Derivatization-Assisted Acoustic Ejection Mass Spectrometry for High-Throughput FAHFA Prescreening

Fatty acid esters of hydroxy fatty acids (FAHFAs) are bioactive lipids with significant structural diversity and potential health benefits, playing crucial roles in metabolic regulation, inflammation, and insulin sensitivity. However, the low abundance of FAHFAs in biological samples (0.1% to 0.001% of free fatty acids) and the wide range of molecular polarities (ClogP values from 0.26 to 18.24) across short-chain to long-chain FAHFAs pose substantial challenges for high-throughput screening. Here, we developed a novel high-throughput prescreening strategy, termed DAEMS (derivatization-assisted acoustic ejection mass spectrometry), which integrates chemical derivatization with acoustic ejection mass spectrometry (AEMS). By leveraging the ultrafast analysis speed of AEMS (1–3 samples per second) and the sensitivity enhancement of N,N-dimethylethylenediamine (DMED) derivatization, DAEMS enables the screening of over 2800 potential FAHFA species in less than 2 h─significantly faster than the conventional LC-MRM MS-based screening approaches. The DAEMS strategy achieved 83% coverage and 79% accuracy in preliminary screening compared to the LC-MRM MS. Furthermore, we also revealed novel oxidized FAHFA species in edible fungi for the first time, suggesting potential biosynthesis involving oxylipins or oxidative modifications. This study demonstrates DAEMS as a promising tool for rapid FAHFA prescreening, and the discovery of oxidized FAHFAs provides new insights into FAHFA diversity and metabolism.