You-Rim Lee^a, Seungyeon Lee^a, Sohyen Kwon^a, Jiyeong Lee^a,b, Hee-Gyoo Kang^a,c

^aDepartment of Senior Healthcare, Graduate School, Eulji University, Uijeongbu, 11759, Republic of Korea
^bDepartment of Biomedical Laboratory Science, College of Health Science, Eulji University, Uijeongbu, 11759, Republic of Korea
^cDepartment of Biomedical Laboratory Science, College of Health Sciences, Eulji University, Seongnam, 13135, Republic of Korea

Corresponding authors: Jiyeong Lee, Hee-Gyoo Kang

Establishing a correlation between environmental variables and chemical change can significantly improve the quality of research in multiple fields. Among various environmental variables, temperature and humidity are closely related to the rate of chemical reactions. This study aimed to confirm changes in metabolite markers that were previously discovered in other temperature and humidity environment conditions and to confirm the possibility that they could act as markers. After blood collection from the subjects and bloodstain preparation, the quantitative values of the bloodstain metabolites were confirmed (when the age of the bloodstain was within a month) under eight environmental conditions (4 °C/30%, 4 °C/60%, 25 °C/30%, 25 °C/60%, 25 °C/90%, 40 °C/30%, 40 °C/60%, and 40 °C/90%). Age-of-bloodstain estimation models were constructed to confirm the applicability of bloodstain metabolites as markers for bloodstain age in various environments. The average concentration of metabolite markers exhibited a decreasing trend with the age of the bloodstain, which transformed into an increasing trend from day 7 onwards. In terms of temperature and humidity, 25 °C and 90%, respectively, showed the most dissimilar metabolite change pattern compared to other conditions. The age-of-bloodstain estimation models developed here have an R-square value of up to 0.92 for each condition and an R-square value of 0.71 when all environmental conditions were combined. The findings herein highlight the immense potential of blood metabolites for field application, confirming the possibility of predicting metabolite changes from the rates of their chemical reactions and validating the importance of metabolites as age-of-bloodstain markers under various environmental conditions.