Development of a tailored sample preparation protocol for untargeted metabolic and lipidomic profiling of post-mortem vitreous humour, and exploration of its metabolomic landscape for forensic applications.

Traditional post-mortem toxicology primarily focuses on intoxications and relies heavily on the measurement of xenobiotics and their subsequent interpretation. However, to resolve broader forensic inquiries, an additional analytical approach is needed in toxicological investigations. Thanatometabolomics, the systematic analysis of small endogenous metabolites (e.g. lipids, amino acids) in post-mortem biofluids, represents an emerging concept aimed at addressing critical challenges, such as accurately determining the post-mortem interval and elucidating biochemical events preceding death. Although blood remains the gold standard in traditional forensic toxicology, it has notable limitations, including microbial degradation and post-mortem redistribution of compounds, which introduces variability dependent on sampling conditions. Vitreous humour, a more metabolite-stable post-mortem biofluid, offers a promising alternative matrix for thanatometabolomic research. However, a thorough review of the literature reveals a significant lack of established protocols for untargeted metabolomic analysis of vitreous humour, hindering progress in advancing the field of post-mortem toxicology.

Three dilution series derived from three established sample preparation procedures: Folch biphasic extraction (CHCl₃: MeOH: H₂O, 8: 4: 3, v/v/v), Bligh-Dyer biphasic extraction (CHCl₃: MeOH: H₂O, 2: 2: 1.8, v/v/v) and a methanol single-phase extraction were evaluated. Additionally, the metabolite recoveries in each sample preparation were assessed through re-extraction of the remaining protein residue. Data acquisition was then performed with four in-house metabolomic platforms, employing liquid chromatography coupled to Quadrupole-Time of Flight high-resolution mass spectrometry. Finally, data preprocessing was conducted with MS-DIAL.

The results of this study showed that low concentrations of lipids in vitreous humour necessitate large sample volumes, while polar metabolites are present in higher concentrations, allowing for smaller sample volumes. A diverse array of metabolites was identified, predominantly associated with pathways involved in energy metabolism (e.g. organic acids, carnitine), amino acid metabolism (e.g. tryptophan, lysine, tyrosine), oxidative stress regulation (e.g. methionine, glutathione), and nucleotide metabolism (e.g. hypoxanthine, cytidine, uridine). The lipid fraction was primarily composed of phospholipids, fatty acids, and sphingomyelins, supporting key functions such as energy production, intercellular communication, and the maintenance of structural integrity. Both, Folch and Bligh-Dyer extractions, proved superior to single-phase extraction, with Bligh Dyer particularly enhancing the detection of lipids in positive ionization mode. Additionally, Folch and Bligh-Dyer extraction revealed a more diverse array of lipid profiles compared to single phase extraction, in both positive and negative ionization modes. Re-extraction provided no additional benefit for either polar metabolites or lipids in the biphasic extractions, except for lipids in the single-phase extraction.

The interpretation of post-mortem vitreous humour metabolomics data remains challenging due to the limited scope of prior research and the absence of standardized protocols and guidelines for pre-analytical sample handling. Critical pre-analytical factors, such as short-term and long-term stability and the influence of different collection containers, remain unexplored, complicating the evaluation of these preliminary findings.

The study demonstrates that biphasic extractions are essential for effectively detecting the sparse lipid content in vitreous humour, ensuring comprehensive profiling of vitreous humour for further forensic applications. Nevertheless, a diverse range of metabolites and lipids can be detected in post-mortem vitreous humour, underscoring its potential as a valuable alternative matrix for post-mortem investigations. However, standardization and further investigation regarding pre-analytical factors is required for in depth assessment of post-mortem vitreous humour metabolomic and lipidomic data.