LC-MS/MS法测定大鼠血浆中黄酮醇糖苷及其主要代谢物  

Simultaneous determination of flavonol glycoside and its major metabolites by LC-MS/MS in rat plasma

作　　者：董磊 刘金秋 李亮 陈笑艳 单伟光[1] 钟大放[1,2] DONG Lei;LIU Jin-qiu;LI Liang;CHEN Xiao-yan;SHAN Wei-guang;ZHONG Da-fang(College of Pharmaceutical Science,Zhejiang University of Technology,Hangzhou 310014,China;Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203,China)

机构地区：[1]浙江工业大学药学院,浙江杭州310014 [2]中国科学院上海药物研究所,上海201203

出　　处：《药学学报》2018年第8期1344-1350,共7页Acta Pharmaceutica Sinica

摘　　要：黄酮醇糖苷是处于临床试验阶段的新药,拟用于治疗高脂血症。本文建立了液相色谱-串联质谱(LC-MS/MS)法测定SD大鼠血浆中的黄酮醇糖苷(M0)及其代谢物苷元(M1)和葡萄糖醛酸结合物(M2):采用d_6-黄酮醇糖苷作为内标,血浆样品经甲醇(含0.2%甲酸)沉淀蛋白后,通过XDB C_(18)(50 mm×4.6 mm,1.8μm)色谱柱分离,以去离子水(含0.2%甲酸)-甲醇为流动相梯度洗脱,色谱运行时间为4.5 min。采用电喷雾电离源(ESI),以多反应监测模式(MRM)检测。用于定量分析M0、M1和M2的离子对分别为m/z 461.3→m/z 299.1、m/z 299.1→m/z 283.1和m/z 475.0→m/z 299.1,用于定量分析内标d_6-黄酮醇糖苷的离子对为m/z 467.3→m/z305.1。本方法经验证后,成功应用于SD大鼠药动学研究。SD大鼠灌胃给予黄酮醇糖苷30 mg·kg^(-1)后,M0的C_(max)为(341±106)ng·m L^(-1)、AUC_(0-t)为(1 960±725)h·ng·m L^(-1),M1在血浆中的含量低于分析方法的定量下限2 ng·m L^(-1),无法测定相应浓度计算C_(max)和AUC_(0-t),M2的C_(max)为(1 720±843)ng·m L^(-1)、AUC_(0-t)为(8 510±2 920)h·ng·m L^(-1),M2的C_(max)约为M0的5.0倍,AUC_(0-t)约为M0的4.3倍。结果表明,SD大鼠灌胃给予黄酮醇糖苷后,在体内主要以原形M0和代谢物M2的形式存在,且M2的暴露量显著高于M0,推测黄酮醇糖苷经口服给药后在体内经肠菌群水解成苷元形式被吸收,经历较强的首过代谢后,M1进一步生成葡萄糖醛酸结合物。本文首次测定了黄酮醇糖苷在大鼠血浆中的主要代谢物,为临床药动学试验设计提供了依据。Flavonol glycoside is in clinical trials for treatment of hyperlipidemia.An accurate and sensitive liquid chromatography-tandem mass spectrometry（LC-MS/MS）method was developed and validated for the simultaneous determination of flavonol glycoside（M0）,aglycone（M1）and glucuronide conjugate（M2）in rat plasma.d6-Flavonol glycoside was used as internal standard（IS）.After extraction from the plasma by protein precipitation,the analytes and internal standard were separated on a XDB C18 column（50 mm×4.6 mm,1.8μm）using a gradient elution procedure.The mobile phase consisted of methanol and water（0.2%formic acid）at a flow rate of 0.6 m L·min^-1.The total run time was 4.5 min.Positive electrospray ionization was performed using multiple reaction monitoring（MRM）with transitions of m/z 461.3→m/z 299.1 for M0,m/z 299.1→m/z283.1 for M1,m/z 475.0→m/z 299.1 for M2,and m/z 467.3→m/z 305.1 for d6-flavonol glycoside.The method was validated and successfully applied to the pharmacokinetics study of flavonol glycoside in SD rats which were given flavonol glycoside（30 mg·kg^-1）by gavage.The Cmaxof M0 is（341±106）ng·m L^-1 and AUC0-t is（1 960±725）h·ng·m L^-1,while the Cmaxof M2 is（1 720±843）ng·m L^-1and AUC0-t is（8 510±2 920）h·ng·m L^-1.The results suggest that flavonol glycoside existed mainly in the form of M0 and M2 in rats.After flavonol glycoside being hydrolyzed by the intestinal flora,it was absorbed in the form of aglycone and further metabolized to M2 after the first-pass effect.In this paper,the main metabolites of flavonol glycoside in rat plasma were determined for the first time,which provided a basis for the design of clinical pharmacokinetic experiment.

关 键 词：液相色谱-串联质谱 黄酮醇糖苷 葡萄糖醛酸结合物 药动学 

分 类 号：R917[医药卫生—药物分析学]