Abstract
A liquid chromatography-mass spectrometry (LC-MS) method was used for separation of lipid classes as well as both qualitative and semiquantitative detection of individual lipids in biological samples. Data were acquired using high-resolution full-scan MS and high-energy collisional dissociation (HCD) all ion fragmentation. The method was evaluated for efficient separation and detection in both positive and negative ionization mode using standards spanning six lipid classes. Platform linearity and robustness, related to the mitochondrial lipid cardiolipin (CL), were assessed using extracted ion chromatograms with mass tolerance windows of 5 ppm or less from full scan exact mass measurements. The platform CL limit of detection was determined to be 5 pmol (0.9 μM) on the column, with mass accuracy <1.5 ppm, retention time coefficients of variation (CV) < 0.5%, and area CV < 13%. This mass accuracy was critical to the identification of unknown CL species in mitochondria samples, through the elimination of false positives. In addition to detection and relative quantitation of CL species in mitochondria, CL structures were characterized through the use of alternating HCD scans at different energies to produce diagnostic fragmentations on all ions in the analysis. The developed lipid profiling method was applied to mitochondrial samples from an animal study related to the linkages between diet, mitochondrial function, and disease. The analysis identified 28 unique CL species and two monolysocardiolipin species that are often associated with mitochondrial stress and dysfunction.