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Otsuki T.,Anti Doping Research Laboratory | Kishikawa Y.,Anti Doping Research Laboratory | Suzuki H.,Nippon Medical School | Ueki M.,Anti Doping Research Laboratory
Forensic Toxicology | Year: 2014

Erythropoietin (EPO) is a glycoprotein that stimulates production of red blood cells. After the patents for recombinant EPO (epoetin) expired in 2007, biosimilar erythropoiesis-stimulating agents (ESAs) have become widely circulated in the black market. However, the heterogeneity of ESAs in the post-translational glycosylation hinders the identification of these compounds. In the present study, after cleaving N-linked glycans from tryptic peptides of ESAs with N-glycosidase F, we analyzed the resulting eight glycan-free peptides by liquid chromatography-electrospray ionization-tandem mass spectrometry in the multiple reaction monitoring mode. It has been confirmed that all ESAs studied gave rise to common signature peptides SLTTLLR, VYSNFLR, and YLLEAK. In darbepoetin alfa (DPO), glycan-free tryptic peptide GQALLVNSSQVNETLQLHVDK was detected instead of GQALLVNSSQPWEPLQLHVDK in the epoetin. One of the signature peptides VNFYAWK was found to be significantly suppressed in continuous erythropoietin receptor activator (CERA), indicating pegylation at Lys52. Our method allowed simultaneous identification of ESAs including epoetin, DPO, CERA, and the biosimilars. © 2014 Japanese Association of Forensic Toxicology and Springer.

Bosch J.,Bioanalysis Group | Ueki M.,Anti doping Research Laboratory | Such-Sanmartin G.,Bioanalysis Group | Segura J.,Bioanalysis Group | And 3 more authors.
Analytica Chimica Acta | Year: 2012

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22. kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20. kDa hGH isoforms and also a ratio 22/20. kDa is derived. Using a single set (nine healthy male subjects, 7 days, 0.026. mg/kg/day of rhGH, 2 week wash out period) both approaches were compared. To quantify the agreement between the immunoassays, B.A. (Bland-Altman) analysis and P.r. (Pearson correlation) were used. To fully understand the assay readings, all relevant antibodies were characterised by surface plasmon resonance (SPR).In either approach the ratio numerator produces similar results and the denominator determines both signal-amplitude and time-frame of possible application. The rec vs pit approach displays a higher distinctive capacity to detect hGH abuse but the complex binding properties of the capture antibodies make it very difficult to evaluate the precise contributions of the individual hGH variants to the assay result. In the 22 vs 20 approach, the 20 kDa hGH concentration measures determine its applicability. Both approaches are based on a different principle, should be preferably applied within 24 h after rhGH administration, and are perfectly comparable given the results obtained. The reduced time frame of application indicates that their principle application should be preferably in an out-of-competition setting. © 2012 Elsevier B.V.

Ueki M.,Anti doping Research Laboratory
Bunseki Kagaku | Year: 2014

Abuses of performance-enhancing agents and the manipulation or cheating of test systems are prohibited in order to protect fairness in sports and athletes' health according to ethical considerations. Doping control is applied worldwide at all levels of sports. Recent trends of test results show that doping with naturally occurring substances, such as androgenic steroids, the pro-hormones and the metabolites, peptide hormones, blood and blood components etc. are increasing, so that the testing methods for origin identification must fit for the purpose depending on the property of the target compounds. This review refers to up-dated information concerning the procedures for origin identification of doping agents with special focus on GC-online isotope ratio mass spectrometry. © 2014 The Japan Society for Analytical Chemistry.

Aoki K.,Anti doping Research Laboratory | Aoki K.,Nihon Pharmaceutical University | Shinohara H.,Anti doping Research Laboratory | Tanaka H.,Anti doping Research Laboratory | Ueki M.,Anti doping Research Laboratory
Forensic Toxicology | Year: 2014

When testing a urine sample for testosterone abuse, a ratio of testosterone glucuronide (T) to epitestosterone glucuronide (ET) of 4.0 or above is considered suspicious. A degree of variation, however, has been observed in T/ET ratio between individuals from both the same and different ethnic backgrounds. The majority of this variation might be due to UGT2B17 deletion genotype (UGT2B17 deletion-type). The aim of this study was to investigate the use of the same urine sample for the analysis of T/ET ratio and UGT2B17 deletion-type. Japanese men were deletion-typed via a UGT2B17 copy number assay using DNA from blood. Urinary T and ET levels were determined using gas chromatography-mass spectrometry before (n = 112) and after a testosterone injection (n = 25). Basal T level and the increase in T/ET ratio after injection were dependent on UGT2B17 deletion-type, being lower in subjects with deletion (del/del) than nondeletion (ins/del or ins/ins) genotype. UGT2B17 deletion-typing was first performed using DNA from urine cryopreserved for 1-1.5 years (n = 66). The concentration of DNA required for discrimination between the deletion and nondeletion genotype by copy number assay was more than 0.1 ng/ml urine. Discrimination was possible in 94.0 % of urine samples (5-7 ml each). These findings show that T/ET ratio and UGT 2B17 deletion-type can be analyzed exclusively via urine samples, removing the need for the collection of other samples, such as blood or buccal cells. The combination of T/ET ratio and UGT 2B17 deletion-type may help inform decisions regarding a genotype-specific T/ET cutoff ratio. © 2013 Japanese Association of Forensic Toxicology and Springer.

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