Qualitative and Quantitative Study of Glycosphingolipids in Human Milk and Bovine Milk Using High Performance Liquid Chromatography–Data-Dependent Acquisition–Mass Spectrometry

Abstract

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography–tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9–17.4 µg Crb/mL and 1.3–3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8–12.0 and 14.3–16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

Keywords

• high performance liquid chromatography–tandem mass spectrometry
• glucosylceramide
• galactosylceramide
• lactosylceramide
• bovine milk
• human milk