Forensic science plays a critical role in criminal investigations by assisting in personal identification and reconstruction of crime-related events.^1,2 Although DNA analysis remains the gold standard for forensic identification, biological evidence recovered from crime scenes is frequently exposed to environmental conditions that may compromise DNA integrity.^3,4 Consequently, forensic researchers continue to explore alternative biological markers capable of providing reliable information when conventional methods are insufficient.^5,6

Serum is a biologically rich fluid containing numerous metabolites, enzymes, lipids, proteins, and oxidative stress markers that reflect an individual's physiological and biochemical state.^7,8 Advances in analytical biochemistry have demonstrated that metabolomic profiles can generate unique biochemical fingerprints influenced by genetic background, nutrition, environmental exposure, and metabolic activity.^9,10 These characteristics suggest potential forensic applications in individual differentiation.

In addition to identification, estimation of the post-crime interval (PCI) remains one of the most challenging aspects of forensic investigations.¹¹ Traditional methods often rely on physical observations and environmental assessments, which may be subjective and influenced by external factors. Biochemical degradation patterns occurring after blood deposition may offer a more objective approach for estimating the time elapsed since sample deposition.

Oxidative stress biomarkers, including malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), undergo predictable changes following environmental exposure.¹² Similarly, metabolic indicators such as glucose and lactate exhibit time-dependent alterations resulting from ongoing biochemical reactions. Monitoring these changes may facilitate the development of predictive forensic models capable of estimating post-crime intervals.

Therefore, the present study was designed to investigate serum metabolomic signatures as a novel forensic biochemical approach for personal identification and estimation of post-crime interval using a cohort of 120 participants.

Study Design: A laboratory-based cross-sectional analytical study. Study Population The study included 120 healthy adult volunteers (60 males and 60 females) aged between 20 and 60 years. Inclusion Criteria • Healthy adults aged 20–60 years. • No history of chronic metabolic disorders. • No acute infections within the preceding four weeks. Exclusion Criteria • Liver disease. • Kidney disease. • Diabetes mellitus. • Current use of antioxidant supplements. Sample Collection Five milliliters of venous blood were collected from each participant using sterile vacutainer tubes. Blood samples were centrifuged at 3000 rpm for 10 minutes to obtain serum. Biochemical Analysis The following parameters were measured: • Glucose • Lactate • Urea • Creatinine • Uric acid • Total cholesterol • Triglycerides • ALT • AST • MDA • SOD • CAT Commercial diagnostic kits and spectrophotometric methods were used for analysis. Environmental Exposure Experiment Aliquots of serum samples were maintained at room temperature (25°C ± 2°C) and analyzed at: • 0 hours • 24 hours • 48 hours • 72 hours Statistical Analysis Data were analyzed using SPSS version 26. Continuous variables were expressed as mean ± SD. Repeated-measures ANOVA evaluated temporal changes, while discriminant function analysis assessed identification accuracy. A p-value <0.05 was considered statistically significant.

Table 1. Baseline Biochemical Characteristics of Study Participants (n=120)

Table 2. Changes in Serum Biochemical Markers During Environmental Exposure

Table 3. Forensic Predictive Performance of Serum Metabolomic Profiling

The present study demonstrates the forensic applicability of serum metabolomic profiling for individual identification and post-crime interval estimation. Distinct biochemical signatures composed of metabolic, enzymatic, and oxidative stress markers enabled differentiation among participants with high accuracy. These findings suggest that serum metabolites may serve as individualized biochemical fingerprints, providing valuable forensic information beyond traditional DNA-based methods. Environmental exposure produced predictable alterations in several biomarkers. Lactate accumulation and progressive increases in MDA indicated ongoing metabolic activity and oxidative degradation, while reductions in SOD and CAT reflected declining antioxidant defense mechanisms. These temporal biochemical changes allowed the construction of a predictive model capable of estimating post-crime interval with considerable accuracy. The identification accuracy of 91.7% observed in this study highlights the potential utility of serum metabolomics in situations where DNA evidence is degraded or unavailable. Furthermore, the integration of oxidative stress markers into forensic investigations may improve the objectivity of temporal estimations. The study supports the growing role of biochemistry and metabolomics in modern forensic science and provides a foundation for future research involving larger populations and advanced analytical technologies.

Serum metabolomic profiling represents a promising and innovative forensic biochemical approach for both personal identification and post-crime interval estimation. Distinct metabolite signatures and predictable degradation patterns of oxidative stress biomarkers demonstrated high forensic utility among 120 participants. These findings suggest that serum-based biochemical fingerprinting could serve as a valuable complementary tool in forensic investigations, particularly when conventional DNA evidence is compromised or unavailable.

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