Detecting the Administration of Endogenous Anabolic Androgenic Steroids

Abstract

The detection of the administration of an androgen such as testosterone that could be present normally in human bodily fluids is based upon the methodical evaluation of key parameters of the urinary profile of steroids, precisely measured by GC/MS. Over the years, the markers of utilization were identified, the reference ranges of diagnostic metabolites and ratios were established in volunteers and in populations of athletes, and their stability in individual subjects was studied. The direct confirmation comes from the measurement of delta (13)C values reflecting their synthetic origin, ruling out a potential physiological anomaly. Several factors may alter the individual GC/MS steroid profile besides the administration of a testosterone-related steroid, the nonexhaustive list ranging from the microbial degradation of the specimen, the utilization of inhibitors of 5alpha-reductase or other anabolic steroids, masking agents such as probenecid, to inebriating alcohol drinking. The limitation of the testing strategy comes from the potentially elevated rate of false negatives, since only the values exceeding those of the reference populations are picked up by the GC/MS screening analyses performed by the laboratories on blind samples, excluding individual particularities and subtle doping. Since the ranges of normal values are often described from samples collected in Western countries, extrapolating data to all athletes appears inefficient. Furthermore, with short half-life and topical formulations, the alterations of the steroid profile are less pronounced and disappear rapidly. GC/C/IRMS analyses are too delicate and fastidious to be considered for screening routine samples. An approach based upon the individual athlete's steroid profiling is necessary to pick up variations that would trigger further IRMS analysis and investigations.

Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction

“…There is limited published data on serum/plasma concentrations of some of the anabolic steroids, with generally more focus on measurement of these compounds in urine samples, usually for the purposes of detecting use as performance-enhancing drugs in competitive athletics [55-57]. Of the anabolic steroids for which serum/plasma concentrations are available, methyltestosterone appears to be the one most likely to impact testosterone immunoassay measurements in males.…”

Confounding factors and genetic polymorphism in the evaluation of individual steroid profiling

“…However, the phenotype of an individual is also regulated strongly by the exogenous factors, which may temporarily interfere with the homeostasis and the metabolic routes of endogenous steroids. The effect of various pharmaceutical preparations (eg, endogenous and exogenous steroids, oral contraceptives, human chorionic gonadotropin, LH and glucocorticosteroids) on T/E and steroid profile is extensively summarised earlier, especially by Mareck et al 36 and also elsewhere,14 87–91 emphasising the alterations in androgen synthesis arising from the feedback received via hypothalamic–pituitary axis.…”

“…The first application in doping was published in 1994 by Becchi et al ,28 who employed GC coupled to IRMS for the determination of carbon isotope ratio of T extracted from human urine. This significant work was then followed by numerous studies that explored doping detection based on the carbon isotopic ratio of endogenous hormones linked to T metabolism 7 14 134–147. Recently, some T preparations were reported as having a similar carbon isotopic ratio compared with T produced endogenously,148 149 pushing scientists to find alternative methods based on hydrogen and deuterium ratio to discern naturally produced T from synthetic formulations 150–152.…”

“…Phase I and phase II metabolic reactions are responsible for, respectively, functionalisation and addition of conjugates (ie, glucuronides or sulfates)8–11 to steroids, thereby increasing their hydrophilicity and allowing their dissolution and elimination in urine mixture. Since steroid conjugates analysis is not compatible with GC-MS, the only analytical technique recognised by WADA for endogenous steroids quantification in urine,12 deconjugation of the conjugated moiety by enzymatic hydrolysis (β-glucuronidase) is a crucial step during sample preparation and prior to GC-MS measurement 13 14…”

History of Doping and Doping Control

“…ketoconazol administration, the "steroid profile") and later the application of isotope ratio mass spectrometry, IRMS (Ayotte 2009) have been included as additional means to suspect, to detect or to prove the misuse of testosterone and other -mainly endogenous -anabolic steroids.…”