中国药师
中國藥師
중국약사
CHINA PHARMACIST
2014年
11期
1804-1807,1808
,共5页
雷旭伟%王双虎%胡国新%周云芳
雷旭偉%王雙虎%鬍國新%週雲芳
뢰욱위%왕쌍호%호국신%주운방
细胞色素P450 2C9%UPLC-ESI-MS/MS%甲苯磺丁脲%体外活性分析
細胞色素P450 2C9%UPLC-ESI-MS/MS%甲苯磺丁脲%體外活性分析
세포색소P450 2C9%UPLC-ESI-MS/MS%갑분광정뇨%체외활성분석
CYP2C9%UPLC-ESI-MS/MS%Tolbutamide%In vitro activity analysis
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 UPLC? BEH 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CYP2C91,2,3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,10000 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=0.9998),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。CYP2C91,2,3和13孵育甲苯磺丁脲后结果显示突变体CYP2C92,3,13的体外酶学活性分别为野生型CYP2C91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。
目的：建立一種超高效液相串聯三重四級桿質譜法快速測定CYP2C9體外酶學活性檢測的新方法。方法：色譜柱為ACQUITY UPLC? BEH C18柱(100 mm ×2.1 mm,1.7μm)；流動相為乙腈-水(含0.1%甲痠和0.5%氨水)(40∶60),流速為0.2 ml·min-1,柱溫30℃,內標為氯磺丙脲；質譜條件：電噴霧離子化源(ESI),正離子檢測模式；用實驗室製備的CYP2C91,2,3和13酶在37℃孵育甲苯磺丁脲後,加入800μl冰乙痠乙酯終止反應,10000 g離心後取有機層于氮吹儀下吹榦併用200μl流動相複溶後上樣。結果：4-羥基甲苯磺丁脲的保留時間為1.21 min,線性範圍為0.05~5 ng·μl-1(r=0.9998),最低定量限為0.01 ng·μl-1,迴收率為99.3%~100.3%。4-羥基甲苯磺丁脲的日內、日間RSD均<5%,孵育體繫中的其他內源性物質不榦擾測定。CYP2C91,2,3和13孵育甲苯磺丁脲後結果顯示突變體CYP2C92,3,13的體外酶學活性分彆為野生型CYP2C91的47.3%,11%和0.3%。結論：該方法快速、穩定,該方法操作簡便,適于CYP2C9的快速活性檢測及抑製藥等的相關性研究。
목적：건립일충초고효액상천련삼중사급간질보법쾌속측정CYP2C9체외매학활성검측적신방법。방법：색보주위ACQUITY UPLC? BEH C18주(100 mm ×2.1 mm,1.7μm)；류동상위을정-수(함0.1%갑산화0.5%안수)(40∶60),류속위0.2 ml·min-1,주온30℃,내표위록광병뇨；질보조건：전분무리자화원(ESI),정리자검측모식；용실험실제비적CYP2C91,2,3화13매재37℃부육갑분광정뇨후,가입800μl빙을산을지종지반응,10000 g리심후취유궤층우담취의하취간병용200μl류동상복용후상양。결과：4-간기갑분광정뇨적보류시간위1.21 min,선성범위위0.05~5 ng·μl-1(r=0.9998),최저정량한위0.01 ng·μl-1,회수솔위99.3%~100.3%。4-간기갑분광정뇨적일내、일간RSD균<5%,부육체계중적기타내원성물질불간우측정。CYP2C91,2,3화13부육갑분광정뇨후결과현시돌변체CYP2C92,3,13적체외매학활성분별위야생형CYP2C91적47.3%,11%화0.3%。결론：해방법쾌속、은정,해방법조작간편,괄우CYP2C9적쾌속활성검측급억제약등적상관성연구。
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 UPLC? BEH 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 condi-tions 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 000g, 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 reten-tion 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-1 with 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 tol-butamide 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.
