Determination of phospholipids in milk samples by means of hydrophilic interaction liquid chromatography coupled to evaporative light scattering and mass spectrometry detection

The combined use of the state-of-the-art hybrid mass spectrometers together with high efficient liquid chromatography could surely be a useful tool for such a challenging task, as phospholipids (PLs) analysis. In this research, we used hydrophilic interaction liquid chromatography (150 mm×2.1 mm I.D., 2.7 μm d.p. partially porous column) to achieve the separation of major PLs classes in cow's and donkey's milk samples. Solid-phase extraction (SPE) was performed in order to pre-concentrate minor PLs from non polar lipids (triacylglycerols) and the recovery for the extraction method was assayed on a milk sample, fortified with 5 μg/mL of SM pure standard, and analyzed in triplicate. A value of 89.99% was calculated, with a coefficient of variation (CV%) of 1.93. A 70-min long stepwise gradient of water/acetonitrile afforded baseline separation of PLs classes, at 50 μL/min flow rate. Accurate detection by an ion trap-time of flight (IT-TOF) mass spectrometer (in both positive and negative ionization mode) allowed to fully characterize the distinctive phospholipid profile and fatty acid composition of cow's and donkey's milk, the latter being analyzed for the first time. Evaporative light scattering detection was further employed to attain the quantitative evaluation of major PLs classes identified, by the external calibration method using reference material solutions in the 5-200 μg/mL concentration range. Major difference between the two analyzed samples consisted in the total PLs amount, which in cow's milk was determined as over 20-fold higher than the donkey's.