Article Analysis of Tetracyclines in Medicated Feed for Food Animal Production by HPLC-MS/MS Rosa Elvira Gavilán 1,†, Carolina Nebot 1,†,*, Jose Manuel Miranda 1,†, Yolanda Martín-Gómez 2,†, Beatriz Vázquez-Belda 1,†, Carlos Manuel Franco 1,† and Alberto Cepeda 1,† Received: 31 July 2015; Accepted: 15 December 2015; Published: 24 December 2015 Academic Editor: Christopher C. Butler 1 Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Veterinary Medicine, University of Santiago de Compostela, 27002 Lugo, Spain; [email protected] (R.E.G.); [email protected] (J.M.M.); [email protected] (B.V.-B.); [email protected] (C.M.F.); [email protected] (A.C.) 2 Laboratorio de Sanidad Animal, Lugar Barrio Jove de Arriba, 0 S/N, 33290 Gijón, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-982-822-454; Fax: +34-982-254-592 † These authors contributed equally to this work. Abstract: The use of medicated feed is a common practice in animal food production to improve animal health. Tetracyclines and β-Lactams are the groups that are most frequently added to this type of feed. The measurement of the concentration of the analytes in these types of samples is sometimes due to the matrix characteristic, and manufacturers are demanding fast, precise and reproducible methods. A rapid confirmatory method based on a simple extraction protocol using acidified methanol and followed by high performance liquid chromatography coupled to a tandem mass spectrometer for the quantification of four tetracyclines in feed is presented. Validation was performed following the guidelines of Decision 2002/657/EC. Results indicated that the four tetracyclines can be identified and quantified in a concentration range between 50 and 500 mg/kg with recoveries between 84% and 109% and RSD for precision under reproducible conditions between 12% and 16%. Satisfactory results were also obtained with interlaboratory studies and by comparing the method with an HPLC-Fluorescent method. Keywords: feed; antimicrobial; tetracyclines; HPLC-MS/MS; Decision 2002/657/EC 1. Introduction Meat consumption increases each year and, consequently, so does food production of animal origin [1]. To increase production and reduce cost, animals are raised intensively in farms; big farms require greater control of animal health because illnesses can be easily transmitted from one animal to another and cause large economic losses. Therefore, the use of veterinary drugs in food production is very important for controlling and improving animal health. These substances are not only used for therapeutic treatment but also for prophylaxis. Pharmaceuticals can be administered to animals in various forms including tablets, suspensions, emulsions, injections, implants and creams. A common practice is the administration of pharmaceuticals for prophylactic purposes through food. Intensively produced animals are often fed with concentrated feed, which are a mixture of various materials (oats, wheat, barley, rye, cottonseed, and crambe) and additives. Various classes of veterinary medicines are administered through feed as prophylactic treatment, including antibiotics (sulphonamides, tetracyclines and β-Lactams) and anti-parasitic agents (coccidiostats and ivermectins). As in humans, the pharmaceutical dose will depend on Antibiotics 2016, 5, 1; doi:10.3390/antibiotics5010001 www.mdpi.com/journal/antibiotics Antibiotics 2016, 5, 1 2 of 10 the species of animal. Given that the same maker produces feed for various species of animals, an exhaustive quality control of the concentration of the pharmaceutical in the feed should be carried out. According to Kools et al. (2008), the estimation of pharmaceuticals used in food production was 6.051 t of active substance for the European Union. Antibiotics was the most frequently used group (5393 t) followed by anti-parasitic agents (194 t) [2]. The study also indicated that within the antibiotics class, tetracyclines and β-lactams were the group used in the highest amounts. Most controls related to veterinary drugs in animal food production are performed on the final food (egg, milk, muscle, liver, etc.). Some controls are also conducted on water for animals, their food, and their faeces, but most of the analyses are for the evaluation of the presence of substances such as pesticides [3,4], nitrofurans [5] and mycotoxins [6–8]. The presence of substances such as antibiotics in feed samples is normally assessed by the manufacturer and generally with screening methods. Few analytical methods can be found in the literature for antimicrobials in medicated feed [9–11] and most of the methods reported describe analytical techniques for non-medicated feed or residues of antibacterial in food [12–14]. Administration of medicated feed to animal food production can only be conducted under veterinary prescription and vigilance therefore control of the correct antimicrobial concentration in the feed is vital to avoid further feed safety problems. Additionally, it should be highlighted that residue of tetracycline in feed for animal food production are not permitted at any concentration. Based on the common use of tetracycline in food production, the low number of methods available for their analysis in feed for animal food production samples and the manufacture demand, the aim of this research work is to present an reliable and producible HPLC-MS/MS method for the analysis of tetracyclines in medicated feed samples. 2. Results To optimize each tetracycline standard solutions of individual compounds at 1 mg/L in 0.1% formic acid in methanol were infused into the MS. This helped to select the precursor and product ions of each of the tetracyclines and the internal standard (IS). The cone voltage and collision energy were optimised to obtain the most intense signal for each ion. Table1 shows precursor and product ions selected for tetracycline identification, as well as the cone voltage and collision energy employed for each transition. The transition between the precursor ion and product ion 1 and 2 was employed for confirmation of the analytes, and the transition between the precursor ion and product ion 1 was employed for quantification. Table 1. Retention time, cone voltage, collision energy, precursor and product ions employed for ion identification. Retention Precursor > Cone Voltage Collision Time (min) Product Ion (V) Energy 445 > 410 30 29 Tetracycline 11.71 445 > 154 30 27 445 > 226 30 27 445 > 428 30 20 Doxycycline 13.41 445 > 201 30 27 445 > 153 30 20 479 > 260 30 23 Clortetracycline 11.84 479 > 286 30 23 479 > 305 30 23 461 > 426 30 20 Oxytetracycline 12.61 461 > 443 30 20 461 > 408 30 20 465 > 448 30 17 Democlociclina 12.00 465 > 288 30 17 Antibiotics 2016, 5, 1 3 of 10 Antibiotics 2016, 5, 0001 3 of 10 AntibioticsThe 2016 selection, 5, 0001 of the HPLC column and the chromatography method was based on previously3 of 10 The selection of the HPLC column and the chromatography method was based on previously describedThe selection work [15 of]; however,the HPLC the column gradient and wasthe chro modifiedmatography to achieve method better was resolution based on between previously the described work [15]; however, the gradient was modified to achieve better resolution between the eluteddescribed peaks. work [15]; however, the gradient was modified to achieve better resolution between the eluted peaks. elutedThe peaks. best results were achieved with the simplest extraction protocol which employed acidified The best results were achieved with the simplest extraction protocol which employed acidified methanol;The best this results protocol were was achieved previously with described the simplest by AOAC extraction forthe protocol extraction which of employed one tetracycline acidified in methanol; this protocol was previously described by AOAC for the extraction of one tetracycline in feedmethanol; samples this [16 protocol]. Once thewas method previously was described selected, the by validationAOAC forprocedure the extraction was of conducted. one tetracycline in feed samples [16]. Once the method was selected, the validation procedure was conducted. feed Thesamples validation [16]. Once was conducted the method following was select theed, requirement the validation included procedure in the Decision was conducted. 2002/657/EC [17]. The validation was conducted following the requirement included in the Decision 2002/657/EC A totalThe of 20validation feed samples was wereconducted analysed following to determine the re selectivity/specificity.quirement included in The the successful Decision quantification 2002/657/EC [17]. A total of 20 feed samples were analysed to determine selectivity/specificity. The successful of[17]. tetracyclines A total of 20 and feed the samples absence were of interfering analysed to peaks determine at their selectivity/specificity. retention times demonstrated The successful the quantification of tetracyclines and the absence of interfering peaks at their retention times selectivity/specificityquantification of tetracyclines of the method. and Figuresthe absence1 and2 showof interfering SRM chromatograms peaks at their of tetracyclines retention times in a demonstrated the selectivity/specificity of the method. Figures 1 and 2 show SRM chromatograms of blankdemonstrated sample and the inselectivity/specificity a fortified sample at of 50 the mg/kg. method. Figures 1 and 2 show SRM chromatograms of tetracyclinestetracyclines inin aa blankblank samplesample andand inin