Liquid chromatography-electrospray ionization mass spectrometry (LC-MS) is an effective and popular technique used in lipid metabolomic studies. Although many LC-MS methods enabling the determination of sphingolipid molecular species have been reported, they do not cover a broad range of sphingolipid metabolites with expanding glycerophospholipids (GPLs) and diacylglycerol (DAG). In this study, we developed an approach for the comprehensive analysis of sphingolipids, GPLs and DAG molecular species in a biological sample, without alkaline hydrolysis. After validating the reliability of this approach, we analyzed tissue lipids of sphingomyelin synthase 2-knockout mice and found that changes in sphingolipid metabolism in the liver affect the level of docosahexaenoic acid-containing GPLs. Our method analyzes GPLs and DAG, as well as sphingolipids within biological samples and, thus, will facilitate more comprehensive studies of sphingolipid metabolism in pathology and diagnostics.