| Desmopressin is one of the essence of synthetic vasopressin analogue,because of its lead to the blood—thining effect,it may masking some doping illegal behavior,that is the reason why World Anti Doping Agency(WADA)added desmopressin into the masking agent part of Porhibited List in 2011.Desmopressin is one of small peptid,and efficient and accurate detection of small peptides from human urine or plasma is one of heavy problem in the field of anti-doping field,there was no literature reported inland and abroad only Germany and Belgium had reported some analytical method.At the same time,it was still not reported that after using desmopressin,the testing window period and taking endogenous parameters effect of human body,the extension of detection window period will greatly increase the detection rate of prohibit drugs and unuaually changes of endogenous parameter to the human body will become the important indirect evidence whether athletes using doping,therefore,for the research of this two tests is very important and imperative.There are many parts of test for this study:First of all,this study using liquid chromatography tandem mass spectrorietry to explore the detection method of Desmopressin in human urine and plasma:Through optimized of the pretreatment which including the buffer solution,elution buffer and program,etc.;the chromatographic condition(including the chromatographic column,mobile phase system,HPLC flow,gradient elution program,etc.);mass spectrometry condition(including the ion acquisition mode,fragmentor,collision energy,drying gas temperature,etc.,developed the detection method of Desmopressin by LC-MS/MS.Second the study operated method validation for the detection method mentioned above,the content.of validation including that limit of detection(LOD),limit of quantitation(LOQ),range and relationship of linear,intraday precision and interday precision,rate of recovery,matrix effect experiment.Then this study collected and detected positive urine sample,and made the metabolic curve by the prototype,understood the general metabolic rule of desmopressin and confirmed the analytical window period by this analysis method.The secound one is using gas chromatography tandem mass spectrometry to detected the human endogenous parametric,contrast and discover the unusual changes of data by ABPS,accumulated the data and experience for deeply test and study in future.The last part of this study is collection plasma which was the volunteer used the desmopressin,recorded the hematological parameters and determine degree of suspicious by ABPS.This study developed one of the detection method to confirmed desmopressin from human urine and plasma,and the method was efficient,specificity and consistent with the actual situation of our laboratory.The study improved pretreatment experiment and the working hour was reduced 6N8 hours than beforek,at the same time the validation was meet the general standard:The correlation coefficients of linear relationship are greater than 0.99;intra-day and inter-day RSD was almost less than 20%;the recovery of method was higher than 50%and the matrix interference less than 20%;the limit of detection in human urine is 100pg/mL and in human plasma was 2000pg/mL,its accordanced with WADA MRPL.At the same time,this study used to establish detection methods in eight subjects to return positive urine samples were detected out desmopressin,the longest detection window period can reach 13 hours after taking the drug.Experiment not detected in plasma samples desmopressin prototype metabolites or debris metabolite signals.This study detection and statistics endogenous parameter of urine after the volunteer taking drug,found that after taking desmopressin,T/E value fluctuates significantly,and larger ABPS software display abnormal suspicious,T/A and A/E value at the same time also have certain fluctuations,numerical results of the above three directions desmopressin endogenous parameters have certain influence to the human body.This study also for drug blood sample after the subjects compared with the blank urine sample,in nine regular blood parameters were found no obvious changes,input parameters to ABPS software system for result determination,also did not show the obvious exception.This research established an effective pretreatment and detection method to detect desmopressin which was specificity and accord with the actual situation of our laboratory,from all the volunteers urine samples success of detected desmopressin prototype,at the same time using LC-MS/MS detection results for the first time mapped the metabolic curve,to some extent,understanding the metabolism of the drug in the body and eventually determine the method of detection window period,the above aspects the research blank in make up for the drug doping detection ability at the same time as the doping test provides a data basis;This research for human endogenous parameter of preliminary experimental results show that taking desmopressin will lead to a certain degree of abnormal fluctuations endogenous parameter data,but due to fewer subjects,the research also need further experiment and discussion;Finally,this study after taking drugs for the hematology parameters of the research results show that the conventional single take 0.2 mg doses,water quantity of 300 ml/min 30 cases,induced by the drug pharmacology of blood dilution had no obvious on blood parameters in abnormal fluctuations. |
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