Journal of Chromatography B, 902 (2012) 42–46 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B jo ur n al hom ep age: www.elsevier.com/locate/chromb Direct urine analysis for the identification and quantification of selected benzodiazepines for toxicology screening Sevasti Karampela, Ioanna Vardakou, Ioannis Papoutsis, Artemis Dona, Chara Spiliopoulou, Sotirios Athanaselis, Constantinos Pistos ∗ Department of Forensic Medicine and Toxicology, School of Medicine, National and Kapodistrian University of Athens, Mikras Asias 75, Athens 11527, Greece a r t i c l e i n f o Article history: Received 5 March 2012 Accepted 14 June 2012 Available online 20 June 2012 Key words: Benzodiazepines Human urine LC/MS Direct injection Screening a b s t r a c t A simple and rapid LC/MS method with direct injection analysis was developed and validated for the identification and quantification of ten benzodiazepines (flunitrazepam, nordiazepam, diazepam, 7- aminoflunitrazepam, flurazepam, bromazepam, midazolam, alprazolam, temazepam and oxazepam) in human urine using diazepam-d5 as internal standard (IS). The main advantage of the proposed method- ology is the minimal sample preparation procedure, as diluted urine samples were directly injected into LC/MS system. Electrospray ionization in positive mode using selected ion monitoring was chosen for the identification and quantification of the analytes. The linear range was 50–1000 ng/mL for each analyte, with square correlation coefficient (r 2 ) ≥ 0.981. Interday and intraday errors were found to be ≤5.72%. The LC/MS method was applied at ten real samples found initially to be positive and negative, using immunoassay technique. Finally the results were confirmed with GC/MS. The method demonstrates simplicity and fast sample preparation, accuracy and specificity of the analytes which make it suitable for replacement of immunoassay screening in urine avoiding thus false negative/false positive results. Using this method, laboratories may overcome the problem of high cost instrumentation such as LC–MS/MS by providing similar sensitivity and specificity with other methods. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Benzodiazepines are widely prescribed as anxiolytics, sedative hypnotics, anticonvulsants or muscle relaxants [1,2]. Clinical pop- ularity of benzodiazepines has been ascribed to the wide safety margin of their therapeutic index, minimal serious adverse side- effects and low potential for physical dependence [3]. Apart from their therapeutic applications, benzodiazepines are often abused by drug addicts. As a consequence, these drugs are frequently involved in both clinical and forensic cases [2]. A number of studies have been reported in the literature on the determination of benzodiazepines and their metabolites in biologi- cal fluids. These studies use either immunological methods [4–6] or chromatographic techniques [7–13]. Enzyme immunoassay assays (EIAs) are known to provide rapid results for preliminary screen- ing in urine for the presence of benzodiazepines with usual cut-off level of 100 ng/mL; however, specificity remains a significant dis- advantage of such methods [14] where this class of drugs appears to cross react with other substances in a number of cases [15–17]. In one study, 16 of 50 positive benzodiazepine EIAs were submitted by ∗ Corresponding author. Tel.: +30 2107462433; fax: +30 2107716098. E-mail address: cpistos@med.uoa.gr (C. Pistos). patients taking sertraline who were not ingesting benzodiazepines. In a review phase of the same study, false-positive benzodiazepine EIAs caused by sertraline were found in 26% of 2447 patients taking sertraline urine specimens [17]. Gas chromatography, coupled with mass spectrometry, is not applicable to the determination of the entire range of benzodi- azepine panel, because of the thermal instability or scarce volatility shown by some of them, even after derivatization [18]. In contrast, methods based on liquid chromatography (LC) hyphenated with mass spectrometry (MS) are successfully employed for all benzo- diazepines [19]. LC–MS/MS appears as the most eligible technique for the simul- taneous determination of several benzodiazepines [20]. However, and despite the increased use of LC–MS/MS instrumentation in tox- icological laboratories, the high cost of the instrument remains a main problem that make it not always available. Most of the reported LC–MS/MS methods [1,21,11,22,19,23,24] in the litera- ture appear to use traditional solid phase (SP) or liquid–liquid (LL) extraction techniques which both require costly consumables such as cartridges and solvents. Two other articles present the deter- mination of selected benzodiazepines using column switching [25] and on line extraction [26] that require particular system configu- ration which are not always available in a toxicological laboratory. Additionally, de Jager and Bailey [26] determined only a small 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2012.06.012