Applications of Tandem Mass Spectrometry (LC–MSMS) in estimating the post-mortem interval using the biochemistry of the vitreous humour Elena Lendoiro a , Cristina Cordeiro b,c , Marı ´a S. Rodrı ´guez-Calvo d , Duarte N. Vieira b,c , Jose ´ M. Sua ´ rez-Pen ˜ aranda d , Manuel Lo ´ pez-Rivadulla a,d , Jose ´ I. Mun ˜ oz-Baru ´s a,d, * a Institute of Forensic Science, University of Santiago de Compostela, Spain b National Institute of Legal Medicine and Forensic Sciences of Portugal – Centre Branch, Portugal c Centre of Forensic Sciences (CENCIFOR), University of Coimbra, Portugal d Department of Pathology and Forensic Science, University of Santiago de Compostela, Spain 1. Introduction In the field of forensic pathology one of the most important issues is the correct estimation of the post mortem interval (PMI). This is of particular relevance in criminal investigation, where recourse to a method that provides a PMI whose precision can be upheld in a court of law can be crucial [1,2]. It is widely accepted that the quantification of potassium concentrations ([K+]) and hypoxanthine ([Hx]) in the vitreous humour is useful in estimating the time of death within a recent time interval [3–7]. The importance of this problem is demonstrated by the fact that it has become the subject of numerous publications in leading journals in this field, which highlights the difficulties of this approach, and has led to further comparative studies [8]. It has been reported that urea (U) may modify the relationship between the concentration of vitreous potassium and PMI [4,9,10] and that the cause of death itself can also modify this relationship [10,11]. Different analytical methods for quantification of potassium and [Hx] have been developed in order to improve accuracy in estimating the time of death [12,13]. Although an inverse prediction by changing the variables is the statistical method of choice when using linear regression to relate the [K+] or [Hx] in the vitreous with PMI [4,14], other more flexible methods of regression have been developed which by including all the possible variables [K+], [Hx], [U], in the vitreous humour together with the cause of death, provide a more accurate estimate [11]. Although the calculation is much more complex than the simple application of the typical single-line regression formula, a free to use statistical package has been presented which provides a fast and intuitive result [15]. Despite all the advances made in this area, it is well recognized that the difficulties in calibration, validation, different methodolo- gies and instrumentation from different laboratories may lead to obtaining different concentrations from the same sample [16,17]. It has also been demonstrated that pre-treatment of samples before analysis may lead to some variations in the determination of potassium and urea [17]. However, not only pre-treatment of the sample, but the extraction of the vitreous humour itself should be carried out with a precise technique, taking care during the procedure to avoid causing any vascular injury which might lead to haematic contamination. Any fluid that is not clear could produce erroneous results and should be discarded [2,4,9,18]. Forensic Science International 223 (2012) 160–164 A R T I C L E I N F O Article history: Received 10 April 2012 Received in revised form 27 June 2012 Accepted 22 August 2012 Available online 13 September 2012 Keywords: Tandem mass spectrometer Post mortem interval Hypoxanthine Vitreous humour A B S T R A C T It is widely accepted that the quantification of potassium concentrations ([K+]) and hypoxanthine ([Hx]) in the vitreous humour is useful in estimating the time of death within a recent time interval. Despite all the advances made in this area, it is well recognized that difficulties in calibration, validation and the use of different methodologies and instrumentation from different laboratories may lead to obtaining different concentrations from the same sample. The extraction of the vitreous humour itself should also be carried out with a precise technique, taking care during the procedure to avoid causing any vascular injury that might lead to haematic contamination. Any fluid that is not clear could lead to erroneous results and should be discarded. We present a new LC–MSMS method developed for quantitative and qualitative analysis of [Hx] (valid also for guanine and xanthine), and qualitative determination of uric acid in vitreous humour. We also introduce a methodology to assess haematic contamination in order to improve the estimation of time since death. The method was fully validated in terms of linearity, sensitivity, imprecision, analytical recovery, extraction and process efficiency and matrix effect. ß 2012 Elsevier Ireland Ltd. All rights reserved. * Corresponding author at: Institute of Forensic Science, University of Santiago de Compostela, Spain. Tel.: +34 981 812 325; fax: +34 981 812 459. E-mail address: joseignacio.munoz.barus@usc.es (J.I. Mun ˜ oz-Baru ´ s). Contents lists available at SciVerse ScienceDirect Forensic Science International jou r nal h o mep age: w ww.els evier .co m/lo c ate/fo r sc iin t 0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2012.08.022