Talanta 79 (2009) 1094–1099 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Estimation of postmortem interval by hypoxanthine and potassium evaluation in vitreous humor with a sequential injection system Marieta L.C. Passos a , Ana M. Santos a , Ana I. Pereira a , J. Rodrigo Santos a , Agostinho J.C. Santos b,c , M. Lúcia M.F.S. Saraiva a,∗ , José L.F.C. Lima a a REQUIMTE, Servic ¸o de Química-Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal b Servic ¸ o de Medicina Legal, Faculdade de Medicina, Universidade do Porto, Portugal c Servic ¸o de Patologia Forense, Instituto Nacional de Medicina Legal, I.P. – Delegac ¸ ão Norte, Portugal article info Article history: Available online 13 March 2009 Keywords: Sequential injection analysis Hypoxanthine Xanthine oxidase Potassium tubular electrode Vitreous humor Postmortem interval abstract The estimation of the time since death known as postmortem interval (PMI) is a main issue in the field of forensic science and legal medicine. In this work it is proposed a sequential injection system for the deter- mination of hypoxanthine and potassium in the same sample of vitreous humor since the concentrations of both parameters change with PMI and the vitreous humor has been regarded as the ideal extracel- lular fluid for these kinds of determinations. By measuring both parameters the accuracy of estimation of PMI can be increased, and the effects of factors which influence the values in postmortem chemistry minimized. Hypoxanthine determination is based on its oxidation to uric acid (290 nm), catalyzed by immobilized xanthine oxidase, and the quantification of potassium levels in vitreous humor was performed using a tubular potassium ion-selective electrode. With a unique analytical cycle both analytes were evaluated being potassium levels determined during the degradation of hypoxanthine in the enzymatic reactor. Working concentration ranges between 6.04–40.00 mol L -1 and 7.00 × 10 -5 to 1.00 × 10 -1 mmol L -1 were obtained, for hypoxanthine and potassium, respectively. The method proved to be reproducible with R.S.D. <5% for hypoxanthine and <3% for potassium. Sam- pling rate was approximately 30 per hour for the sequential determination of both parameters being 15 and 60 determinations per hour if hypoxanthine or potassium, where evaluated independently. Statis- tical evaluation at the 95% confidence level showed good agreement between the results obtained, for the vitreous humor samples, with both the SIA system and the comparison batch procedures. Moreover the methodology has low environmental impact in agreement with the demands of green analytical chemistry as only 2.7mL of chemical waste is produced during both determinations. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The estimation of the time since death known as postmortem interval (PMI) is a main issue in the field of forensic science and legal medicine being one of the most difficult and challenging problem for forensic pathologists. Numerous methods have been reported for the determination of PMI by chemical means. While some studies were carried out on blood and cerebrospinal fluid (CSF), nowadays research have been performed on vitreous humor (VH) [1]. Analytical determinations in vitreous humor have advan- tages over those in blood and CSF. Vitreous humor is easier to collect, is isolated and well-protected anatomically, is preserved postmortem even in cases of severe head trauma and is less subject ∗ Corresponding author. Tel.: +351 222078939; fax: +351 222004427. E-mail address: lsaraiva@ff.up.pt (M.L.M.F.S. Saraiva). to contamination and putrefaction than blood and CSF. Additionally, in vitreous humor, chemical changes occur at a slow rate extending the period of time during which it may be used for PMI estimation purposes [2]. The postmortem increase of potassium concentrations in vitre- ous humor, first described by Sturner [3] is the most extensively studied parameter for estimating the PMI [1] being well known that the increasing concentration of this ion results from the energy breakdown and the related cessation of the active transport and the selective membrane permeability [4]. Several formu- lae have been proposed for estimating PMI on the basis of postmortem potassium levels in vitreous humor [4–11], using different kinds of methodologies, as capillary electrophoresis [4,6,8], low pressure ion chromatography [7] or flame photometry [12]. However, the correlation between potassium concentration in vitreous humor and PMI depends on several factors, related with 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.02.054