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, 1.8 μm),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 C18色谱柱(50 mm×2.1 mm,1.7 μm),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, 1.8 μm) 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 mL·min-1. ESI with positive and negative ions pattern was used. After intragastric administration, serum, bile, urine, and feces of rats were collected for analyzing. 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 standard. UPLC® BEH C18(50 mm×2.1 mm, 1.7 μm) 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, glycyrrhetinic 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 have Ⅰ phase (demethylation, oxidation) metabolism and Ⅱ phase (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 relationships were good (r>0.995 0), 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 in Ⅱ phases. Meanwhile, the plasma concentration 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 studied the processes of Xiaoer Fengye Kechuanping mixture in the body and provide theoretical basis for exploring the pharmacodynamic material basis.
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