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

Bacterial lipopolysaccharide (LPS) responsible for endotoxin effect induces inflammatory reactions. The endotoxins are difficult to separate from the gram-negative polysaccharide (PS) during poly-saccharide 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 meningo-coccal 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/mu g in meningococcal C and Y serogroup poly-saccharides in comparison to higher endotoxin 177-523 Eu/mu g in meningococcal A, W and X ser-ogroups. 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. (c) 2021 Elsevier B.V. All rights reserved.

Automated summary

The study investigated a new approach for quantifying endotoxin content using gas chromatography-mass spectrometry (GC-MS) to estimate lipopolysaccharide (LPS) levels. Quantitative estimation of endotoxin was achieved by detecting 3-O-acetylated myristic acid, showing GC-MS results were in agreement with the traditional Limulus amebocyte lysate (LAL) test method.