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

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Abstract
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/106 cells of C16:0 lysoPAF and C18:0 lysoPAF, respectively. Rat macrophages synthesized 15.9 ng/106 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.