RP-HPLC法同时测定大鼠血浆中4种CYP450探针药物的浓度

目的: 建立大鼠血浆中4种细胞色素P450亚型酶(CYP1A2、CYP2C9、CYP2D6和CYP3A4)的探针药物咖啡因、甲苯磺丁脲、美托洛尔和氨苯砜同时测定的方法,为相关研究提供方法学参考。 方法: 用一种提取溶剂系统对4种药物同时提取,并用反相高效液相色谱法(RP-HPLC)同时测定,选用Diamonsil C₁₈色谱柱(4.6 mm×200 mm,5 μm);流动相为甲醇-水-冰乙酸-三乙胺(40: 60: 0.4: 0.5);检测波长:咖啡因271 nm,甲苯磺丁脲229 nm,美托洛尔225 nm,氨苯砜295 nm;流速在0~16 min时为0.5 mL·min^-1,16 min后为1.4 mL·min^-1;柱温35℃。 结果: 咖啡因、甲苯磺丁脲、美托洛尔和氨苯砜的线性范围分别为0.10~100.0 μg·mL^-1(r=0.9956),0.25~25.0 μg·mL^-1(r=0.9997),0.050~20.0 μg·mL^-1(r=0.9995),0.050~50.0 μg·mL^-1(r=0.9997);方法回收率分别为71.43%~87.17%,86.22%~97.80%,77.29%~91.26%,67.46%~98.47%。 结论: 所建立的方法准确、便捷、灵敏度高,可在同一个试验过程中,同时测定大鼠血浆中4种CYP450亚型酶探针药物(咖啡因、甲苯磺丁脲、美托洛尔和氨苯砜)的浓度,可用于同时测定某些受试药物对CYP450 的4个主要亚型酶(CYP1A2、CYP2C9、CYP2D6和CYP3A4)的调控作用及相关研究。

Objective: To establish a method for simultaneously determining the concentrations of four probe drugs of CYP450 including caffeine,tolbutamide,metoprolol and dapsone in rat plasma. Methods: The four drugs were extracted with one solvent system and simultaneously determined by RP-HPLC on a Diamonsil C₁₈ column(4.6 mm×200 mm,5 μm) with the mobile phase consisting of methanol-water-glacial acetic acid-triethylamine(40: 60: 0.4: 0.5) at the flow rate of 0-16 min 0.5 mL·min^-1,after 16 min 1.4 mL·min^-1.The detection wavelength for caffeine,tolbutamide,metoprolol and dapsone were at 271 nm,229 nm,225 nm and 295 nm respectively,and the column temperature was 35℃. Results: The four drugs and the internal standard(phenacetin) were separated well under the above RP-HPLC conditions.There were good linearity of caffeine,tolbutamide,metoprolol,and dapsone in the ranges of 0.10-100.0 μg·mL^-1 (r=0.9956),0.25-25.0 μg·mL^-1 (r=0.9997),0.050-20.0 μg·mL^-1 (r=0.9995),0.050-50.0 μg·mL^-1(r=0.9997).The recoveries were 71.43%-87.17%,86.22%-97.80%,77.29%-91.26%,67.46%-98.47%,respectively. Conclusion: The method is reliable,accurate,sensitive,and suitable for determining plasma concentrations of the four probe drugs(caffeine,tolbutamide,metoprolol and dapsone) in rats in a single run,which could be employed in evaluating the effects of drugs on CYP450 subtype enzymes activities and in relatived studies.