Excretion of 19-norandrosterone after consumption of boar meat

The consumption of the offal of noncastrated pigs can lead to the excretion of 19-norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the method of choice to differentiate between an endogenous or exogenous origin of urinary NorA. In some cases, after the consumption of wild boar offal, the delta C-13 values of urinary NorA fulfill the criteria of an adverse analytical finding due to differing food sources of boar and consumer. However, consumption of wild boar's offal is not very common in Germany, and thus, the occurrence of such an analytical finding is unlikely. In contrast, the commerce with wild boar meat has increased in Germany within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In order to probe for the probability of the occurrence of urinary NorA after consumption of wild boar meat, human urine samples were tested following the ingestion of commercially available game. In approximately half of the urine samples, traces of NorA were detected postadministration of 200 to 400 g boar meat. The highest urinary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h after the meal. delta C-13 values ranged from -18.5 parts per thousand to -23.5 parts per thousand, which would have led to at least two adverse analytical findings if the samples were collected in an antidoping context. IRMS analysis on German boar tissue samples showed that delta C-13 values for wild boar's steroids are unpredictable and may vary seasonally

Investigations on the in vivo metabolism of 5 alpha-androst-2-en-17-one

Rationale The anabolic steroid 5 alpha-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as a steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2 beta,3 alpha-dihydroxy-5 alpha-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance. Methods The metabolism of 2EN was reinvestigated after oral administration of twofold-deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high-accuracy/high-resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9 days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations. Results More than 15 different metabolites still containing the deuterium label were detected after administration. The presence of steroids exhibiting a 5 beta-configuration was unexpected as the administered 2EN features a 5 alpha-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5 beta-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9 days after administration. Conclusions The new metabolites identified in this study enable the detection of the misuse of 2EN for up to 9 days. The conversion of a 5 alpha-steroid to urinary metabolites with 5 beta-configuration has not been reported so far and should be further investigated

Sensitive detection of testosterone and testosterone prohormone administrations based on urinary concentrations and carbon isotope ratios of androsterone and etiocholanolone

The testing strategy for the detection of testosterone (T) or T-prohormones is based on the longitudinal evaluation of urinary steroid concentrations accompanied by subsequent isotope ratio mass spectrometry (IRMS)-based confirmation of samples showing atypical concentrations or concentration ratios. In recent years, the IRMS methodology focussed more and more on T itself and on the metabolites of T, 5 alpha- and 5 beta-androstanediol. These target analytes showed the best sensitivity and retrospectivity, but their use has occasionally been challenging due to their comparably low urinary concentrations. Conversely, the carbon isotope ratios (CIR) of the main urinary metabolites of T, androsterone (A) and etiocholanolone (EITO), can readily be measured even from low urine volumes; those however, commonly offer a lower sensitivity and shorter retrospectivity in uncovering T misuse. Within this study, the CIRs of A and ETIO were combined with their urinary concentrations, resulting in a single parameter referred to as 'difference from weighted mean' (DWM). Both glucuronidated and sulfated steroids were investigated, encompassing a reference population (n = 110), longitudinal studies on three individuals, influence of ethanol in two individuals, and re-analysis of several administration studies including T, dihydrotestosterone, androstenedione, epiandrosterone, dehydroepiandrosterone, and T-gel. Especially DWM calculated for the sulfoconjugated steroids significantly prolonged the detection time of steroid hormone administrations when individual reference ranges were applied. Administration studies employing T encompassing CIR common for Europe (-23.8 parts per thousand and -24.4 parts per thousand) were investigated and, even though for a significantly shorter time period and less pronounced, DWM could demonstrate the exogenous source of T metabolites

Mass spectrometric characterization of urinary hydrafinil metabolites for routine doping control purposes

Little information on the human metabolism and urinary elimination of hydrafinil (9-fluorenol) exists. In order to support preventive anti-doping activities concerning compounds such as hydrafinil, a pilot elimination study was conducted with three healthy male volunteers receiving a single oral dose of 50 mg of hydrafinil. Urine samples were collected prior to and up to 72-h post-administration and were subjected to both gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, which allowed for the identification of the intact drug as well as Phase I and Phase II metabolites, primarily hydroxylated and/or glucuronidated or sulfo-conjugated hydrafinil. The identity of these metabolites was corroborated by high-resolution/high-accuracy tandem mass spectrometry, and the applicability of routine doping control workflows for the detection of hydrafinil and its main metabolites was assessed. Therefore, two findings of hydrafinil and its metabolites were recorded, which concerned out-of-competition doping control samples and, hence, were not pursued with confirmatory analyses. Yet, the initial testing procedure results indicate that hydrafinil might require consideration in sports drug testing programs to ensure its detection, if classified as prohibited by the World Anti-Doping Agency (WADA)

Detecting the misuse of 7‐oxo‐DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis

Rationale The misuse of 7-oxo-DHEA (3 beta-hydroxyandrost-5-ene-7,17-dione) is prohibited according to the World Anti-Doping Agency (WADA) code. Nevertheless, it is easily available as a dietary supplement and from black market sources. In two recent doping control samples, significant amounts of its main metabolite 7 beta-OH-DHEA were identified, necessitating further investigations. Methods As both 7-oxo-DHEA and 7 beta-OH-DHEA are endogenously produced steroids and no concentration thresholds applicable to routine doping controls exist, the development and validation of a carbon isotope ratio (CIR) mass spectrometry method ha been desirable. Excretion studies encompassing 7-oxo-DHEA, 7-oxo-DHEA-acetate, and in-house deuterated 7-oxo-DHEA were conducted and evaluated with regard to urinary CIR and potential new metabolites of 7-oxo-DHEA. Results Numerous urinary metabolites were identified, some of which have not been reported before, while others corroborate earlier findings on the metabolism of 7-oxo-DHEA. The CIRs of both 7-oxo-DHEA and 7 beta-OH-DHEA were significantly influenced for more than 50 h after a single oral dose of 100 mg, and a novel metabolite (5 alpha-androstane-3 beta,7 beta-diol-17-one) was found to prolong this detection time window by approximately 25 h. Applying the validated method to routine doping control specimens presenting atypically high urinary 7 beta-OH-DHEA levels clearly demonstrated the exogenous origin of 7-oxo-DHEA and 7 beta-OH-DHEA. Conclusions As established for other endogenously produced steroids such as testosterone, the CIR allows for a clear differentiation between endo- and exogenous sources of 7-oxo-DHEA and 7 beta-OH-DHEA. The novel metabolites detected after administration may help to improve the detection of 7-oxo-DHEA misuse and simplify its detection in doping control specimens

Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis

Rationale The misuse of 7-oxo-DHEA (3 beta-hydroxyandrost-5-ene-7,17-dione) is prohibited according to the World Anti-Doping Agency (WADA) code. Nevertheless, it is easily available as a dietary supplement and from black market sources. In two recent doping control samples, significant amounts of its main metabolite 7 beta-OH-DHEA were identified, necessitating further investigations. Methods As both 7-oxo-DHEA and 7 beta-OH-DHEA are endogenously produced steroids and no concentration thresholds applicable to routine doping controls exist, the development and validation of a carbon isotope ratio (CIR) mass spectrometry method ha been desirable. Excretion studies encompassing 7-oxo-DHEA, 7-oxo-DHEA-acetate, and in-house deuterated 7-oxo-DHEA were conducted and evaluated with regard to urinary CIR and potential new metabolites of 7-oxo-DHEA. Results Numerous urinary metabolites were identified, some of which have not been reported before, while others corroborate earlier findings on the metabolism of 7-oxo-DHEA. The CIRs of both 7-oxo-DHEA and 7 beta-OH-DHEA were significantly influenced for more than 50 h after a single oral dose of 100 mg, and a novel metabolite (5 alpha-androstane-3 beta,7 beta-diol-17-one) was found to prolong this detection time window by approximately 25 h. Applying the validated method to routine doping control specimens presenting atypically high urinary 7 beta-OH-DHEA levels clearly demonstrated the exogenous origin of 7-oxo-DHEA and 7 beta-OH-DHEA. Conclusions As established for other endogenously produced steroids such as testosterone, the CIR allows for a clear differentiation between endo- and exogenous sources of 7-oxo-DHEA and 7 beta-OH-DHEA. The novel metabolites detected after administration may help to improve the detection of 7-oxo-DHEA misuse and simplify its detection in doping control specimens

Case Study: Atypical delta C-13 values of urinary norandrosterone

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 delta C-13 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 delta C-13 values of such a scenario, urine samples collected after consumption of a wild-boar-testicle meal were analyzed. IRMS revealed highly enriched delta C-13 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-3-based forage, probably in winter and spring, to a C-4-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 delta C-13 values for NorA of -18 parts per thousand, 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

Risk of unintentional antidoping rule violations by consumption of hemp products

Consumption of hemp products is continuously growing, with an expanding scope of applications. Suppliers operate through different distribution channels, but the Internet is a major retail platform. Hemp products are prepared from cannabis plants and, therefore, might contain a variety of different natural cannabinoids. According to the regulations of the World Anti-Doping Agency, all natural and synthetic cannabinoids are prohibited in-competition, with the explicit exemption of cannabidiol. Therefore, an investigation of 23 hemp products for the presence of cannabinoids was performed to determine the likelihood of unintentional violations of anti-doping regulations. An assay for the detection of 16 cannabinoids in nutritional supplements was developed and validated. The sample preparation consisted of QuEChERS extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry. All 23 commercially available hemp products were analyzed, and assay characteristics such as selectivity, limit of detection, limit of identification, limit of quantification, linearity, imprecision, recovery, and accuracy were determined. Twenty of 23 hemp products included a variety of cannabinoids at, occasionally, substantial concentrations, with four products covering the entire spectrum of tested cannabinoids. An ethics committee-approved single-dose administration study was conducted with the commercially available hemp products, investigating the presence of 16 cannabinoids in urine collected pre- and post-consumption. Variable patterns of cannabinoids or their metabolites in urine were observed. In 30% of the urine samples collected 8 h after ingestion, the presence of a prohibited cannabinoid would have resulted in an unintentional violation of anti-doping regulations

Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use

According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/ml. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA's Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of antidoping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products, were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the minimum required performance level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete's in-competition urine sample represents a substantial risk of an antidoping rule violation

Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination - Insights from metandienone metabolism

The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on metandienone focused on the identification of 17 beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one (20 beta OH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner-Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13- trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC-MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize metandienone leading to the formation of 11 beta-hydroxymetandienone and 18-hydroxymetandienone. Following Wagner-Meerwein rearrangement, the resulting products could be assigned to 20 beta OH-NorMD and 11 beta OH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of metandienone was confirmed by analysis of post-administration samples of metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20 beta OH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes (C) 2012 Elsevier Ireland Ltd. All rights reserved