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Prospective Study for Discrimination of Pork Contamination and IllicitAdministration of Clenbuterol in Doping ControlClenbuterol is a kind of classical anabolic agent steroids banned by the World Anti Doping Agency(WADA),it is mainly used in the treatment of asthma in clinic.Due to its anabolic function,it is also abused as lean meat in animal husbandry.Therefore,animals reared with clenbuterol produce tissue residues in their bodies,furthermore,it will cause the second pollution or even poisoning problems.From 2011 to 2016,the results of clenbuterol positive from ADAMS system of WADA illustrated that the number of clenbuterol positive is on rise.In addition,the official website of China Anti-Doping Agency issue a notice of strengthening the risk of prevention and control of clenbuterol in meat products,which stressed clenbuterol positive cases of athletes caused by ingesting meat contaminated by clenbuterol with an upward tendency.Contamination of clenbuterol in meat products is not only existed in China,but also existed in other countries,such as Italy,Mexico,Spain,Portugal and France.Therefore,how to distinguish meat contamination and illicit administration of clenbuterol by means of doping analysis is a worldwide problem.There is a chiral carbon in the chemical structure of clenbuterol,so it has a pair of optical isomers(R-(-)-clenbuterol and S-(+)-clenbuterol).Therapeutic clenbuterol consists of a racemic mixture of R-(-)-clenbuterol and S-(+)-clenbuterol.It is reported that the ratio of R-(-)-clenbuterol to S-(+)-clenbuterol has differences in the residues of the edible tissues of pigs,which is no more than 1.Based on the above research foundation,this study has discussed the analysis method of distinction the illicit clenbuterol administration with food contamination by using pork which is the largest meat consumption for Chinese athletes as the object.The starting point of this study is to compare the differences of RS ratio in human urine between therapeutic clenbuterol preparations intaking and inadvertent ingestion via pork.Objectives:Scientific data will be provided to support the results of doipng analysis for the distinction of contamination and illicit use by doping analysis technique.Methods:LC-MS/MS analytical methods were established and validated to separate R-(-)-clenbuterol and S-(+)-clenbuterol in urine,blood and tissues,respectively.Pharmacokinetics and tissue distributions of two enationmers in Bama minipigs and urine samples from 26 volunteers after oral administration of clenbuterol with pork origin or drug origin were studied by these methods.A judgment basis was concluded through the comparison between one group of human experiment which was given clenbuterol by therapeutic preparation and the other group of human experiment which was given clenbuterol in meat.Validation parameters of these methods for R-(-)-clenbuterol and S-(+)-clenbuterol in the blood,urine and tissue established in this paper were all in accordance with the technical guidelines for the study of drug non clinical pharmacokinetics(2014 Edition)and the international standard of laboratory issued by WADA.Results:After intragastric administration of clenbuterol to Bama pig,the ratio of R/S in blood was more than I,and the ratio of R/S in urine was less than 1,and the residual R/S ratio in the edible muscle tissue was less than 1.Twenty-six volunteers were treated with meat contamined by clenbuterol and clenbuterol preparation,respectively.The R/S ratio in urine was 0.76 + 0.09 and 1.09 + 0.07,respectively.Conclusions:Through the above research results,we can get the following R/S ratio reference:(1)when 0.67?R/S?0.85,it should be judged as a source of pork.(2)When 0.85<R/S<1.02,more evidence should be provided.(3)When 1.02<R/S<1.16,it should be judged as a source of clenbuterol preparation.(4)When R/S<0.67 or R/S>1.16,it should be judged as a source of non-clenbuterol preparation.This discrimination method of pork contamination and illicit administration of clenbuterol will provide a scientific basis for the athletes who is positive in doping control induced by inadvertently intaking clenbuterol from contaminated meat.Part ? The Study for Metabolites of Methasterone in Human Urine in Doping ControlFrom 2012 to 2016,the greatest progress of doping analysis technology was the detection of long-term metabolites of steroids.Among the re-analysis results of the athletes urine samples from 2008 and 2012 Olympic Games,the most positive substances reported were long-term metabolites of steroids.Therefore,the detection of long-term metabolites of steroids is a topic of general interests in the field of doping.Methasterone is a substance with high positive frequency which is prohibited by World Anti-Doping Agency(WADA)with no reports of long-term metabolites so far.Objectives:Long-term metabolites in urine will be found to prolong the time window for doping control after oral administration of methasterone.Methods:LC-QTOF-MS was used to look for glucuronide conjugated and sulfate conjugated metabolites,and a LC cleanup procedure was employed to isolate these metabolites and the LC-fraction containing relative conjugated metabolites was subjected to enzymatic hydrolysis and TMS derivatization before GC-MS analysis.The detailed structural information in steroidal skeleton was based on GC-MS analysis.Results:Fifteen metabolites including seven glucuronide-conjugated and six free metabolites together with two sulfate-conjugated ones were found for methasterone in human urine after a single dose of 40 mg.In order to estimate the utility of all metabolites to prolong and/or improve the detection of methasterone misuse,the detection windows of all metabolites described above were appraised in administration study urine samples.Detection times commonly ranged from 3 days to approximately 10 days.G1 and G2 clearly extend the detection window up to 10 days indicating the longest retrospectivity for the detection of methasterone misuse.Conclusions:According to the international standard of WADA laboratory,the long-term metabolites of methasterone found in this study can be used as a quality control substance to confirm the positive of the substance in the urine samples of the athletes.Therefore,the long-term metabolites found in this study have important practical significance in the field of doping control. |
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