Affinity ultrafiltration and UHPLC-HRMS based screening of tyrosinase inhibitors from wolfberry

Tyrosinase, the pivotal rate-limiting enzyme in melanogenesis, is regarded as a primary target for managing skin hyperpigmentation. Despite the potent inhibitory activity of phenolic amides from wolfberry (Lycium barbarum L.), their high-fidelity screening is frequently hindered by intricate structural isomerism and mass spectrometric interference. To address these analytical bottlenecks, a multidimensional platform was established by coupling ligand fishing (LF) with offline two-dimensional supercritical fluid chromatography cross ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry technology (2D-SFC × UHPLC-HRMS). Leveraging superior system orthogonality, complex isomers intractable for conventional reversed-phase methods were successfully resolved. Characterization reliability was further fortified by building block-based molecular networking (BBMN) and the removal of in-source fragments (RISF), enabling the unambiguous identification of 198 compounds (131 spermidine, 40 spermine, and 27 tyramine). Ultimately, 11 tyrosinase inhibitors were discovered. This study provides a high-throughput paradigm for the rapid identification of enzyme inhibitors from complex functional foods.