Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.

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Over the last decade the number of laboratories accredited by the International Olympic Committee (IOC) has grown to 25. Nearly half of the ∼90 000 samples tested annually are collected on short notice—the most effective means to deter the use of anabolic androgenic steroids (AAS). The major urinary metabolites of AAS have been characterized and are identified by their chromatographic retention times and full or partial mass spectra. The process of determining if an athlete has used testosterone (T) begins with finding a T to epitestosterone (E) ratio >6 and continues with a review of the T/E–time profile. For the user who discontinues taking T, the T/E reverts to baseline (typically ∼ 1.0). For the extremely rare athlete with a naturally increased T/E ratio, the T/E remains chronically increased. Short-acting formulations of T transiently increase T/E, and E administration lowers it. Among ancillary tests to help discriminate between naturally increased T/E values and those reflecting T use, the most promising is determination of the carbon isotope ratio.

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Background: Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical. Methods: Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the δ13C values for 5β-androstane-3α,17β-diyl diacetate (5βA), 5α-androstane-3α,17β-diyl diacetate (5αA), and 5β-pregnane-3α,20α-diyl diacetate (5βP) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6. Results: The within-assay precision SDs for 5βA, 5αA, and 5βP were ± 0.27‰, ± 0.38‰, and ± 0.28‰, respectively. The between-assay precision SDs ranged from ± 0.40‰ to ± 0.52‰. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean δ13C (SD) values were −25.69‰ (± 0.92‰), −26.35‰ (± 0.68‰), and −24.26‰ (± 0.70‰), for 5βA, 5αA, and 5βP, respectively. 5βP was greater than 5βA and 5αA (P <0.01), and 5βA was greater than 5αA (P <0.01). The means − 3 SD were −28.46‰, −28.39‰, and −26.37‰ for 5βA, 5αA, and 5βP, respectively. The maximum difference between 5βP and 5βA was 3.2‰, and the maximum 5βA/5βP was 1.13. Three athletes with chronically elevated T/Es had δ13C values consistent with testosterone administration and three did not. Conclusions: This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, ±3 SDs, and ranges of δ13C values in a control group are established. In comparison, testosterone users have low 5βA and 5αA, large differences between 5βA or 5αA and 5βP, and high 5βA/5βP and 5αA/5βP ratios.