A new LC-MS/MS confirmation method for the determination of 17 drugs of abuse in oral fluid and its application to real samples Elisa Bassotti a , Giuseppe Maria Merone b , Annachiara D’Urso c , Fabio Savini d , Marcello Locatelli e , Angela Tartaglia e , Paolo Dossetto f , Cristian D’Ovidio g , Ugo de Grazia c, * a R&D Department Eureka Lab Division, Chiaravalle, Italy b Department of Neuroscience, Imaging and Clinical Sciences, University of Chieti–Pescara “G. d’Annunzio”, 66100 Chieti, Italy c Fondazione IRCCS Istituto Neurologico Carlo Besta, Laboratory of Neurological Biochemistry and Neuropharmacology, Via Celoria 11, Milano 20133, Italy d Laboratory of Pharmatoxicology - Hospital “Santo Spirito”, Pescara 65124, Italy e Department of Pharmacy, University of Chieti–Pescara “G. d’Annunzio”, Chieti 66100, Italy f Sciex, Milano, Italy g Department of Medicine and Aging Sciences, Section of Legal Medicine, University of Chieti–Pescara “G. d’Annunzio”, Chieti 66100, Italy A R T I C L E I N F O Article history: Received 17 January 2020 Received in revised form 4 May 2020 Accepted 5 May 2020 Available online 12 May 2020 Keywords: Forensic toxicology Oral fluid Drugs of abuse Hyphenated configuration Confirmation method A B S T R A C T A new liquid chromatography–tandem mass spectrometry (LC–MS/MS) confirmation method for the direct analysis of 17 drugs starting from 200 mL of diluted oral fluid (OF), in a single chromatographic run, was developed and validated. Cocaine, benzoylecgonine (BEG), cocaethylene, D-9-tetrahydrocannabinol (D-9-THC), buprenorphine, 6-acetylmorphine (6AM), morphine, codeine, methadone, 2-ethylidene-1,5- dimethyl-3,3-diphenylpyrrolidine (EDDP), amphetamine, methamphetamine, 3,4-methylenedioxyme- thamphetamine (MDMA), methylenedioxyamphetamine (MDA), 3,4-Methylenedioxy-N-ethylamphet- amine (MDE), ketamine, N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) were determined in a chromatographic run of 12 min only with no sample pre-treatment, after the addition of 15 different internal standards (ISs). The method met all requirements in terms of linearity, accuracy (precision and trueness), recovery, and stability requested by FDA guidelines. Carry-over and interferences were negligible, as well as the matrix effects. LLOQs are below the limits defined by European guidelines and Italian national laws. The original oral fluid collections are stable at least six months at 20  C and one week at +4  C. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Drug of abuse represent a worldwide problem. Illicit drugs use may lead to sub-optimal or erroneous driving behavior resulting in accidents and sometimes in death of the involved subjects. In such cases or in routine road checks carried out by the police consider the collection of a salivary sample, which is analyzed with first level screening methods. These semi-quantitative results must be confirmed with confirmatory techniques, such as liquid chroma- tography combined with mass spectrometry. Oral fluids (OF) has been introduced recently as a biological matrix suitable for on-road illicit drug determination because time course of drug in oral fluid may resemble that in plasma. Moreover OF can be considered a valid alternative specimen for confirmation testing as in OF drugs are excreted mainly as parent compound [1–4]. In fact police officers, without medical supervision, cannot use invasive methods while are authorized to collect OF samples. In new Italian traffic laws, OF has been considered as an alternative biological specimen for the determination of drug of abuse [5]. A very comprehensive review about analysis of DOA in OF is the paper published by Reinstadler et al. [6]. A number of studies have been published that highlight how both the sample treatment process [7] and the use of hyphenated instrumentation [8,9] are important in order to obtain analytical performances that satisfy the current regulations in terms of sensitivity, selectivity and fast confirmatory analysis. The aim of this study is the development and validation of a quantitative (and confirmatory) method for the analysis of the major substances of abuse founded nowadays during checks by law enforcement, on a salivary sample with LC–MS/MS systems, with a minimal sample pre-treatment, in a single chromatographic run, and with analytical performances able to respect the actual laws. * Corresponding author. E-mail address: ugo.degrazia@istituto-besta.it (U. de Grazia). http://dx.doi.org/10.1016/j.forsciint.2020.110330 0379-0738/© 2020 Elsevier B.V. All rights reserved. Forensic Science International 312 (2020) 110330 Contents lists available at ScienceDirect Forensic Science International journal homepage: www.elsevier.com/locat e/f orsciint