小儿风叶咳喘平合剂3个成分在大鼠体内的药代动力学研究

Pharmacokinetics study of three active components from Xiaoer Fengye Kechuanping mixture after oral administration in rats

目的:对小儿风叶咳喘平合剂中3个主要药效成分在大鼠体内的代谢产物以及药代动力学进行研究。方法:基于超高效液相色谱-电喷雾飞行时间质谱(UHPLC-ESI TOF MS)方法,采用ACQUITY UPLC?HSS T3色谱柱(100 mm×2.1 mm, 1.8μm),以0.1%甲酸水和乙腈为流动相进行梯度洗脱,流速为0.3 mL·min^(-1),质谱采用电喷雾离子源(ESI),以正负离子模式采集多级质谱碎片信息,对大鼠灌胃小儿风叶咳喘平合剂后的血清、胆汁、尿液和粪便进行分析,结合离子碎片进行代谢产物鉴定分析;同时采用超高效液相色谱串联质谱(UHPLC-MS/MS)的技术进行药代动力学研究。以格列齐特为内标物,采用ACQUITY UPLCⓇ BEH C_(18)色谱柱(50 mm×2.1 mm, 1.7μm),以0.1%甲酸水和乙腈为流动相进行梯度洗脱。血浆样品采用甲醇沉淀蛋白,采用ESI及多反应监测(MRM),选择监测离子(麻黄碱m/z 166.2→148.1、甘草次酸m/z 471.2→453.2、对香豆酸m/z 162.9→119.1、内标m/z 322→170)。小儿风叶咳喘平合剂分别按2、5、10 mL·kg^(-1)灌胃,测定其中3个成分的质量浓度,并用DAS 3.1软件计算药代动力学参数。结果:共鉴定出3个原型及10个代谢产物[Ⅰ相(去甲基化、氧化)代谢、Ⅱ相(甲基化、硫酸酯化、葡萄糖醛酸苷化结合)代谢];3个成分在血浆中无干扰,专属性符合要求;规定范围内线性关系良好(r均>0.995 0);提取回收率和基质效应在接收范围之内;常温、进样器、冻融稳定性均符合要求;3个成分均符合二室模型。结论:采用UHPLC-ESI-TOF的方法鉴定了小儿风叶咳喘平合剂中3个主要药效成分的代谢产物,同时采用UHPLC-MS/MS的技术对3个成分在大鼠体内的血药浓度进行检测,并计算药代动力学参数,为进一步了解小儿风叶咳喘平合剂体内过程,探索功效物质基础提供理论依据。

Objective:To study the metabolites and pharmacokinetics of three main pharmacodynamic components of Xiaoer Fengye Kechuanping mixture in rats.Methods:UHPLC-ESI TOF MS and ACQUITY UPLCⓇHSS T3(100 mm×2.1 mm,1.8μm)column with a 0.1%formic acid water and acetonitrile as mobile phase were used.Gradient elution was applied and the flow rate was 0.3 m L·min^(-1).ESI with positive and negative ions pattern was used.After intragastric administration,serum,bile,urine,and feces of rats were collected for analy-zing.The metabolites were identified by analyzing the mass-to-charge ratios,retention times,and fragments of ions.Meanwhile,UHPLC-MS/MS was used in pharmacokinetic study.Gliclazide were used as internal stand-ard.UPLCⓇ BEH C_(18)(50 mm×2.1 mm,1.7μm)column was used.The mobile phase of 0.1%formic acid water and acetonitrile was gradient eluted and the plasma samples were precipitated with methanol.The ESI and MRM were used for quantification.The selected monitoring ions were ephedrine m/z 166.2→148.1,glycyrrhetin-ic acid m/z 471.2→453.2,p-coumaric acid m/z 162.9→119.1,and internal standard m/z 322→170.Xiaoer Fengye Kechuanping mixture was gavaged by 2,5 and 10 mL·kg^(-1),respectively.DAS 3.1 software was used to calculate the pharmacokinetic parameters after the plasma concentrations of three components were determined.Results:3 prototypes and 10 metabolites which haveⅠphase(demethylation,oxidation)metabolism andⅡphase(methylation,esterification and glucuronic acid sulfate)metabolism were identified.Meanwhile,the specificity of the three components in plasma met the requirements without interference,and all linear relation-ships were good(r>0.9950),the extraction recovery and matrix effect were within the requested range.The stability of different conditions met the requirements.All three components fit the two-compartment model.Conclusion:The three main efficacy components of Xiaoer Fengye Kechuanping mixture are identified by the UHPLC-ESI TOF MS.The p-coumaric acid mainly metabolizeds inⅡphases.Meanwhile,the plasma concen-tration of three components in rats are analyzed by UHPLC-MS/MS to calculate pharmacokinetic parameters.It is found that the absorption rate of glycyrrhetinic acid is slower than that of other two components.This paper stud-ied the processes of Xiaoer Fengye Kechuanping mixture in the body and provide theoretical basis for exploring the pharmacodynamic material basis.