Discovery and validation of metabolite markers in bloodstains for bloodstain age estimation

The age estimation of bloodstains measures the time-dependent changes in the levels of bloodstain biomolecules. Although several studies have identified bloodstain metabolites as markers for estimating bloodstain age, none has consider gender and age-related metabolomic differences or long-time bloodstain age. Therefore, we aimed to identify metabolite markers for estimating the age of bloodstains at weekly intervals within 28 d and validate them through the MRM technique. Interestingly, adenosine 5′-monophosphate (5′-AMP), choline and pyroglutamic acid were selected as markers. We validated a total of 7 metabolites, including 5 previously reported metabolites, ergothioneine, hypoxanthine, L-isoleucine, L-tryptophan and pyroglutamic acid. Choline and hypoxanthine might be used to differentiate between day 0 and day 14 at weekly intervals, while L-isoleucine and L-tryptophan might help distinguish between 7 d before and 14 d after. Evaluation of the changes in the levels of metabolites according to gender and age, revealed that the average level of all 7 metabolites was higher in women on day 0. Moreover, the level of ergothioneine was significantly higher in the elderly than the youth under all time conditions. In this study, we confirmed the potential effectiveness of metabolites in bloodstains as forensic markers, and provided a new perspective on metabolomic approaches linked to forensic science.