Quantitation of endotoxin by gas chromatography-mass spectrometry in Neisseria meningitidis serogroups A, C, W, Y and X during polysaccharide purification used in conjugate vaccine

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 209; p. 114536
• Main Authors: Shende, Niraj, Karale, Abhijeet, Marne, Kishor, Deshpande, Hrishikesh, Belapurkar, Hrushikesh, Mallya, Asha D., Dhere, Rajeev M.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 05.02.2022
• Subjects: Endotoxin; Endotoxins - analysis; Fatty acid methyl ester; Gas Chromatography-Mass Spectrometry; GC-MS; LAL; Lipopolysaccharide; N. meningitidis; Neisseria meningitidis; Polysaccharide; Polysaccharides; Serogroup; Vaccine; Vaccines, Conjugate; GC-MS; LAL; Lipopolysaccharide; Vaccine; N. meningitidis; Endotoxin; Fatty acid methyl ester; Polysaccharide; LPS
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2021.114536

•Transesterification and acetylation of lipopolysaccharide in meningococcal PS results in 3-O-acetyl tetradecanoic acid.•3-hydroxyl fatty acid methyl ester serves as the marker for LPS quantitation in the meningococcal polysaccharide.•GC-MS provides valuable complimentary tool to monitor the endotoxin content in intermediate and purified polysaccharide.•GC-MS can be used to quantitate LPS in other gram-negative bacteria, lipids and proteins. Bacterial lipopolysaccharide (LPS) responsible for endotoxin effect induces inflammatory reactions. The endotoxins are difficult to separate from the gram-negative polysaccharide (PS) during polysaccharide purification. The most common method to quantify LPS is the limulus amebocyte lysate (LAL) test which interferes with the agents used during PS purification. The gas chromatography-mass spectrometry (GC-MS) provides a suitable alternative by estimating lipid-A chain anchored 3-hydroxy fatty acid methyl ester (FAME) to estimate LPS however, there are no reports of its application in natural polysaccharides used for vaccine preparation. The transesterification of LPS and meningococcal PS yielded primary target 3-O-acetylated myristic acid which was detected by GC-MS and provided quantitative estimation of endotoxin. The GC-MS method was found in agreement with the LAL values showing lower endotoxin content< 10Eu/µg in meningococcal C and Y serogroup polysaccharides in comparison to higher endotoxin 177–523 Eu/µg in meningococcal A, W and X serogroups. The high endotoxin content in purified polysaccharide was attributed to it being detected in its intermediate stage by GC-MS unlike the LAL test. Thus GC-MS serves as a valuable method for endotoxin monitoring and quantitation in gram-negative meningococcal intermediate and purified PS during vaccine preparation.