did not respond to presumed T administrations by elevated concentration ratios in urine but only by significantly elevated T concentrations found in serum [6]
In order to prevent time-consuming follow-up investigations on athletes found with abnormal serum steroid concentrations, an isotope ratio mass spectrometry (IRMS)-based confirmation method to differentiate between T administrations and other possible confounding factors will be beneficial

Summary

Introduction

Since decades the detection of testosterone (T) or testosterone prohormone misuse in sports drug testing relies on the Published in the topical collection Recent Trends in (Bio)Analytical Chemistry with guest editors Antje J. The limitations of the urinary steroid profile were demonstrated by data obtained on two female athletes, who did not respond to presumed T administrations by elevated concentration ratios in urine but only by significantly elevated T concentrations found in serum [6]. These results were corroborated by clinical trials encompassing lowdose administrations of T to female volunteers [7, 8]. In order to prevent time-consuming follow-up investigations on athletes found with abnormal serum steroid concentrations, an IRMS-based confirmation method to differentiate between T administrations and other possible confounding factors will be beneficial

Methods

Results

Conclusion