Platelet‐Activating Factor Quantification Using Reversed Phase Liquid Chromatography and Selected Reaction Monitoring in Negative Ion Mode

Abstract

Platelet-activating factor (PAF) is a potent biologically active phospholipid that mediates human physiological and pathophysiologic responses. PAF levels increase transiently and are typically assessed by techniques with limitations related to expense, sensitivity, pre-analysis derivatization and interference with isobaric molecules. This study elucidates a facile, accurate liquid chromatography-mass spectrometry analytical method for PAF. In negative ion mode using electrospray ionization, collisionally-activated dissociation analysis showed a unique product ion for acetate adducts of PAF molecular species representing the loss of methyl acetate from the polar head group and loss of a part of the acetate group from the sn-2 position. This product ion was exploited for selected reaction monitoring of PAF molecular species following separation by reversed-phase liquid chromatography. Standard calibration responses were determined, and this method was able to detect as low as 100 fmol of PAF. Finally, PAF molecular species were quantified in human neutrophils and monocytes.

Forty Years Since the Structural Elucidation of Platelet-Activating Factor (PAF): Historical, Current, and Future Research Perspectives

“…For instance, the mass spectrometry analysis of plasma oxPL in diabetes patients [265], the level of plasma oxPL in Alzheimer’s disease patients treated with a carotenoid supplement [266], or PAFLL in coronary artery disease patients at risk of cognitive decline due to depression [267]. However, one of the current major challenges of oxPL, PAF, and PAFLL research is the development of simplified mass spectrometric procedures for high-throughput and affordable analysis [268,269]. Certainly, there are several limitations to PAF quantification including sensitivity, pre-analysis derivatization, interference with isobaric molecules, and the fact that it is expensive to conduct [269].…”

“…However, one of the current major challenges of oxPL, PAF, and PAFLL research is the development of simplified mass spectrometric procedures for high-throughput and affordable analysis [268,269]. Certainly, there are several limitations to PAF quantification including sensitivity, pre-analysis derivatization, interference with isobaric molecules, and the fact that it is expensive to conduct [269]. Therefore, further research is required to develop reliable, inexpensive, and reproducible methods to further advance this research field.…”