Off-line HPLC method combined to LC–MS for the determination of sildenafil and its active metabolite in post-mortem human blood according to confirmation criteria Constantinos Pistos * , Ioannis Papoutsis, Artemis Dona, Maria Stefanidou, Sotiris Athanaselis, Constantinos Maravelias, Chara Spiliopoulou University of Athens, Medical School, Laboratory of Forensic Medicine and Toxicology, 75M. Asias Str., Goudi 11527, Athens, Greece Received 17 November 2007; received in revised form 2 March 2008; accepted 24 March 2008 Available online 16 May 2008 Abstract A simple HPLC method has been validated for the determination of sildenafil and its active metabolite (N-desmethylsildenafil) in human blood, using an octadecyl silica (ODS) hypersil column. The chromatographic run time is less than 25 min using a mobile phase of 35:65 (v/v) acetonitrile–0.015 M disodium hydrogen phosphate (Na 2 HPO 4 ), triethylamine 0.1%, pH 7.4 at 1 mL/min flow rate and UV–vis detection at 230 nm. The method is linear in the concentration range of 10–500 ng/mL (r > 0.999, n = 5) for each analyte, with relative standard deviation (R.S.D.) less than 5.05%. Interday and intraday errors were found to be 11.94%. The limits of detection and quantitation for both analytes were 5.0 ng/mL (s/n > 3) and 10.0 ng/mL (s/n > 10), respectively. The method was applied in two post-mortem human blood samples, concerning two fatal cases from sildenafil citrate use, reported for the first time in Greece, and the results were further confirmed with LC–MS. The method is proposed as supplementary to LC–MS when inadequate mass fragmentation does not provide information appropriate to meet confirmation criteria. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: HPLC UV–vis; Confirmation criteria; Sildenafil; N-Desmethylsildenafil 1. Introduction Sildenafil (S) (1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo- 3-propyl-1H-pyrazolo-[4,3-d]pyrimidin-5-yl) phenylsulpho- nyl]-4-methylpiperazine) (Fig. 1a) has been widely prescribed for erectile dysfunction [1–4] and its bioavailabilty, metabo- lism, elimination route and pharmacokinetics have been extended reported [3,5]. Mean maximum sildenafil plasma concentrations measured after a single oral dose of 100 mg to healthy male volunteers is 450 ng/mL plasma. The lower therapeutic concentrations in human plasma after a 25 mg single oral dose are approximately 7 ng/mL [5]. Many analytical methods, using high performance liquid chromatography (HPLC), have been published for quantification of the parent drug sildenafil in plasma and not for its active metabolite, using ultraviolet visible (UV–vis) detector [6,7], or a liquid chromatography system combined with a triple quadru- pole mass spectrometry detector [8], as well as in oral fluids using a liquid chromatography single mass spectrometry system [9]. Lewis and Johnson [10] reported the detection of both sildenafil and N-desmethylsildenafil in post-mortem fluids and tissues, while Weinmann et al. [11] reported their detection in urine and tissue samples using liquid chromatography tandem mass spectrometry (LC–MS/MS) system. Al-Ghazawi et al. [12] developed a method for the determination of both analytes in plasma using electrochemical detection, Cooper et al. [13] in plasma using UV–vis detector and Saisho et al. [14] in human hair by GC–MS. Although most of these methods are sensitive and accurate, none of them describe how to overcome the inadequate LC–MS fragmentation of both sildenafil and its active metabolite in human blood. According to our experiments, the proposed methods in the literature for the determination of both compounds in plasma appear to be problematic when they are applied in human blood. Considering that at a number of cases, www.elsevier.com/locate/forsciint Available online at www.sciencedirect.com Forensic Science International 178 (2008) 192–198 * Corresponding author. Tel.: +30 210 7462433; fax: +30 210 7716098. E-mail address: cpistos@med.uoa.gr (C. Pistos). 0379-0738/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2008.03.018