ANALYSIS OF A MONOPHOSPHORYL LIPID-A IMMUNOSTIMULANT PREPARATION FROMSALMONELLA-MINNESOTA R595 BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

MPL(R) 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 mixt ure of MLA congeners that contain 4, 5, and 6 fatty acids. In this pap er, we report a new high-performance liquid chromatography (HPLC) meth od for analyzing the congener composition and purity of MPL. MPL is fi rst derivatized with dinitrobenzyloxyamine (DNBA), resulting in incorp oration of the dinitrobenzyl chromophore at the reducing end of all ML A congeners. DNBA-MPL is then analyzed by reversed-phase HPLC on a Wat ers NovaPak C-18, 4 mu m particle size, 300 mmX3.9 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 dihy drogenphosphate (TBAP), in acetonitrile-water (95:5, v/v), 5 mM TBAP, over 45 min. A synthetic compound, corresponding to a hexaacyl MLA con gener, is used for determination of the detector response factor, allo wing the MLA content of MPL (i.e., purity) to be determined. Overall, this method provides better separation, higher sensitivity, and is fas ter and safer than previous methods used for the analysis of MPL.