液相色谱-质谱联用法测定人血浆中氯吡格雷及其3种代谢产物的浓度
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篇名: 液相色谱-质谱联用法测定人血浆中氯吡格雷及其3种代谢产物的浓度
TITLE:
摘要: 目的:建立同时测定人血浆中氯吡格雷(CLP)及其中间代谢产物2-氧-氯吡格雷(2-O-CLP)、非活性代谢产物氯吡格雷羧酸代谢物(CLPCA)和活性代谢产物氯吡格雷硫醇代谢物(CLPTM)浓度的方法。 方法:选取陆军军医大学第一附属医院确诊为脑卒中的90名患者,早晨空腹口服1片氯吡格雷片(75 mg/片),于服药2 h后采集血样,将CLPTM经2-溴-3’-甲氧基苯乙酮衍生形成CLPTM-D后与其余3种待测物一起通过乙腈蛋白沉淀提取后,采用液相色谱-串联质谱法(LC-MS/MS)测定其浓度。色谱柱为Agilent poroshell 120 EC-C18,流动相为0.1%甲酸的乙腈溶液-0.1%甲酸水溶液(90 ∶ 10,V/V),采用多反应监测模式进行正离子检测,检测离子对分别为CLPCA 质荷比(m/z) 308.1→198.1、CLP m/z 322.3→212.0、2-O-CLP m/z 338.3→155.0、CLPTM-D m/z 504.4→354.1、内标噻氯吡啶m/z 264.0→154.1。结果:CLPCA、CLP、2-O-CLP、CLPTM-D和内标的保留时间分别为2.01、3.32、2.83、2.68、1.87 min,CLPCA、CLP、2-O-CLP、CLPTM-D检测质量浓度的线性范围分别为100~10 000、0.2~20、0.3~30、0.5~50 ng/mL(r均≥0.999 5),日内、日间精密度试验的RSD均≤9.5%(n=5),准确度为93.5%~98.9%(n=5),提取回收率为85.4%~95.9%(n=5),基质效应的CV为2.7%~6.2%(n=5)。稳定性(-80 ℃放置3个月、3次冷冻-解冻循环、4 ℃放置8 h)试验中,CLPCA、CLP、2-O-CLP和CLPTM-D的RE均≤10.0%(n=5)。结论:建立的LC-MS/MS法特异性强,测定结果准确可靠,可用于检测人血浆中CLP及其3种代谢产物的浓度。
ABSTRACT: OBJECTIVE: To establish the method for simultaneous determination of clopidogrel (CLP), its intermediate metabolite (2-O-CLP), inactive metabolite (CLPCA) and active metabolite (CLPTM) in human plasma. METHODS: Totally 90 patients diagnosed as stroke were selected from the First Affiliated Hospital of Army Medical University. They were given one CLP tablet (75 mg/tablet) orally on an empty stomach in the morning. Blood samples were collected 2 h after taking the tablet. CLPTM- D was formed by derivation of CLPTM with 2-bromo-3’-methoxyacetophenone and extracted by precipitation of acetonitrile protein together with the other three substances to be measured. LC-MS/MS method was adopted. The determination was performed on Agilent poroshell 120 EC-C18 column with mobile phase consisted of acetonitrile (0.1% formic acid) and water (0.1% formic acid) (90 ∶ 10, V/V). The quantitation analysis was performed using multiple reaction monitoring at the specific ion transitions of m/z 308.1→198.1 (CLPCA), 322.3→212.0 (CLP), 338.3→155.0 (2-O-CLP), 504.4→354.1 (CLPTM-D) and 264.0→154.1 (ticlopidine, internal standard), respectively. RESULTS: The retention time of CLPCA, CLP, 2-O-CLP, CLPTM-D and internal standard were 2.01, 3.32, 2.83, 2.68, 1.87 min, respectively. The linear range of CLPCA, CLP, 2-O-CLP and CLPTM-D were 100-10 000, 0.2-20, 0.3-30, 0.5-50 ng/mL (all r≥0.999 5). The intra-day and inter-day RSD were all less than 9.5% (n=5). Accuracy ranged from 93.5%-98.9% (n=5), and extraction recovery was from 85.4% to 95.9% (n=5). The matrix effect ranged from 2.7%-6.2% (n=5). In stability tests (storing at -80 ℃ for 3 months, 3 freeze-thaw cycles, storing at 4 ℃ for 8 h), RE of CLP, CLPCA and CLPTM-D were all lower than 10.0% (n=5). CONCLUSIONS: Established LC-MS/MS method has the advantages of high specificity, accuracy and reliability, and can be used to detect the concentration of CLP and its three metabolites in human plasma.
期刊: 2019年第30卷第21期
作者: 刘职瑞,姚璞,杨波,王瑜,冯伟,孙凤军
AUTHORS: LIU Zhirui,YAO Pu,YANG Bo,WANG Yu,FENG Wei,SUN Fengjun
关键字: 液相色谱-串联质谱法;氯吡格雷;代谢产物;血浆浓度
KEYWORDS: LC-MS/MS; Clopidogrel; Metabolites; Plasma concentration
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