Case Study: Atypical δ¹³C values of urinary norandrosterone

Abstract

Isotope ratio mass spectrometry (IRMS) has been established in doping control analysis to identify the endogenous or exogenous origin of a variety of steroidal analytes including the 19-norsteroid metabolite norandrosterone (NorA). NorA can be found naturally in human urine in trace amounts due to endogenous demethylation or in situ microbial degradation. The administration of nortestosterone (nandrolone) or different prohormones results in the excretion of urinary NorA. Usually, this can be detected by IRMS due to differing δ C values of synthetic 19-norsteroids compared to endogenous reference compounds. The consumption of uncastrated pig edible parts like offal or even meat may also lead to a urinary excretion of NorA. In order to determine the δ C values of such a scenario, urine samples collected after consumption of a wild-boar-testicle meal were analyzed. IRMS revealed highly enriched δ C values for urinary NorA, which could be related to the completely corn-based nutrition of the animal. Isotopic analysis of the boar's bristles demonstrated a dietary change from C -based forage, probably in winter and spring, to a C -based diet in the last weeks to months prior to death. These results supported the interpretation of an atypical test result of a Central European athlete's doping control sample with δ C values for NorA of -18 ‰, most probably caused by the consumption of a wild boar ragout. As stated before, athletes should be fully aware of the risk that consumption of wild boar may result in atypical or even adverse analytical findings in sports drug testing.

Dietary Supplement and Food Contaminations and Their Implications for Doping Controls

“…The maximal values for 19-noretiocholanolone were 0.5-1.2 ng/mL. In sports drug testing, IRMS is routinely employed to demonstrate the exogenous origin of 19-norandrosterone detected in an athlete's urine sample at low concentrations between 2.5 and 15 ng/mL [46,122]. As such urine levels would also be observed after the consumption of edible tissue from non-castrated pigs, another administration study was conducted in 2018, in order to clarify which impact the ingestion of boar offal has on the δ 13 C values of urinary 19-norandrosterone [122].…”

“…In sports drug testing, IRMS is routinely employed to demonstrate the exogenous origin of 19-norandrosterone detected in an athlete's urine sample at low concentrations between 2.5 and 15 ng/mL [46,122]. As such urine levels would also be observed after the consumption of edible tissue from non-castrated pigs, another administration study was conducted in 2018, in order to clarify which impact the ingestion of boar offal has on the δ 13 C values of urinary 19-norandrosterone [122]. Two male healthy volunteers consumed a meal prepared from wild boar testicles and subsequently collected urine samples for a period of 24 h. Approximately 4 h following administration, maximum 19-norandrosterone concentrations of 4 and 8 ng/mL were detected employing GC-MS, and IRMS analysis yielded highly enriched δ 13 C values, which would constitute an AAF.…”

“…Dietary supplements contaminated with prohormones of nandrolone (e.g., 19-norandrostenedione) Supplement consumption n/a [42,43,47,50] Dietary supplement contaminated with prohormones of testosterone (e.g., 4-androstenedione) Supplement consumption n/a [43] Musk pod formulations naturally containing different anabolic-androgenic steroids Treatment with traditional Asian medicine yes [103] Meat contaminated with clenbuterol Food intake yes [106,109,110] no [111,116] Meat contaminated with clostebol Food intake yes [118] Meat contaminated with nandrolone Food intake yes [118] Meat contaminated with metenolone Food intake yes [119] Offal and meat from non-castrated pigs/boars naturally enriched with different steroid hormones Food intake yes [121,122] Dietary supplement contaminated or adulterated with stanozolol Supplement consumption yes [54] Dietary supplements contaminated or adulterated with ostarine Supplement consumption yes [58][59][60][61][62][63][64] Dietary supplements contaminated with hydrochlorothiazide Supplement consumption yes [90] NSAID contaminated with hydrochlorothiazide Administration of an analgesic yes [97] Malaria chemoprophylaxis drug containing proguanil In vesica conversion of proguanil metabolite yes [98] Dietary supplement containing N,N-dimethyl-2-phenylpropan-1-amine & β-methylphenethylamine Supplement consumption no [85] Dietary supplement containing N-ethyl-α-ethyl-phenylethylamine Supplement consumption yes [87] Dietary supplement containing octodrine Supplement consumption no [88] Poppy seeds naturally containing high amounts of morphine Food intake no [123,125]…”

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…Brailsford et al recently reported on the detection of four nandrolone ester formulations, which were found to yield δ 13 C values for nandrolone between −21.5 and − 22.5‰, thus precluding the corroboration of an ADVR by means of IRMS in various instances. The situation has gained further complexity by the fact that nandrolone, ingested by athletes via offal or meat obtained from uncastrated pigs, can exhibit a broad range of carbon isotope signatures as presented by Hülsemann et al In the course of follow‐up studies concerning ATF/AAF case investigations, the seasonal variability of δ 13 C values of wild boar‐derived edible tissue and, consequently, also the contained nandrolone, was reported. Hence, depending on the athlete's ERC δ 13 C values, a doping control urine sample test result can either be atypical/inconclusive (i.e.…”

The 36th Manfred Donike workshop on doping analysis

“…Differentiating natural endogenous steroids from synthetic analogs is routinely accomplished by IRMS. Here, studies by Brailsford et aland Huelsemann et al were found to be particularly noteworthy as atypical carbon isotope signatures of nandrolone and its main metabolite 19‐norandrosterone were investigated. Apparently, synthetic preparations comprising ‘human’ carbon isotope signatures were identified, which complicates the currently employed strategy of routine doping controls.…”