Quantitation and characterization of phospholipids in pharmaceutical formulations by liquid chromatography-mass spectrometry

A simple and fast method for phospholipid analysis was developed using high-performance liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface. Separation of the phospholipid molecular species was achieved using a linear gradient of a mixture of chloroform-10 mM ammonium acetate-methanol (30:5:65) on a silica column. Optimization of the mass spectrometer conditions has allowed the method to separate and detect the phospholipids mainly as protonated molecular species. In comparison to existing LC-MS methods, improvement in the total analysis time and sensitivity were achieved. Separation of all major phospholipid molecular classes was achieved in less than 6 min. Marked improvement was observed in the linearity of the response of the phospholipids studied providing a linear response over three orders of magnitude. Data supporting the validation of this method for the characterization of major phospholipids molecular species are also presented.