Improved method for the quantification of lysophospholipids including enol ether species by liquid chromatography-tandem mass spectrometry

James G Bollinger,Hiromi Ii,Martin Sadilek,Michael H Gelb

doi:10.1194/jlr.d000885

Abstract

LC/ESI-MS/MS has been previously demonstrated to be a powerful method to detect and quantify molecular species of glycerophospholipids including lysophospholipids. In this study, we provide an improved pre-mass spectrometry lipid extraction procedure that avoids the acid-catalyzed decomposition of plasmenyl phospholipids that is problematic with previously reported methods. We show that the use of lysophospholipid internal standards with perdeuterated fatty acyl chains avoids isobar problems associated with the use of internal standards containing odd carbon number fatty acyl chains. We also show that LC prior to MS is required to avoid numerous problems associated with isobars and with MS in-source decomposition of lysophosphatidylserine. The reported method of using normal phase chromatography/ESI-MS is used to quantify lysophospholipids in serum and to quantify lysophospholipids produced in mammalian cells by human group X secreted phospholipase A(2). The latter shows that group X phospholipase A(2) added exogenously to cells generates a different set of lysophospholipids compared with enzyme produced endogenously in cells, which supports earlier studies showing that this phospholipase A(2) can act on cell membranes prior to externalization from cells.

Highlights

LC/ESI-MS/MS has been previously demonstrated to be a powerful method to detect and quantify molecular species of glycerophospholipids including lysophospholipids
We show that lysophosphatidylserine species break down in the mass spectrometer source to generate lysophosphatidic acid species, which underscores the need for LC separation of these species prior to mass spectrometry
The new method results in high recovery of the major types of lysophospholipid species including lysophosphatidic acid (LPA) and LPI, which are generally more difficult to isolate than the more hydrophobic species

Summary

Introduction

LC/ESI-MS/MS has been previously demonstrated to be a powerful method to detect and quantify molecular species of glycerophospholipids including lysophospholipids. The problem with acidification during extraction is that plasmalogens are rapidly degraded at low pH due to spontaneous cleavage of their sn-1-enol ether chain to give the corresponding sn-1 hydroxyl group [4]. This leads to formation of sn-2-acyl-lysophosphatidylcholine and sn-2-acyl-lysophosphidylethanolamine and possible other species. These sn-2 lysophospholipids behave to the corresponding sn-1-acyl lysophospholipids when examined by ESI-MS/MS because they are isobaric and often give rise to the same major fragment ions This acid-catalyzed breakdown leads to an underestimation of the amount of enol ether-lysophospholipid species and an overestimation of the amount of lysophospholipid species.

Methods

Results

Conclusion