Expanding Lipidomics Coverage: Effective Ultra Performance Liquid Chromatography-high Resolution Mass Spectrometer Methods for Detection and Quantitation of Cardiolipin, Phosphatidylglycerol, and Lysyl-phosphatidylglycerol

Introduction: Lipidomics can reveal global alterations in a broad class of molecules whose functions are innately linked to physiology. Monitoring changes in the phospholipid composition of biological membranes in response to stressors can aid the development of targeted therapies. However, exact quantitation of cardiolipins is not a straightforward task due to low ionization efficiencies and poor chromatographic separation of these compounds.

Objective: The aim of this study was to develop a quantitative method for the detection of cardiolipins and other phospholipids using both a targeted and untargeted analyses with a Q-Exactive.

Methods: HILIC chromatography and high-resolution mass spectrometry with parallel reaction monitoring was used to measure changes in lipid concentration. Internal standards and fragmentation techniques allowed for the reliable quantitation of lipid species including: lysyl-phosphatidylglycerol, phosphatidylglycerol, and cardiolipin.

Results: The untargeted analysis was capable to detecting 6 different phospholipid classes as well as free fatty acids. The targeted analysis quantified up to 23 cardiolipins, 10 phosphatidylglycerols and 10 lysyl-phosphatidylglycerols with detection limits as low as 50 nM. Biological validation with Enterococcus faecalis demonstrates sensitivity in monitoring the incorporation of exogenously supplied free fats into membrane phospholipids. When supplemented with oleic acid, the amount of free oleic acid in the membrane was 100 times greater and the concentration of polyunsaturated cardiolipin increased to over 3.5 µM compared to controls.

Conclusions: This lipidomics method is capable of targeted quantitation for challenging biologically relevant cardiolipins as well as broad, untargeted lipid profiling.