Twelve-month tracking of metabolomic changes in ex vivo bloodstains: the analytical validity window of bloodstain metabolites.

A comprehensive understanding of chemical changes in blood and bloodstains exposed to ex vivo environments remain limited. In this study, we evaluated the analytical validity window of metabolite analysis in ex vivo bloodstains by tracking chemical changes over 12 months. Bloodstains from a discovery set of 14 participants were analyzed at monthly intervals from Month 0 to Month 12 using LC-MS/MS nontargeted analysis. A total of 2,210 molecular features (MFs) were filtered, of which 270 MFs persisted continuously throughout the 12-month period. Principal component analysis, hierarchical clustering, and correlation analysis showed that the most pronounced changes to MF-levels occurred within the first two months, with the majority of baseline-derived MFs disappearing between Month 0 and Month 2. Five identified metabolites (choline, 5'-AMP, pyroglutamic acid, aspartic acid, and ornithine) were then quantified by targeted analysis in an independent validation set of 40 participants, with concentrations expressed as median [IQR]. Choline increased significantly from Month 0 and remained elevated throughout the 12 months, supporting its potential utility as a candidate long-term marker for distinguishing fresh blood from aged bloodstains. 5'-AMP changed significantly only within the first four months; the remaining three metabolites showed only minor fluctuations with no significant pairwise differences. These findings indicate that MF-level changes primarily reflect loss of baseline-derived MFs and were most pronounced within the initial two months. Individual targeted metabolites follow distinct trajectories and more gradual, irregular changes continue thereafter. This study provides foundational insights for the application of metabolomics to forensic bloodstain analysis.