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.