Patrycja Mojsak ¹ Paulina Samczuk ^1,2* Paulina Klimaszewska ¹ Michał Burdukiewicz ^1,3 Jaroslaw Chilimoniuk ^1* Krystyna Grzesiak ⁴ Karolina Pietrowska ¹ Justyna Ciborowska ^5* Anna Niemcunowicz-Janica ^1* Adam Kretowski ^1* Michal Ciborowski ^1* Michal Szeremeta ^1*

• ¹ Medical University of Bialystok, Bialystok, Podlaskie Voivodeship, Poland
• ² Central Police Forensic Laboratory (CLKP), Warsaw, Masovian, Poland
• ³ Autonomous University of Barcelona, Barcelona, Catalonia, Spain
• ⁴ University of Wrocław, Wrocław, Silesian, Poland
• ⁵ Forensic Laboratory of the Voivodeship Police Headquarters in Bialystok, Białystok, Poland

Accurate post-mortem interval (PMI) estimation is essential in forensic investigations.Although various methods for PMI determination have been developed, only an approximate estimation is still achievable, and an accurate PMI indication is still challenging. Therefore, in this study, we employed gas chromatography-mass spectrometry (GC-MS)-based metabolomics to assess post-mortem changes in porcine blood samples collected with and without the addition of anticoagulant (EDTA). Our study aimed to identify metabolites dependent on the EDTA addition and time (taking into account the biodiversity of the studied organism) and those that are time-dependent but resistant to the addition of an anticoagulant.The experiment was performed on blood samples collected from 16 animals (domestic pig, breed: Polish Large White), 8 with and 8 without EDTA addition. The moment of death (time 0) and 15 additional time points (from 3 to 168 hours after death) were selected to examine changes in metabolites' levels in specific time intervals. We employed linear mixed models to study the relationship between metabolite intensities, time and presence of EDTA while accounting for the effect of individual pigs.We confirmed that the intensity of 16 metabolites (mainly amino acids) significantly depends on PMI and the presence of EDTA. However, the intensity of the ideal biomarker(s) for PMI estimation should be determined only by the time after death and not by external factors such as the presence of the anticoagulant agent. Thus, we identified 41 metabolites with time-dependent intensities that were not susceptible to EDTA presence. Finally, we assessed the performance of these metabolites in a PMI predictive model. Citraconic acid yielded one of the lowest errors in general PMI estimation (32.82 h). Moreover, similar errors were observed for samples with and without EDTA (33.32 h and 32.34 h, respectively). Although the small sample size and information leak in predictive modelling prevent drawing definite conclusions, citraconic acid shows potential as a robust PMI estimator.