Lipopolysaccharide determination by reversed-phase high-performance liquid chromatography after fluorescence labeling
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Lipopolysaccharide determination by reversed-phase high-performance liquid chromatography after fluorescence labeling. / Parlesak, Alexandr; Bode, Christiane.
In: Journal of Chromatography A, Vol. 711, No. 2, 1995, p. 277-288.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Lipopolysaccharide determination by reversed-phase high-performance liquid chromatography after fluorescence labeling
AU - Parlesak, Alexandr
AU - Bode, Christiane
N1 - (Ekstern)
PY - 1995
Y1 - 1995
N2 - 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 β-hydroxymyristic acid and β-hydroxylauric acid by reversed-phase HPLC. β-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-β-hydroxytridecanoic acid [β-OH(13:0)] and β-hydroxypentadecanoic acid [β-OH(15:0)] ethyl esters were used. The limits of detection of β-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.
AB - 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 β-hydroxymyristic acid and β-hydroxylauric acid by reversed-phase HPLC. β-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-β-hydroxytridecanoic acid [β-OH(13:0)] and β-hydroxypentadecanoic acid [β-OH(15:0)] ethyl esters were used. The limits of detection of β-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.
U2 - 10.1016/0021-9673(95)00518-R
DO - 10.1016/0021-9673(95)00518-R
M3 - Journal article
C2 - 7581848
AN - SCOPUS:0029087177
VL - 711
SP - 277
EP - 288
JO - Journal of Chromatography
JF - Journal of Chromatography
SN - 0301-4770
IS - 2
ER -
ID: 317459334