An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

John S Owen,Robert L Wykle,Michael P Samuel,Michael J Thomas

doi:10.1194/jlr.d400029-jlr200

Abstract

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.

Highlights

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS)
Owen et al Improved assay for platelet-activating factor 375 vantage of the fact that PAF, as a choline-containing phospholipid, yields abundant positive ions that efficiently fragment in collision-induced dissociation to give the characteristic product ion at m/z 184 (e.g., m/z 524 → 184 for 16:0 PAF)
When d3-16:0 PAF and unlabeled 16:0 PAF standards were coinjected on normal-phase high-performance liquid chromatography-mass spectrometry (LC-MS)/ MS with polymorphonuclear neutrophil (PMN) lipid extract, the m/z 524 → 184 channel gave two peaks (Fig. 1A)

Summary

Introduction

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoylformyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. The requirement for derivatization makes these assays time-consuming, expensive, and susceptible to losses and artifacts Such drawbacks have prompted the development of PAF assays using HPLC coupled to MS or tandem MS (LC-MS or LC-MS/MS, respectively) [29,30,31,32,33,34].

Methods

Results

Conclusion