HPLC法同时测定大鼠血浆中3种CYP 450 探针药物的浓度

Simultaneous determination of three probe drugs of CYP450 in rat plasma by HPLC

关键词:  细胞色素P450  高效液相色谱法  非那西丁  甲苯磺丁脲  咪达唑仑

Key words:  CYP450  HPLC  phenacetin  tolbutamide  midazolam

摘要:

建立同时测定大鼠血浆中3种细胞色素P450（CYP450）亚型酶( CYP1A2、CYP2C9、CYP3A4)活性的Cocktail探针药物的高效液相色谱（HPLC）检测方法，为相关肝药酶代谢研究提供方法学参考。分别取非那西丁，咪达唑仑和甲苯磺丁脲三种探针药物，制成混合探针溶液，用一种提取溶剂系统对3种探针药物同时提取，并用高效液相色谱法（HPLC）同时测定该3种探针药物的血药浓度，并对其进行专属性，线性范围及检测限，精密度，回收率，稳定性的验证。色谱条件选用Diamonsil C18色谱柱（4.6 mm×250 mm，5 μm）；流动相为甲醇-50 mM磷酸盐缓冲对（45:55；pH3.2）；检测波长：230 nm；流速1.0 mL/min；柱温35 ℃。结果：非那西丁，咪达唑仑，甲苯磺丁脲的线性范围分别为0.10～10.0 μg/mL(r=0.9993)，0.05～5.0 μg/mL(r=0.9999)，0.10～50.0 μg/mL(r=0.9991)；方法回收率分别为79.28 %～87.91 %，76.43 %～85.36 %，82.79 %～90.61 %。该HPLC检测方法快捷，准确，灵敏度高，可以在同一次试验中，同时测定大鼠血浆中3种CYP450亚酶探针药物（非那西丁，甲苯磺丁脲，咪达唑仑）的浓度，各探针药物的吸收峰分离完全，无干扰，可用于开展某些受试药物对大鼠CYP450的3个主要亚型酶（CYP1A2，CYP2C9，CYP3A4）调控作用的相关研究。

Abstract:

To provide a methodological reference for the study of metabolism of liver enzymes, an HPLC method for simultaneously determining three cytochrome P450(CYP) (CYP1A2, CYP2C9, CYP3A4) probe drugs in rat plasma was established. Cocktail probe solution was prepared by mixing three specific probe drugs of CYP1A2, 2C9 and 3A4（phenacetin, tolbutamide and midazolam, respectively）. The three drugs were extracted with one solvent system, determined in rat plasma by HPLC, and verified the specificity, linear range, detection limit, precision, recovery and stability. The isolation was performed on a Diamonsil C18 column (4.6 mm × 250 mm, 5 μm) with mobile phase consisting of methanol -50 mM phosphate buffer (45:55; pH3.2); the detection wavelength was 230 nm; the flow rate was 1.0 mL/min and the column temperature was 35 ℃. Results: There were good linearity of phenacetin, midazolam and tolbutamide in the ranges of 0.10~10.0 μg/mL(r=0.9993), 0.05~5.0 μg/mL(r=0.9999), 0.10~50.0 μg/mL(r=0.9991). The recoveries were 79.28 %~87.91 %, 76.43 %~85.36 %, 82.79 %~90.61 %, respectively. The method is rapid, accurate, sensitive and suitable for simultaneously determining the concentrations of three CYP450 probe drugs(phenacetin, tolbutamide and midazolam) in rat plasma, the three drugs and the internal standard(carbamazepine) were separated well under the above HPLC conditions, which could be employed in evaluating the effects of drugs on rat’s CYP450 subtype enzymes（CYP1A2, CYP2C9, CYP3A4） activities and in relatived studies.