UHPLC-MS/MS同时检测大鼠血浆中5种CYP450探针药物

Determination of Five CYP450 Probe Drugs in Rat Plasma by UHPLC-MS/MS

关键词:  超高效液相色谱-串联质谱法  CYP450酶  探针药  药动学

Key words:  UHPLC-MS/MS  CYP450  probe drug  pharmacokinetics

摘要:

目的 采用UHPLC-MS/MS同时检测大鼠血浆中非那西丁、甲苯磺丁脲、奥美拉唑、美托洛尔、咪达唑仑的血药浓度。方法 血浆样品经乙腈沉淀，采用Agilent ZORBAX Eclipse Plus C₁₈色谱柱（2.1 mm×50 mm，1.8 μm）；流动相为乙腈-含0.1%甲酸的水，梯度洗脱，流速为0.4 mL·min^-1。检测采用电喷雾离子源，多反应监测。非那西丁：[M+H]⁺，m/z 180.1→109.9；甲苯磺丁脲：[M+H]⁺，m/z 271.1→91.0；奥美拉唑：[M+H]⁺，m/z 346.1→135.9；美托洛尔：[M+H]⁺，m/z 268.2→115.0；咪达唑仑：[M+H]⁺，m/z 326.1→290.8；内标卡马西平：[M+H]⁺，m/z 237.1→194.0。6只♂ SD大鼠，单剂量口服灌胃10 mg·kg^-1非那西丁，1 mg·kg^-1甲苯磺丁脲，10 mg·kg^-1奥美拉唑，10 mg·kg^-1美托洛尔和10 mg·kg^-1咪达唑仑，分别在给药后多点尾静脉采血。用DAS计算药动学参数。结果 血浆中非那西丁、甲苯磺丁脲、奥美拉唑、美托洛尔和咪达唑仑在各自浓度范围内线性关系良好。日内及日间RSD均<15%，提取回收率>75%，稳定性考察结果良好。非那西丁的AUC_0-t为（5 868.30±2 062.87）ng·mL^-1·h；甲苯磺丁脲的AUC_0-t为（58 056.34±15 569.16）ng·mL^-1·h；奥美拉唑的AUC_0-t为（14 181.67±4 085.40）ng·mL^-1·h；美托洛尔的AUC_0-t为（1 123.67±180.469）ng·mL^-1·h；咪达唑仑的AUC_0-t为（946.91±322.03）ng·mL^-1·h。结论 该方法灵敏度高、操作方便、结果准确，可作为CYP450酶活性及相关研究的测定方法。

Abstract:

OBJECTIVE To establish a UHPLC-MS/MS method for simultaneously determination of phenacetin, tolbutamide, omeprazole, metoprolol and midazolam in rat plasma. METHODS The plasma sample was prepared by acetonitrile precipitation. The analytical column was Agilent ZORBAX Eclipse Plus C₁₈(2.1 mm×50 mm, 1.8 μm). The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile with gradient elution. The flow rate was 0.4 mL·min^-1. Detection equipped with electrospray ionization source with multiple reaction monitoring. The tandem mass ion transitions monitored were m/z 180.1→109.9 for phenacetin([M+H]⁺), m/z 271.1→91.0 for tolbutamide, m/z 346.1→135.9 for omeprazole ([M+H]⁺), m/z 268.2→115.0 for metoprolol([M+H]⁺), m/z 326.1→290.8 for midazolam([M+H]⁺), and m/z 237.1→194.0 for carbamazepine(IS,[M+H]⁺). Blood sample was collected of the tail vein at multiple time point after a single oral dose of administration of phenacetin(10 mg·kg^-1), tolbutamide (1 mg·kg^-1), omeprazole(10 mg·kg^-1), metoprolol(10 mg·kg^-1) and midazolam(10 mg·kg^-1) in six male SD rats. The pharmacokinetic parameters were calculated by DAS. RESULTS The calibration curve was linear over the range of curve for the five probe drugs. Intra-day and inter-day RSDs for assaying the plasma sample were both <15%, the recovery rate was >75% for the five drugs. The phenacetin AUC_0-t was (5 868.30±2 062.87) ng·mL^-1·h; the tolbutamide AUC_0-twas (58 056.34±15 569.16) ng·mL^-1·h; the omeprazole AUC_0-t was (14 181.67±4 085.40) ng·mL^-1·h; the metoprolol AUC_0-t was (1 123.67±180.469) ng·mL^-1·h; the midazolam AUC_0-twas (946.91±322.03) ng·mL^-1·h. CONCLUSION The methods is proved to be sensitivity, simple and accuracy, and can be used as an analytical method for the study of CYP450 enzyme activity and related research.