Phosphorus-31 nuclear magnetic resonance (³¹P NMR) for quantitative measurements of phospholipids derived from natural products: effect of analysis conditions

Abstract

In the present study the running conditions (sample pH, scan number, relaxation delay and NMR solvent) for the quantification of phospholipids by ³¹P NMR using commercial pure standards were optimised. A mixture of pure standards was run at different pH (7.3, 7.8, 9.5 and 12), scan numbers (64, 128, 192 and 512), relaxation delay times (1s, 2s, 3.5s and 10s) and NMR solvents (D₂O/EDTA/sodium cholate or CDCl₃/CD₃OD/EDTA) to achieve this goal. Best ³¹P NMR conditions were found to be pH 7.3, a scan number of 192 and a relaxation delay of 3.5s, which were used for the quantification of natural samples (Krill oil, soybean lecithin and egg phospholipid) in D₂O/EDTA/sodium cholate or CDCl₃/CD₃OD/EDTA. Phospholipids and lysophospholipids were highly resolved and detected in D₂O/EDTA/sodium cholate compared to CDCl₃/CD₃OD/EDTA. A number of phospholipids (lysophoshatidylglycerol-2, LPG-2; lysophosphatidylethanolamine, LPE; N-acyl phosphatidylethanolamine, APE) were not detected in CDCl₃/CD3₃OD/EDTA, but they were detected in D₂O/EDTA/sodium cholate. Therefore, it is concluded that D₂O/EDTA/sodium cholate is a better detection solvent for ³¹P NMR quantification of phospholipids extracted from natural sources.