UPLC-MS/MS检测长白山白眉蝮蛇蛇毒中的类凝血酶  

Determination of Thrombin-like Enzyme from Venom of Agkistrodon Halys Pallas by UPLC-MS/MS

作　　者：咸瑞卿 王聪聪[1] 巩丽萍 张迅杰 彭丽 石峰 XIAN Rui-qing;WANG Cong-cong;GONG Li-ping;ZHANG Xun-jie;PENG Li;SHI Feng(Shandong Institute for Food and Drug Control,NMPA Key Laboratory for Research and Evaluation of GenericDrugs,Shandong Research Center of Engineering and Technology for Consistency Evaluation of Generic Drugs,Jinan 250101,China;School of Pharmacy,Shandong University,Jinan 250012,China;Department of QualityControl,China Resources Double-Crane Limin Pharmaceutical(Jinan)Co.,Ltd.,Jinan 250200,China)

机构地区：[1]山东省食品药品检验研究院,国家药品监督管理局仿制药研究与评价重点实验室,山东省仿制药一致性评价工程技术研究中心,山东济南250101 [2]山东大学药学院,山东济南250012 [3]华润双鹤利民药业(济南)有限公司质量部,山东济南250200

出　　处：《食品与药品》2022年第4期323-327,共5页Food and Drug

基　　金：国家药典委员会药品标准制修订研究课题(编号:2019H04)。

摘　　要：目的基于具有种属特异性的长白山白眉蝮蛇类凝血酶特征肽,建立超高效液相色谱-三重四极杆质谱(UPLC-MS/MS)检测蛇毒种属来源及类凝血酶含量的方法。方法样品经还原、烷基化、胰蛋白酶消化后,利用UPLC-MS/MS对类凝血酶特征肽进行检测。色谱柱:Waters ACQUITY UPLC BEH C18(50 mm×2.1 mm,1.7µm);流动相:0.1%甲酸溶液-0.1%甲酸乙腈,梯度洗脱;流速:0.2 ml/min;以m/z 877.4(双电荷)→892.4,550.2作为检测离子,采用ESI+模式进行多反应监测(MRM)。结果特征肽在20~400 ng/ml范围内浓度与色谱峰面积呈良好的线性关系(r=0.9990),3个水平的加标回收率为91.2%~107.8%,精密度和重复性均良好。结论本方法准确、可靠,可用于长白山白眉蝮蛇蛇毒中类凝血酶的定性定量分析,为蛇毒血凝酶类药物的质量控制提供技术支持。Objective To establish a method for the determination of species source and content of thrombin-like enzyme from venom of Agkistrodon halys Pallas by UPLC-MS/MS based on the species-specific maker peptide of thrombin-like enzyme of Agkistrodon halys Pallas.Methods After reduction,alkylation and trypsin digestion,the maker peptide of thrombin-like enzyme was detected by UPLC-MS/MS.Waters ACQUITY UPLC BEH C18(50 mm×2.1 mm,1.7µm)chromatographic column was adopted,using 0.1%formic acid solution-0.1%formic acid acetonitrile at a flow rate of 0.2 ml/min as mobile phase of gradient elution.The positive ion mode of electrospray ionization(ESI+)and multiple reaction monitoring(MRM)were adopted.The m/z 877.4(double charges)→892.4,550.2 were chosen as the detection ion pairs.Results A good linear relationship was established between the concentration of maker peptide and the peak area in the range of 20-400 ng/ml(r=0.9990).The recoveries of spiked samples at three levels were 91.2%-107.8%,demonstrating good precision and repeatability.Conclusion The method was accurate and reliable,and can be used for qualitative and quantitative analysis of thrombin-like enzyme from venom of Agkistrodon halys Pallas,providing technical support for the quality control of snake venom hemocoagulase drugs.

关 键 词：长白山白眉蝮蛇 特征肽 类凝血酶 超高效液相色谱-串联质谱 

分 类 号：R927.2[医药卫生—药学]