色谱 ›› 2019, Vol. 37 ›› Issue (2): 155-161.DOI: 10.3724/SP.J.1123.2018.08006

• 研究论文 • 上一篇    下一篇

液相色谱-串联质谱法测定百日咳和百白破疫苗中百日咳杆菌气管细胞毒素

龙珍1, 卫辰2, 郭志谋3, 马霄2, 李月琪1, 姚劲挺1, 冀峰1, 李长坤1, 黄涛宏1   

  1. 1. 岛津企业管理(中国)有限公司, 北京 100020;
    2. 中国食品药品检定研究院, 卫生部生物技术产品检定方法及其标准化重点实验室, 北京 102629;
    3. 中国科学院分离分析化学重点实验室, 中国科学院大连化学物理研究所, 辽宁 大连 116023
  • 收稿日期:2018-08-22 出版日期:2019-02-09 发布日期:2014-12-31
  • 通讯作者: 郭志谋,E-mail:guozhimou@dicp.ac.cn;李月琪,E-mail:fxlyq@shimadzu.com.cn

Determination of tracheal cytotoxin in pertussis and diphtheria tetanus acellular pertussis vaccines using liquid chromatography-tandem mass spectrometry

LONG Zhen1, WEI Chen2, GUO Zhimou3, MA Xiao2, LI Yueqi1, YAO Jinting1, JI Feng1, LI Changkun1, HUANG Taohong1   

  1. 1. Shimadzu Scientific Instrument Company, Beijing 100020, China;
    2. National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 102629, China;
    3. Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
  • Received:2018-08-22 Online:2019-02-09 Published:2014-12-31

摘要:

百日咳杆菌气管细胞毒素(TCT)是一种引起百日咳及百日咳相关疫苗不良反应的毒性糖肽。尽管各国药典均规定了百日咳疫苗产品中TCT的含量限度,但均没有推荐TCT的含量测定方法。该研究发展了一种液相色谱-串联质谱法用于TCT的含量测定。实验优化了包括色谱柱类型和流动相组成在内的TCT色谱条件。虽然TCT在反相色谱模式和亲水作用色谱(HILIC)模式下均具有较好的保留,但HILIC模式与蛋白质沉淀的前处理方法兼容性更好,因此采用HILIC模式分离TCT。优化所得的方法具有较宽的线性范围(5.76~369 ng/L),良好的重复性(峰面积的相对标准偏差不大于3.9%),各种基质中均有较好的回收率(96.4%~102.5%),且定量限是药典要求的TCT最高限量的1/1279。将本方法用于共纯化百日咳疫苗、组分百日咳疫苗、共纯化无细胞百白破疫苗和组分无细胞百白破疫苗中TCT的检测,所有产品均未检出TCT,表明被检样品具有较好的工艺条件可避免TCT在产品中的残留。

关键词: 百白破疫苗, 百日咳杆菌气管细胞毒素, 百日咳疫苗, 液相色谱, 质谱

Abstract:

Tracheal cytotoxin (TCT) is a toxic glycopeptide, which contribute to the adverse effects of pertussis toxin (PT) and related vaccines. Although pharmacopeias limit the amount of TCT in PT product, there is no recommended TCT determination method in any pharmacopeia. In this study, a liquid chromatography-tandem mass spectrometry method was developed to determine TCT. Chromatographic conditions, including column-type and mobile-phase composition, were optimized. According to the literature reports, both reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) can provide a good retention for TCT. A large amount of organic solvent is usually used for protein precipitation, which may affect the RPLC mode, leading to peak distortion, while such effects were not observed in HILIC mode. Thus, HILIC mode was used to analyze TCT in this study. The developed method had a wide linear range (5.76-369 ng/L), good precision (no more than 3.9%), satisfied recoveries in various matrices (96.4%-102.5%). The limit of quantification (LOQ) of the developed method was 1279 times lower than the one required by Chinese Pharmacopeia, wherein the required amount of TCT should be less than 2 pmol per dose. The developed method was used to detect TCT in pertussis vaccine (acellular component), pertussis vaccine (acellular, co-purified), co-purified diphtheria tetanus pertussis vaccine, and component diphtheria tetanus acellular pertussis vaccine. As a result, TCT was not detected in any of the selected samples indicating the safety of these vaccines and PT products.

Key words: diphtheria tetanus pertussis acellular vaccine (DTaP), liquid chromatography (LC), mass spectrometry (MS), pertussis vaccine (PT), tracheal cytotoxin (TCT)

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