The Study Of Detection Method Of Hair Doping By Liquid Chromatography Tandem Mass Spectrometry

Purpose This dissertation focused on the detection and evaluation study of doping agents that is abused or uneasily detected(containing parent drug, its esters and metabolites) in hair using specific and sensitive liquid chromatograph-mass spectrometry. the new analysis method is proved to be valid by testing beta-blockers(metopropol and bispropol) and beta2-agonists(clenbuterol) in hair of hypertension patients and elite athletes, This research provides a technical support for detection of hair doping in doping control.Methods50mg of decontaminated hair was incubated in100u1Proteinase K,100μ1Dithiothreitol and tris-HCl buffer solution,2h at55℃, in the presence of5u1of testosterone-d3used as internal standard. After pentane/ether was added, agitated, centrifuged and frozen, pour the upper organic phase. Phosphate buffer,5μ1of internal standard and chloroform/isopropanol were added, when the lower aqueous phase was melted, agitated, centrifuged and the upper organic phase was absorbed, then two organic phase was evaporated to dryness. Finally, methanol, mobile phase A and n-hexane were used to remove grease, The extracted residue was reconstituted with solution (90%mobile phase A), A10μl aliquot of the solution was injected into the LC-MS/MS system. Because of reconstituted solution was different between esters and nonesters, they were separately detected.50mg of hair was incubated in100μ1Proteinase K,100μ1Dithiothreitol and tris-HCl buffer solution,2h at55℃for testing esters, in the presence of10u1of testosterone-d3used as internal standard, After pentane was added, agitation, centrifuged and frozen, evaporated to dryness. The extracted residue was reconstituted with solution (90%mobile phase B), A20u1aliquot of the solution was injected into the LC-MS/MS system.Results and discussion Through optimizing chromatographic conditions and mass conditions of hair testing, X Bridge RP18column (3.5μm,100mm X2.1mm) and Agilent ZORBAX XDB-C18column (3.5μ m,50mmX2. lmm) were separately chose for testing esters and nonesters, The flow rate through the column were separately0.2ml/min and0.4ml/min,10μ1and20μ1were separately injected, and2.5mM ammonium formate-0.2%formic acid methanol/acetonitrile (v:v=7:3) system and2.5mM ammonium formate-0.2%formic acid-methanol system were separately used to test esters and nonesters. The method of hair sample treatment was also optimized. Pentane/ether and chloroform/isopropanol two-step liquid-liquid extraction was used to analyze nonesters, simultaneously pentane liquid-liquid extraction was used to analyze esters. Correlation coefficient, LOD and LOQ were appropriate for routine quantitative and sensitivity in detection of doping in hair. The intra-day and inter-day RSD for most of doping agents both were less than20%, recovery mostly is above50%and matrix effect mostly range from80%to120%. When the method for simultaneous detection of forty-three doping was used to detect beta-blockers in hair of hypertension patients and clenbuterol in hair of athletes, drugs was successfully detect.Conclusions The method for simultaneous detection of42doping and esters detection method for distinguishing between exogenous and endogenous agents were established, then they were proved to be effective by method validation and testing hair of hypertension patients and athletes. This research provides technical support for detection of hair doping in doping control.