Analysis of platelet activating factor in human saliva by gas chromatography/mass spectrometry

Platelet activating factor (PAF) bioactivity has been demonstrated in saliva from normal volunteers. We sought structural confirmation and evidence of heterogeneity in the 1-O-alkyl chain of the acetyl glyceryl ether phosphoryl choline (AGEPC) extracted from saliva by employing stable isotope dilution techniques in conjunction with gas chromatography/mass spectrometry. The method described involves removal of the polar phosphocholine moiety, accounts for acetyl group migration, and allows for acylation of the resultant free hydroxyl with pentafluorobenzoyl chloride. Thin-layer chromatography (TLC) purification is undertaken after phospholipase C cleavage and again after pentafluorobenzoyl chloride derivatization. The majority of the ion current is represented in the molecular anion, allowing measurement of 50 pg in biological fluid with a signal-to-noise ratio of greater than or equal to 9. In one subject with markedly increased salivary PAF levels, we found evidence for molecular heterogeneity of AGEPC with production of not only C16:0 but also C18:0 and C18:1 in the alkyl chain. This technique, by using TLC in lieu of high-performance liquid chromatography, avoids potentially confounding trace contamination effects, produces spectra with few interfering signals and increases sample throughput.