Analysis of a monophosphoryl lipid A immunostimulant preparation from Salmonella minnesota R595 by high-performance liquid chromatography

MPL immunostimulant, a 4'-monophosphoryl lipid A (MLA) preparation obtained from the lipopolysaccharide of Salmonella minnesota R595, is being developed for several clinical indications. MPL comprises a mixture of MLA congeners that contain 4, 5, and 6 fatty acids. In this paper, we report a new high-performance liquid chromatography (HPLC) method for analyzing the congener composition and purity of MPL. MPL is first derivatized with dinitrobenzyloxyamine (DNBA), resulting in incorporation of the dinitrobenzyl chromophore at the reducing end of all MLA congeners. DNBA-MPL is then analyzed by reversed-phase HPLC on a Waters NovaPak C18, 4 microns particle size, 300 mm x 3.9 mm column. Optimal separation of DNBA-MLA species is obtained using a linear gradient of 10% to 80% isopropanol-water (95:5, v/v), 5 mM tetrabutylammonium dihydrogenphosphate (TBAP), in acetonitrile-water (95:5, v/v), 5 mM.TBAP, over 45 min. A synthetic compound, corresponding to a hexaacyl MLA congener, is used for determination of the detector response factor, allowing the MLA content of MPL (i.e., purity) to be determined. Overall, this method provides better separation, higher sensitivity, and is faster and saler than previous methods used for the analysis of MPL.