Analysis of effector cell-derived lyso platelet activating factor by electron capture negative ion mass spectrometry

Quantification of 1-O-alkyl-2-lyso-sn-3-glycero-phosphocholine (lysoPAF) and determination of the different molecular species released by cells has been hampered by the molecules's lack of intrinsic bioactivity, unavailability of a suitable internal standard, and reliance on derivatives requiring electron impact techniques. We have synthesized trideuterated internal standards (labeled on the terminal carbon of the alkyl chain) for both C16:0 and C18:0 lysoPAF. Using these standards, we isolated and quantified lysoPAF released from A23187-stimulated human neutrophils and rat alveolar macrophages. Extracted lysoPAF was purified by solid-phase extraction and thin-layer chromatography. The polar phosphorylcholine group was removed with 29 M HF or phospholipase C. The two free hydroxyl groups were derivatized with pentafluorobenzoyl chloride. The resultant bis-pentafluorobenzoyl derivative, analyzed by gas chromatography/electron capture negative ion mass spectrometry, underwent substantial fragmentation. Lowering of the ion source temperature resulted in a dramatic increase in signal-to-noise ratio, with the vast majority of the ion current carried in the molecular anion. Stimulated neutrophils released 16.3 and 10.2 ng/10(6) cells of C16:0 lysoPAF and C18:0 lysoPAF, respectively. Rat macrophages synthesized 15.9 ng/10(6) cells of C16:0 lysoPAF, but C18:0 lysoPAF was variably detected at low levels. We conclude that use of the bispentafluorobenzoyl ester derivative of lysoPAF allows facile quantification of this autacoid metabolite in biological matrices.