以非那西丁为探针采用高效液相色谱法快速测定细胞色素P4501A2的酶活性

Rapid Determination of Cytochrome P450 1A2 Activity with Phenacetin Probe Using High-performance Liquid Chromatography

目的建立一种快速、高效的以非那西丁作为探针药物评价细胞色素P4501A2（CYP1A2）酶活性的高效液相色谱-紫外检测方法。方法色谱柱为Agilent Zorbax SB-C18柱（50mm×4．6mmI．D．，5μm）；流动相为乙腈-水（含0．01％七氟丁酸），梯度洗脱，流速2．0ml·min^-1；检测波长为244nm。非那西丁与人CYP1A2酶在37℃温孵适当时间后，加入50μl乙腈终止反应，10000g离心后取上清液进样分析测定。结果对乙酰氨基酚的保留时间为2．85min，线性范围为1．00～200μmaol·L^-1（r=0．9999），最低定量限（LLOQ）为1．00μmol·L^-1，回收率为99．8％-102．3％；非那西丁的保留时间为3．68min，线性范围为1．00～200μmol·L^-1（r=0．9999），最低定量限（LLOQ）为1．00μmol·L^-1，回收率为98．3％～101．1％。两者的日内、日间相对标准偏差均小于15％，温孵体系中的其他内源性物质不干扰测定。结论该方法快速、稳定、灵敏度高，适合体外非那西丁及其代谢物对乙酰氨基酚的测定，可应用于体外CYP1A2酶活性的评价及酶动力学的研究．

Objective To develop a method for the evaluation of cytochrome P450 1A2 activity using phenacetin as the probe compound in vitro by high - performance liquid chromatography - ultraviolet detection （ HPLC - UV）. Methods An Agilent Zorbax SB - C18（50 mm ×4.6 mm I. D. , 5 μm） column was used as stationary phase. The gradient mobile phase consisted of acetonitrile and water （containing 0.01% heptafluorobutyric acid）. The flow rate was 2.0 ml · min^-1 , and the UV detector was set at 244 nm. Firstly, phenacetin was incubated with human CYP 450 1A2 enzyme in vitro at 37℃ and stopped by addition of 50 μl acetonitrile and centrifuged （10 000 g） for 3 min. Finally, the supernatant was analyzed by RP- HPLC. Results The retention time of acetamidophenol was 2.85 min. The linear calibration curves of acetamidophenol were obtained in the concentration range of 1.00 - 200 μmol · L ^-1 （ r = 0.999 9 ）. The lower limit of quantification （LLOQ） of acetamidopbenol was 1.00 μmol · L ^-1. The average recovery was 99.8%- 102.3%. The retention time of phenacetin was 3.68 min. The linear calibration curves of phenacetin were obtained in the concentration range of 1.00 - 200 μmol · L^-1 （ r = 0. 999 9 ）. The lower limit of quantification （LLOQ） of acetamidophenol was 1.00 μmol · L^-1. The average recovery was 98.3% -101.1%. The intra- and inter- day relative standard deviations were all less than 15%. There were no endogenous substances existing in the incubation system which interference the determination of the analytes of interest. Conclusion The method is simple, rapid and suitable for the evaluation of eytochrome P450 1 A2 activity in vitro.