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