摘要
目的:建立液相色谱串联质谱(LC-MS/MS)法同时测定人血浆中阿托伐他汀及2个活性相关的羟基他汀酸代谢物和3个毒性相关的他汀内酯型代谢物的浓度,并应用于健康人药代动力学研究和患者血药浓度分析。方法:血浆样本酸化后通过蛋白沉淀法处理。液相色谱分离采用Zorbarx SB-C_(18)(50 mm×2.1 mm,5μm)色谱柱,以含0.05%甲酸的甲醇-乙腈(1∶1)和水-甲醇-乙腈(9∶0.5∶0.5)为流动相,梯度洗脱,流速0.35 mL·min^(-1)。采用电喷雾电离源,正离子模式、多反应监测扫描;检测离子对m/z分别为阿托伐他汀559.3→440.2、邻羟基阿托伐他汀酸(2-HAT)和对羟基阿托伐他汀酸(4-HAT)575.1→440.3、阿托伐他汀内酯(ATL)540.9→448.2、邻羟基阿托伐他汀内酯(2-HATL)和对羟基阿托伐他汀内酯(4-HATL)557.2→448.2及内标匹伐他汀422.2→290.0。对分析方法进行全面验证后检测健康受试者及临床患者服用阿托伐他汀钙片后血浆样品,分析阿托伐他汀和5个代谢产物的药代代动力学特征。结果:阿托伐他汀及其代谢物浓度在0.1~25 nmol·L^(-1)范围内线性关系良好,日内、日间精密度的RSD及准确度的RE均<15%,各种条件下稳定性良好。健康受试者口服20 mg阿托伐他汀钙片后,阿托伐他汀、2-HAT、4-HAT、ATL、2-HATL和4-HATL的C_(max)均值分别为11.48、4.71、0.28、1.71、2.52和2.31 nmol·L^(-1);AUC_(0-∞)均值分别为87.31、58.79、8.60、28.75、45.76、31.49 nmol·h·L^(-1);t_(1/2)均值分别为7.96、7.93、19.58、8.76、8.98和21.37 h。患者服药12 h后阿托伐他汀、2-HAT、4-HAT、ATL、2-HATL和4-HATL的血药浓度分别为(4.16±1.31)nmol·L^(-1)、(2.65±1.33)nmol·L^(-1)、(1.15±1.16)nmol·L^(-1)、(2.96±1.83)nmol·L^(-1)、(4.27±2.00)nmol·L^(-1)和(3.70±1.74)nmol·L^(-1)。结论:本研究建立的人血浆中阿托伐他汀及5个代谢物同时定量检测方法准确、快捷、灵敏、稳定,可用于临床药代动力学研究和血药浓度监测。临床试验结果表明毒性相关内酯型代谢物具有较高暴露水平,需关注可能带来的副反应风险。
Objective:To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the simultaneous determination of atorvastatin, two activity-related hydroxy statin metabolites and three toxicity-related statin lactones in human plasma, and its application to the study of pharmacokinetics in healthy subjects and the analysis of concentrations in patients. Methods: After acidification, plasma samples were treated by protein precipitation. The LC separation was performed on a Zorbarx SB-C_(18)(50 mm×2.1 mm, 5 μm) column. Methanol-acetonitrile(1∶1) water-methanol-acetonitrile(9∶0.5∶0.5) containing 0.05% formic acid were used as the mobile phases for gradient elution, and the flow rate was 0.35 mL·min^(-1). The electric spray ionization source, positive ion mode and multi-reaction monitoring scanning were adopted for MS detection. The m/z of each targeted analyte was 559.3→440.2 for atorvastatin, 575.1→440.3 for 2-hydroxy atorvastatin acid(2-HAT) and 4-hydroxy atorvastatin acid(4-HAT), 540.9→448.2 for atorvastatin lactone(ATL), 557.2→448.2 for 2-hydroxy atorvastatin lactone(2-HATL) and 4-hydroxy atorvastatin lactone(4-HATL), and 422.2→290.0 for the internal standard of pitavastatin. After a full method validation, the developed LC-MS/MS method was used to determine the plasma samples of healthy subjects and patients after taking atorvastatin calcium tablets, and the pharmacokinetic characteristics of atorvastatin and five metabolites were analyzed. Results: The calibration curves of atorvastatin and its metabolites presented a good linear relationship in the range of 0.1-25 nmol·L^(-1). The RSD of intra-and inter-day precision and the RE of accuracy were all less than 15%, and the stability was well tolerated under different conditions. In healthy subjects after oral administration of 20 mg atorvastatin calcium tablets, the respective mean values of C_(max) for atorvastatin, 2-HAT, 4-HAT, ATL, 2-HATL and 4-HATL were 11.48, 4.71, 0.28, 1.71, 2.52 and 2.31 nmol·L^(-1), AUC_(0-∞) were 87.31, 58.79, 8.60, 28.75, 45.76, 31.49 nmol·h·L^(-1), t_(1/2) were 7.96, 7.93, 19.58, 8.76, 8.98 and 21.37 h. After 12 h of administration, the average blood concentrations of atorvastatin, 2-HAT, 4-HAT, ATL, 2-HATL and 4-HATL in the patient were(4.16±1.31) nmol·L^(-1),(2.65±1.33) nmol·L^(-1),(1.15±1.16) nmol·L^(-1),(2.96±1.83) nmol·L^(-1),(4.27±2.00) nmol·L^(-1) and(3.70±1.74) nmol·L^(-1). Conclusion: The method for the simultaneous quantitative determination of atorvastatin and five metabolites in human plasma established in this study is accurate, rapid, sensitive and stable, and can be used for clinical pharmacokinetics research and plasma drug concentration monitoring. The clinical studies revealed that toxicity related lactone metabolites have a high level of exposure in humans, which requires attention to the possible risk of side effects.
作者
宋玉晨
宫晓
易欢
张颖
郭春莉
SONG Yu-chen;GONG Xiao;YI Huan;ZHANG Ying;GUO Chun-li(Beijing Key Laboratory of Pharmacology of Chinese Materia Medica,Institute of Basic Medical Sciences of Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;Department of Neurology,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2024年第1期58-67,共10页
Chinese Journal of Pharmaceutical Analysis
基金
国家自然科学基金项目(81873179)
中国中医科学院科技创新工程项目(CI2021A04906,CI2021A01303)。
