Establishment And Application Of A New Method For The Determination Of Prednisolone And Prednisone Stimulants By Online Two-dimensional Liquid Chromatography With Isotopic Fingerprinting

Objective:This thesis aims to establish a gas phase combustion isotope ratio mass spectrometry（GC-C-IRMS）detection technique for prednisone and prednisolone by developing an efficient two-dimensional liquid phase separation method for glucocorticoids,which are prohibited substances in stimulants as stipulated by the International Anti Doping Agency（WADA）,and conduct carbon isotope fingerprint tracing research to complete the identification of the source of pseudo endogenous glucocorticoid stimulants.At the same time,mass spectrometry technology is used to quantitatively detect urine samples of oral glucocorticoids and isotope ratio mass spectrometry to analyze the concentration level and carbon isotope ratio change trend of various steroid hormones in the body,and further study the impact of glucocorticoids on the determination of steroids in the body.Methods:1.Carry out mass spectrometry ion pair collection for glucocorticoids,complete the optimization of mass spectrometry conditions,establish a screening window for glucocorticoids,establish a liquid chromatography mass spectrometry（LC-MS）quantitative detection method,and conduct methodological validation,including,linearity,minimum detection limit,quantitative limit,precision,accuracy,matrix effect,extraction recovery rate,and other indicators.2.To reduce wastage and enrich the target substance,the sample pretreatment method was optimized,and a two-dimensional liquid chromatography analysis method was established to separate glucocorticoids.3.Establish GC-C-IRMS technology and detect glucocorticoid test samples,conduct carbon isotope fingerprint tracing research,and complete the source identification of pseudo endogenous glucocorticoid stimulants.4.Analyze the impact of oral glucocorticoid drugs on the metabolic concentration and isotope ratio of steroid hormones based on mass spectrometry quantitative and isotope ratio mass spectrometry detection results.5.Establish a DWM model by measuring the targetΔδ¹³C value,combined with the substance quantitative results and isotope ratio mass spectrometry results,forms a new statistical indicator DWM,which is applied to the analysis of samples taking pseudo endogenous steroid stimulants and is intended to serve as a supplementary indicator for doping detection.Results:1.Establish four quantitative detection methods for glucocorticoids,including cortisone（CT）,hydrocortisone（HCT）,prednisone（PS）,and prednisolone（PSL）,using liquid chromatography mass spectrometry（LC-MS）.The linear range is 2-300 ng/m L,the minimum detection limit for CT,HCT,and PSL is 1ng/m L,the minimum detection limit for PS is 2ng/m L,the quantitative limit for CT,HCT,and PSL is 2 ng/m L,and the quantitative limit for PS is 5ng/m L,with a precision of less than 20%,and a matrix effect of 96%to 120%,The extraction recovery rate is greater than 86%.This quantitative method has passed methodological validation and is used to detect glucocorticoid test samples.PS and PSL peak in 3-5 hours and complete elution within10 hours,while CT and HCT peak in 20-25 hours after administration.After glucocorticoid testing,the concentrations of the male steroid hormone testosterone and its metabolites in the sample were significantly inhibited,and after 45 hours,they returned to baseline values.2.Successfully established a two-dimensional online liquid chromatography separation and detection method for glucocorticoids,which improves separation efficiency,reduces sample loss,and is conducive to enriching the target analyte compared to traditional two offline liquid phase LC-MS separations.3.Successfully established a gas phase combustion isotope ratio mass spectrometry（GC-C-IRMS）detection technique for glucocorticoids.4.Detection of carbon isotope ratios in glucocorticoid subjects using gas phase combustion isotope ratio mass spectrometry（GC-C-IRMS）technologyδ¹³C and difference changes,plottingδ¹³C andΔδ¹³C value versus time,the analysis of the results showed that prednisone and prednisolone in urine samples within 24 hours after administration belonged to the detection window period,and the test result was positive.When assessing the impact of glucocorticoid administration on the results of male steroid isotope testing,after taking glucocorticoid alone,the results of male steroid isotope ratios were negative;When taking glucocorticoid and testosterone undecanoate simultaneously,the metabolism of male steroid hormones is still inhabited.When the abnormal standard of WADA for testosterone and epitestosterone ratio T/E>4 is not met,the isotope ratio of the sample is measured and the result is positive.5.Calculate and analyze the DWM value of the supplemental indicator measured by the test sample,and provide the recommended DWM thresholds for men and women taking prednisolone and prednisolone,respectively:the absolute DWM（PS）value of prednisolone in male subjects is within 2.50,and the absolute DWM（PSL）value is within 4.00;The absolute value of DWM（PS）in female prednisolone subjects was within 4.00,and the absolute value of DWM（PSL）was within 8.00;The absolute value of DWM（PS）in male prednisone subjects was within 2.00,and the absolute value of DWM（PSL）was within 3.00;In prednisone female subjects,the absolute value of DWM（PS）was within 1.50,and the absolute value of DWM（PSL）was within 5.00.Conclusion:In this study,a mass spectrometry detection technique was established to accurately quantify glucocorticoids;The method based on online two-dimensional liquid chromatography and isotope ratio mass spectrometry can be used to determine the concentrations of prednisone,prednisolone,pregnanediol,and pregnanetriolδ¹³C andΔδ¹³C value;Taking glucocorticoids for a short period of time can cause significant increases in the concentrations of CT,HCT,PS,and PSL,which inhibits the metabolism of male steroid testosterone and its metabolites during protein assimilation;If both glucocorticoids and testosterone undecanoate are taken at the same time,the quantitative test result shows that T/E is less than 4,and the isotope mass spectrometry determination of the sample shows a positive result.It is recommended that when abnormal glucocorticoid testing occurs,isotope ratio mass spectrometry can be performed on the protein assimilating male steroid testosterone and its metabolites simultaneously to further determine doping violation factors.