代谢分析

人血浆中阿托伐他汀和5个代谢产物LC-MS/MS分析方法的建立及在药代动力学研究中的应用*

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  • 1.中国中医科学院 西苑医院 基础医学研究所 北京市中药药理重点实验室,北京 100091;
    2.中国中医科学院西苑医院 脑病科,北京 100091
第一作者 Tel:18510100379;E-mail:1980919247@qq.com
**张 颖 Tel:(010)62835639;E-mail:zhyingde@sina.com;郭春莉 Tel:(010)62835301;E-mail:guospringrry@163.com

修回日期: 2023-12-17

  网络出版日期: 2024-06-21

基金资助

*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)

Establishment of an LC-MS/MS method for the determination of atorvastatin and five metabolites in human plasma and its application in pharmacokinetics*

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  • 1. 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;
    2. Department of Neurology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China

Revised date: 2023-12-17

  Online published: 2024-06-21

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-C18(50 mm×2.1 mm,5 μm)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Cmax均值分别为11.48、4.71、0.28、1.71、2.52和2.31 nmol·L-1;AUC0-∞均值分别为87.31、58.79、8.60、28.75、45.76、31.49 nmol·h·L-1; t1/2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。

本文引用格式

宋玉晨, 宫晓, 易欢, 张颖, 郭春莉 . 人血浆中阿托伐他汀和5个代谢产物LC-MS/MS分析方法的建立及在药代动力学研究中的应用*[J]. 药物分析杂志, 2024 , 44(1) : 58 -67 . DOI: 10.16155/j.0254-1793.2024.01.06

Abstract

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-C18(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 Cmax 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, AUC0-∞ were 87.31, 58.79, 8.60, 28.75, 45.76, 31.49 nmol·h·L-1, t1/2 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.

参考文献

[1] 孙哲, 杨文娟, 王瑞麟, 等. 基于回顾性队列的阿托伐他汀钙仿制药与原研药安全性和经济性评价及危险因素评估[J]. 中国医院药学杂志, 2022, 42(14): 1447
SUN Z, YANG WJ, WANG RL, et al. Retrospective comparative study on safety economy and risk assessment of the generic and branded atorvastatin calcium[J]. Chin J Hosp Pharm, 2022, 42(14): 1447
[2] 惠春, 林大专, 孙莹. 阿托伐他汀致不良反应24例文献分析[J]. 中国药房, 2010, 21(44): 4189
HUI C, LIN DZ, SUN Y. Literature analysis of 24 adverse drug reactions cases caused by atorvastatin[J]. China Pharm, 2010, 21(44): 4189
[3] GHIM JL, PHUONG NTT, KIM MJ, et al. Pharmacokinetics of fixed-dose combination of atorvastatin and metformin compared with individual tablets[J]. Drug Des Devel Ther, 2019, 13: 1623
[4] TURNER RM, FONTANA V, BAYLISS M, et al. Development, validation and application of a novel HPLC-MS/MS method for the quantification of atorvastatin, bisoprolol and clopidogrel in a large cardiovascular patient cohort[J]. J Pharm Biomed Anal, 2018, 159: 272
[5] HERMANN M, BOGSRUD MP, MOLDEN E, et al. Exposure of atorvastatin is unchanged but lactone and acid metabolites are increased several-fold in patients with atorvastatin-induced myopathy[J]. Clin Pharmacol Ther, 2006, 79(6): 532
[6] 易志恒, 李筱旻, 申秋莹, 等. LC-MS-MS法测定人血浆中阿托伐他汀、邻位阿托伐他汀和对位阿托伐他汀的浓度[J]. 中南药学, 2021, 19(12): 2564
YI ZH, LI YW, SHEN QY, et al. LC-MS-MS determination of atorvastatin,ortho-hydroxy atorvastatin and para-hydroxy atorvastatin in human plasma[J]. Cent South Pharm, 2021, 19(12): 2564
[7] CHOI YK, PARK SE, KIM EY, et al. Pharmacokinetics of atorvastatin and sustained-release metformin fixed-dose combination tablets: two randomized, open-label, 2-way crossover studies in healthy male subjects under fed conditions[J]. Transl Clin Pharmacol, 2017, 25(4): 190
[8] 梁美芳, 陈庆状, 杨沛群, 等. 基于真实世界的阿托伐他汀仿制药和原研药防治缺血性脑卒中/短暂性脑缺血发作的有效性和安全性比较[J]. 中国临床药理学与治疗学, 2022, 27(7): 785
LIANG MF, CHEN QZ, YANG PQ, et al. Efficacy and safety of generic and branded atorvastatin in patients with ischemic stroke/transient ischemic attack: a real-world study[J]. Chin J Clin Pharmacol, 2022, 27(7): 785
[9] KNEBEL W, GASTONGUAY MR, MALHOTRA B, et al. Population pharmacokinetics of atorvastatin and its active metabolites in children and adolescents with heterozygous familial hypercholesterolemia: selective use of informative prior distributions from adults[J]. J Clin Pharmacol, 2013, 53(5): 505
[10] 吕斌, 寻添荣, 吴树龙, 等. HPLC-MS/MS同时检测大鼠血浆中阿托伐他汀和伏立康唑的浓度[J]. 南方医科大学学报, 2019, 39(3): 337
LÜ B, XUN TR, WU SL, et al. Interaction between atorvastatin and voriconazole in rat plasma: a HPLC-MS/MS based study[J]. J South Med Univ, 2019, 39(3): 337
[11] 易小翠. 基于液相色谱-质谱联用法的苯二氮类药物分析方法的建立及在大鼠体内药代动力学的研究[D]. 宜春: 宜春学院, 2021
YI XC. Establishment of Analytical Method for Benzodiazepine and Study of Pharmacokinetics in Rats by LC-MS[D]. Yichun: Yichun University, 2021
[12] HWANG JG, YU KS, LEE S. Comparison of the pharmacokinetics of highly variable drugs in healthy subjects using a partial replicated crossover study: a fixed-dose combination of fimasartan 120 mg and atorvastatin 40 mg versus separate tablets[J]. Drug Des Devel Ther, 2020, 14: 1953
[13] ANDERSON K, NELSON CH, GONG Q, et al. Assessment of the effect of filgotinib on the pharmacokinetics of atorvastatin, pravastatin, and rosuvastatin in healthy adult participants[J]. Clin Pharmacol Drug Dev, 2022, 11(2): 235
[14] FDA. Bioanalytical-Method-Validation-Guidance-Industry[S]. 2018
[15] HUANG F, MARZIN K, KOENEN R, et al. Effect of steady-state faldaprevir on pharmacokinetics of atorvastatin or rosuvastatin in healthy volunteers: a prospective open-label, fixed-sequence crossover study[J]. J Clin Pharmacol, 2017, 57(10): 1305
[16] 罗楠, 谭力, 张玫, 等. 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
LUO N, TAN L, ZHANG M, et al. Determination of atorvastatin and its active metabolites in plasma of rat by LC-MS/MS[J]. Chin J Mod Appl Pharm, 2019, 36(9): 1029
[17] 孙斌, 魏振满, 郭晓东, 等. LC-MS/MS定量测定人体血浆中阿托伐他汀浓度[J]. 现代生物医学进展, 2013, 13(13): 2565
SUN B, WEI ZM, GUO XD, et al. Quantitative determination of the atorvastatin concentration in healthy human plasma by LC-MS/MS[J]. Prog Mod Biomed, 2013, 13(13): 2565
[18] 姜楠, 杨永革, 许雪廷, 等. LC-MS-MS法测定人血浆中阿托伐他汀浓度[J]. 药学实践杂志, 2011, 29(1): 15
JIANG N, YANG YG, XU XY, et al. Determination of atorvastatin in human plasma by LC-MS-MS[J]. J Pharm Pract, 2011, 29(1): 15
[19] 陈菡菁, 徐红蓉, 苑菲, 等. LC-MS/MS法同时测定人血浆中阿托伐他汀及其活性代谢物的浓度[J]. 中国临床药理学杂志, 2017, 33(6): 542
CHEN HQ, XU HR, YUAN F, et al. Simultaneous determination of atorvastatin and its active metabolites in human plasma by LC-MS/MS[J]. Chin J Clin Pharmacol, 2017, 33(6): 542
[20] 丁杨明, 王晓雪, 陈瑶, 等. UPLC-MS/MS法同时测定人血浆样本中瑞舒伐他汀、阿托伐他汀及其代谢物的浓度[J]. 国际药学研究杂志, 2020, 47(3): 236
DING YM, WANG XX, CHEN Y, et al. Simultaneous determination of rosuvastatin,atorvastatin and their metabolites in human plasma by UPLC-MS/MS[J]. J Int Pharm Res, 2020, 47(3): 236
[21] BAGGETT MC, NYKAMP D. Statin-associated bilateral foot myopathy[J]. J Pharm Pract, 2020, 33(6): 899
[22] LINS RL, MATTHYS KE, VERPOOTEN GA, et al. Pharmacokinetics of atorvastatin and its metabolites after single and multiple dosing in hypercholesterolaemic haemodialysis patients[J]. Nephrol Dial Transplant, 2003, 18(5): 967
[23] HERMANN M, BOGSRUD MP, MOLDEN E, et al. Exposure of atorvastatin is unchanged but lactone and acid metabolites are increased several-fold in patients with atorvastatin-induced myopathy[J]. Clin Pharmacol Ther, 2006, 79(6): 532
[24] GIBSON DM, BRON NJ, RICHENS A, et al. Effect of age and gender on pharmacokinetics of atorvastatin in humans[J]. J Clin Pharmacol, 1996, 36(3): 242
[25] SKOTTHEIM IB, BOGSRUD MP, HERMANN M, et al. Atorvastatin metabolite measurements as a diagnostic tool for statin-induced myopathy[J]. Mol Diagn Ther, 2011, 15(4): 221
[26] AMSDEN GW, KUYE O, WEI GC. A study of the interaction potential of azithromycin and clarithromycin with atorvastatin in healthy volunteers[J]. J Clin Pharmacol, 2002, 42(4): 444
[27] PATEL AM, SHARIFF S, BAILEY DG, et al. Statin toxicity from macrolide antibiotic coprescription: a population-based cohort study[J]. Ann Intern Med, 2013, 158(12): 869
[28] LENNERNÄS H. Clinical pharmacokinetics of atorvastatin[J]. Clin Pharmacokinet, 2003, 42(13): 1141
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