Post-mortem changes of ions in the body fluids have been proposed as an objective tool for inferring the time of death. In particular, the post-mortem increase of potassium concentrations in the vitreous humour has gained great attention in the literature. On the other hand, ammonium, another ion released in post-mortem processes, has received much less attention, potentially due to unresolved analytical issues using current clinical chemistry methods. This paper presents an application of a new analytical approach based on capillary electrophoresis providing the simultaneous analysis of potassium and ammonium ions in the vitreous humour. In addition, to assess the consistency of the post-mortem increase of ammonium concentrations in the vitreous humour, the determination of this ion in the vitreous humour of the two eyes of the same body at the same post-mortem interval has been verified. Vitreous humour was collected from 33 medico-legal cases where the time of death was known exactly. Prior to analysis, all samples were diluted 1:20 with a 40 μg/mL solution of BaCl2 (internal standard). In the study of the variability of the ammonium concentration between the two eyes, no statistically significant differences were found, supporting the hypothesis of an even post-mortem increase of the ion concentrations in this particular biological fluid. Significant correlations of potassium and ammonium ions with the post-mortem interval were found, with r2 of 0.75 and 0.70, respectively.
Background
Although the post-mortem increase of ammonium in biological fluids is well known, ammonium analysis in vitreous humour has never been used in recent times for the determination of the post-mortem interval. The present work represents a new application of capillary electrophoresis with indirect UV detection in the field of forensic analysis.
Methods
The electrophoretic separation was carried out in a running buffer made of 5 mM imidazole, 5 mM 18-crown-6 ether and 6 mM d,l-α-hydroxybutyric acid (HIBA). To overcome the lack of optical absorption of ammonium, indirect UV detection was applied. The used wavelength was 214 nm.
Results
The method showed good linearity in the concentration range from 0.16 to 5.0 mM. The limit of detection, 0.039 mmol/L, was established on the basis of the linearity curve. Precision and bias studies carried out on the pure ammonium solutions and in real biological samples, revealed %RSDs well below 20%. A preliminary application to real cases where the death time was precisely known (14 bodies) was carried out plotting vitreous humour ammonium vs. post-mortem interval with a resulting good linear correlation until 100 h post-mortem.
Conclusions
After validation in real cases, the present method can become a powerful tool to unravel one of the most challenging issues of forensic investigation: determination of the time of death.
A gas chromatography‐mass spectrometry method was developed to determine the chemical composition of alcoholic beverages. Extraction was performed with 280 μl of butyl acetate added to 1.7 ml of the sample and placed into 2 ml gas chromatograph auto‐injector vial. One microliter from the upper organic layer was analyzed. Natural constituents (i.e., phenylethyl alcohol, 4‐hydroxybenzoate) found in beers and wines were profiled. Age markers for wines aged in contact with oak wood were studied. Artificial flavors and preservatives were identified in low quality and fruit wines (i.e., methyl anthranilate, triacetin). Esters of phthalic acids and squalene were identified as main polluters. Beers were contaminated to a lesser extent, while wines contained slightly higher concentrations, and distillated had higher concentrations of contaminants. The concentration of contaminants was related to the degree of alcohol and the quality of the beverages. No contamination was found in alcoholic beverages stored in polyethylene terephthalate containers.
Practical applications
The present study corresponds to a broad characterization of alcoholic beverages based on an optimized gas chromatography‐mass spectrometry micro‐extraction method to identify natural volatile and semi‐volatile constituents as well as other substances such as artificial flavors, preservatives, and contaminants. The study has the potential to inform at the level of food safety, alcohol authenticity, and food toxicology.
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