摘要
目的利用高效液相色谱串联质谱(HPLC-MS/MS)系统建立三羧酸循环主要有机酸的测定方法,探究孕期邻苯二甲酸二(2-乙基己基)酯(DEHP)暴露小鼠血清、肝脏和胎盘中富马酸、苹果酸、琥珀酸、α-酮戊二酸、顺乌头酸、枸橼酸6种三羧酸循环有机酸成分变化。方法孕鼠血清、肝脏和胎盘样品经过前处理后,以含10 mmol/L乙酸铵的水溶液和5μmol/L亚甲基二膦酸作流动相A和含10 mmol/L乙酸铵的90%乙腈水溶液和5μmol/L亚甲基二膦酸作流动相B,流速为0.35 ml/min,采用梯度洗脱程序通过Waters ACQUITY UPLC BEH Amide Column,130,1.7μm,(2.1 mm×150 mm)色谱柱进行洗脱,质谱检测系统采用电喷雾离子源,负离子多反应监测。结果三羧酸循环相关6种有机酸代谢物的标准曲线相关系数r在定量范围内均大于0.996,所建立的方法准确度为97.14%~108.26%,低、中、高浓度日间和日内精密度相对标准偏差(RSD)为1.35%~6.73%,基质效应为93.29%~107.47%,提取回收率为94.82%~112.57%。与对照组相比,孕期DEHP暴露组小鼠不同组织样本中富马酸、苹果酸、α-酮戊二酸、顺乌头酸、枸橼酸均降低(P<0.05)。结论本实验建立HPLC-MS/MS方法检测孕期DEHP暴露小鼠血清、肝脏和胎盘中6种三羧酸循环有机酸成分,方法稳定、灵敏度高、选择性强。孕期DEHP暴露引起小鼠肝脏、血清、胎盘中的三羧酸循环有机酸代谢物水平变化,提示孕期DEHP暴露可能会影响线粒体三羧酸循环过程,在孕期DEHP暴露所致疾病的诊断和治疗中具有潜在价值。
Objective To establish a method for measuring major organic acids in the tricarboxylic acid cycle using a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)system,and to investigate the changes in six tricarboxylic acid cycle organic acids(fumaric acid,malic acid,succinic acid,α-ketoglutaric acid,cis-aconitic acid,and citric acid)in the serum,liver,and placenta of mice exposed to di(2-ethylhexyl)phthalate(DEHP)during pregnancy.Methods The serum,liver and placental samples from pregnant mice were processed and eluted through a Waters ACQUITY UPLC BEH Amide Column(130,1.7μm,2.1 mm×150 mm)using a gradient elution program.Mobile phase A comprised an aqueous solution of 10 mmol/L ammonium acetate and 5μmol/L methanephosphonic acid,while mobile phase B consisted of a 90%acetonitrile aqueous solution containing 10 mmol/L ammonium acetate and 5μmol/L methanephosphonic acid,with a flow rate maintained at 0.35 ml/min.The mass spectrometry detection system utilized an electrospray ionization technique with negative ion mode for multiple reaction monitoring.Results The correlation coefficients of the standard curves for the six tricarboxylic acid cycle organic acid metabolites were all above 0.996 within the quantitative range.The method’s accuracy ranged from 97.14%to 108.26%,with inter-day and intra-day precision relative standard deviation between 1.35%and 6.73%.The matrix effect was between 93.29%and 107.47%,and the extraction recovery rate ranged from 94.82%to 112.57%.Analysis of six tricarboxylic acid cycle organic acids in the liver,serum,and placenta of DEHP-exposed mice during pregnancy showed significant reductions in fumaric acid,malic acid,α-ketoglutaric acid,cis-aconitic acid,and citric acid compared to the control group(P<0.05).Conclusion The HPLC-MS/MS method established in this study for detecting six tricarboxylic acid cycle organic acids in the serum,liver,and placenta of DEHP-exposed pregnant mice is stable,highly sensitive and selective.Prenatal DEHP exposure induced alterations in the levels of tricarboxylic acid(TCA)cycle organic acid metabolites in the liver,serum,and placenta of mice,suggesting that DEHP exposure during pregnancy may interfere with mitochondrial TCA cycle processes.These findings indicate potential value in the diagnosis and treatment of diseases associated with prenatal DEHP exposure.
作者
陶文康
张伦
黄倩倩
余芸
蒋月
侯梦贞
凌旭涛
谢芳
王建青
Tao Wenkang;Zhang Lun;Huang Qianqian;Yu Yun;Jiang Yue;Hou Mengzhen;Ling Xutao;Xie Fang;Wang Jianqing(School of Pharmaceutical Sciences,Anhui Medical University,Hefei 230032;Dept of Pharmacy,The First Affiliated Hospital of Anhui Medical University North District,Hefei 230012;Anhui Public Health Clinical Center,Hefei 230012)
出处
《安徽医科大学学报》
北大核心
2025年第5期897-905,共9页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金项目(编号:82073566)
安徽省公共卫生临床中心安徽医科大学第一附属医院北区科研培育基金项目(编号:2023YKJ14、2023YKJ06、2023YKJ11)
安徽医科大学青年科学基金项目(编号:2022xkj043)
安徽省卫生健康科研项目(编号:AHWJ2023A20294)。
关键词
三羧酸循环
三羧酸循环代谢物
高效液相色谱-质谱联用系统
孕鼠
方法学
DEHP
tricarboxylic acid cycle
tricarboxylic acid cycle metabolites
high performance liquid chromatography-mass spectrometry system
pregnant mice
methodology
DEHP
