The review, conducted by researchers at the Universidade Federal do Pará in Brazil, searched the Scopus, Web of Science, and PubMed databases for English-language articles published between 2014 and 2024, excluding reviews and meta-analyses. After removing duplicates from an initial pool of 414 articles and screening titles, abstracts, and full texts, 54 studies made the final cut. From these, the team extracted data on study objectives, main results, and recommendations for collection, storage, and biomarker detection. The overarching conclusion is clear: no single sample tells the whole story. Instead, laboratories should analyze multiple matrix types and multiple biomarkers, collect samples in duplicate, refrigerate or freeze them depending on storage duration, use preservatives, and ensure that staff are properly trained.

The central problem the review addresses is microbial fermentation. After death, bacteria and yeasts can convert glucose in the body into ethanol and carbon dioxide, a process that produces so-called post-mortem ethanol. This neoformation is not limited to visibly decomposing bodies; it can occur in specimens kept under inadequate conditions, such as high temperatures or the absence of preservatives like sodium fluoride. The result can be false-positive blood alcohol values that have nothing to do with what the deceased actually drank. Conversely, ethanol already present can be lost through oxidation into acetaldehyde, producing false negatives. Pathological conditions complicate matters further: individuals with diabetes, whose bodies carry excess glucose, can generate significant post-mortem blood ethanol even when they consumed no alcohol at all.

To disentangle drinking from fermentation, forensic toxicologists increasingly rely on non-oxidative metabolites of ethanol, chiefly ethyl glucuronide (EtG) and ethyl sulfate (EtS). Because these compounds are direct products of the body’s metabolism of alcohol, they cannot be manufactured by microbes fermenting glucose. Their presence in post-mortem samples is therefore directly tied to alcohol consumed before death. Studies cited in the review demonstrated a selective association between ante-mortem drinking and EtG and EtS detection: the biomarkers were absent in ethanol-negative samples from both deceased and living individuals, but appeared and then declined over time in ethanol-positive ones. Notably, EtG and EtS are not spontaneously formed even under post-mortem hyperglycemic conditions, so their detection supports metabolic activity before death, including in diabetic cases.

But the two biomarkers behave differently as bodies decompose. EtG is more sensitive yet less stable, and can be degraded during advanced decomposition, risking false negatives. EtS, by contrast, remains reliable even in severely putrefied blood, particularly at high temperatures, making it the more dependable marker for excluding pre-death drinking when results conflict. One strategy the review highlights is measuring EtG in vitreous humor, the clear fluid inside the eye, which is more sterile than blood and better protected from decomposition. Interpretation, the authors stress, must always account for the matrix, preservation conditions, and degree of decomposition.

Acetaldehyde, ethanol’s oxidative metabolite, is another piece of the puzzle, though a treacherous one. Its origin is difficult to pin down: it may arise from bacterial activity, yeast fermentation, ethanol oxidation, putrefaction, or poor blood preservation. The review notes that in well-preserved bodies and living individuals, acetaldehyde concentrations stayed within a stable range of 0.14 milligrams per milliliter or less, while highly putrefied bodies showed significantly higher values, indicating an association with putrefaction. Abnormally high acetaldehyde in the presence of ethanol and higher alcohols such as 1-propanol and 1-butanol may signal microbial neoformation. Conversely, acetaldehyde appearing without ethanol or 1-propanol may indicate that ethanol was oxidized and lost, particularly under extreme storage conditions. Higher alcohols themselves, produced when amino acids released by protein degradation ferment, appear at far lower concentrations than ethanol; a blood 1-propanol cutoff of 0.104 milligrams per deciliter has been proposed as a criterion for identifying post-mortem microbial ethanol production, though some researchers question its reliability.

When it comes to choosing samples, the review finds strong support for a multi-matrix approach. Peripheral blood, generally drawn from the femoral vein to avoid contamination and post-mortem redistribution, remains the primary specimen. Urine is closely associated with blood and, in healthy individuals, contains no glucose, reducing fermentation risk, though diabetic victims or those with urinary infections can confound this advantage. Vitreous humor emerges as a standout: anatomically isolated, minimally susceptible to decomposition and glycosuria, easy to collect from both eyes, and stable after death. Its inclusion as a routine matrix is strongly recommended, especially for putrefied bodies, outdoor deaths, and diabetic individuals. Cerebrospinal fluid, particularly from the spinal canal rather than the cerebral ventricles, offers another protected alternative. More exotic options are also on the table: dried blood spots for stable metabolites, costal cartilage ground in liquid nitrogen, brain tissue, hair for chronic abuse markers, and even necrophagous insect larvae, in which EtS proved a promising and stable indicator of alcohol ingestion.

Preservation practices emerge as the make-or-break factor in sample integrity. Refrigeration at 4 degrees Celsius is the minimum requirement, but samples stored longer than 48 hours should ideally be frozen, preferably at minus 80 degrees. Ethanol losses at room temperature can reach 15 to 16 percent, and even at minus 20 degrees, variations of 8 percent over 30 days have been recorded. Because oxidation is a primary cause of ethanol loss, samples should be collected in duplicate so that unopened tubes remain available for reanalysis; previously opened tubes showed greater losses in long-term studies. Tubes should be filled more than halfway to limit oxygen headspace, sealed tightly, made of sterile glass, and homogenized gently before storage. Fluoride preservatives, at concentrations of roughly 1 to 2 percent sodium or potassium fluoride, are essential: they inhibit both microbial growth and the enzyme enolase, curbing glycolysis and endogenous ethanol production. Anticoagulants such as EDTA or potassium oxalate may accompany them, while lithium heparin alone proved ineffective even at minus 80 degrees.

The stakes of all this extend well beyond the laboratory bench. Blood alcohol levels influence medicolegal interpretation, investigative conclusions, and judicial decisions, and the integrity of the evidence chain from collection to courtroom underpins due process itself. The review’s authors argue that protocols enabling clearer correlations between results and real conditions can prevent or detect alterations in ethanol levels, ensuring validity, traceability, and a fairer trial. They call for integrating ethanol metabolite analysis and multiple matrices into routine laboratory protocols, supported by standard operating procedures, investment in technology, and ongoing staff training. Emerging frontiers, from epigenetic markers of alcohol use disorder to ethyl esters revealing combined use of alcohol and synthetic cannabinoids, promise to widen the analytical toolkit further. For now, the message to forensic science is one of disciplined redundancy: sample widely, preserve rigorously, and never trust a single number to tell the story of how alcohol entered a body, or when.

Subject of Research: Post-mortem ethanol detection and the selection, collection, and storage of biological matrices in forensic toxicology

Article Title: Biological matrices for determining the origin of post-mortem ethanol: Recommendations for collection and storage

Article References: da Paz, A. P. S., Nunez, L. B. B., Jr, & de Mello, V. J. (2026). Biological matrices for determining the origin of post-mortem ethanol: Recommendations for collection and storage. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03994-9

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03994-9

Keywords: forensic toxicology, post-mortem ethanol, ethyl glucuronide, ethyl sulfate, vitreous humor, microbial fermentation, sodium fluoride, blood alcohol, sample preservation, forensic autopsy, biomarkers, chain of custody

The conventional toolbox for estimating the postmortem interval, or PMI, has long relied on physical signs of decomposition: body temperature cooling curves, the onset and resolution of rigor mortis, lividity patterns, and in later stages, insect colonization and microbial succession. Each of these approaches has well-documented weaknesses. Ambient temperature swings, morgue storage, wrappings and encasement, and individual variation in physiology can all distort the timelines inferred from these markers. Researchers have even shown that rigor mortis can persist far longer than classic models assume when bodies are kept cold, and that maggot development continues during refrigerated storage, complicating entomological estimates. The result, as the review’s authors note, is that conventional methods are often compromised by environmental factors and variable preservation conditions, restricting their accuracy and practical utility.

Vitreous humor offers a way around many of these problems, and the reason is anatomical. The fluid is sequestered behind the blood-retinal barrier, a selective gatekeeper that tightly controls which substances pass between the bloodstream and the eye. Because of this isolation, the vitreous humor exhibits relative metabolic stability after death and resists contamination and bacterial invasion far better than blood, cerebrospinal fluid or tissue samples. It is also less susceptible to the postmortem redistribution of drugs and metabolites that plagues toxicological interpretation. These properties make it, in the words of the review, an optimal biological matrix for PMI estimation, one that can extend the effective time window during which reasonably precise estimates are possible.

The scientific pedigree of vitreous humor analysis stretches back more than a century, to structural studies of the gel in the 1860s, but its forensic career truly began in the 1960s. In 1963, investigators reported that potassium concentrations in the vitreous humor rise steadily after death, a finding quickly confirmed by parallel studies showing that the correlation could serve as a clock for the postmortem interval. The mechanism is elegantly grim: after circulation stops, cells lose their ability to maintain ion gradients, and potassium leaks from intracellular stores into the surrounding fluid at a roughly predictable rate. Later work added hypoxanthine, a breakdown product of ATP metabolism that accumulates as oxygen deprivation sets in, and showed that combining hypoxanthine with potassium and ambient temperature improves estimates further.

Beyond potassium and hypoxanthine, the review catalogues a rich cast of biochemical characters whose postmortem trajectories correlate with time since death. Sodium and chloride concentrations shift in patterns that have proven useful both for PMI estimation and for distinguishing saltwater drowning from immersion deaths unrelated to drowning. Calcium and magnesium rise as cellular membranes fail. Glucose, urea nitrogen, creatinine and uric acid provide complementary information, though glucose is complicated by the fact that it can be elevated in conditions such as ethylene glycol poisoning. Ammonium accumulates as proteins degrade, and has even been measured at crime scenes using microfluidic paper-based devices designed to bring thanatochemistry out of the autopsy suite and into the field. Free amino acids, lactate, and protein fragments analyzed by high-performance liquid chromatography and mass spectrometry round out an expanding analytical repertoire.

Historically, the mathematical treatment of these signals relied on linear regression equations, most famously the potassium-based formulas refined across generations of studies. These equations correct for ambient temperature, age and other covariates, and software tools have been built to make them accessible to practicing pathologists. Yet the underlying biology is not strictly linear. Electrolyte diffusion, enzymatic degradation, temperature-dependent reaction kinetics and individual variation all interact in complicated ways, and single-marker equations carry confidence intervals that can span hours or days. Flexible regression models and chemometric approaches have pushed accuracy further, but the review argues that the real leap forward comes from artificial intelligence.

Machine learning entered the field in earnest in 2002, when researchers paired capillary zone electrophoresis measurements of vitreous electrolytes with artificial neural networks to estimate the PMI, demonstrating that nonlinear models could capture dependencies that simple equations missed. Since then, the approach has gathered momentum. Neural networks and other algorithms have been applied to sodium and potassium data, to metabolomic profiles of ocular fluids, and to microbiome succession data from animal models. Recent studies combining the human metabolome with machine learning have reported substantially improved PMI predictions, and systematic reviews now document machine learning and metabolomics applications across multiple tissue types. The review emphasizes that these models do more than enhance predictive accuracy; they enable robust nonlinear time series analysis of the complex, interacting biochemical changes that unfold after death.

The authors’ forward-looking vision is a multimodal fusion framework that integrates multiple streams of information: vitreous electrolytes, hypoxanthine and lactate kinetics, amino acid and peptide degradation patterns, imaging data such as the postmortem changes visible in the eye on computed tomography, and contextual variables like temperature and body weight. By training machine learning models on such heterogeneous datasets, the framework would hedge against the weaknesses of any single marker while exploiting the complementary strengths of many. The review also points to enabling technologies that could make this vision practical at scale, including tandem mass spectrometry, capillary electrophoresis, electrochemical biosensors for hypoxanthine, and rapid field-deployable microfluidic devices. Such a framework, the authors argue, is poised to become an indispensable tool for PMI estimation in forensic investigations and medicolegal proceedings.

Challenges remain before the vision becomes routine practice. Machine learning models are only as good as the data they are trained on, and the forensic literature contains decades of measurements gathered with different instruments, protocols and populations. Standardization of sampling and analytical methods will be essential, as will validation across diverse climatic conditions, causes of death and antemortem physiological states, since factors such as pre-death hypoxia can independently elevate hypoxanthine levels. Interpretability is another concern; courts and defense attorneys will demand to know why a model produced a particular estimate. Still, the review makes a compelling case that the convergence of a uniquely stable biological matrix, a maturing analytical toolkit and powerful computational methods is transforming one of forensic science’s oldest quests. What began with a simple potassium measurement in the 1960s is evolving into a data-rich, algorithm-driven science of the dead, one in which the fluid of the eye may soon testify, with unprecedented precision, about the moment life ended.

The choice of vitreous humor over alternative biological matrices reflects a broader pattern in postmortem biochemistry, where researchers have explored pericardial fluid, cerebrospinal fluid, and even synovial fluid as potential sources of time-dependent markers. Each fluid has its own advantages and drawbacks. Cerebrospinal fluid, for instance, contains promising markers such as glial fibrillary acidic protein, neuron-specific enolase and S100B, but its biochemical composition can vary depending on the sampling site along the spinal column, a source of variability that complicates comparisons across studies. Pericardial fluid has proven valuable for metabolomic investigations and for detecting markers of antemortem drug exposure, yet it is more exposed to contamination and postmortem diffusion from adjacent tissues than the sequestered ocular environment.

Practical considerations also favor the eye in casework. Vitreous humor can be collected with a simple needle aspiration through the sclera, typically from both eyes, and the small sample volumes required are compatible with standard clinical biochemistry analyzers as well as more sophisticated platforms. The fluid’s clarity and low protein content reduce matrix effects in spectrophotometric and chromatographic assays, which is one reason potassium-based methods achieved early reproducibility. At the same time, forensic researchers have cautioned that sampling technique, the interval between collection and analysis, and storage temperature can all influence measured concentrations, underscoring the need for harmonized protocols.

Imaging-based approaches add a complementary, non-destructive dimension. Studies of postmortem computed tomography have documented progressive changes in the eye, and measurements of the radiodensity of vitreous humor and cerebrospinal fluid have been proposed as indicators of the time since death. Because such imaging can be performed before autopsy without altering the specimen, radiological markers could eventually be integrated alongside biochemical ones in the multimodal frameworks the review envisions.

The pursuit of a single universal formula for the postmortem interval has long been viewed with skepticism among forensic scientists, and this skepticism shapes the review’s argument for data-driven fusion. Rather than seeking one equation that fits all circumstances, the field appears to be moving toward context-aware models that weigh temperature history, individual physiology and multiple analytes simultaneously. If validated rigorously, such models would not replace the forensic pathologist’s judgment but would supply a quantified, uncertainty-aware estimate that can be communicated transparently in medicolegal settings, marking a meaningful shift in how time since death is established in practice.

Subject of Research: Postmortem interval estimation using vitreous humor biochemistry and machine learning

Article Title: From biochemical markers to machine learning: a review of postmortem interval estimation via vitreous humor analysis

Article References: Rao, M., Wang, C., Tian, Y., Tao, H., Liu, L., & Zeng, X. (2026). From biochemical markers to machine learning: a review of postmortem interval estimation via vitreous humor analysis. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03979-8

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03979-8

Keywords: vitreous humor, postmortem interval, forensic science, machine learning, potassium, hypoxanthine, blood-retinal barrier, thanatochemistry, forensic toxicology, artificial neural networks, multimodal fusion, death investigation