摘要
目的：建立测定人肝微粒体孵育体系中氯吡格雷及其活性代谢产物（AM）和非活性代谢产物（CCAM）药物浓度的检测方法,并将其应用于氯吡格雷的体外代谢研究。方法：AM含有巯基,需用衍生化试剂2-溴-3＇-甲氧基苯乙酮（MPB）将肝微粒体孵育体系中的AM衍生化成MP-AM进行测定。分别采用蛋白沉淀法和液液萃取法处理人肝微粒体孵育体系样品,测定氯吡格雷原药及MP-AM和CCAM药物浓度。氯吡格雷原药浓度的测定在HPLC仪上进行,采用Accurasil C18色谱柱（250 mm×4.6 mm,5μm）,流动相为甲醇-水（磷酸调p H 2.85）（22∶78）,流速1 m L·min-1,检测波长235 nm;MP-AM和CCAM的浓度测定在UPLC-MS/MS仪上进行,采用Thermo C18色谱柱（100 mm×2.1 mm,5μm）,流动相为乙腈（含0.1%甲酸）-水（含0.1%甲酸）（9∶1）,流速200μL·min-1,正离子模式多反应监测（MRM）扫描分析,离子通道分别为MP-AM m/z503.6→353.8,CCAM m/z 307.7→197.8,内标氯雷他定m/z 382.8→336.9。结果：人肝微粒体孵育体系中氯吡格雷,MP-AM和CCAM线性范围分别为0.5～200μmol·L-1、1～200 ng·m L-1和10～2 000 ng·m L-1,定量下限分别为0.5μmol·L-1、1 ng·m L-1和10 ng·m L-1,回收率分别在95.4%～98.8%、59.4%～73.4%和54.8%～72.8%之间,氯吡格雷原药的批内、批间精密度RSD均小于9.4%,MP-AM的批内、批间精密度RSD均小于11.4%,CCAM的批内、批间变异均小于9.7%。内标氯雷他定的回收率分别为96.7%和66.7%。稳定性试验中,在各种贮存条件下氯吡格雷、MP-AM和CCAM均稳定性良好。CCAM生成的酶促反应动力学参数Km值为（15.86±1.83）μmol·L-1,Vmax值为（620.2±19.74）pmol·min-1·mg-1,Clint值为（39.15±2.06）μL·min-1·mg-1;AM生成开始随着底物浓度的增加而增加,在底物浓度为20μmol·L-1时其生成量达到最大,随后随着底物浓度的增加其生成量反而降低。结论：所建立的测定方法操作简便快速,重复性好,特异
Objective： To establish an HPLC method for the determination of clopidogrel and an UPLC-MS / MS method for the determination of its active metabolite（ AM） and inactive metabolite clopidogrel carboxylic acid metabolite（ CCAM） in human liver microsomes（ HLMs） incubation system,and to investigate the enzyme kinetics of clopidogrel in HLMs. Methods： AM contained a thiol group,thus requiring stabilization in biological samples. The alkylating reagent 2-bromo-3＇-methoxyacetophenone（ MPB） was used to stabilize AM in HLMs to MPB-derivatized clopidogrel active metabolite（ MP-MA）. The concentration of clopidogrel was determined by HPLC. The HLMs processing procedure involved single-step protein precipitation. Then the samples were chromatographed on an Accurasil C18（ 250 mm × 4. 6 mm,5 μm） using a mobile phase consisting of methanol and water（ p H was adjusted to 2. 85 with phosphoric acid）（ 22∶ 78） at a flow rate of 1 m L·min- 1. The concentrations of MP-AM and CCAM were determined by UPLC-MS / MS. The HLMs processing procedure involved a liquid-liquid extraction. Then the chromatography was conducted on a Thermo C18column（ 100 mm × 2. 1 mm,5 μm） using a mobile phase consisting of acetonitrile（ 0. 1% formic acid） and water（ 0. 1% formic acid）（ 9∶ 1） at a flow rate of 200 μL·min- 1. The protonated ions of analytes were detected in positive ionization in multiple reaction monitoring mode（ MRM）. The mass transition pairs of m / z 503. 6 →353. 8,m / z 307. 7 →197. 8,m / z 382. 8 →336. 9 were used to detect MP- AM,CCAM and loratadine respectively. Results： The calibration curves were linear over a concentration range of 0. 5-200 μmol·L- 1for clopidogrel,1-200 ng·m L- 1for MP-AM and 10-2 000 ng·m L- 1for CCAM and the low limits of quantization were 0. 5 μmol·L- 1,1ng·m L- 1and 10 ng·m L- 1,respectively. The extraction recovery rates ranged from 95. 4%-98. 8% for clopidogrel,59. 4%-73. 4% for MP-AM and 54. 8%-72. 8% for CCAM. The intra-day and i
出处
《药物分析杂志》
CAS
CSCD
北大核心
2015年第10期1741-1750,共10页
Chinese Journal of Pharmaceutical Analysis
基金
国家自然科学基金(项目编号:81173132)
