LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures

Catherine E. Costello,Francine K. Welty,Haya Herscovitz,Ulf Sommer

doi:10.1194/jlr.m500506-jlr200

Catherine E. Costello, Francine K. Welty

Open Access

https://doi.org/10.1194/jlr.m500506-jlr200

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Abstract

A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. After an optional initial fractionation on Silica 60, normal-phase HPLC-MS on a YMC PVA-Sil column is used first for class separation, followed by reversed-phase LC-MS or LC-tandem mass spectrometry using an Atlantis dC18 capillary column, and/or nanospray MS, to fully characterize the individual lipids. The methodology is applied here for the analysis of human apolipoprotein B-associated lipids. This approach allows for the determination of even low percentages of lipids of each molecular species and showed clear differences between lipids associated with apolipoprotein B-100-LDL isolated from a normal individual and those associated with a truncated version, apolipoprotein B-67-containing lipoproteins, isolated from a homozygote patient with familial hypobetalipoproteinemia. The methods described should be easily adaptable to most modern MS instrumentation.

Highlights

A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated
We compared the lipids associated with normal apolipoprotein B-100 (B100)-containing LDL with those associated with mutant apolipoprotein B-67 (B67)-containing lipoproteins, which are found in certain cases of familial hypobetalipoproteinemia [7]
For the primary separation step, we chose the route of separation by compound classes by normal-phase LC-MS

Summary

Introduction

A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. The methodology is applied here for the analysis of human apolipoprotein Bassociated lipids This approach allows for the determination of even low percentages of lipids of each molecular species and showed clear differences between lipids associated with apolipoprotein B-100-LDL isolated from a normal individual and those associated with a truncated version, apolipoprotein B-67-containing lipoproteins, isolated from a homozygote patient with familial hypobetalipoproteinemia. We demonstrate here a simple, robust, and reproducible methodology for lipid analysis, which has been achieved by adapting to LC-MS several separation systems described in the literature for the thin-layer and liquid chromatography of lipids. After an optional initial cleanup and prefractionation on Silica 60, we use normal-phase HPLC-MS for class separation first, an optional reversed-phase LC-MS or LC-tandem mass spectrometry (MS/MS) system for further analysis. We compared the lipids associated with normal apolipoprotein B-100 (B100)-containing LDL with those associated with mutant apolipoprotein B-67 (B67)-containing lipoproteins, which are found in certain cases of familial hypobetalipoproteinemia [7]

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Conclusion