LC-MS/MS法同时测定大鼠血浆中4种CYP450探针药物浓度

中国临床药理学与治疗学 ›› 2017, Vol. 22 ›› Issue (2): 144-150.

LC-MS/MS法同时测定大鼠血浆中4种CYP450探针药物浓度

刘晶晶^1,2, 梁智^1,2, 罗详冲³, 何功浩¹, 冯恩富¹, 徐贵丽¹

¹解放军昆明总医院药学部，昆明 650032，云南； ²昆明医科大学，昆明 650500，云南；³昆明医科大学第三附属医院胸外科，昆明 650118，云南

收稿日期:2016-10-14 修回日期:2017-01-04 出版日期:2017-02-26 发布日期:2017-03-02
通讯作者: 徐贵丽，女，博士，教授，博士生导师，研究方向：药代动力学。 Tel：0871-64774784 E-mail:kmxuguili@126.com
作者简介:刘晶晶，女，硕士研究生，研究方向：药代动力学。 Tel：15288142287 E-mail:kmljj2287@126.com
基金资助:
国家自然科学基金项目（81673683）

Simultaneous determination of four probe drugs of CYP450 in rat plasma by LC-MS/MS

LIU Jingjing ^1,2, LIANG Zhi ^1,2, LUO Xiangchong³, HE Gonghao¹, FENG Enfu ¹, XU Guili¹

¹Department of Pharmacy, Kunming General Hospital of PLA, Kunming 650032, Yunnan, China; ²Kunming Medical University, Kunming 650500, Yunnan, China;³ The Second Department of Thoracic Surgery, the Third Affiliated Hospital of Kunming Medical University, Kunming 650118, Yunnan, China

Received:2016-10-14 Revised:2017-01-04 Online:2017-02-26 Published:2017-03-02

摘要/Abstract

摘要：

目的:建立高效液相-串联质谱（LC-MS/MS）法同时测定大鼠血浆中非那西丁(1A2),甲苯磺丁脲(2C9)，右美沙芬(2D6)，咪达唑仑(3A4) 4 种探针底物浓度。方法:采用symmetry C18柱(2.1 mm×50 mm,3.5 μm)，流动相为甲醇：0.1％甲酸水溶液(67∶33,V/V)，流速为 0.2 mL/min，柱温35 ℃。电喷雾离子化，检测方式为多反应离子监测。以卡马西平为内标，同时检测大鼠血浆中非那西丁、甲苯磺丁脲、右美沙芬、咪达唑仑的药物浓度，并进行其专属性、精密度、准确度、稳定性的验证。结果:非那西丁( 10～5 000 ng/mL )、甲苯磺丁脲( 20～10 000 ng/mL )、右美沙芬( 4～1 000 ng/mL )、咪达唑仑( 4～1 000 ng/mL )线性关系良好，R²>0.99，准确度为95.6%～105.2%，日内和日间精密度RSD(%)<10%，提取回收率>77%，RSD(%)<7%、基质效应在90.7%～100.3%之间，RSD(%)<5.9%，稳定性好。结论:建立了同时快速测定大鼠血浆中4种探针药物（非那西丁、甲苯磺丁脲、右美沙芬、咪达唑仑）浓度的高效液相-串联质谱分析方法，准确度和灵敏度高，专属性强。血浆样品的稳定性好，适用于CYP450酶活性的研究。

关键词: LC-MS/MS, 细胞色素P450酶, cocktail

Abstract:

AIM:To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of four probe drugs of phenacetin (1A2), tolbutamide (2C9), dextromethorphan (2D6) and midazolam (3A4) in rat plasma. METHODS:Chromatographic separation was achieved on a 2.1 mm×50 mm, 3.5 μm particle, symmetry C18 column at 35 ℃. The mobile phase was constituted of methanol and 0.1% formic acid in water (67∶33,V/V), running at a flow rate of 0.2 mL/min. Mass spectrometry was performed under the multiple reaction monitoring (MRM) mode with an ESI source. With the internal standard carbamazepin, the specific probe substrates of phenacetin, tolbutamide, dextromethorphan and midazolam could be detected in rat plasma. This method was applied into the analysis of plasma samples in rat and was evaluated in terms of recovery, stability, precision and accuracy, and. RESULTS:A good linear relationship was found in phenacetin (10-5 000 ng/mL), tolbutamide (20-10 000 ng/mL), dextromethorphan (4-1 000 ng/mL) and midazolam (4-1 000 ng/mL). The correlation coefficiency (R²) was greater than 0.99. The accuracy of data ranged from 95.96% to 103.10%. RSD(%) of intra-day and inter-day precision was less than 10%. The extraction recoveries of probe drugs were greater than 77% and RSD(%) less than 7%. The range of matrix effect was 90.7% to 100.3% and RSD(%) less than 5.9%. The data exhibited excellent stability. CONCLUSION: The established method is accurate, sensitive, and suitable for determining plasma concentrations of the four probe drugs (phenacetin, tolbutamide, dextromethorphan and midazolam).The plasma samples demonstrate excellent stability and it could be used for evaluating the CYP1A2, CYP2C9, CYP2D6 and CYP3A4 activities in the drug-drug interaction studies.

Key words: LC-MS/MS, CYP450, cocktail

中图分类号:

R965.2

刘晶晶, 梁智, 罗详冲, 何功浩, 冯恩富, 徐贵丽. LC-MS/MS法同时测定大鼠血浆中4种CYP450探针药物浓度[J]. 中国临床药理学与治疗学, 2017, 22(2): 144-150.

LIU Jingjing, LIANG Zhi, LUO Xiangchong, HE Gonghao, FENG Enfu, XU Guili. Simultaneous determination of four probe drugs of CYP450 in rat plasma by LC-MS/MS[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2017, 22(2): 144-150.

参考文献

［1］Kennedy DA, Seely D. Clinically based evidence of drug-herb interactions: a systematic review［J］. Expert Opin Drug Saf, 2010, 9(1): 79-124.
［2］Ingelman-Sundberg M, Oscarson M, Mclellan RA, et al. Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment［J］.Trends Pharmacol Sci，1999, 20(8): 342-349.
［3］Pirmohamed M, Park BK.Cytochrome P450 enzyme polymorphisms and adverse drug reactions［J］. Toxicology, 2003, 192(1): 23-32.
［4］Pelkonen O, Turpeinen M, Hakkola J, et al. Inhibition and induction of human cytochrome P450 enzymes: current status［J］. Arch Toxicol, 2008, 82(10): 667-715.
［5］Zhang L, Reynolds KS, Zhao P, et al. Drug interactions evaluation: An integrated part of risk assessment of therapeutics［J］. Toxicol Appl Pharmacol, 2010, 243(2): 134-145.
［6］FDA.Guidance for Industry. Drug Interaction Studies-Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations. Draft Guidance［EB/OL］.2012: 43-44. http://www. fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM292362.pdf
［7］Yan J, He X, Feng S, et al. Up-regulation on cytochromes P450 in rat mediated by total alkaloid extract from Corydalis yanhusuo［J］. BMC Complement Altern Med, 2014, 14: 306.
［8］Huang Y, Zheng SL, Zhu HY, et al. Effects of aescin on cytochrome P450 enzymes in rats［J］. J Ethnopharmacol, 2014, 151(1): 583-590.
［9］Xu BB, Xu ZS, Zheng SL, et al. Effect of colchicine on rat hepatic cytochrome P450 enzymes by cocktail probe drugs［J］. Pharmazie, 2014, 69(1): 43-47.
［10］Cheng Y, Huang Y, Tian Y, et al. Assessment of the effects of Radix Bupleuri and vinegar-baked Radix Bupleuri on cytochrome 450 activity by a six-drug cocktail approach［J］. Chin J Nat Med, 2013, 11(3): 302-308.
［11］Zheng L, Lu Y, Cao X, et al. Evaluation of the impact of Polygonum capitatum, a traditional Chinese herbal medicine, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs［J］. J Ethnopharmacol, 2014, 158: 276-282.
［12］朱冬春, 程钢, 孙旭群,等. HPLC法同时测定大鼠血浆中CYP450酶探针药物氯唑沙宗、甲苯磺丁脲和咪达唑仑浓度［J］.安徽医药, 2015, 19(7): 1260-1263.
［13］武洁, 钟荣玲, 王大为,等. HPLC-MS-MS法测定大鼠血浆中6种CYP450探针药物及甘草对CYP450酶活性的影响［J］.中国实验方剂学杂志, 2014, 20(23): 110-116.
［14］陆苑, 谢玉敏, 潘洁,等. 超高效液相色谱法检测6种探针底物代谢产物并评价人细胞色素P450同工酶活性［J］.贵阳医学院学报, 2015, 40(6): 560-565.
［15］European Medicines Agency. Guideline on the Investigation of Drug Interactions［EB/OL］.2012: 49-50 http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/ WC500129606.pdf.
［16］Vuckovic D. Current trends and challenges in sample preparation for global metabolomics using liquid chromatography-mass spectrometry［J］. Anal Bioanal Chem, 2012, 403(6): 1523-1548.
［17］Liu Y, Jiao J, Zhang C, et al. A Simplified method to determine five cytochrome p450 probe drugs by hplc in a single run［J］ Biol Pharm Bull, 2009, 32(4): 717-720.
［18］国家药典委员会. 中华人民共和国药典［S］.四部. 北京: 中国医药出版社, 2015: 通则363-368.

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