A new method for the determination of ammonium in the vitreous humour based on capillary electrophoresis and its preliminary application in thanatochemistry

Abstract

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.

Lactate determination in human vitreous humour by capillary electrophoresis and time of death investigation

“…Finally, considering other PMI biochemical markers, literature describes a positive correlation of potassium and ammonium [23–25]. Further investigation including La is suggested to verify if a combination of more than one of these parameters could increase the accuracy of PMI determination.…”

Simultaneous analysis of potassium and ammonium ions in the vitreous humour by capillary electrophoresis and their integrated use to infer the post mortem interval (PMI)

“…The details of the validation of the analytical methods for the determination of NH 4 + and K + in vitreous humour used in the present work were reported in previous papers. 17,21…”

“…Surprisingly, it was neglected by most of authors for quite obscure reasons, but probably related to well-known analytical problems in clinical chemistry and laboratory medicine. 12–18 To the best of our knowledge, a single paper did appear in 1978 reporting an ammonium increase in the vitreous humour in correlation with the post mortem interval. In this article, a colorimetric method based on the Nessler’s reagent was used, without any separation step.…”

“…16 Only recently, a CE method for the determination of ammonium in the vitreous humour with indirect ultraviolet (UV) detection has been reported, showing a potential for use in this context. 17 This simple CE separation method had previously proved suitable for the separation and quantitative determination of potassium ions in the vitreous humour. 9 In addition, two more methodologies developed by Musile et al., a gas diffusion device and a microfluidic paper-based device, were recently developed in our research group.…”

Optical coherence tomography in forensic sciences: a review of the literature

“…Although several studies of forensic pathology have focused on the eye, to date the main area of forensic speculation has dealt with the variation of post mortal biochemistry of the vitreous humor, or at most of the aqueous humor [23][24][25][26][27][28].…”