A method for the identification and quantification of macrolides (erythromycin, josamycin, tilmicosin, tylosin, spiramycin and neospiramycin) in tilapia fillets by liquid chromatography coupled to quadrupole time of flight mass (LC-QToF) spectrometry is presented. Sample preparation was quite simple and low cost: proteins were precipitated and the analytes were extracted with ethanol, extract was defatted with hexane and concentrated by solvent evaporation. The matrix effect was statistically demonstrated during method validation, in which matrix-matched calibration was applied. Matrix effect mechanism was clarified thanks to the capability of QToF mass spectrometer of generating full scan spectra with accurate mass measurement. The limits of quantification were at least 45% lower than the maximum residue limits. The method was able to identify the studied macrolides with relative m/z errors lower than 2.5. ppm and to monitor two fragment ions per analyte, which is in accordance with the European Community recommendations for the analysis of contaminants in foods. Samples from the retail market of São Paulo State, Brazil, were analyzed by the developed method and none of them presented positive results for the macrolides studied. © 2014 Elsevier Inc.342153162Barcelo, D., Petrovic, M., Challenges and achievements of LC-MS in environmental analysis: 25 years on (2007) TRAC Trends in Analytical Chemistry, 26, pp. 2-11(2009) Veterinary Drug Residues in Food - Maximum Residue Limits Home Page, Rome, Italy, , http://www.codexalimentarius.net/mrls/vetdrugs/jsp/vetd_q-e.jsp, Retrieved from:(20.06.11), Codex AlimentariusCommission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002) Official Journal of the European Communities, 221, pp. 8-36. , EC, European Commission(2011) European Medicines Agency. Maximum Residue Limits Reports Home Page, London, England, , http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/landing/vet_mrl_search.jsp&murl=menus/regulations/regulations.jsp&mid=WC0b01ac058006488e, Retrieved from:(20.06.11)., EMA(2009) Food and Agriculture Organization of the United Nations, , ftp://ftp.fao.org/docrep/fao/012/i1013t/i1013t.pdf, Fishery and Aquaculture Statistics, Rome, Italy, Retrieved from:(17.11.10), FAO(2010) Food and Agriculture Organization of the United Nations. Cultured Aquatic Species Information Programme - Oreochromis niloticus, , http://www.fao.org/fishery/culturedspecies/Oreochromis_niloticus/en#tcN90112, Retrieved from:(17.11.10), FAOGobel, A., McArdell, C.S., Suter, M.J., Giger, W., Trace determination of macrolide and sulfonamide antimicrobials, a human sulfonamidemetabolite, and trimethoprim in wastewater using liquid chromatography coupled to electrospray tandem mass spectrometry (2004) Analitical Chemistry, 76, pp. 4756-4764Hernández, F., Ibáñez, M., Sancho, J.V., Pozo, O.J., Comparison of different mass spectrometric techniques combined with liquid chromatography for confirmation of pesticides in environmental water based on the use of identification points (2004) Analytical Chemistry, 76, pp. 4349-4357Horie, M., Takegami, H., Toya, K., Nakazawa, H., Determination of macrolide antibiotics in meat and fish by liquid chromatography-electrospray mass spectrometry (2003) Analytica Chimica Acta, 492, pp. 187-197Horie, M., Chemical analysis of macrolides antibiotics (1995) Chemical Analysis for Antibiotics Used in Agriculture, pp. 165-205. , AOAC International, Arlington, VA, USA, H. Oka, H. Nakazawa, K. Harada, J.D. MacNeil (Eds.)(2010) The Japan Food Chemical Research Foundation. Positive List System for Agricultural Chemical Residues in Food Home Page, Osaka, Japan, , http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/pages/MRLs-p, Retrieved from:(20.06.11), JFCRFJo, M., Lee, H., Lee, T., Park, K., Oh, E., Kim, P., Horie, M., Simultaneous determination of macrolide residues in fish and shrimp by liquid chromatography-tandem mass spectrometry (2011) Food Science and Biotechnology, 20, pp. 823-827Kanfer, I., Skinner, M.F., Walker, R.B., Analysis of macrolide antibiotics (1998) Journal of Chromatography A, 812, pp. 255-286Lucchetti, D., Fabrizi, L., Esposito, A., Guandalini, E., Di Pasquale, M., Coni, E., Simple confirmatory method for the determination of erythromycin residues in trout: a fast liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry (2005) Journal of Agricultural and Food Chemistry, 53, pp. 9689-9694Brazilian Ministry of Agriculture, Livestock and Supply (2009) Secretariat of Agriculture and Livestock Defense Normative Instruction No. 24 of July 14th Official Union Journal, Section 1:7, July 22nd, , MAPABrazilian Ministry of Agriculture, Livestock and Supply (2010) Secretariat of Agriculture and Livestock. Defense Normative Instruction No. 8 of April 29th. Official Union Journal, Section 1:27, May 3rd, , MAPAMercado Común del Sur (2000) Resolution N° 054/2000, Brasilia, Brazil, , http://200.40.51.218/SAM/GestDoc/PubWeb.nsf/Normativa?ReadForm&lang=POR&id=48287E473D6D9BD8032576AA0055A7F6, Retrieved from:(07.01.11)., MERCOSURMiller, J.C., Miller, J.N., (1988) Statistics for Analytical Chemistry, , Ellis Horwood/John Wiley & Sons, Chichester/New YorkList of antimicrobials of veterinary importance (2007) Paris, France, , http://www.oie.int/downld/Antimicrobials/OIE_list_antimicrobials.pdf, Retrieved from:(13.12.2010), OIE, World Organization for Animal HealthRibani, M., Bottoli, C.B.G., Collins, C.H., Jardim, I.C.S.F., Melo, L.F.C., Validação em métodos cromatográficos e eletroforéticos (2004) Química Nova, 27, pp. 771-780Thompson, M., Stephen, L.R., Wood, R., Laboratory validation of methods of analysis (IUPAC Technical Report) (2002) Pure Applied Chemistry, 74, pp. 835-855(2010) Code of Federal Regulations. Title 21: Food and Drugs, Part 556: Tolerances for residues of new animal drugs in food, pp. 370-386. , http://www.gpoaccess.gov/cfr/index.html, USDA, Washington, DC, Retrieved from:(20.06.2011), USDA, United States Department of Agriculture(2011) Aquaculture Approved Drugs, , http://www.fda.gov/AnimalVeterinary/DevelopmentApprovalProcess/Aquaculture/ucm132954.htm, USFDA, Washington, DC, Retrieved from:(20.06.11)Wang, J., Leung, D., Analysis of macrolide antibiotic residues in eggs, raw milk, and honey using both ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry (2007) Rapid Communication Mass Spectrometry, 21, pp. 3213-3222Wang, J., Analysis of macrolide antibiotics using liquid chromatography-mass spectrometry in food, biological and environmental matrices (2009) Mass Spectrometry Reviews, 28, pp. 50-92