Multiclass screening method to detect more than fifty banned substances in bovine bile and urine Simone Moretti a , Francesca Lega b , Lorenzo Rigoni b , Giorgio Saluti a , Danilo Giusepponi a , Antimo Gioiello c , Elisabetta Manuali a , Rosanna Rossi a , Roberta Galarini a, * a Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”, Via G. Salvemini 1, 06123 Perugia, Italy b Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Universit a 10, 35020 Legnaro, PD, Italy c Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo, 1, 06123 Perugia, Italy highlights graphical abstract  A screening method for banned sub- stances was developed in bovine urine and bile.  First method fully validated for the simultaneous detection of 53 substances.  LC-Q-Orbitrap was applied with par- allel reaction monitoring acquisition.  A novel sample clean-up was described to include this wide range of analytes.  The CCbs were assured irrespective to sample-to-sample variability. article info Article history: Received 13 March 2018 Received in revised form 8 June 2018 Accepted 12 June 2018 Available online xxx Keywords: Bile Urine Cattle Anabolic drugs Liquid-chromatography coupled to hybrid high-resolution mass spectrometry abstract A multiclass screening method to detect fifty-three forbidden substances by liquid-chromatography coupled to hybrid high-resolution mass spectrometry (LC-Q-Orbitrap) was developed and validated in bovine bile and urine. Eight classes of compounds were included in the method's scope (b-agonists, corticosteroids, nitroimidazoles, progestins, resorcylic acid lactones (RALs), sedatives, steroids and stil- benes) plus chloramphenicol and dapsone. After hydrolysis, the sample was divided in two aliquots, which followed two parallel purification steps. The reunified extracts were injected and two chro- matographic runs performed in positive and negative ionization mode, respectively. The validation data (60 different samples per matrix) proved that the method was fit for purpose with detection capabilities lower than 1 mgL 1 in both matrices. The combined application of accurate mass acquisition and two- stage mass spectrometry (parallel reaction monitoring) was crucial to achieve suitable selectivity, which is the most critical parameter mainly for urines. Finally, the long-standing problem of the high rate of false positive results for RALs, due to the natural ingestion of mycotoxin, zearalenone, was taken on including all their labelled standards. That allowed a very satisfactory management of this screening test. © 2018 Elsevier B.V. All rights reserved. 1. Introduction Treatments in farm with growth-promoting agents has been prohibited in European Union for over 25 years [1 ,2], although the various commercial controversies and debates, like the so-called * Corresponding author. E-mail address: r.galarini@izsum.it (R. Galarini). Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca https://doi.org/10.1016/j.aca.2018.06.037 0003-2670/© 2018 Elsevier B.V. All rights reserved. Analytica Chimica Acta xxx (2018) 1e12 Please cite this article in press as: S. Moretti, et al., Multiclass screening method to detect more than fifty banned substances in bovine bile and urine, Analytica Chimica Acta (2018), https://doi.org/10.1016/j.aca.2018.06.037