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
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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1/1 279。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。
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 1 279 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.
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 1 279 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.
引文
[1]Ye J K,Li K L,Xu D S,et al.Chinese Journal of Vaccines and Immunization,2017,23(5):481叶家楷,李克莉,许涤沙,等.中国疫苗和免疫,2017,23(5):481
[2]Cookson B T,Cho H L,Herwaldt L A,et al.Infect Immun,1989,57(7):2223
[3]Long Z,Li J,Guo Z,et al.J Pharm Biomed Anal,2018,155:253
[4]Adin D M,Engle J T,Goldman W E,et al.J Bacteriol,2009,191(7):2012
[5]Dong S,Yan Z,Yang H,et al.Anal Sci,2017,33:293
[6]Long Z,Guo Z,Acworth I N,et al.Talanta,2016,151:239
[7]Long Z,Guo Z,Liu X,et al.Talanta,2016,146:423
[8]Long Z,Jin Y,Liu X D,et al.Chinese Journal of Chromatography,2015,33(9):938龙珍,金燕,刘晓达,等.色谱,2015,33(9):938
[9]Castells C B,Castells R C.J Chromatogr A,1998,805(1):55
[10]Kozlowski E S,Dalterio R A.J Sep Sci,2007,30(14):2286
[11]Layne J,Farcas T,Rustamov I,et al.J Chromatogr A,2001,913(1):233
[12]Ruta J,Rudaz S,Mccalley D V,et al.J Chromatogr A,2010,1217(52):8230
[13]Janvier S,De S E,Wynendaele E,et al.Talanta,2017,174:562
[14]Ruta J,Rudaz S,McCalley D V,et al.J Chromatogr A,2010,1217(52):8230
[15]Gamache P H.Charged Aerosol Detection for Liquid Chromatography and Related Separation Techniques.New York:John Wiley&Sons,Inc.,2017
[16]Keshishian H,Addona T,Burgess M,et al.Mol Cell Proteomics,2009,8:2339
[17]Long Z,Guo Z,Acworth I,et al.Talanta,2016,151:239
[18]Kim Y A,Abd A M,Radman M M,et al.J Chromatogr B,2018,1076:130
[2]Cookson B T,Cho H L,Herwaldt L A,et al.Infect Immun,1989,57(7):2223
[3]Long Z,Li J,Guo Z,et al.J Pharm Biomed Anal,2018,155:253
[4]Adin D M,Engle J T,Goldman W E,et al.J Bacteriol,2009,191(7):2012
[5]Dong S,Yan Z,Yang H,et al.Anal Sci,2017,33:293
[6]Long Z,Guo Z,Acworth I N,et al.Talanta,2016,151:239
[7]Long Z,Guo Z,Liu X,et al.Talanta,2016,146:423
[8]Long Z,Jin Y,Liu X D,et al.Chinese Journal of Chromatography,2015,33(9):938龙珍,金燕,刘晓达,等.色谱,2015,33(9):938
[9]Castells C B,Castells R C.J Chromatogr A,1998,805(1):55
[10]Kozlowski E S,Dalterio R A.J Sep Sci,2007,30(14):2286
[11]Layne J,Farcas T,Rustamov I,et al.J Chromatogr A,2001,913(1):233
[12]Ruta J,Rudaz S,Mccalley D V,et al.J Chromatogr A,2010,1217(52):8230
[13]Janvier S,De S E,Wynendaele E,et al.Talanta,2017,174:562
[14]Ruta J,Rudaz S,McCalley D V,et al.J Chromatogr A,2010,1217(52):8230
[15]Gamache P H.Charged Aerosol Detection for Liquid Chromatography and Related Separation Techniques.New York:John Wiley&Sons,Inc.,2017
[16]Keshishian H,Addona T,Burgess M,et al.Mol Cell Proteomics,2009,8:2339
[17]Long Z,Guo Z,Acworth I,et al.Talanta,2016,151:239
[18]Kim Y A,Abd A M,Radman M M,et al.J Chromatogr B,2018,1076:130