Shinji Kageyama

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.

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.

Doping control of biosimilar epoetin kappa and other recombinant erythropoietins after intravenous application

Since the expiration of patent protection, a number of new recombinant erythropoietin (rEPO) biosimilars have appeared on the worldwide market. In 2010, epoetin kappa, which is biosimilar to epoetin alfa, was clinically approved in Japan. Currently, both isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) are approved by the World Anti-Doping Agency (WADA) for detection of rEPO doping. Because it was unclear whether epoetin kappa could be detected by WADA-accredited detection methods, intravenous administration studies of epoetin kappa, epoetin alfa, and epoetin beta were performed to test the applicability of these methods. The isoform bands of epoetin kappa expanded more widely towards the basic area and the profile appeared to be composed of at least eight bands, which were clearly different from those of other epoetins. The results showed that epoetin kappa also contains isoforms of higher molecular masses than those of originator epoetins on SDS-PAGE; the mass distribution was confirmed by electrospray ionization time-of-flight mass spectrometry. We clearly detected epoetin kappa after its administration up to 10 h by IEF-PAGE and 24 h by SDS-PAGE; the detection window of the SDS-PAGE is longer than that of the IEF-PAGE. SDS-PAGE compensates for the disadvantages of IEF-PAGE in detecting urinary epoetin kappa. We also concluded that athletes abusing rEPO might move to intravenous injections for shorter clearance times instead of subcutaneous injections. In conclusion, out-of-competition tests need to be applied more frequently to improve the effectiveness of the rEPO detection.

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.

Comparison of urine analysis and dried blood spot analysis for the detection of ephedrine and methylephedrine in doping control

When the misuse of stimulants is determined in doping control tests conducted during the in-competition period, athletes are asked to account for the violation of the rules. This study was designed to evaluate whether the urinary threshold values (10 µg/mL) for ephedrine and methylephedrine set by the World Anti-Doping Agency (WADA) can be exceeded after the oral administration of each substance (25 mg). In addition, the study describes the validity of a liquid chromatography-tandem mass spectrometric method using dried blood spot testing to detect ephedrine and methylephedrine by comparing it to a quantitative laboratory urine assay. After administration of ephedrine, the urinary concentration of ephedrine did not exceed the threshold at 4-10 h in two subjects, whereas the threshold was exceeded in both the subjects at 12 h after administration. For methylephedrine, the urinary concentrations of all the subjects failed to reach the threshold for up to 10 h after administration. The concentrations reached the threshold at 12-24 h after administration in some volunteers. In contrast, the blood concentrations of ephedrine and methylephedrine reached their maximum levels at 2-8 h after administration. The blood concentrations showed a low inter-individual variability, and the results suggested that the urinary excretion of ephedrine and methylephedrine can be strongly affected by urine pH and/or urine volume. These facts suggest that urinary concentrations cannot reflect the psychoactive level of ephedrines in circulation. Thus, dried blood analysis might be suitable for the adequate detection of stimulants during in-competition testing.

Possibility of analytical finding of glycerol caused by self‐catheterization in doping control

Glycerol is listed on the World Anti-Doping Agency (WADA) prohibited list as a masking agent principally because the administration of glycerol increases plasma volume and decreases the concentration of haemoglobin and the value of haematocrit in blood. Glycerol is a naturally occurring substance; therefore, the threshold is set as 1.0 mg/mL in the WADA technical document (WADA TD2013DL). In a WADA-accredited doping control laboratory, three doping control urine specimens collected from impaired athletes were determined to contain a high concentration of glycerol (>1.0 mg/mL); two of these specimens were considered adverse analytical findings (AAFs). Self-catheterization is necessary for athletes with neurological disorders such as neurogenic bladder dysfunction. We conducted a simple simulation of self-catheterization as an experimental test using urethral catheters with an antiseptic agent containing glycerol to confirm the influence of this antiseptic agent on the quantitative value of glycerol in doping control analysis. Some users employ a catheter with glycerol solution (ca. 1 mL) to avoid pain during use. The urine sample passed through such a catheter exhibited a glycerol concentration (4.94 mg/mL) greater than the threshold level. In September 2014, the threshold for glycerol will change from 1.0 to 4.3 mg/mL (WADA TD2014DL); however, a possibility exists for the quantitative value of glycerol in doping control analysis to exceed the threshold because of the use of an antiseptic agent containing glycerol for self-catheterization. The AAF for glycerol for impaired athletes, particularly those who participate in Paralympic sports, should account for the use of a catheter with glycerol.

Mass spectrometric characterisation of darbepoetin alfa biosimilars with C-terminal arginine residues

Human erythropoietin (EPO) and recombinant human EPO (rEPO) are approximately 30-kDa glycosylated proteins comprising 165 amino acids. Darbepoetin alfa (NESP) is a glycosylated protein encompassing five changes in the amino acid sequence of human EPO, which contains two extra sugar chains. NESP is under patent protection in the USA until May 2024 and in Europe until July 2016, which suggests that the number of NESP biosimilars might substantially increase. The detailed characterisation of biosimilar products are required to ensure the identity and purity of the biosimilar products in terms of safety and efficacy for patients. In this study, a mass spectrometric characterisation of NESP biosimilar products is demonstrated. The study comprises a time-of-flight mass spectrometry characterisation for the asialo-NESPs after sialidase digestion and primary structure characterisation using bottom-up analysis after endoproteinase Glu-C digestion of the core protein. The study revealed that there was a wide range of glycoforms spaced by 365 Da intervals, namely, HexHexNAc units, which indicated that NESP biosimilars likely contained more N-acetyllactosamine units in their molecules. The bottom-up analysis also showed that the NESP biosimilars, as well as a rEPO biosimilar, contain not only the des-arginine product but also the C-terminal arginine product comprising 166 amino acids, whereas the innovator products contain des-arginine EPO comprising only 165 amino acids. The C-terminal arginine EPO would be used as a potential marker for doping with EPO bisimilaras. These findings also point to a need for the investigation of immunogenicity and comparability for the biosimilar products. Copyright

Analytical detection of trimetazidine produced by metabolic conversion of lomerizine in doping control analysis

The identification of trimetazidine in urine samples might result from administration of the permitted drug lomerizine. Laboratories are therefore urged to carefully investigate suspicious cases where trimetazidine is detected. Differentiation of abuse of the banned substance trimetazidine from use of the permitted drug lomerizine would be supported by analysis of the intact drug lomerizine and/or specific metabolites. Copyright

Effects of intravenous infusion of glycerol on blood parameters and urinary glycerol concentrations

In sports, the oral intake and intravenous administration of glycerol as a potential masking agent have been prohibited. The effect of glycerol on blood parameters was investigated by comparing the intravenous administration of glycerol (20g/200mL) with that of an electrolyte (8g glucose/200mL) as a comparator (n=7, fixed-dose-rate i.v. infusion, 200mL in 1h). This study was also designed to evaluate whether the urinary concentrations reached the positivity threshold after the intravenous infusion of glycerol. Significant decreases of the haemoglobin (HGB, g/dL), haematocrit (HCT, %) and OFF-h Score (OFF-score) values were observed after the infusion of glycerol (P<0.05 at 1-6h). The differences in the HGB, HCT and OFF-score between pre- and post-administration were -0.49±0.23g/dL (2h), -1.54±0.73% (2h) and -3.89±3.66 (2h), respectively. Glycerol infusion significantly increased the plasma volume by 12.1% (1h), 6.3% (2h) and 5.7% (3h) compared with the initial values. The infusion of the comparator also increased the plasma volume by 9.6% (1h), 5.8% (2h) and 4.9% (3h) compared with the values before infusion. There were no significant differences in the change of the plasma volume between the intravenous infusions of glycerol and the glucose-based electrolyte (as the comparator) (P≥0.05). This finding might indicate that glycerol itself only exhibited limited effects on the expansion of plasma. After administration of glycerol, the urinary glycerol concentrations increased from 0.0013±0.0004mg/mL to 6.86±2.86mg/mL at 1h and 6.45±3.08mg/mL at 2h. The intravenous infusion of glycerol can most likely be detected using the current urine analysis; however, the dependence of the concentration of urinary glycerol on the urine volume should be considered.

Determination of mepitiostane metabolites in human urine by liquid chromatography/tandem mass spectrometry for sports drug testing.

Mepitiostane (2α,3α-epithio-17β-(1-methoxycyclopentyloxy)-5α-androstane), which is a prodrug of epitiostanol (2α,3α-epitio-5α-androstane-17β-ol), is an epitiosteroid having anti-estrogenic and weak androgenic anabolic activities. The World Anti-Doping Agency prohibits the misuse of mepitiostane by athletes. Detection of the urinary metabolites epitiostanol sulfoxide and epitiostanol was studied using liquid chromatography/mass spectrometry (LC-MS) for doping control purposes. The use of LC-MS provided advantages over gas chromatography/mass spectrometry for detecting heat labile steroids because epitiostanol and epitiostanol sulfoxide were primarily pyrolized to 5α-androst-2-en-17β-ol. The method consists of enzymatic hydrolysis using β-glucuronidase (Escherichia coli), liquid-liquid extraction, and subsequent ultra-performance liquid chromatography/electrospray-tandem mass spectrometry. Epitiostanol sulfoxide was determined at urinary concentrations of 0.5-50ng/mL, recovery was 76.2-96.9%, and assay precision was calculated as 0.9-1.7% (intra-day) and 2.0-6.6% (inter-day). Epitiostanol was determined at urinary concentrations of 0.5-50ng/mL, recovery was 26.1-35.6% and assay precision was calculated as 4.1-4.6% (intra-day) and 3.3-8.5% (inter-day). The limits of detection for epitiostanol sulfoxide and epitiostanol were 0.05ng/mL and 0.10ng/mL, respectively. Epitiostanol sulfoxide and epitiostanol, as their gluco-conjugates, were identified in human urine after oral administration of 10mg mepitiostane. Epitiostanol sulfoxide and epitiostanol could be detected up to 48h and 24h after administration, respectively. The results showed that the detection window of epitiostanol is much shorter than that of epitiostanol sulfoxide. The LC-MS detection of urinary epitiostanol sulfoxide, a specific metabolite with a sulphur atom in its molecular structure, is likely to be able to identify the abuse of mepitiostane.