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)