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.