Stable carbon isotope ratio profiling of illicit testosterone preparations – domestic and international seizures

Abstract

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport. It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products. Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. δ(13) C ≥ -25.8 ‰). In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of 'enriched' substrates--which if administered to human subjects would be considered problematic for the use of current GC-C-IRMS methodologies for the doping control of testosterone in sport. The distribution of δ(13) C values for this illicit testosterone sample population (n = 283) ranged from -23.4 ‰ to -32.9 ‰ with mean and median of -28.6 ‰--comparable to previous work. However, only 13 out of 283 testosterone samples (4.6 %) were found to display δ(13) C values ≥ -25.8 ‰, confirming that in the vast majority of cases of illicit testosterone administration, current GC-C-IRMS doping control procedures would be capable of confirming misuse.

IRMS delta values (13C) of nandrolone and testosterone products available in the UK: Implications for anti‐doping

“…14 As GC-C-IRMS analysis involves combustion of a target substance to form CO 2 no spectral information is available to facilitate analyte identification and help identify interfering peaks. 12 The availability of synthetic T and N preparations with endogenous δ 13 C values on the market, as has been shown in this study and those of others, [17][18][19]22 further complicates substantiating evidence of their misuse. 20 This step is also important to monitor co-elution of interferents with the target steroids which, if present, may alter the measured carbon isotope value.…”

“…10,19 All δ 13 C values for steroids reported in this manuscript are for the free steroid, with correction for the influence of the acetate moiety done using a mass balance approach. [17][18][19]22 In doping-control analysis, detecting the misuse of steroids produced naturally by humans (i.e. 10 Splitting of GC eluents into the MSD and IRMS alleviates this problem by allowing simultaneous acquisition of full scan mass spectra of chromatographic peaks along with isotope ratio analysis.…”

“…collection post-administration22 could also influence a negative or positive outcome since the δ 13 C value will normalize to the athlete's baseline level after a washout time, that is either a normalization of the δ 13 C value back to endogenous in the case of T 27 or a reduction in 19-NA concentration such that it is below the measurement capability of the assay.On the other hand, misuse of steroid preparations that have δ 13 C values further into the exogenous range (less than −29‰) remain easy to detect. For instance, administration of the most 13 C-depleted products (−32.7‰) represent the most likely to yield positive results by GC-C-IRMS analysis as demonstrated in previous studies 28.…”

Multidimensional isotope analysis of carbon, hydrogen and oxygen as tool for identification of the origin of ibuprofen

“…There are two main techniques for measuring stable isotope 53 ratios of organic compounds: isotope ratio mass spectrometry (IRMS) and nuclear magnetic 54 resonance spectrometry (NMR). IRMS is routinely applied for the measurement of 2 H/ 1 H (D/H), 55 13 C/ 12 C, 15 N/ 14 N, 18 O/ 16 O and 34 S/ 32 S ratios in pure substances, or measurement of 2 H/ 1 H, 56 13 C/ 12 C, 15 N/ 14 N of mixtures, when gas chromatography is used or 13 C/ 12 C of mixtures with 57 liquid chromatography. This technique is providing the bulk isotope composition of a chemical 58 compound.…”

“…The techniques has been used for -fingerprinting‖ of different active pharmaceutical M a n u s c r i p t 4 ingredients and illicit drugs, for example, tropicamide, hydrocortisone, quinine hydrochloride, 60 tryptophan, naproxen, topiramate, methylamphetamine, ephedrine, testosterone, cocaine. [2, 3, 7, 61 9, [13][14][15] NMR allows the analysis of site-specific natural isotope fractionation (SNIF -NMR), 62 which provides information on the element for each of the sites of the molecule. This enables a 63 better discrimination between samples and thus yields more data and specific information on 64 isotope composition of samples compared to bulk isotope analysis.…”