Quantification of glycerophospholipids and sphingomyelin in human milk and infant formula by high performance liquid chromatography coupled with mass spectrometer detector

Phospholipids and sphingomyelin have a central role in infant nutrition, phospholipid acting as a nutrient carrier of long chain polyunsaturated fatty acids and sphingomyelin having an important role in cognitive function. However, analytical methods to precisely characterize and quantify these compounds in maternal milk are needed. Phospholipids and sphingomyelin were extracted using chloroform and methanol and separated on Polaris 3 Si column 250 × 2.0 mm from Varian and analyzed by high performance liquid chromatography (HPLC) coupled with mass spectrometer detector (MS). The analytical method was validated and repeatability, intermediate reproducibility, and recovery values were calculated. The relative standard deviation of repeatability (CV(r)) and intermediate reproducibility (CV(iR)) values ranged between 2.3 and 7.2% and 9.5 and 17.8%, respectively and the recovery values between 96 and 109%. Finally, the validated method was tested on human milk samples and on infant formula which were analysed also by HPLC coupled with evaporative light scattering detector (ELSD). In human milk, sphingomyelin (9.28 mg 100 mL−1) was the most abundant compound, followed by phosphatidylcholine (5.39 mg 100 mL−1), phosphatidylethanolamine (2.85 mg 100 mL−1) and phosphatidylinositol (1.82 mg 100 mL−1).