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 (endotox ins), has been developed. The determination of lipid A was based on th e quantitative measurement of beta-hydroxymyristic acid and beta-hydro xylauric acid by reversed-phase HPLC. beta-Hydroxy acids were liberate d from ester and amide linkages in endotoxins by acid catalyzed methan olysis. The resulting methyl esters were derivatized with 9-anthracene -carboxyl chloride, 9-fluorene-carboxyl chloride and 4-(1-pyrenyl)buty ri acid chloride and quantified with a fluorescence detector. The effe ctiveness of the three derivatizing agents was compared. As internal s tandards-beta-hydroxytridecanoic acid [beta-OH(13:0)] and beta-hydroxy pentadecanoic acid [beta-OH(15:0)] ethyl eaters were used. The limits of detection of beta-hydroxymyristic acid were 0.5 pg for the 9-anthro yl and 2 pg for the fluorenoyl and 4-(1-pyrenyl)butyroyl eater per sam ple (signal-to-noise ratio of 3). The detection limits of lipopolysacc haride from a smooth strain (Escherichia coli 0111:B4) was 20 pg, whil e that from two rough strains (E. coli Nissle 1917 and Salmonella typh imurium SL 1181) was 5 pg per sample after methanolysis and derivatiza tion with 9-anthroyl chloride.