Determination of endotoxin by the measurement of the acetylated methyl glycoside derivative of Kdo with gas-liquid chromatography-mass spectrometry.
A gas-liquid chromatographic-mass spectrometric (GLC-MS) method was applied to the detection of 3-deoxy-d-manno-2-octulosonic acid (Kdo), a constituent of bacterial lipopolysaccharide (LPS, endotoxin). Samples containing LPS were dried, methanolyzed with 2 M HCl in methanol at 60 degrees C for 1 h and acetylated with acetic anhydride and pyridine (1:1, v/v) solution at 100 degrees C for 30 min, then the products were analyzed by GLC-MS or GLC-MSMS. Four acetylated methylglycoside methyl ester derivatives of Kdo are formed in these conditions, namely one with pyranose ring (Kdo1), two derivatives in the furanose form (Kdo2 and 3) and one derivative of anhydro Kdo (Kdo4), as results from their mass fragmentation patterns. Synthetic Kdo produced mainly Kdo4 derivative, whereas Kdo1 of pyranose ring shape was the predominating derivative formed from LPS. The ion fragment of m/z 375 was selected for the specific detection of this Kdo1 derivative, which might be applied for the endotoxin determination. That approach was used for the analysis of preparations of bacteria, bacteriophages and samples of animal sera. In order to ensure the removal of phosphate substitutions from Kdo, methanolyzed samples can be treated with alkaline phosphatase (2.6 U, pH 9.2, 37 degrees C, 15 min), what was elaborated on Vibrio LPS preparation.
Rybka
J
J
L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland.
Gamian
A
A
D000107:Acetylation; D000818:Animals; D017122:Bacteriophage T4 / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000032:analysis; D004731:Endotoxins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D000432:Methanol; D008757:Methylglucosides / Q000378:metabolism; D051381:Rats; D013400:Sugar Acids / Q000032:analysis; D014734:Vibrio cholerae / Q000737:chemistry
D017077:Culture Media, Conditioned; D004731:Endotoxins; D008757:Methylglucosides; D013400:Sugar Acids; D000432:Methanol; C120356:3-deoxy-manno-oct-2-ulopyranosonic acid