Beyond the first cup: Untargeted metabolomics reveals metabolite depletion and persistence across multiple coffee...

Coffee brewing is a dynamic solid-liquid extraction process, yet the fate of metabolites beyond the conventional single brewing cycle remains unclear. Here, untargeted UPLC-QTOF-MS/MS-based metabolomics was applied to systematically profile metabolite depletion and persistence across multiple brewing cycles at three roasting levels. Brewing cycle was a major source of metabolite variation across roasting levels, with the first cycle differing most from subsequent cycles. A total of 1030 negative and 360 positive features differed significantly across brewing cycles. The most significantly depleted metabolites were confirmed as phloretic acid, shikimic acid, quinic acid, and caffeine using authentic standards. Notably, the second cycle was dominated by caffeoylquinic acids and their derivatives, caffeine, cysteinesulfenic acid, and levoglucosan. This study provides chemical evidence consistent with conventional single-cycle brewing while highlighting post-brewing coffee residues as potential sources for targeted recovery and waste valorization.