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An Improved Method for Separating Cardiolipin by HPLC
Author(s) -
BarcelóCoblijn Gwendolyn,
Murphy Eric J.
Publication year - 2008
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-008-3212-3
Subject(s) - chromatography , chemistry , cardiolipin , high performance liquid chromatography , thin layer chromatography , hexane , solvent , elution , phospholipid , biochemistry , membrane
Herein we report an improved method to separate cardiolipin (Ptd 2 Gro) 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 Ptd 2 Gro 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 Ptd 2 Gro was well resolved from other phospholipids. This method is useful to separate and quantify Ptd 2 Gro from small tissue samples thereby avoiding the variability associated with TLC methods.