Makoto Ueki

ANALYSIS OF EXOGENOUS DEHYDROEPIANDROSTERONE EXCRETION IN URINE BY GAS CHROMATOGRAPHY/ COMBUSTION/ISOTOPE RATIO MASS SPECTROMETRY

A detailed procedure for the analysis of exogenous dehydroepiandrosterone (DHEA) in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) has been established for detecting doping with DHEA. The average delta-value (parts per thousand difference of (13)C/(12)C ratio from the isotope ratio standard) of 26 synthetic steroids commercially available was -30.1 +/- 2.6, and was significantly lower than that of human endogenous DHEA in urine of the world class athletes who had participated in the XVIIth Olympic Winter Games (-20.3 +/- 2.1, n = 446). Although large inter-individual variations of urinary DHEA excretion were observed following a single oral administration of 50 mg of DHEA, no significant inter-individual difference was found when the excretion of exogenous DHEA was monitored in terms of delta-values using GC/C/IRMS; the minimum delta-values were observed around 6-8 h after the administration, and the values returned to the base level at over 72 h after the dosing. Thus, the deviations in delta-values of DHEA and its diol metabolites are considered to be conclusive evidence for detecting doping with DHEA. Some successful cases of detection of doping with DHEA from athletes are also reported.

20K-GH and its use in detecting GH abuse

Following the successful production of recombinant 20K-GH, several studies investigating the physiology of this GH isoform have been undertaken. In this report, we review studies of its biological effect, measurement and secretion. To use serum 20K-GH level in detecting GH abuse, new method has been established and serum 22K-GH, 20K-GH were measured in normal subjects and athletes, and no abnormal results were found among athletes. Another study confirmed that serum 22K-GH increased remarkably and 20K-GH decreased following the exogenous administration of 22K-GH. The duration was relatively short, approximately 24-36h in our and other studies. The increase of the ratio, 22K-GH/20K-GH was the most suitable indicator of GH abuse. Studies supported by the WADA were undertaken in collaboration with an Australian Group. A new approach for the GH isoform assay by beads assay platform is being developed. It is concluded that the direct measurement of 20K-GH is a valid scientific approach, for detecting GH abuse, although the duration of the positive results is short. Our method will be useful in combination with the marker method, an out-of-competition test or test for target cases. Furthermore, its application for the doping test passport is considered to be a possible future strategy.

Doping with naturally occurring steroids

AbstractThe current definition of doping involves the restriction of the use of naturally occurring compounds The typical examples of such dope agents are testosterone and the related steroid hormones.1) One difficulty of dope analysis stems from the origin of the dope agents As the detection of synthetic anabolic steroids became relatively easy, and since there was no procedure available that could differentiate exogenous natural hormones, testosterone and its esters became a popular substitute for steroid abusers. Testosterone is the worst of the popular anabolic steroids that are abused by sportsmen, according to the laboratory statistics of 1995 issued by the Medical Commission of the International Olympic Committee (MC-IOC).Recent incidents in sport also involve doping cases with other testosterone-related compounds Doping test results from the Asian Games in Hiroshima, 1994, involved 16 positive cases of 5α-dihydrotestosterone (DHT) and the results proved its abuse by world class athletes.2)Some nat...

Detection of 1-phenyl-N-methyl-1,2,3,4-tetrahydroisoquinoline and 1-phenyl-1,2,3,4-tetrahydroisoquinoline in human brain by gas chromatography-tandem mass spectrometry

1-Phenyl-N-methyl-1,2,3,4-tetrahydroisoquinoline and 1-phenyl-1,2,3,4-tetrahydroisoquinoline were detected for the first time in parkinsonian human brain using gas chromatography-tandem mass spectrometry (GC-MS-MS). Since these compounds are structural analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that produces parkinsonism in humans, they might be candidates for endogenous MPTP-like neurotoxins.

Structure-based comprehensive identification of erythropoiesis-stimulating agents and their biosimilars

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.

Determination of bicozamycin and its benzoylester derivative in yellowtail tissues by high performance liquid chromatography

Abstract A unique and practical method was developed for quantitative analysis of Bicozamycin(BCM) and its benzoylester(BCM-BZ) from yellowtail(Seriola quinqueradiata) tissues(blood, muscle, liver, kidney) by High Performace Liquid Chromatography(HPLC). Tissues were homogenized and then deprotenized with acetonitrile or a mixture of acetonitrile and water. The extract was evaporated, and the residue was dissolved in water and partitioned by addition of chloroform or carbontetrachloride. The aqueous solution was taken up into a BOND ELUT C18 cartridge column, after washing with water, BCM was eluted by methanol then the eluent was evaporated to dryness. After that the residue was dissolved in methanol/ethyl acetate/hexane (0.3:3:10) and the solution was applied to a BOND ELUT DI0L(or SEP PAK-DIOL)column, BCM eluted with acetone was dissolved in the mobile phase and analyzed by HPLC on two C18 reversed phase columns. BCM-BZ extracted from tissues was also cleaned up with BOND ELUT DIOL, and analyzed by HPLC...

Trends in Drug Abuse in Compelltive Sports, and the International Anti-Doping Movement

AbstractThe results of our twelve years drug abuse survey in competitive sports is discussed. The most typically abused drug among the Japanese general public is methamphetamine, and the problem has been escalating during the last few years especially among female adolescents. Throughout our survey period, the major dope agents abused by sportsmen have been ephedrines and injectable anabolic steroids. As detection of synthetic anabolic steroid is relatively easy, doping with naturally-occurring steroids has become the major doping agent in sports.In some countries, a governmental or non-governmental co-ordination agency has been founded to promote, develop and co-ordinate the national anti-doping program in sports, and out-of-competition testing (OOCT) is now frequently conducted by the agency independently from sports federation. It is thought that it would be helpful for society to be ready for the growing problem of hormone abuse among the general public if the international anti-doping movement agains...