Original article 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) Covadonga Palacio 1 , Rossella Gottardo 1 , Vito Cirielli 2 , Giacomo Musile 1,3 , Yvane Agard 1 , Federica Bortolotti 1 and Franco Tagliaro 1,3 Abstract 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 lg/mL solution of BaCl 2 (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 r 2 of 0.75 and 0.70, respectively. Keywords Ammonium, capillary electrophoresis, post mortem interval, potassium, vitreous humour Introduction The estimation of time of death remains one of the major challenges in criminal investigations and is often an issue that is debated in the courts. In the past decades, active research has been carried out to identify sound and objective parameters upon which the estimation of the post mortem interval (PMI) could be grounded. 1,2 In fact, PMI estimation is traditionally based on the evaluation of the body’s physical changes occurring in the ‘early post-mortem period’, comprising ‘rigor mortis’, lividities, body cooling and, in some cases, the so-called supravital phenomena. This approach, however, shows important points of weakness, since lividities, rigor mortis and, to some extent, supravital phenomena suffer from high variability and often sub- jectivity of recording. Also, body cooling is greatly affected by several confounding factors (e.g. environ- mental temperature and humidity, clothing, posture, 1 Department of Diagnostics and Public Health, Section of Forensic Medicine, University of Verona, Italy 2 Department of Prevention, Unit of Forensic Medicine, Vicenza, Italy 3 Institute of Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Russia Corresponding author: Rossella Gottardo, Department of Diagnostics and Public Health, Section of Forensic Medicine, University of Verona, Policlinico G.B. Rossi, Piazzale L.A. Scuro 10, 37134 Verona, Italy. Email: rossella.gottardo@univr.it Medicine, Science and the Law 0(0) 1–9 ! The Author(s) 2020 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/0025802420934239 journals.sagepub.com/home/msl