| 引用本文: | 罗楠,谭力,张玫,阿基业,孙润彬,冯冬,齐谢敏,邹秉杰,周国华,黄青,宋沁馨.LC-MS/MS测定大鼠血浆中阿托伐他汀及其活性代谢产物[J].中国现代应用药学,2019,36(9):1029-1036. |
| LUO Nan,TAN Li,ZHANG Mei,A Jiye,SUN Runbin,FENG Dong,QI Xiemin,ZOU Bingjie,ZHOU Guohua,HUANG Qing,SONG Qinxin.Determination of Atorvastatin and Its Active Metabolites in Plasma of Rat by LC-MS/MS[J].Chin J Mod Appl Pharm(中国现代应用药学),2019,36(9):1029-1036. |
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| LC-MS/MS测定大鼠血浆中阿托伐他汀及其活性代谢产物 |
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罗楠1,2, 谭力2, 张玫2, 阿基业3, 孙润彬3, 冯冬3, 齐谢敏4, 邹秉杰4, 周国华4, 黄青2, 宋沁馨1
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1.中国药科大学 药物质量与安全预警教育部重点实验室, 南京 210009;2.江苏省食品药品监督检验研究院检验技术研究中心, 南京 210000;3.中国药科大学 药物代谢与动力学重点实验室, 南京 210009;4.南京大学医学院附属金陵医院药理科, 南京 210002
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| 摘要: |
| 目的 建立LC-MS/MS同时测定大鼠血浆中阿托伐他汀、邻羟基阿托伐他汀及对羟基阿托伐他汀的方法，并应用于CYP3A酶诱导模型大鼠和正常大鼠体内阿托伐他汀的药动学研究。方法 采用甲基叔丁基醚-乙酸乙酯（50︰50）液液萃取法提取大鼠血浆中药物。使用XBridge C18（2.1 mm×250 mm，3.5 μm）色谱柱，柱温35℃；流动相为0.1%甲酸-乙腈（40：60），等度洗脱4.4 min，流速0.2 mL·min-1；进样体积10 μL。质谱采用电喷雾离子（ESI）源，以正离子多反应监测（MRM）模式进行定量分析，选择m/z 559.1→440.1（阿托伐他汀），m/z 575.3→440.2（邻羟基阿托伐他汀/对羟基阿托伐他汀），m/z 564.3→445.3（阿托伐他汀-d5，内标）作为检测离子对。以地塞米松80 mg·kg-1·d-1连续灌胃给药4 d，建立CYP3A酶诱导模型，取正常及模型大鼠给药后0，0.083，0.17，0.25，0.33，0.5，0.75，1，1.5，2，3，4，6 h血样于肝素抗凝管中，离心收集血浆，冷冻保存直到进行测定。结果 阿托伐他汀及其2种代谢产物在0.49~500.00 ng·mL-1内均有良好的线性关系（r2>0.99）；批内、批间精密度RSD<15%（n=6）；方法的提取回收率和基质效应均满足生物样品的检测要求；含药血浆在室温放置4，24 h、4℃放置3 d稳定。诱导组大鼠血浆中阿托伐他汀血药浓度达峰时间（Tmax）提前，血药浓度-时间曲线下面积（AUC0-t）显著低于正常组，消除速率常数K和清除率CL略高于正常组。结论 新建立的方法简便、稳定、灵敏，能够用于大鼠血浆内阿托伐他汀及其活性代谢产物的浓度测定和药动学研究。进入CYP3A酶诱导模型大鼠与正常大鼠体循环的活性药物成分差异显著。 |
| 关键词: 阿托伐他汀 羟基阿托伐他汀 液相色谱-串联质谱法 药动学 CYP3A酶 液液萃取法 |
| DOI：10.13748/j.cnki.issn1007-7693.2019.09.001 |
| 分类号:R917 |
| 基金项目:国家自然科学基金项目（81603196，81673390）；江苏省重点研发计划（社会发展）项目（BE2016745）；药物质量与安全预警教育部重点实验室资助项目（DQCP2017MS01）；江苏省青蓝工程 |
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| Determination of Atorvastatin and Its Active Metabolites in Plasma of Rat by LC-MS/MS |
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LUO Nan1,2, TAN Li2, ZHANG Mei2, A Jiye3, SUN Runbin3, FENG Dong3, QI Xiemin4, ZOU Bingjie4, ZHOU Guohua4, HUANG Qing2, SONG Qinxin1
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1.China Pharmaceutical University, Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education, Nanjing 210009, China;2.Center of Inspection and Technical Research, Jiangsu Institute for Food and Drug Control, Nanjing 210000, China;3.China Pharmaceutical University, Key Laboratory of Drug Metabolism and Pharmacokinetics, Nanjing 210009, China;4.Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China
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| Abstract: |
| OBJECTIVE To establish a LC-MS/MS method for simultaneous determination of atorvastatin, o-hydroxyatorvastatin and p-hydroxyatorvastatin in rat plasma, and apply to the pharmacokinetic study of atorvastatin in CYP3A-induced model rats and normal rats. METHODS MTBE-ethyl acetate(50︰50) was used to extract the drug in rat plasma in addition to the selection of XBridge C18(2.1 mm×250 mm, 3.5 μm) as the HPLC column. The column was kept at a constant temperature(35℃). The mobile phase was 0.1% formic-acetonitrile(40:60), isocratic elution for 4.4 min at a flow rate of 0.2 mL·min-1 and the injection volume was 10 μL. The detection of analytes was achieved by tandem mass spectrometry with electrospray ionization(ESI) interface in positive ion mode. The following multiple reaction monitoring(MRM) transitions were selected:m/z 559.1→440.1(atorvastatin), m/z 575.3→440.2(o-hydroxyl atorvastatin/p-hydroxyl atorvastatin), m/z 564.3→445.3 (atorvastatin-d5, IS). The rats randomly selected as CYP3A-induced models were administrated with an oral dose of 80 mg·kg-1·d-1 dexamethasone for 4 d. Blood samples of normal and model rats were collected in heparinized tubes before, and at 0.083, 0.17, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6 h after atorvastatin administration. Plasma was harvested by centrifugation and stored frozen until analysis. RESULTS Atorvastatin and its metabolites showed a wide linear range from 0.49-500.00 ng·mL-1(r2>0.99). The analytical method presented the RSD of intra-day precision and inter-day precision were <15%(n=6); the extraction recovery and matrix effect satisfied the determination standard for biological samples. Futhermore, the drug contained plasma samples stayed stable in 4, 24 h in room temperature and 3 d in 4℃. In pharmacokinetic parameters, the Tmax and AUC0-t of rats in induced group were lower than those of normal group, while the K and CL were higher. CONCLUSION The newly developed method is convenient, stable and sensitive. It can be applied to the simultaneous determination of atorvastatin and its active metabolites in rat plasma for the pharmacokinetics study. The active atorvastatin components entering the general circulation demonstrated significant differences between the CYP3A-induced model rats and normal rats. |
| Key words: atorvastatin hydroxyl atorvastatin LC-MS/MS pharmacokinetics CYP3A enzyme liquid-liquid extraction |
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