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目的:建立同时测定人血浆中氯吡格雷(CLO)及其活性代谢产物(CATM)、非活性代谢产物(CCAM)浓度的方法,并用于药动学研究。方法:取烷化剂2-溴-3′-甲氧基苯乙酮(MPB)保护的血浆样品,经乙腈沉淀蛋白后,以卡马西平为内标,采用超高效液相色谱-串联质谱(UPLC-MS/MS)法测定。色谱柱为Waters ACQUITY UPLC HSS T3,流动相为水(含0.1%甲酸)-乙腈(含0.1%甲酸),梯度洗脱,流速为0.50 ml/min。采用电喷雾电离源(ESI),多反应监测(MRM)方式进行正离子监测,用于定量分析的离子对分别为m/z 322.1→211.8(CLO)、m/z 504.1→155.0(CATM烷化衍生物,CATMD)、m/z 308.3→198.0(CCAM)、m/z 273.2→194.3(内标)。结果:CLO、CATMD、CCAM血药浓度分别在0.03~20.00、0.30~200.00、10.00~10 000.00 ng/ml范围内线性关系良好;日内、日间RSD<15%,相对误差(RE)为-3.5%~5.7。5名健康受试者单剂量口服CLO 300 mg后,CLO、CATM、CCAM的cmax分别为(7.89±5.46)、(15.58±8.08)、(8 023.33±1 047.39)ng/ml,tmax分别为(1.25±0.43)、(1.25±0.43)、(1.67±0.29)h,t1/2分别为(2.31±0.61)、(0.64±0.08)、(6.53±2.55)h,AUC0-t分别为(17.19±14.59)、(21.39±9.58)、(30 648.85±8 026.63)ng·h/ml。结论:该方法操作简便、灵敏度高、分析时间短,适用于CLO及其代谢物血药浓度测定及药动学研究。
Objective: To establish a method for simultaneous determination of clopidogrel (CLO) and its active metabolites (CATM) and non-active metabolites (CCAM) in human plasma and its pharmacokinetics. Methods: Plasma samples of alkylating agent 2-bromo-3’-methoxyacetophenone (MPB) were collected and precipitated with acetonitrile. Carbamazepine was used as an internal standard and analyzed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) method. The column was a Waters ACQUITY UPLC HSS T3 with a mobile phase of water (containing 0.1% formic acid) -acetonitrile (containing 0.1% formic acid), gradient elution at a flow rate of 0.50 ml / min. The positive ions were monitored by electrospray ionization source (ESI) and multi-reaction monitoring (MRM). The ion pairs for quantitative analysis were m / z 322.1 → 211.8 (CLO), m / z 504.1 → 155.0 Derivatives, CATMD), m / z 308.3 → 198.0 (CCAM), m / z 273.2 → 194.3 (internal standard). Results: The linearity of CLO, CATMD and CCAMA was linear in the range of 0.03-20.00,0.30-200.00 and 10.00-10 000.00 ng / ml respectively. The intra-day and inter-day RSD was less than 15% and the relative error (RE) was -3.5 % ~ 5.7.5 After a single oral dose of 300 mg CLO, the cmax of CLO, CATM and CCAM were (7.89 ± 5.46), (15.58 ± 8.08), (8 023.33 ± 1047.39) ng / ml, tmax were (1.25 ± 0.43), (1.25 ± 0.43) and (1.67 ± 0.29) h respectively, and t1 / 2 were (2.31 ± 0.61), (0.64 ± 0.08) and (17.19 ± 14.59), (21.39 ± 9.58) and (30 648.85 ± 8 026.63) ng · h / ml, respectively. Conclusion: The method is simple and convenient to operate with high sensitivity and short analysis time. It is suitable for the determination of blood concentration and pharmacokinetics of CLO and its metabolites.