Parlesak A, Bode C
Hohenheim University, Stuttgart, Germany.
J Chromatogr A. 1995 Sep 22;711(2):277-88. doi: 10.1016/0021-9673(95)00518-r.
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.
已开发出一种用于定量测定脂多糖（内毒素）中共价结合的疏水成分脂多糖A的灵敏方法。脂多糖A的测定基于通过反相高效液相色谱法定量测量β-羟基肉豆蔻酸和β-羟基月桂酸。通过酸催化的甲醇解作用，从内毒素中的酯键和酰胺键释放出β-羟基酸。所得甲酯用9-蒽甲酰氯、9-芴甲酰氯和4-(1-芘基)丁酸氯进行衍生化，并使用荧光检测器进行定量。比较了三种衍生化试剂的有效性。使用β-羟基十三烷酸[β-OH(13:0)]乙酯和β-羟基十五烷酸[β-OH(15:0)]乙酯作为内标。每个样品中，9-蒽甲酰酯形式的β-羟基肉豆蔻酸的检测限为0.5 pg，芴甲酰酯和4-(1-芘基)丁酰酯形式的为2 pg（信噪比为3）。经甲醇解并用9-蒽甲酰氯衍生化后，光滑菌株（大肠杆菌0111:B4）的脂多糖检测限为每个样品20 pg，而两种粗糙菌株（大肠杆菌Nissle 1917和鼠伤寒沙门氏菌SL 1181）的为每个样品5 pg。
