Expanded coverage of N-acyl amino acids using one-pot micro synthesis-chemical derivatization-LC-MS strategy: A key...

N-acyl amino acids (N-acyl AAs) are potential bioactive compounds in fermented soybean (FS), but their accurate annotation is challenging due to their structural diversity, the existence of various isomers and wide polarity differences. Herein, we established a novel One-pot Micro Synthesis-Chemical Derivatization (OMSCD)-LC-MS analytical approach for comprehensive profiling of N-acyl AAs. First, the rapid synthesis of 142 N-acyl AA standards combined with in situ DIAAA derivatization in a one-pot manner significantly enhanced ionization and separation efficiency, particularly enabling excellent discrimination of isomers. Next, using this method, 222 N-acyl AAs were rapidly identified in FS, of which 200 were reported for the first time. Among them, N-oleoyl AAs, present at high contents in FS, demonstrated significant anti-liver cancer activity in a cell model, with MAPK1 acting as a promising candidate target. Overall, the OMSCD-LC-MS strategy facilitates the N-acyl AAs profiling, offering new preliminary insights into the functional components of FS.