Internal standard metabolites for estimating origin blood volume of bloodstains

The volume of blood leaked from blood vessels may change due to evaporation of water under the natural influence of the external environment. Bloodstains and dried blood spots (DBS), which describes blood dried in the external environment, are similar in their production and their metabolite quantification profiles. In both bloodstain metabolite analysis in the forensic science field and DBS metabolite analysis in the clinical field, it is important to determine the volume of the origin blood as this affects metabolite quantification results. Therefore, the purpose of this study is to discover the internal standard metabolites that have quantitatively proportional relationships with origin blood volume and maintain constant concentrations even as the age of the bloodstain increases. As a result, the concentrations of L-isoleucine and L-phenylalanine increased in proportion to the origin blood volume of the bloodstain. The differences in concentration of L-isoleucine were significant in all volume comparisons except in the comparison between 65 μL and 85 μL. The differences in concentration of L-phenylalanine were significant in all volume comparisons except between 65 μL and 45 μL and between 65 μL and 85 μL. In addition, it was confirmed that both metabolites tended to maintain constant concentrations without being affected by bloodstain age as the volume became smaller. These internal standard metabolites can be used for estimating the origin blood volume of bloodstains during metabolite analysis of bloodstains and DBS and could provide a volume criterion for standardization when comparing metabolite quantification between samples.