Comprehensive pseudotargeted metabolomics analysis based on two-phase liquid extraction-UHPLC-MS/MS for the...

Pseudotargeted analysis combines the advantages of untargeted and targeted metabolomics methods. This study proposed a comprehensive pseudotargeted metabolomics method based on two-phase liquid extraction using ultra-high-performance liquid chromatography-tandem mass spectrometry. Two-phase liquid extraction, composed of both aqueous and organic phases, extracted a wide range of metabolites from polar to nonpolar in plasma samples. Besides, the two phases were combined and detected in a single injection to save analytical time. A total of 486 potential metabolites were detected by the developed approach. Compared with the conventional methanol-based protein precipitation method, the two-phase liquid extraction method significantly increased the metabolite coverage by 20.29%. Besides, the proposed pseudotargeted metabolomics method exhibited higher sensitivity and better repeatability than the untargeted method. Finally, we applied the established pseudotargeted method to the metabolomics study of depressive rats and screened 53 differential variables. Sixteen determined differential metabolites were mainly in four metabolic pathways including glycerophospholipid, arachidonic acid, sphingolipid metabolisms, pentose and glucuronate interconversions. The results indicated that the pseudotargeted method based on two-phase liquid extraction broadened the metabolite coverage with good sensitivity and repeatability, exhibiting significant potential for discovering differential metabolites in metabolomics studies.