Comprehensive approach to the quantitative analysis of mitochondrial phospholipids by HPLC–MS

A normal-phase HPLC–MS method was established to analyze mitochondrial phospholipids quantitatively as well as qualitatively. An efficient extraction procedure and chromatographic conditions were developed using twelve standardized phospholipids and lysophospholipids. The chromatographic conditions provided physical separation of phospholipids by class, and efficient ionization allowed detection of low abundance phospholipids such as phosphatidylglycerol and monolysocardiolipin. The chromatographic separation of each class of phospholipid permitted qualitative identification of molecular species without interference from other classes. This is advantageous for mitochondrial lipidomics because the composition of mitochondrial phospholipids varies depending on tissue source, pathological condition, and nutrition. Using the method, seven classes of phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, and monolysocardiolipin) were detected in rat heart and skeletal muscle mitochondria and all but phosphatidylserine were quantified. The concentration was calculated using standard curves with an internal standard generated for each class of phospholipid. The method was validated for intraday and interday variation and showed excellent reproducibility and accuracy. This new method, with each step documented, provides a powerful tool for accurate quantitation of phospholipids, a basic structural component of mitochondrial membranes.