Introduction This study investigates the intraindividual variability and ex-vivo stability of vitreous humor (VH) analytes for forensic thanatochemistry. VH, an anatomically isolated and robust postmortem fluid, provides valuable insights into the postmortem interval (PMI) and potential antemortem medical conditions.

Materials and methods A total of 207 VH samples were collected from 36 cases and divided into three cohorts: serial sampling across three days postmortem, intraindividual variability assessment using triplicates, and ex-vivo stability evaluation through freeze-thaw cycles.

Results Linear relationships between PMI and potassium, calcium, creatinine, and lactate were identified, but their non-linear trends limited PMI estimation accuracy. Sodium, chloride, and glucose demonstrated variations linked to cardiovascular and infectious conditions, while elevated urea and creatinine correlated with renal or multi-organ failure. The study highlighted the importance of interpreting these biochemical markers in conjunction with clinical and environmental factors to improve reliability.

Discussion Ex-vivo stability testing revealed relevant variations in VH levels, emphasizing the need for standardized pre-analytical handling. Overall, while VH analytes offer promising forensic applications, their use for PMI estimation and medical diagnoses requires cautious interpretation within a multidisciplinary context.