Evaluation of sulfate metabolites as markers of intramuscular testosterone administration in Caucasian and Asian populations

Abstract

The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed‐mode solid‐phase extraction and analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Urine samples after a single IM injection (100 mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre‐ and post‐administration samples. For Caucasian volunteers, a separation between pre‐ and post‐administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A‐S/T‐S) which prolonged the DT 1.2–2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3–1.5 times in two Asian volunteers. Other ratios between A‐S or etiocholanolone sulfate and E‐S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T‐S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended.

Urinary steroid profile in relation to the menstrual cycle

“…The proportion of sulfate conjugated E compared with glucuronide conjugated E did not differ throughout the menstrual cycle 5 ; that is, there seem to be no alteration in excretion routes during the menstrual cycle. It is possible that sulfate metabolites may be useful additional biomarkers of T doping, but notably, all studies available have been conducted in men only 28,30,31 …”

“…It is possible that sulfate metabolites may be useful additional biomarkers of T doping, but notably, all studies available have been conducted in men only. 28,30,31 Six out of the 17 women (35%) had undetectable T glucuronide levels in their urine samples. This may be due to a double deletion of the UGT2B17 gene coding for the major enzyme responsible for glucuronidation of T. 32 As T was undetectable in all the >120 urine samples in these six women, it is likely that all of them carry the UGT2B17 del/del polymorphism.…”

Causal Effects of Genetically Determined Metabolites on Risk of Polycystic Ovary Syndrome: A Mendelian Randomization Study

“…Based on 15 genetic scores with different degrees of specificity to EPIA-S, we demonstrated that high levels of EPIA-S are genetically associated with a higher risk of developing PCOS. Despite a lack of information on its effects, EPIA-S has recently been proposed as a marker for oral or intramuscular testosterone administration (32,33). This was also proposed for the administration of other steroid hormones, such as dehydroepiandrosterone (DHEA), 4-androstenedione, and dihydrotestosterone (34).…”

Current Insights into the Steroidal Module of the Athlete Biological Passport

“…The steroidal module in its present form is a powerful tool to detect the misuse of endogenous steroids that is based on scientific research and method development conducted in the last four decades. Multifaceted research was conducted in order to further improve the detectability focussing mainly on the implementation of additional endogenous metabolites and their ratios [84][85][86][87][88][89][90]. In many of these cases, slight improvements in sensitivity or retrospectivity were demonstrated but this seemed not to justify spending the effort necessary to implement these metabolites into routine screening procedures world-wide.…”