体内、体外相结合测定及评价CYP2C19 酶活性研究的方法建立

目的:建立一种快速、准确的以奥美拉唑(Omeprazole,OPZ)作为“探针药物”评价或测定细胞色素P450 2C19(CYP2C19)酶活性的高效液相色谱(HPLC)-紫外检测(VWD)方法.方法:采用的色谱柱为Agilent Extend-C18柱(4.6 mm×100 mm,5μm),流速1.0 mL·min-1,紫外检测波长290 nm,柱温40℃.体内实验:大鼠静脉注射OPZ(20 mg· kg-1),按时间点眼眶取血,检测OPZ与其代谢产物5’-羟基奥美拉唑(5’-hydroxy omeprazole,5 '-OHOPZ)的血浆药物浓度.体外实验:不同浓度OPZ与大鼠肝微粒体温孵后,检测温孵体系中5’-OHOPZ的浓度,以其生成的速率来评价CYP2C19酶的活性.结果:体内实验:内标非那西丁、OPZ与5’-OHOPZ保留时间(tR)分别为8.91,10.26,14.93 min,OPZ线性范围为0.28 ～18.0 mg·L-1,最低定量限(lower limit of quantitation,LLOQ)为0.28 mg·L-1,高、中、低浓度提取回收率分别为91.13％,97.21％,96.55％,OPZ的日内、日间相对标准偏差(relative standard deviation,RSD)小于10％;5’-OHOPZ线性范围为0.19 ～6.25 mg·L-1,LLOQ为0.19 mg·L-1,高、中、低浓度提取回收率分别为94.36％,97.57％,90.64％,5’-OHOPZ的日内、日间RSD小于10％.体外实验:内标非那西丁、OPZ与5’-OHOPZ的保留时间(tR)分别为8.91,10.26,14.93 min,5’-OHOPZ的线性范围为0.06 ～2.0 mg·L-1,LLOQ为0.06 mg·L-1,高、中、低浓度提取回收率分别为96.53％,99.40％,98.00％,日内、日间RSD差小于10％.结论:大鼠血浆及肝微粒体温孵体系中的其他内源性物质不干扰待测物的测定.该方法快速、稳定、灵敏度高,适合OPZ及其代谢产物5’-OHOPZ的测定,该文建立了体内、体外相结合测定及评价CYP2C19酶活性研究的方法.

In vivo and in vitro Research method for Determination and Evaluation of CYP2C19 Activity

Objective:To establish a rapid and accurate method with omeprazole(OPZ) as a ’probe drug’ in order to evaluate or determine the activity of cytochrome(CYP2C19) by the high-performance liquid chromatography(HPLC)-UV detection(VWD).Method: The analytical column was used in accordance with Agilent Extend-C18 column(4.6 mm ×100 mm,5 μm),flow rate at 1.0 mL · min-1,UV detection at 290 nm,and column temperature at 40 ℃.In vivo study: 20 mg · kg-1 of OPZ was given to the rats via the tail vein.Blood samples were collected at designed time points post dose and plasma samples were obtained.OPZ and 5′-hydroxy omeprazole(5′-OHOPZ) were determined by an HPLC method.In vitro study:After incubation,the formation rate of 5′-OHOPZ was used to evaluate the function of CYP2C19.Result: In vivo: the internal standard,OPZ and 5′-OHOPZ retention times were 8.91,10.26,14.93 min.The linear range of OPZ was 0.28-18.0 mg · L-1,and the lower limit of quantitation(LLOQ) was 0.28 mg · L-1.Extraction recovery at low,medium and high concentrations were 91.13%,97.21% and 96.55%,respectively.The intra-and inter-day relative standard deviation(RSD) was less than 10%.The linear range of 5′-OHOPZ was 0.19-6.25 mg · L-1,and the lower limit of quantitation(LLOQ) was 0.19 mg · L-1.Extraction recovery at low,medium and high concentrations were 94.36%,97.57% and 90.64%,respectively.The intra-and inter-day relative standard deviation(RSD) was less than 10%.In vitro: The internal standard,OPZ and 5′-OHOPZ retention times were 8.91,10.26,14.93 min.The linear range of 5′-OHOPZ was 0.06-2.0 mg · L-1,and the lower limit of quantitation(LLOQ) was 0.06 mg · L-1.Extraction recovery at low,medium and high concentrations were 96.53%,99.40% and 98.00%,respectively.The intra-and inter-day relative standard deviation(RSD) was less than 10%.Conclusion: Other endogenous substances in the plasma and incubation system do not interfere with the determination.The method is rapid,stable and highly sensitive.It is suitable for determination of OPZ and its metabolites 5′-OHOPZ and can be used for in vivo and in vitro determination of CYP2C19 activity and research of enzyme kinetics.