Electrophoresis 2018, 0, 1–8 1 D´ ebora Cecatto Ferreira Ana L ´ ucia de Toffoli Edvaldo Vasconcelos Soares Maciel Fernando Mauro Lanc ¸as Institute of Chemistry of S ˜ ao Carlos, University of S ˜ ao Paulo, S˜ ao Carlos, SP, Brazil Received February 1, 2018 Revised June 6, 2018 Accepted June 7, 2018 Research Article Online fully automated SPE-HPLC-MS/MS determination of ceftiofur in bovine milk samples employing a silica-anchored ionic liquid as sorbent Solid-phase extraction coupled online with high performance liquid chromatography and tandem mass spectrometry was successfully applied to determine low concentrations of ceftiofur antibiotic in bovine milk samples. A silica-anchored ionic liquid was applied as sorbent material to be used as extraction phase in the proposed online system. The mate- rial was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. In order to improve the system reproducibility, the following experimental parameters were optimized: organic solvent percentage, time and sample loading flow rate. Subsequently, the method was validated presenting satisfactory results as adequate selectivity, good linearity and correlation coefficient higher than 0.98. The limit of detection and quantification were 0.1 and 0.7 g/L, respectively. The precision of the methodology was evaluated as repeatability and intermediate precision, with relative standard deviation values lower than 15%. The accuracy of the method ranged from 72.8 to 137% and the minimum and maximum recovery values were 73.4 and 111.3%, respectively. After the validation, seven milk samples were analyzed and although ceftiofur was not detected in any of them the method was demonstrated to be efficient when applied to the analysis of milk samples fortified with the pollutant of interest. Keywords: ceftiofur / ionic liquid / milk analysis / online SPE / residues DOI 10.1002/elps.201800053  Additional supporting information may be found online in the Supporting Infor- mation section at the end of the article. 1 Introduction The fast world population increasing demands a permanent raise on the production of food from animal sources. As a consequence the use of antimicrobials to increase animal Correspondence: Professor Fernando Mauro Lanc ¸ as, Institute of Chemistry at S ˜ ao Carlos, University of S ˜ ao Paulo, Postal code 780, 13560–970 Sao Carlos, SP, Brazil Fax: +(55) 16 3373 9984 E-mail: flancas@iqsc.usp.br Abbreviations: ANVISA, National Sanitary Inspection Agency; C18, octadecyl-silica; ICH, International Conference on Harmonization; ILs, ionic liquids; MERCOSUL, Southern Common Market; MRL, maximum residue limits; MRM, mul- tiple reaction monitoring; NST, Nano Separation Technol- ogy; PAMVET, Waste Analysis Program of Veterinary Medic- inal Products in Foods of Animal Origin; QuEChERS, Quick, Easy, Cheap, Effective, Rugged and Safe; SPE, solid phase extraction production and aiming the treatment of their diseases has also been increased. Besides that, the use of different types of antibiotics as growth promoters leads to an evolution and selection of antibiotic-resistant microorganism in the animals [1, 2]. The antibiotic investigated in the present work belongs to the -lactam family, and to the cephalosporins class [3, 4]. Ceftiofur hydrochloride is a third-generation cephalosporin with a broad spectrum, possessing activity against Gram- positive and Gram-negative bacteria [5, 6]. The molecule has a 3-position linkage of furoic acid thioester that differenti- ates it from other third generation cephalosporins [3]. When the drug is applied, the thioester bond is cleaved to form the active metabolite desfuroylceftiofur, conjugated amino acids and disulfide dimers. The active metabolite has the beta-lactam ring, which is related to the third-generation cephalosporins [7]. Color Online: See the article online to view Figs. 1 and 3 in color. C  2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com