摘要
利用酶催化反应,结合在线SPE/液相色谱-质谱联用分析系统,建立了一种通过定量酶反应过程测定生物效价的分析方法,用于评价肝素类药物抗IIa因子效价和抗Xa因子效价。本方法采用在线C 18柱富集纯化样品后,在另一根C 18柱上,以水-乙腈体系为流动相进行分离,并采用电喷雾正离子模式,以多反应检测(MRM)方式进行定量分析。方法学考察结果表明,酶反应产物对硝基苯胺(pNA)在3~1000μg/L浓度范围内线性关系良好,方法的定量限为3μg/L,准确度(相对回收率)为88.8%~100.6%,日内与日间精密度(相对标准偏差)分别为1.1%~5.4%(n=6)和3.8%~10.9%(n=18),无明显基质效应。以肝素钠标准品为对照品,测定了2个肝素钠供试品的生物效价,均达到药典标准要求。本方法较紫外分光光度法具有更好的灵敏度和良好的重现性,酶试剂及底物用量更少,可作为测定肝素类药物生物效价的新方法,为肝素类药物生产过程中的质量控制提供技术支持。
An analytical method based on online solid-phase extraction(SPE)coupled with liquid chromatography-tandem mass spectrometry was established for determination of biological potency of heparin,including anti-Factor IIa activity and anti-Factor Xa activity.The online SPE was conducted with ten-port switching valve by equipping a C 18 column as the SPE column,where the samples were cleaned up in on-line mode.Subsequently,the analyte was eluted from the SPE column onto the analytical column(C 18 column)by water-acetonitrile for LC-MS/MS analysis performed in the positive mode and multiple reaction monitoring(MRM)mode.The analytical method was validated to demonstrate specificity,linearity,accuracy,precision,LLOQ,stability and matrix effect.In the linearity range of 3-1000μg/L,the LLOQ for p-nitroaniline(pNA)in the enzyme reaction were 3μg/L,the intra-day and inter-day precisions were 1.1%-5.4%(n=6)and 3.8%-10.9%(n=18),respectively,and the accuracy was 88.8%-100.6%.Minimal ion suppression was observed for pNA.Comparing to the reference standard,the biological potencies of two heparin sodium samples were assessed using this method.The method showed better sensitivity,no need for large of enzyme and substrate,and good reproducibility than UV spectrophotometry method.Therefore,it could be applied to the determination of biological potency of heparin,contributing to the quality control of pharmaceutical products.
作者
刘若男
朱黎
刘堂荣
邹旋
许哲
管华诗
LIU Ruo-Nan;ZHU Li;LIU Tang-Rong;ZOU Xuan;XU Zhe;GUAN Hua-Shi(School of Medicine and Pharmacy,Ocean University of China,Key Laboratory of Marine Drugs,Chinese Ministry of Education,Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering,Qingdao 266003,China;Innovation Center for Marine Drugs Screening and Evaluation,Laboratory for Marine Drugs and Bioproducts,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao 266237,China;Marine Biomedical Research Institute of Qingdao,Qingdao 266071,China)
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2019年第8期1267-1275,共9页
Chinese Journal of Analytical Chemistry
基金
国家自然科学基金项目(No.21505124)
中央高校基本科研业务费专项资金
中国博士后科学基金(No.2015M582144)
青岛市博士后应用研究项目资助~~
关键词
质谱
生物效价
肝素
在线固相萃取
高效液相色谱
Mass spectrometry
Biological potency
Heparin
Online solid-phase extraction
High performance liquid chromatography
