Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[S]

• Published in: Journal of lipid research Vol. 56; no. 7; pp. 1363 - 1369
• Main Authors: Pais de Barros, Jean-Paul, Gautier, Thomas, Sali, Wahib, Adrie, Christophe, Choubley, Hélène, Charron, Emilie, Lalande, Caroline, Le Guern, Naig, Deckert, Valérie, Monchi, Mehran, Quenot, Jean-Pierre, Lagrost, Laurent
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-07-2015; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology; Elsevier
• Subjects: Animals; Biochemistry, Molecular Biology; Blood Chemical Analysis - methods; Chromatography, High Pressure Liquid; diagnostic tool; Female; Horseshoe Crabs; human; Humans; inflammation; Life Sciences; lipid transfer protein; Lipopolysaccharides - blood; Lipopolysaccharides - chemistry; Lipopolysaccharides - metabolism; lipoprotein; liquid chromatography tandem mass spectrometry; Male; mass spectrometry; Membrane Proteins - metabolism; Methods; Mice; Middle Aged; mouse; sepsis; systemic inflammatory response syndrome; Tandem Mass Spectrometry; mouse; systemic inflammatory response syndrome; liquid chromatography tandem mass spectrometry; inflammation; lipoprotein; diagnostic tool; mass spectrometry; lipid transfer protein; human; sepsis
• DOI: 10.1194/jlr.D059725
• EISSN: 1539-7262
• ISSN: 0022-2275

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.