A novel method for the analysis of platelet-activating factor: direct derivatization of glycerophospholipids

A novel, facile, and sensitive method for the quantitative and complete structure-proof analysis of platelet-activating factor (PAF) and other glycerophospholipids is described. 1-O-Alkyl/acyl-2-acyl-3-glycerophospholipids were treated with heptafluorobutyric anhydride in a one-step reaction to yield 1-O-alkyl/acyl-2-acyl-3-heptafluorobutyroyl-sn-glycerols as gas-liquid chromatography (GLC)-compatible derivatives. Furthermore, the components of the polar head group were also analyzed from the aqueous extract of the same reaction mixture as t-butyldimethylsilyl derivatives. Thus, this new method eliminates the need for phospholipase C treatment and subsequent purification procedures. Moreover, the direct derivatization of PAF homologs and analogs with hepatofluorobutyric anhydride does not result in positional isomerization of the product, providing increased specificity for gas-liquid chromatography-mass spectrometric (MS) analysis. It has also been shown that the heptafluorobutyroyl (HFB) derivative can easily be converted to the respective t-butyldimethylsilyl analog in a one-step reaction using t-butyldimethylsilyl chloride/imidazole reagent. Analogous to the formation of heptafluorobutyroyl derivatives, PAF also was reacted with pentafluorobenzoyl chloride to generate the pentafluorobenzoyl derivative. Therefore, this method has wide applicability for the formation of GLC-compatible derivatives of various glycerophospholipids. Our successful HFB derivatization and GLC-MS detection of subnanogram quantities of PAF indicate that this analytical procedure will greatly facilitate complete and quantitative identification of each of the molecular species of biologically derived PAF.