Abstract
An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidified triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidified triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2)(2)(C18:1)(2) versus (C18:2)(3)(C18:0)(1). These compounds were then analyzed by HPLC-MS(3) to examine specific diacylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined.
Highlights
MATERIALS AND METHODS2-Propanol, triethylamine, and glacial acetic acid were purchased from Fisher Scientific (Cleveland, OH)
An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented
We demonstrated the feasibility of characterizing cardiolipin molecular species by reverse-phase HPLC-MS/MS using a quadrupole ion trap mass spectrometer and recursive mass spectrometry (MS, MS/MS, and MS3) [10,11,12]
Summary
2-Propanol, triethylamine, and glacial acetic acid were purchased from Fisher Scientific (Cleveland, OH). Acetonitrile was purchased from Jade Scientific (Canton, MI). HPLC grade water was prepared with a Synergy® Reagent-Grade water system (Millipore, Billerica, MA). 1,1’,2,2’-tetramyristoyl cardiolipin, and 1,1’,2,2’-tetraoleoyl cardiolipin were purchased from Avanti Polar Lipids (Alabaster, AL). Solutions were prepared in chloroform (7.3, 13.1, and 9.4 mM, respectively). Bovine heart cardiolipin was purchased from SigmaAldrich (St. Louis, MO) and a 10 mM solution (in chloroform) was prepared
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