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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。
OBJECTIVE: To establish a rapid and accurate method for the determination of cytochrome P450 2C19 (CYP2C19) activity using Omeprazole (OPZ) as “probe drug” and high performance liquid chromatography (HPLC) (VWD) method. Methods: The Agilent Extend-C18 column (4.6 mm × 100 mm, 5 μm) was used with a flow rate of 1.0 mL · min-1. The UV detection wavelength was 290 nm and the column temperature was 40 ℃. In vivo experiments: OPZ (20 mg.kg-1) was intravenously injected into the rat’s eyes and blood was collected from the orbit of the eye at the time point to detect the expression of OPZ and its metabolites 5’-hydroxy omeprazole (5’-OHOPZ) Plasma drug concentration. In vitro experiments: After different concentrations of OPZ and rat liver microsome were incubated, the concentration of 5’-OHOPZ in the incubation system was detected and the activity of CYP2C19 enzyme was evaluated at the rate of its formation. Results: In vivo experiments, the internal standard of phenacetin, OPZ and 5’-OHOPZ retention time (tR) were 8.91,10.26,14.93 min, OPZ linear range of 0.28 ~ 18.0 mg.L-1, the lower limit of quantification limit of quantitation (LLOQ) was 0.28 mg.L-1, and the recoveries of high, middle and low concentrations were 91.13%, 97.21% and 96.55% respectively. The relative standard deviations (RSDs) Less than 10%. The linear range of 5’-OHOPZ was 0.19 ~ 6.25 mg.L-1 and the LLOQ was 0.19 mg.L-1. The recoveries of high, middle and low concentrations were 94.36%, 97.57%, 90.64%, 5 ’-OHOPZ day, day RSD less than 10%. In vitro experiments, the internal standard phenacetin, OPZ and 5’-OHOPZ retention time (tR) were 8.91,10.26,14.93 min, 5’-OHOPZ linear range of 0.06 ~ 2.0 mg.L-1, LLOQ 0.06 mg.L-1. The recoveries of high, medium and low concentrations were 96.53%, 99.40% and 98.00%, respectively. The intra-day and inter-day RSDs were less than 10%. CONCLUSIONS: Other endogenous substances in rat plasma and liver microsomal incubation systems do not interfere with the determination of analytes. The method is rapid, stable and sensitive and suitable for the determination of 5’-OHOPZ in OPZ and its metabolites. The method for the determination of CYP2C19 activity in vitro and in vivo was established.