液液萃取LC-MS/MS法同时测定人血浆中甘草酸及甘草次酸  

Simultaneous determination of glycyrrhizic acid and glycyrrhetinic acid in human plasma by liquid-liquid extraction LC-MS/MS method

作　　者：骆献丽 陈晓英 李原 孙冬梅 张明辉 刘山崎 LUO Xian-li;CHEN Xiao-ying;LI Yuan;SUN Dong-mei;ZHANG Ming-hui;LIU Shan-qi(Lepu Pharmaceuticals,Inc.,Zhengzhou 45000,China)

机构地区：[1]乐普药业股份有限公司,郑州450000

出　　处：《药物分析杂志》2024年第9期1504-1512,共9页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:建立液液萃取液相色谱-串联质谱法(LC-MS/MS)同时测定人血浆中甘草酸、甘草次酸的浓度。方法:在0.1 mL人空白血浆中加入甘草酸、甘草次酸及内标物Apixaban-13C-d_(3)(IS),离子化试剂(20%甲酸溶液)50μL,以乙酸乙酯490μL、甲基叔丁基醚210μL为萃取剂,萃取后上清液氮气吹干,再用含0.2%甲酸的[乙腈-水(1∶1)]溶液200μL复溶,进行LC-MS/MS分析。色谱条件:采用Boston Mni C_(18)(50 mm×2.1 mm,3μm)色谱柱,流动相为0.2%甲酸水溶液-0.2%甲酸乙腈溶液,梯度洗脱,流速0.6 mL·min^(-1),柱温40℃,进样体积5μL,进样器温度4℃;质谱条件:采用电喷雾离子源(ESI+),选择多反应监测模式(MRM)进行定量分析,检测离子对分别为m/z 823.4→453.3(甘草酸)、m/z 471.4→189.0(甘草次酸)和m/z 464.3→447.1(IS)。结果:血浆中甘草酸和甘草次酸质量浓度分别在0.5~80 ng·mL^(-1)和2~800 ng·mL^(-1)范围内线性关系良好(r>0.99),最低定量浓度分别为0.5 ng·mL^(-1)和2 ng·mL^(-1)。批内、批间精密度(RSD)均<6.8%,准确度在92.3%~104.2%;甘草酸和甘草次酸的提取回收率分别约为28.0%和40.0%,IS的提取回收率约为65%,RSD均小于7.9%;甘草酸和甘草次酸的内标归一化基质因子均约为1,RSD均小于7.3%。结论:该方法灵敏、准确、简便、快速,可用于人血浆中甘草酸及甘草次酸的同时测定。Objective:To establish a liquid-liquid extraction liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for simultaneous determination of the concentration of glycyrrhizic acid and glycyrrhetinic acid in human plasma.Methods:Glycyrrhizic acid,glycyrrhetinic acid and the internal standard Apixaban-~(13)C-d_(3)(IS)were added to 0.1 mL of human blank plasma.50μL of ionization reagent(20%formic acid solution),490μL of ethyl acetate and 210μL of methyl tert-butyl ether were used as extractant.The supernatant was dried by nitrogen,and the residue was dissolved with 200μL acetonitrile-water(1∶1)containing0.2%formic acid.And 5μL of resulting solution was injected to the LC-MS/MS for analysis.Chromatographic conditions:the saparation was performed on a BostonΜni C_(18)(50 mm×2.1 mm,3μm)column with mobile phase consisting of 0.2%formic acid aqueous solution(mobile phase A)and 0.2%formic acid acetonitrile solution(mobile phase B)by gradient elution.The flow rate was 0.6 mL·min^(-1),the temperature of column was 40℃.The sample volume was 5μL,and the temperature of the sampler was 4℃.Mass spectrometry conditions:multiple reaction montoring(MRM)was performed on a triple quadrupole mass spectrometer equipped with a ESI source in the positive mode.The detection ion pairs were m/z 823.4→453.3(glycyrrhizic acid),m/z 471.4→189.0(glycyrrhetinic acid)and m/z 464.3→447.1(IS)respectively.Results:The calibration curves were linear over the concentrion ranges of 0.5-80 ng·mL^(-1)for glycyrrhizic acid and 2-800 ng·mL^(-1)for glycyrrhetinic acid(r>0.99)in the plasma,the lower limits of quantifications(LLOQ)were 0.5 ng·mL^(-1)(glycyrrhizic acid)and 2 ng·mL^(-1)(glycyrrhetinic acid),respectively.Inter-and intra-batch precisions(RSDs)were less than 6.8%,and the accuracy ranged from 92.3%to 104.2%.The recovery rates of glycyrrhizic acid and glycyrrhetinic acid were about 28.0%and 40.0%separately,and the recoveries of IS were about 65.0%,the precision(RSDs)were less than 7.9%.The normalized matrix factors of g

关 键 词：液液萃取 甘草酸苷 甘草酸 甘草次酸 液质联用 复方甘草酸苷片 代谢物 

分 类 号：R917[医药卫生—药物分析学]