Quantitative characterization of phospholipids in milk fat via 31P NMR using a monophasic solvent mixture

The phospholipids (PL) occurring in both ewe and cow milk fat globule membrane were identified and quantitatively determined using 31P NMR spectroscopy with inverse gated decoupled sequences, which allowed a rigorous quantitative analysis. A strict relation between amount and distribution of PL and type of feeding was found. The method was calibrated over a mixture of PL standards. A recently introduced solvent constituted by a monophasic dimethylformamide/triethylamine/guanidinium hydrochloride solvent mixture was used. Compared to the traditional chloroform/methanol/water-EDTA solvent, the new solvent mixture shows very similar accuracy and precision from a quantitative point of view. The monophasic solvent overcomes the partition problems related to a biphasic system, and slightly enlarges the range of 31P NMR chemical shifts, thus improving the resolution. In addition, the new solvent apparently displays a lower chemical shift dependence on the various PL concentrations. The limit of the method is mainly determined by the formation of adducts between triethylamine and some PL, namely, PE, monomethylphosphatidylethanolamine, phosphatidylethanolamine plasmalogens, and some lyso-PL. However, the new 31P NMR signals arising from these adducts could be easily quantified in the determination of PE.