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Methodological Study On UPLC-MS/MS Technology For Determination Of Endogenous Corticosteroids

Posted on:2021-08-21Degree:MasterType:Thesis
Country:ChinaCandidate:M Z LiFull Text:PDF
GTID:2491306548484624Subject:Chemical Engineering
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The content of adrenal cortex hormones in human body is related to some diseases such as hypertension and hyperglycemia.They are detection indexes for endocrine diseases such as adrenal hyperplasia,Cushing’s syndrome and hyperaldosteronism.The research direct at common detection indicators of adrenal endocrine diseases,using aldosterone,cortisol,corticosterone and cortisone in human serum as the research objects.In this study,isotope dilution(Isotope Dilution,ID)method was used to establish a quantitative analysis method for human serum corticosteroids based on ultra performance liquid chromatography-triple quadrupole mass spectrometry(UPLCMS/MS).The research content of this article is divided into three parts,the specific content as follows.First,established corticosteroids UPLC-MS/MS analysis method.Determined and optimized the corticosteroids and isotope internal standard mass spectrometric parameters.Screened suitable column for corticosteroids separation,established and optimized UPLC chromatographic separation conditions.The optimal chromatographic conditions as follows: ACQUITY UPLC BEH C18 column,the column temperature was 30 ℃,the flow rate was 0.3 m L/min,the mobile phase was acetonitrile/0.01 m M ammonia solution.The corticosteroid analysis method of mass spectrometry was established in this content.It has high sensitivity and strong selectivity characteristics.Secondly,on the basis of the ID-UPLC-MS/MS instrument method,further studied on serum samples pretreatment method.Established a two-step human serum corticosteroid extraction method.It was that zinc sulfate protein precipitation combined with tert-butyl methyl ether liquid-liquid extraction.The optimized human serum samples pretreatment conditions were tert-butyl methyl ether pure solvent and zinc sulfate solution 0.3 mol/L.Tert-butyl methyl ether extractant volume was 1000 μL,protein precipitant volume was 5 μL.The human serum sample pretreatment method is simple and short time in operation.At last,verified the human serum corticosteroids ID-UPLC-MS/MS quantitative method,to investigated the method applicability and reliability,including precision,linear range,quantitation limit and detection limit of the instrument and specificity,detection limit,quantitation limit,linear range,recovery rate,matrix effect,stability of pretreatment method.The results showed that Cortisol,corticosterone and cortisone’s linear range were from 0.02 ng/m L to 20 ng/m L,aldosterone’s linear range was from0.05 ng/m L to 20 ng/m L,the linear correlation coefficient was above 0.995,serum corticosteroids extraction recovery rate was between 55.5-90.8%.The method recovery rate was from 91.0% to 101.2%,the intra-day precision was less than 13.1%,and the intra-day precision was less than 17.8%,matrix effect was from 85.9% to 108.5%,freeze-thaw stability was from 90.2% to 109.6%,solution stability was from 82.0% to104.0%.In this study,the ID-UPLC-MS/MS quantitative detection method has high accuracy,good stability,and weak matrix effects.It can be used to monitor the content of human serum corticosteroids and provide technical reference for the diagnosis and treatment of endocrine diseases.
Keywords/Search Tags:Corticosteroids, Ultra performance liquid chromatography-triple quadrupole mass spectrometry, Isotope dilution, Liquid-liquid extraction
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