High resolution 31P NMR of extracted phospholipids

The common phospholipids from biological sources were quantitated using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy in conjunction with an analytical reagent composed of two parts: 1) 2 ml of reagent chloroform in which was dissolved 0.01-100 mg of crude tissue lipid extracted from tissue sources using chloroform-methanol 2:1, the extract having been washed with 0.2 vol. of 0.1 M KCl; 2) 1 ml of an aqueous methanol reagent composed of one part 0.2 M (ethylenedinitrilo)-tetraacetic acid in D2O titrated to pH 6 with CsOH and four parts of reagent methanol. In a magnetic field of 11.75 Tesla, the extracted phospholipids yield narrow signals (1.8-3.2 Hz at half-height), corresponding to each generic species, e.g., phosphatidylcholines, phosphatidylethanolamines, etc., permitting resolution among the various phospholipid families and their lyso and plasmalogen derivatives. The reagent permits assays of high precision and accuracy using a modest amount of NMR spectrometer time (ca. 15 min/assay). The procedures described, which are compared to high-performance liquid chromatography, are convenient for the routine analysis of phospholipids from biological sources.