Analysis of endotoxins by capillary electrophoresis.
Endotoxins are part of the outer membrane of gram-negative bacteria such as E. coli. Upon entering the blood stream, they cause a violent, sometimes life-threatening, response of the immune system. Endotoxins are lipopolysaccharides (LPS), lacking optically active groups, and their detection in the underivatized state can be difficult. In this paper the potential of capillary electrophoresis (CE) for LPS analysis is investigated. By using a standard phosphate buffer method, concentrations down to 100 micrograms/mL can be detected within 6 min. The detection limit can be lowered by one order of magnitude by using a sodium dodecyl sulfate (SDS)/borate buffer, pH 9.2. In this buffer, the SDS serves to homogenize the size of the LPS aggregates, while the borate forms complexes with the diol groups of the molecule, thereby enhancing its optical activity. The formation of LPS-affinity complexes with the UV-active polymyxin B or labeling of the LPS with a fluorophore (fluorescein isothiocyanate) was unsuccessful. Best results, in terms of detection limit and speed, were obtained with an indirect UV-detection CE method. By using a strongly UV-active electrophoresis buffer, endotoxins could be detected as "negative" peaks. In this case, a detection limit of 3 micrograms/mL (35 pM) was determined. Proteins and other UV-active substances did not disturb the assay, since they generated no detectable signals. The indirect UV detection was used to quantify the residual LPS content of a DNA preparation from E. coli.
Freitag
R
R
Laboratoire de Biotechnologie Cellulaire, ETH Lausanne, Switzerland.
Fix
M
M
Brüggemann
O
O
D002240:Carbohydrate Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D008070:Lipopolysaccharides / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D014466:Ultraviolet Rays
D008070:Lipopolysaccharides