Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games.

The doping control analyses at the XXXII Olympic Games (July 23 to August 8, 2021) and the XVI Paralympic Games (August 24 to September 5, 2021) held in Tokyo, Japan, after a year of delay due to the COVID-19 pandemic are summarized in this paper. A new satellite facility at the existing World Anti-Doping Agency (WADA)-accredited Tokyo laboratory was established and fully operated by 278 staff, including 33 Tokyo laboratory staff, 49 international experts, and 196 Japanese temporary staff. The numbers of urine samples were 5081 (Olympics) and 1519 (Paralympics), and the numbers of blood samples were 1103 (Olympics) and 500 (Paralympics). The laboratory could prepare for analysis in advance using a paperless chain-of-custody system, allowing for faster turnaround time reporting. For the first time, a new polymerase chain reaction method for detecting erythropoietin (EPO) gene doping was used. The laboratory also analyzed blood samples for detecting steroid esters following the spotting of collected venous EDTA blood onto dried blood spot cards. Moreover, full-scan data acquisition using high-resolution mass spectrometers was performed for all urine samples, allowing for detecting traces of doping substances, which are not currently analyzed in the subsequent data processing. The presence of some prohibited substances was confirmed, resulting in 8 atypical findings (ATFs) and 11 adverse analytical findings (AAFs), including homologous blood transfusion (2 cases) and recombinant EPO in the blood (1 case), at the Olympics, whereas 2 ATFs and 10 AAFs were reported at the Paralympics.

UDP-glucuronosyltransferase 2B17 genotyping in Japanese athletes and evaluation of the current sports drug testing for detecting testosterone misuse.

Ethnicity has been found to influence urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratios among athletes. Uridine diphospho-glucuronosyltransferase 2B17 (UGT2B17) is the most active enzyme in testosterone glucuronidation. UGT2B17 polymorphism analysis is rarely performed in Japanese athletes, and the influence of testosterone administration on steroid profiles and carbon isotope ratios, according to gene polymorphisms, in Asians remains unknown. The prevalence of UGT2B17 genotypes and urinary androgenic steroid profiles, classified according to UGT2B17 genotypes, was investigated in Japanese athletes (255 male and 256 female). Testosterone enanthate (100 mg) was administered intramuscularly to Japanese female volunteers (del/del: n = 6, del/ins: n = 3, ins/ins: n = 1). The distribution rates of the UGT2B17 del/del genotype in Japanese male and female athletes were 74.5% and 60.2%, respectively. The ins/ins genotype was detected in only three male (1.2%) and seven female (2.7%) athletes. The prevalence of the UGT2B17 deletion genotype was extremely high in Japanese athletes. The T/E ratio in the del/del group was significantly lower than that in the other groups. After testosterone was administered to female volunteers, the T/E ratios for the del/del individuals failed to reach the positivity criterion of 4. By contrast, in all of the del/del subjects, the gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis successfully fulfilled the positivity criterion. The overall result has demonstrated the limited effectiveness of population-based T/E ratios in screening tests for testosterone use. Subject-based steroid profiling with UGT2B17 genotyping will be an effective strategy for detecting testosterone misuse.

Determination of growth hormone secretagogue pralmorelin (GHRP-2) and its metabolite in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry.

GHRP-2 (pralmorelin, D-Ala-D-(beta-naphthyl)-Ala-Ala-Trp-D-Phe-Lys-NH(2)), which belongs to a class of growth hormone secretagogue (GHS), is intravenously used to diagnose growth hormone (GH) deficiency. Because it may be misused in expectation of a growth-promoting effect by athletes, the illicit use of GHS by athletes has been prohibited by the World Anti-Doping Agency (WADA). Therefore, the mass spectrometric identification of urinary GHRP-2 and its metabolite D-Ala-D-(beta-naphthyl)-Ala-Ala-OH (AA-3) was studied using liquid chromatography/electrospray ionization tandem mass spectrometry for doping control purposes. The method consists of solid-phase extraction using stable-isotope-labeled GHRP-2 as an internal standard and subsequent ultra-performance liquid chromatography/tandem mass spectrometry, and the two target peptides were determined at urinary concentrations of 0.5-10 ng/mL. The recoveries ranged from 84 to 101%, and the assay precisions were calculated as 1.6-3.8% (intra-day) and 1.9-4.3% (inter-day). Intravenous administration of GHRP-2 in ten male volunteers was studied to demonstrate the applicability of the method. In all ten cases, unchanged GHRP-2 and its specific metabolite AA-3 were detected in urine.

Influence of intravenous administration of growth hormone releasing peptide-2 (GHRP-2) on detection of growth hormone doping: growth hormone isoform profiles in Japanese male subjects.

Administration of exogenous 22 kDa recombinant human growth hormone (rhGH) suppresses the non-22 kDa pituitary growth hormone (GH) secretion by negative feedback; then, the elevated 22 kDa GH to non-22 kDa GH ratio (Rec/Pit ratio) can be utilized to detect doping with rhGH (isoform differential immunoassay). The influence of intravenous administration of growth hormone releasing peptide GHRP-2 on the isoform differential immunoassay for detecting rhGH doping has been investigated.In this study, a reference population (n=100) was used, with 0.04 mg/kg rhGH subcutaneous administration (n=5), 100 µg of GHRP-2 intravenous administration (n=10) and 0.04 mg/kg rhGH combined with 100 µg of GHRP-2 (n=10) in Japanese male subjects. The results indicated that the low dose (0.04 mg/kg) of rhGH led to significantly increased Rec/Pit ratio compared with the Japanese reference limit (P < 0.001). Because GHRP-2 dose led to increases in concentrations of both recombinant GH (recGH) and pituitary GH (pit GH), no significant change in the Rec/Pit ratio was observed (P > 0.05). In a combined administration study, after GHRP-2 dose the Rec/Pit ratios decreased to 39.9-43.9% compared with the elevated ratio caused by the rhGH dose.The results indicated that GHRP-2 administration cannot only be detected by the isoform differential immunoassay but also masks rhGH doping. The analysis of GHRP-2 was found to be suitable for compensating for the disadvantages of the isoform differential immunoassay because GHRP-2 and its metabolite (AA-3) in urine could be detected during the periods of masking of the Rec/Pit ratio by means of liquid chromatography/tandem mass spectrometry.

Analysis of non-ketoic steroids 17alpha-methylepithiostanol and desoxymethyl- testosterone in dietary supplements.

Dietary supplements containing 17alpha-methyl-2,3-epithio-5alpha-androstane-17beta-ol (17alpha-methylepithiostanol), which is a 17-methylated analogue of epithiostanol or a prodrug of desoxymethyltestosterone (17alpha-methyl-5alpha-androst-2-en-17beta-ol), have recently appeared on the Internet. 17alpha-Methylepithiostanol and desoxymethyltestosterone are classified as prohibited substances on the World Anti-Doping Agency (WADA) list. Two preparations, EPISTANE and P-PLEX, were obtained from the Internet so that their contents could be investigated. This study involved gas chromatography/mass spectrometry (GC/MS) analysis after trimethylsilyl (TMS) derivatization, liquid chromatography/mass spectrometry (LC/MS) in atmospheric pressure photoionization (APPI) mode and nuclear magnetic resonance (NMR) spectroscopy. Analysis using LC/MS in APPI mode would be a useful tool for detecting heat-labile and non-polar steroids.Although the labelling of EPISTANE indicates that it contains 17alpha-methyl-2alpha, 3alpha-epithio-5alpha-androstane-17beta-ol only, 17alpha-methyl-2beta,3beta-epithio-5alpha-androstane-17beta-ol and desoxymethyltestosterone were identified in the supplement. The results showed that P-PLEX contained desoxymethyltestosterone and its isomer 17alpha-methyl-5alpha-androst-3-en-17beta-ol. Urine samples can be screened after EPISTANE or P-PLEX administration using the normal screening procedure for anabolic steroids with GC/MS.