LC-MS/MS定量分析小鼠肝组织15种胆汁酸成分研究  

Quantitative profiling of 15 bile acids in mouse liver tissues by using liquid chromatography-tandem mass spectrometry

作　　者：蔡玉莹 殷继明[1] 宁琪琪 高玉雪 杨鹏翔 陈德喜[1] Cai Yuying;Yin Jiming;Ning Qiqi(Beijing Institute of Hepatology,Beijing Precision Medicine and Transformation Engineering,Technology Research Center of Hepatitis and Liver Cancer,You’an Hospital Affiliated to Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学附属北京佑安医院/北京肝病研究所/北京市肝炎与肝癌精准医疗及转化工程技术研究中心,北京市100069

出　　处：《实用肝脏病杂志》2023年第3期320-323,共4页Journal of Practical Hepatology

基　　金：国家自然科学基金资助项目(编号:82073676);北京市属医学科研院所公益发展改革试点项目(编号:京医研2021-10)。

摘　　要：目的 建立快速高效的液相色谱-串联质谱法(LC-MS/MS)同时测定小鼠肝组织15种胆汁酸(BAs)浓度。方法 采用活性炭制备无BAs的空白肝组织,作为制备标准样品和质量控制样品的生物基质。匀浆小鼠肝组织,加入碱性乙腈溶液(5%NH4OH)沉淀蛋白。以^(2)H_(4)-DCA,GUDCA-d_(5)和LCA-d4为内标物,用Agilent Poroshell 120 EC C18色谱柱(100 mm×4.6 mm, 2.7μm)分离,以醋酸铵水溶液和甲醇-乙腈溶液为流动相梯度洗脱,柱温30℃,流速0.3 mL/min,进样量为2μL,采用电喷雾离子源(ESI)负离子模式,多反应监测(MRM)。结果 15种BAs线性关系良好,R^(2)均大于0.993,定量检测限均小于2 ng/mL,基质效应为90.76%~109.25%;日内、日间准确度和精密度均小于15%, 4℃24h、反复冻融、冷冻保存1个月稳定性良好,满足生物样品的分析要求;小鼠肝组织检测结果显示游离型BAs和结合型BAs(G-BAs、T-BAs)都以母体CA为主,TCA含量最高;游离BAs浓度为(723.89±50.65)ng/mL,显著高于G-BAs【(56.90±11.28)ng/mL,P<0.001】,T-BAs浓度为(40322.90±14034.80)ng/mL,显著高于游离BAs(P<0.001)或G-BAs(P<0.001)。结论 建立的LC-MS/MS方法灵敏度高,准确可靠,适用于检测小鼠肝组织BAs浓度。Objective The purpose of this study was to establish a rapid and efficient liquid chromatography tandem mass spectrometry(LC-MS/MS)for simultaneous determination of 15 bile acids in mouse liver tissues.Methods The activated charcoal was utilized to prepare bile acid-free liver,which served as the biological matrix for the preparation of standard and quality control samples.The mouse liver tissue was homogenized,and a basic acetonitrile solution,including 5%NH 4 OH was added to precipitate proteins.The proteins were separated on an Agilent Poroshell 120 EC C18 column(100 mm×4.6 mm,2.7μm)by using ^(2)H_(4)-DCA,GUDCA-d_(5),and LCA-d_(4) as internal standards.The mobile phase is ammonium acetate aqueous solution and methanol acetonitrile mixed solution for gradient elution,the column temperature was 30℃,the flow rate was 0.3mL/min,and the injection volume was 2μL.The electrospray ion source(ESI)was operated in negative ion mode,and in multiple reaction monitoring(MRM).Results The linearity of the 15 bile acids was good with R^(2) greater than 0.993,the limits of determination were less than 2 ng/mL,and the matrix effects were 90.76%-109.25%;the intra-day and inter-day accuracy and precision were less than 15%,and the stability was good under 4℃for 24 h,repeated freeze-thaw,and freeze-storage for one month,meeting the analytical requirements of biological samples;the detection of mouse liver tissues showed that both unconjugated BAs and conjugated BAs(G-BAs,T-BAs)were dominated by maternal CA,with the highest content of TCA;the concentration of unconjugated BAs was(723.89±50.65)ng/mL,significantly higher than that of G-BAs[(56.90±11.28)ng/mL,P<0.001];the concentration of T-BAs was(40322.90±14034.80)ng/mL,significantly higher than unconjugated BAs(P<0.001),and also significantly higher than G-BAs(P<0.001).Conclusion The LC-MS/MS method we established is sensitive,accurate,reliable,and suitable for the determination of bile acids concentrations in mouse liver tissues,which might help for further studies.

关 键 词：肝组织 胆汁酸 液相色谱-串联质谱 小鼠 

分 类 号：O657.63[理学—分析化学] R-332[理学—化学]