Comparison of TLC, HPLC, and direct-infusion ESI-MS methods for the identification and quantification of diacylglycerol molecular species

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.