Lipopolysaccharide determination by reversed-phase high-performance liquid chromatography after fluorescence labeling

A sensitive method for the quantitative determination of lipid A, the covalently bound hydrophobic component of lipopolysaccharides (endotoxins), has been developed. The determination of lipid A was based on the quantitative measurement of beta-hydroxymyristic acid and beta-hydroxylauric acid by reversed-phase HPLC. beta-Hydroxy acids were liberated from ester and amide linkages in endotoxins by acid catalyzed methanolysis. The resulting methyl esters were derivatized with 9-anthracene-carboxyl chloride, 9-fluorene-carboxyl chloride and 4-(1-pyrenyl)butyric acid chloride and quantified with a fluorescence detector. The effectiveness of the three derivatizing agents was compared. As internal standards-beta-hydroxytridecanoic acid [beta-OH(13:0)] and beta-hydroxypentadecanoic acid [beta-OH(15:0)] ethyl esters were used. The limits of detection of beta-hydroxymyristic acid were 0.5 pg for the 9-anthroyl and 2 pg for the fluorenoyl and 4-(1-pyrenyl)butyroyl ester per sample (signal-to-noise ratio of 3). The detection limits of lipopolysaccharide from a smooth strain (Escherichia coli 0111:B4) was 20 pg, while that from two rough strains (E. coli Nissle 1917 and Salmonella typhimurium SL 1181) was 5 pg per sample after methanolysis and derivatization with 9-anthroyl chloride.