Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports

Abstract

The detection of testosterone abuse in sports is routinely achieved through the 'steroidal module' of the Athlete Biological Passport by GC-MS(/MS) quantification of selected endogenous anabolic androgenic steroids (EAAS) from athletes' urines. To overcome some limitations of the "urinary steroid profile" such as the presence of confounding factors (ethnicity, enzyme polymorphism, bacterial contamination, and ethanol), ultrahigh performance liquid chromatography (UHPLC) measurements of blood concentrations of testosterone, its major metabolites, and precursors could represent an interesting and complementary strategy. In this work, two UHPLC-MS/MS methods were developed for the quantification of testosterone and related compounds in human serum, including major progestogens, corticoids, and estrogens. The validated methods were then used for the analyses of serum samples collected from 19 healthy male volunteers after oral and transdermal testosterone administration. Results from unsupervised multiway analysis allowed variations of target analytes to be assessed simultaneously over a 96-h time period. Except for alteration of concentration values due to the circadian rhythm, which concerns mainly corticosteroids, DHEA, and progesterone, significant variations linked to the oral and transdermal testosterone administration were observed for testosterone, DHT, and androstenedione. As a second step of analysis, the longitudinal monitoring of these biomarkers using intra-individual thresholds showed, in comparison to urine, significant improvements in the detection of testosterone administration, especially for volunteers with del/del genotype for phase II UGT2B17 enzyme, not sensitive to the main urinary marker, T/E ratio. A substantial extension of the detection window after transdermal testosterone administration was also observed in serum matrix. The longitudinal follow-up proposed in this study represents a first example of 'blood steroid profile' in doping control analysis, which can be proposed in the future as a complement to the 'urinary module' for improving steroid abuse detection capabilities.

Intranasal delivery of Natesto® testosterone gel and its effects on doping markers

“…This 'missed' sample supports the idea of development of even more sensitive screening methods [19,20] in addition to random IRMS testing. The initial screening method and analysis of the biological passport help to identify 'high risk' samples, which leads to more targeted IRMS testing.…”

Sensitivity of doping biomarkers after administration of a single dose testosterone gel

“…Serum T also increased but only by about 50%. Serum DHT, as well as T, has previously been proposed as good markers for T patch detection by Ponzetto et al , who showed serum T and DHT to be better markers than urinary T/E ratio, even when monitored longitudinally . In addition, ratios between serum T and 17OHP have been shown to be sensitive to oral administration of T as serum T increased while 17OHP decreased .…”

Methods for Doping Detection

“…Even if further investigations must be performed to achieve unequivocal identification of all relevant features, identification of these candidate compounds further reinforces the feasibility of increasing actual detection ability (provided by T/E glucuronide in urine) and prolonging the traceability of T undecanoate misuse by using hydroxylated metabolites. Although all doping controls for steroid abuse are currently performed on urine samples, Ponzetto et al [21] recently generated the first example of a blood 'steroid profile' to confirm the hypothesis that steroid analysis in this matrix represents a complementary strategy to the current urine testing methods. For this purpose, 11 steroid hormones were quantified by UHPLC-MS/MS in serum samples obtained from a clinical study of T administration.…”