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An Improved Method for Separating Cardiolipin by HPLC

  • Original Article
  • Published:
Lipids

Abstract

Herein we report an improved method to separate cardiolipin (Ptd2Gro) from tissue total lipid extracts using a biphasic solvent system combined with high performance liquid chromatography. This method uses a normal phase silica column and two mobile phases: mobile phase A that was n-hexane:2-propanol (3:2 by vol) and mobile phase B that was n-hexane:2-propanol:water (56.7:37.8:5.5 by vol). The initial solvent conditions were 95% A and 5% B, with a flow rate of 1.5 mL/min. The samples were from non-derivatized aliquots of liver, heart, or brain lipid extracts. The peak corresponding to Ptd2Gro appeared at 31 min, was well defined and did not overlap with neighboring peaks. The adjacent peak corresponded to ethanolamine glycerophospholipids and the remaining phospholipids were eluted in a single peak. The identity of the phospholipids separated by this method was verified by thin layer chromatography (TLC) and fatty acid analysis, which confirmed that the Ptd2Gro was well resolved from other phospholipids. This method is useful to separate and quantify Ptd2Gro from small tissue samples thereby avoiding the variability associated with TLC methods.

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Abbreviations

ARA:

Arachidonic acid

DHA:

Docosahexaenoic acid

EtnGpl:

Ethanolamine glycerophospholipids

FAME:

Fatty acid methyl esters

GLC:

Gas liquid chromatography

HPLC:

High performance liquid chromatography

TLC:

Thin layer chromatography

Ptd2Gro:

Cardiolipin

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Acknowledgments

This work was funded in part as a project to EJ Murphy on a COBRE grant from the NIH 1P20 RR117699.

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Author notes

  1. Gwendolyn Barceló-Coblijn

    Present address: Department of Biology, University of the Balearic Islands, Illes Balears, Palma de Mallorca, Spain

Authors and Affiliations

  1. Department of Pharmacology, Physiology, and Therapeutics, School of Medicine and Health Sciences, University of North Dakota, 501 N. Columbia Road, Room 3700, Grand Forks, ND, 58202-9037, USA

    Gwendolyn Barceló-Coblijn & Eric J. Murphy

  2. Department of Chemistry, University of North Dakota, Grand Forks, ND, 58202-9037, USA

    Eric J. Murphy

Authors
  1. Gwendolyn Barceló-Coblijn
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  2. Eric J. Murphy
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Correspondence to Eric J. Murphy.

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Barceló-Coblijn, G., Murphy, E.J. An Improved Method for Separating Cardiolipin by HPLC. Lipids 43, 971–976 (2008). https://doi.org/10.1007/s11745-008-3212-3

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  • DOI: https://doi.org/10.1007/s11745-008-3212-3

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