A convenient method for the relative and absolute quantification of lipid components in liposomes by 1 H- and 31 P NMR-spectroscopy

Liposomes are promising delivery systems for pharmaceutical applications and have been used in medicine in the recent past. Preparation of liposomes requires reliable characterization and quantification of the phospholipid components for which the traditional cumbersome molybdate method is used frequently. The objective was to improve relative and absolute quantification of lipid components from liposomes. A reliable method for quantification of lipid composition in liposome formulations in the 1-10 μmol range with H- and P NMR spectroscopy at 600 MHz has been developed. The method is based on three crystalline small-molecule standards (Ph PO , (Tol) PO , and Ph PO) in CDCl . Excellent calibration linearity and chemical stability of the standards was observed. The method was tested in blind fashion on liposomes containing POPC, PEG-ceramide and a pH-sensitive trans-aminocyclohexanol-based amphiphile (TACH). Relative quantification (percentage of components) as well as determination of absolute lipid amount was possible with excellent reproducibility with an average error of 5%. Quantification (triplicate) was accomplished in 15 min based on H NMR and in 1 h based on P NMR. Very little change in mixture composition was observed over multiple preparative steps. Liposome preparations containing POPC, POPE, DOPC, DPPC, TACH, and PEG-ceramide can be reliably characterized and quantified by H NMR and P NMR spectroscopy at 600 MHz in the μmol range.