Internal standard metabolites for obtaining absolute quantitative information on the components of bloodstains by standardization of samples

•Internal standard metabolites of bloodstains were identified.•Five molecular features having average CVs of≤5% were selected as candidates.•PLS-DA and PCA showed that the effect on the candidates was very low over time.•Stigmasterol exhibited the most stable pattern.•l-methionine remained stable until day 14 and after day 21. Analysis of the components of bloodstains found at crime scenes can provide important information for solving the crime. However, components of blood and bloodstains vary with volume and various other unpredictable factors. Therefore, it is necessary to specify the volume of the initial liquid blood droplet and standardize the analysis. In this study, internal standard metabolites that remained constant in a certain amount of bloodstain, long after deposition of the stain, were identified. Liquid chromatography–electrospray ionization–tandem mass spectrometry of the metabolites extracted from the bloodstain samples at various time points (0, 7, 14, 21, and 28 days) was performed. The coefficient of variation (CV) of the obtained molecular features was calculated for each criterion: time point, subject, and all data (time and subject, triplicate of each). Five molecular features with average CVs of less than or equal to 5% were selected as candidates. Partial least squares discriminant analysis and principal component analysis showed that the effect on the candidates was very low over time. The fold-change value of abundances was confirmed according to time. Stigmasterol exhibited the most stable pattern; l-methionine remained stable until day 14 and after day 21. This study was the first attempt to identify internal standard metabolites that were maintained at a constant level in a bloodstain for a sufficiently long time. Analysis of internal standard metabolites in bloodstains will facilitate determination of the initial blood volume from which the bloodstain was made. Moreover, this method will provide an approach for standardization of bloodstains to obtain absolute quantitative information of bloodstain components at crime scenes.