HPLC-MS-MS法测定大鼠血浆中6种CYP450探针药物及甘草对CYP450酶活性的影响

Simultaneous Determination and Evaluation of Glycyrrhizae Radix on Activity of Six Cytochrome P450 Enzymes in Vivo Using HPLC-MS-MS

摘要:

目的: 建立高效液相色谱-二级质谱联用(HPLC-MS-MS)法同时测定大鼠血浆中6种细胞色素P450(CYP450)探针药物,即非那西丁、甲苯磺丁脲、奥美拉唑、酒石酸美托洛尔、氯唑沙宗和睾酮,并用于体内评价甘草对CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1,CYP3A4酶活性的影响. 方法: 采用汉邦C₁₈柱(2.0 mm×150 mm,5 μm),流动相为甲醇-0.1%甲酸水溶液(70:30),流速为0.2 mL·min^-1,柱温35℃.电喷雾离子化,检测方式为多反应离子监测.大鼠分为2组,分别ig生理盐水和甘草提取液,连续10 d,于第11天给予大鼠探针药物,用HPLC-MS-MS法测定6种探针药物的血药浓度,并分析比较其药动学参数. 结果: HPLC-MS-MS方法的灵敏度、精密度、回收率及稳定性良好.甘草能明显诱导大鼠体内CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1的活性,抑制 CYP3A4的活性. 结论: 本法简便、准确,适用于甘草或其他药物对CYP450同工酶活性的研究.

Abstract:

Objective: An HPLC-MS-MS assay for simultaneous determination of phenacetin, tolbutamide, omeprazole, metoprolol tartaric acid, chlorzoxazone and testosterone simultaneously in rat plasma was established to evaluate Glycyrrhizae Radix on the activity of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). Method: The separation was carried out on a Hanbon C₁₈ column (2.0 mm×150 mm, 5 μm) at 35℃. The mobile phase was methanol-0.1% formic acid (70: 30), running at a flow rate of 0.2 mL· min^-1. Mass spectrometry was performed in the multiple reaction monitoring (MRM) mode with an ESI source. Two groups of rats were orally administered with saline solution and decoction of Glycyrrhizae Radix, respectively, for consecutive 10 days. On the 11th day of administration, a single oral dose of the mixture of 6 probe drugs was given to all rats. The pharmacokinetics of probe drugs in rats was investigated, and the related pharmacokinetic parameters were statistically analyzed. Result: The HPLC-MS-MS method was validated with good sensitivity, precision, recovery and stability. It was found that Glycyrrhizae Radix could remarkably induce the enzyme activity of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP2E1, whereas inhibit the enzyme activity of CYP3A4. Conclusion: The established method is proved to be sensitive, reliable and simple, which is suitable for the study of Glycyrrhizae Radix or other drugs on the activity of CYP450 enzymes.