LC-MS/MS测定大鼠血浆中阿托伐他汀及其活性代谢产物

Determination of Atorvastatin and Its Active Metabolites in Plasma of Rat by LC-MS/MS

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

Key words:  atorvastatin  hydroxyl atorvastatin  LC-MS/MS  pharmacokinetics  CYP3A enzyme  liquid-liquid extraction

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（T_max）提前，血药浓度-时间曲线下面积（AUC_0-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。

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 T_max and AUC_0-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.