Meningococcal X polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection using synthetic N-acetylglucosamine-4-phosphate as standard

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. The polysaccharide is hydrolysed by strong acidic treatment and the peak of glucosamine-4-phosphate (4P-GlcN) detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the commercially unavailable monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was chemically synthesized according to an improved procedure. The method for MenX quantification proposed here is highly selective and sensitive and constitutes an important analytical tool for the development of a conjugate vaccine against MenX.