Development And Validation Of A Uplc-ms/ms Method For The Detection Of Multiple Prohibited Substances And Its Metabolites

In order to keep the fair and clean in sports and protect the athelete from the risk of health problems,doping-control analysis has become an important and necessary step in sports competition.To fullfil the need and desire that identify a continuously growing number of prohibited substances and methods in doping-control,the anti-doping laboratories have to enhance their procedures in terms of comprehensiveness,high-throughout,and sensitivity.This endeavour has been supported by the improved analytical technologies.In the past,high performance liquid chromatography(HPLC),gas chromatography(GC)and gas chromatography-mass spectrometry(GC/MSD)were normally applied for prohibited substance analysis.Currently,the application of LC-MS technique has effectively figure out the issues that occurring in the procedures of sample analysis,such as the derivatization,complicated operation,long detection period and poor sensitivity.In this study,two analytical methods based on HPLC-MS/MS and quadrupole-electrostatic orbital high-resolution mass spectrometry were developed and validated for the simultaneous determination of 12 prohibited parent drugs and metabolites respectively.This investigation was focused mainly on the following two aspects:1.To develop and validate a LC-MS/MS method for the detection of 12 prohibited substances including azosemide(?),buthiazide(?),brinzolamide(?),benzylhydrochlorothiazide(?),chlorazanil(?),cicletanine(?),chlorxolone(?),dorzolamine(?),quinethazone(?),fenquizone(?),meticrane(?)and trimetazidine(?)with limited sample pretreatment.The limit of detections(LODs)for the above described substances ?-? were between 0.01-10ng/mL.The assays were linear over the concentration ranges of 3.0-700 ng/mL?1.5-1000 ng/mL?3.0-4000 ng/mL?3.0-1500 ng/mL?3.0-300 ng/mL?0.3-500 ng/mL?0.03-4000 ng/mL?6.0-5000 ng/mL?6.0-600 ng/mL?30-1600 ng/mL 6.0-5000 ng/mL and 3.0-500 ng/mL in human urine separately.The intra-and inter-day precisions of the method at three concentration levels(low,medium and high)were better than 15%.The established method is precise,sensitive and accurate with minimum sample preparation.It was a fit-for-purpose approach and has been applied in Beijing doping-control laboratory in routine sample analysis.2.Further study on the metabilites identification of azosemide and trimetazidine were carried out by utilizing high-resolution mass spectrometry.Solid phase extraction technique was used for sample preparation.Five azosemide metabilites were identified in rat excretion urine,in which two of them were reported previously and three were detected for the first time.In the case of trimetazidine human excretion study,seven known metabilites and six unknown compounds were found.This study has effectively expanded the detection window for azosemide and trimetazidine.Therefore more positive urine samples could be detected for doping-control purposes.