Quantification of lipopolysaccharides in outer membrane vesicle vaccines against meningococcal disease. High-performance liquid chromatographic determination of the constituent 3-hydroxy-lauric acid.
A high-performance liquid chromatographic (HPLC) assay for quantification of lipopolysaccharides (LPSs, endotoxins) in outer membrane vesicle vaccines against meningococcal disease has been developed. The LPS constituent, 3-hydroxy-lauric acid, served as marker substance for the quantification. LPS from the vaccine was precipitated by ethanol and the fatty acid constituents, including 3-hydroxy-lauric acid, were released by acidic hydrolysis, collected and purified by solid phase extraction on C18 disc-cartridges and converted into phenacyl esters for UV detection at 240 nm. Quantification of the derivatized 3-hydroxy-lauric acid was achieved by HPLC using a Brownlee RP-18 reversed phase column with acetonitrile/water (68:32, v/v) as mobile phase. The method was found to be linear over the range 3-49 microg LPS/ml with a sensitivity of 1.6 (microg/ml)(-1). The repeatability (within-day precision) of the method at three levels (3-49 microg LPS/ml) was 6-14% relative standard deviation and the intermediate (between-day) precision was 7% relative standard deviation (at level 15 microg LPS/ml). The method has been successfully used in the quality control of a meningococcal B outer membrane vesicle vaccine, containing 4-8% LPS relative to protein (w/w), in our laboratory for three years.
Lyngby
Janne
J
Department of Vaccinology, National Institute of Public Health, N-0403 Oslo, Norway.
Olsen
Linda H
LH
Eidem
Tove
T
Lundanes
Elsa
E
Jantzen
Erik
E
D001428:Bacterial Vaccines / Q000737:chemistry; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology; D005227:Fatty Acids / Q000737:chemistry; D006868:Hydrolysis; D007850:Lauric Acids / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D022401:Meningococcal Vaccines / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D011786:Quality Control; D015203:Reproducibility of Results; D013997:Time Factors; D014466:Ultraviolet Rays
C453840:3-hydroxy-lauric acid; D001428:Bacterial Vaccines; D005227:Fatty Acids; D007850:Lauric Acids; D008070:Lipopolysaccharides; D022401:Meningococcal Vaccines; C030358:lauric acid; D000431:Ethanol