LC-MS/MS定量分析小鼠肝组织15种胆汁酸成分研究*

doi:10.3969/j.issn.1672-5069.2023.03.005

实用肝脏病杂志 ›› 2023, Vol. 26 ›› Issue (3): 320-323.doi: 10.3969/j.issn.1672-5069.2023.03.005

LC-MS/MS定量分析小鼠肝组织15种胆汁酸成分研究^*

蔡玉莹, 殷继明, 宁琪琪, 高玉雪, 杨鹏翔, 陈德喜

100069 北京市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

收稿日期:2022-11-10 出版日期:2023-05-10 发布日期:2023-05-08
通讯作者: 陈德喜,E-mail:dexichen@ccmu.edu.cn
作者简介:蔡玉莹,女,26 岁,理学硕士,技师。主要从事代谢小分子质谱研究。E-mail: caiyuying0113@163.com
基金资助:
^*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)

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

Cai Yuying, Yin Jiming, Ning Qiqi, et al.

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

Received:2022-11-10 Online:2023-05-10 Published:2023-05-08

摘要/Abstract

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²H₄-DCA,GUDCA-d₅和 LCA-d₄为内标物,用 Agilent Poroshell 120 EC C18色谱柱(100 mm×4.6 mm,2.7 μm)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2 μL,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,R²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。

关键词: 肝组织, 胆汁酸, 液相色谱-串联质谱, 小鼠

Abstract: 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% NH₄OH was added to precipitate proteins. The proteins were separated on an Agilent Poroshell 120 EC C18 column (100 mm×4.6 mm,2.7 μm) by using ²H₄-DCA, GUDCA-d₅, and LCA-d₄ 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 2 μL. 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 R²greater 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.

Key words: Liver tissues, Bile acids, Liquid chromatography-tandem mass spectrometry, Mouse

蔡玉莹, 殷继明, 宁琪琪, 高玉雪, 杨鹏翔, 陈德喜. LC-MS/MS定量分析小鼠肝组织15种胆汁酸成分研究^*[J]. 实用肝脏病杂志, 2023, 26(3): 320-323.

Cai Yuying, Yin Jiming, Ning Qiqi, et al.. Quantitative profiling of 15 bile acids in mouse liver tissues by using liquid chromatography-tandem mass spectrometry[J]. Journal of Practical Hepatology, 2023, 26(3): 320-323.

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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

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