超高效液相串联质谱法快速测定CYP2C9酶活性
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摘要
目的:建立一种超高效液相串联三重四级杆质谱法快速测定CYP2C9体外酶学活性检测的新方法。方法:色谱柱为ACQUITY UPLCBEH C18柱(100 mm×2.1 mm,1.7μm);流动相为乙腈-水(含0.1%甲酸和0.5%氨水)(40∶60),流速为0.2 ml·min-1,柱温30℃,内标为氯磺丙脲;质谱条件:电喷雾离子化源(ESI),正离子检测模式;用实验室制备的CYP2C9*1,*2,*3和*13酶在37℃孵育甲苯磺丁脲后,加入800μl冰乙酸乙酯终止反应,10 000 g离心后取有机层于氮吹仪下吹干并用200μl流动相复溶后上样。结果:4-羟基甲苯磺丁脲的保留时间为1.21 min,线性范围为0.05~5 ng·μl-1(r=0.999 8),最低定量限为0.01 ng·μl-1,回收率为99.3%~100.3%。4-羟基甲苯磺丁脲的日内、日间RSD均<5%,孵育体系中的其他内源性物质不干扰测定。CYP2C9*1,*2,*3和*13孵育甲苯磺丁脲后结果显示突变体CYP2C9*2,*3,*13的体外酶学活性分别为野生型CYP2C9*1的47.3%,11%和0.3%。结论:该方法快速、稳定,该方法操作简便,适于CYP2C9的快速活性检测及抑制药等的相关性研究。
Objective: To establish an ultra performance liquid chromatography-tandem quadruple mass spectrometry( UPLC-MS /MS) method to determine CYP2C9 activity in vitro. Methods: An ACQUITY UPLCBEH C18( 100 mm ×2.1 mm,1.7 μm) column was used as the stationary phase at 30℃. The mobile phase consisted of acetonitrile-water( containing 0. 1% formic acid and 0. 5%ammonia water)( 40∶ 60,v /v). The flow rate was 0. 2 ml·min- 1. Chlorpropamide was used as the internal standard. The MS conditions were as follows: ESI with positive ion detection mode. Self-prepared CYP2C9* 1,* 2,* 3 and * 13 protein were incubated with tolbutamide at 37℃ and 800μl ethyl acetate was added to stop the reaction. After centrifuged at 10 000 g,the organic layer was then dried using nitrogen,the residue was re-dissolved in 200μl mobile phase and determined by UPLC-MS /MS. Results: The retention time of 4-hydroxytolbutamide was 1. 21 min. An excellent linear calibration curve of 4-hydroxytolbutamide was obtained within the concentration range of 0. 05-5 ng·μl- 1( r =0. 999 8). The lower limit of quantification of 4-hydroxytolbutamide was 0.01 ng·μl- 1with the average recovery of 99. 3%-100. 3%. The intra- and inter-day RSDs were all less than 5%. There was no interference from the endogenous substances existing in the incubation system. The catalytic activity of the variants CYP2C9* 2,* 3 and * 13 after tolbutamide was incubated with CYP2C9* 1,* 2,* 3 and * 13 was 47. 3%,11% and 0. 3% of wild type CYP2C9* 1. Conclusion:The method is simple and stable,and suitable for the fast evaluation of cytochrome CYP2C9 activity in vitro and relevant studies on the inhibitors.
Objective: To establish an ultra performance liquid chromatography-tandem quadruple mass spectrometry( UPLC-MS /MS) method to determine CYP2C9 activity in vitro. Methods: An ACQUITY UPLCBEH C18( 100 mm ×2.1 mm,1.7 μm) column was used as the stationary phase at 30℃. The mobile phase consisted of acetonitrile-water( containing 0. 1% formic acid and 0. 5%ammonia water)( 40∶ 60,v /v). The flow rate was 0. 2 ml·min- 1. Chlorpropamide was used as the internal standard. The MS conditions were as follows: ESI with positive ion detection mode. Self-prepared CYP2C9* 1,* 2,* 3 and * 13 protein were incubated with tolbutamide at 37℃ and 800μl ethyl acetate was added to stop the reaction. After centrifuged at 10 000 g,the organic layer was then dried using nitrogen,the residue was re-dissolved in 200μl mobile phase and determined by UPLC-MS /MS. Results: The retention time of 4-hydroxytolbutamide was 1. 21 min. An excellent linear calibration curve of 4-hydroxytolbutamide was obtained within the concentration range of 0. 05-5 ng·μl- 1( r =0. 999 8). The lower limit of quantification of 4-hydroxytolbutamide was 0.01 ng·μl- 1with the average recovery of 99. 3%-100. 3%. The intra- and inter-day RSDs were all less than 5%. There was no interference from the endogenous substances existing in the incubation system. The catalytic activity of the variants CYP2C9* 2,* 3 and * 13 after tolbutamide was incubated with CYP2C9* 1,* 2,* 3 and * 13 was 47. 3%,11% and 0. 3% of wild type CYP2C9* 1. Conclusion:The method is simple and stable,and suitable for the fast evaluation of cytochrome CYP2C9 activity in vitro and relevant studies on the inhibitors.
引文
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9 Hiratsuka M.In vitro assessment of the allelic variants of cytochrome p450[J].Drug Metab Pharmacokinet,2012,27(1):68-84
10 Minet EF,Daniela G,Meredith C,et al.A comparative in vitro kinetic study of[14C]-eugenol and[14C]-methyleugenol activation and detoxification in human,mouse,and rat liver and lung fractions[J].Xenobiotica,2012,42(5):429-441
11 秦梦楠,刘蕊,刘高峰,等.灯盏花素注射液对大鼠体外肝微粒体细胞色素P450酶活性的影响[J].中国药师,2012,15(2):147-150
2 Van Booven D,Marsh S,McLeod H,et al.Cytochrome P4502C9-CYP2C9[J].Pharmacogenet Genomics,2010,20(4):277-281
3 李智,王果,周宏灏.CYP2C9基因多态性及其功能意义研究进展[J].中国临床药理学与治疗学,2008,13(6):601-609
4 Dorado P,Beltran LJ,Machin E,et al.Losartan hydroxylation phenotype in an Ecuadorian popμlation:influence of CYP2C9 genetic polymorphism,habits and gender[J].Pharmacogenomics,2012,13(15):1711-7
5 李健,文思远,王睿,等.细胞色素P450 CYP2C9基因多态性对甲苯磺丁脲代谢动力学的影响[J].药学学报,2005,40(8):695-699
6 King BP,Khan TI,Aithal GP,Kamali F,Daly AK.Upstream and coding region CYP2C9 polymorphisms:correlation with warfarin dose and metabolism[J].Pharmacogenetics,2004,14:813-822
7 Rao KN,Suman SK,Kiran YR,et al.Co-expression of recombinant human CYP2C9 with human cytochrome P450 reductase in protease deficient S.cerevisiae strain at a higher scale yields an enzyme of higher specific activity[J].Drug Metab Lett,2010,4(4):246-53
8 王双虎,戴大鹏,胡利明,等.细胞色素P450 2C9药物代谢酶体外酶学活性检测新方法的建立[J].医学研究杂志,2013,42(2):23-26
9 Hiratsuka M.In vitro assessment of the allelic variants of cytochrome p450[J].Drug Metab Pharmacokinet,2012,27(1):68-84
10 Minet EF,Daniela G,Meredith C,et al.A comparative in vitro kinetic study of[14C]-eugenol and[14C]-methyleugenol activation and detoxification in human,mouse,and rat liver and lung fractions[J].Xenobiotica,2012,42(5):429-441
11 秦梦楠,刘蕊,刘高峰,等.灯盏花素注射液对大鼠体外肝微粒体细胞色素P450酶活性的影响[J].中国药师,2012,15(2):147-150