Separation of Escherichia coli 055:B5 lipopolysaccharide and detoxified lipopolysaccharide by high-performance capillary electrophoresis.
A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the lipopolysaccharide (LPS) and detoxified LPS (D-LPS), produced by both alkaline treatment in anhydrous conditions and mild acid hydrolysis, from Escherichia coli 055:B5 bacteria. LPS and D-LPS are separated and readily determined within 25 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient greater than about 0.97) was found for the LPS and the two D-LPS species over a wide range of concentrations, from approximately 120 to 360 ng, with a detection sensitivity less than about 100 ng. Furthermore, HPCE was able to separate several molecular species mainly due to the presence of populations with O-specific polysaccharides of distinct and increasing mean chain lengths. This approach could be of great importance for the quantitative determination of LPS and D-LPS during the purification and preparation processes, also considering the importance of D-LPS in the preparation of human vaccines, and for the qualitative evaluation of the heterogeneity of LPS and the O-polysaccharide components.
Volpi
Nicola
N
Department of Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy. volpi@unimo.it
D002021:Buffers; D002240:Carbohydrate Sequence; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000032:analysis*; D008969:Molecular Sequence Data; D019081:O Antigens / Q000032:analysis; D015203:Reproducibility of Results; D012822:Silicon Dioxide
D002021:Buffers; D008070:Lipopolysaccharides; D019081:O Antigens; D012822:Silicon Dioxide