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Multi-Residue Determination of Polar Veterinary Drugs in Livestock and Fishery Products by Liquid Chromatography/ Tandem Mass Spectrometry

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Multi-Residue Determination of Polar Veterinary Drugs in Livestock and Fishery Products by Liquid Chromatography/ Tandem Mass Spectrometry 230 KANDA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 98, NO. 1, 2015 VETERINARY DRUG RESIDUES Multi-residue Determination of Polar Veterinary Drugs in Livestock and Fishery Products by Liquid Chromatography/ Tandem Mass Spectrometry MAKI KANDA, TAKAYUKI NAKAJIMA, HIROSHI HAYASHI, TSUNEO HASHIMOTO, SETSUKO KANAI, CHIEKO NAGANO, YOKO MATSUSHIMA, YUKINARI TATEISHI, SOIchI YOSHIKAWA, YUMI TSURUOKA, TAKEO SASAMOTO, and IchIRO TAKANO Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan Residues of 37 polar veterinary drugs belonging to with microbiological methods, specific chromatographic six families (quinolones, tetracyclines, macrolides, analyses were needed to identify the antibiotics. The identifying lincosamides, sulfonamides, and others) in livestock process was so complicated that it was difficult to identify each and fishery products were determined using a residual drug. validated LC-MS/MS method. There were two key The accuracy of analysis for the residual drugs has been points in sample preparation. First, extraction was required worldwide in recent years. In Japan, the analytic performed with two solutions of different polarity. methodologies used by inspection institutes had to be validated Highly polar compounds were initially extracted until December 13, 2013 according to the notice issued by the with Na2EDTA-McIlvaine’s buffer (pH 7.0). Medium Japanese Ministry of Health, Labour, and Welfare (10, 11). polar compounds were then extracted from the same On the other hand, the simultaneous analysis methodologies samples with acetonitrile containing 0.1% formic for multi-class veterinary drug residues using LC-MS/MS acid. Secondly, cleanup was performed using a have already been reported (8, 9, 12–29). However, the single SPE polymer cartridge. The first extracted trueness and precision of reported analysis using LC-MS/MS solution was applied to the cartridge. Highly polar (8, 9, 12–23, 25, 27) for fluoroquinolones (FQs), tetracyclines compounds were retained on the cartridge. Then, the (TCs), penicillins (PCs), 5-hydroxythiabendazole, and clopidol second extracted solution was applied to the same did not achieve acceptable values according to the “Guidelines cartridge. Both highly and medium polar compounds for the Validation of Analytical Methods for Residual were eluted from the cartridge. This method satisfied Agricultural Chemicals in Food”. Furthermore, the sensitivity the guideline criteria for 37 out of 37 drugs in swine of some analysis was insufficient to detect residual levels of muscle, chicken muscle, bovine muscle, prawn, multi-class veterinary drugs (12, 17–19, 22, 23, 25, 27). On salmon trout, red sea bream, milk, and honey; 35 out the Japanese positive list system, veterinary drugs of which no of 37 in egg; and 34 out of 37 in flounder. The LOQ established maximum residues limits (MRLs) were given the ranged from 0.1 to 5 µg/kg. Residues were detected default regulatory limit (uniform limit of level) at 10 µg/kg. in 24 out of 110 samples and analyzed using the Therefore, the analysis of multi-class drugs needs the LOQ for validated method. each drug to be less than 10 µg/kg. Residues of TCs and FQs have been reported frequently in analyses performed by national institutions in Japan or in the European Union (EU; 30, 31). TC residues were found in swine eterinary drugs are widely used on farms to treat muscle, fish, and honey. The residues of enrofloxacin were found and prevent diseases. However, over-dosing and in shrimp from Asia. Therefore, we need analytical methods to noncompliance with the withdrawal period may V accurately measure the residue concentrations of these drugs. cause drug residues to remain in animal tissues (1, 2). The aim of this study was to determine residues of 37 polar Drug-contaminated livestock and fishery products may have veterinary drugs belonging to six families [quinolones (QLs), a potential risk for the consumer’s health because they can TCs, macrolides (MLs), lincosamides, sulfonamides (SDs), and provoke drug-resistant pathogenic strains of bacteria, allergic others] in livestock and fishery products using a validated LC- reactions, and toxicity (3, 4). Therefore, it is necessary MS/MS method. to monitor livestock and fishery products for the residual By addressing the following five points, we improved veterinary drugs using accurate analysis. We have used two pretreatment procedures and LC-MS/MS conditions: major analytical strategies to measure residual substances, (1) Simple and rapid analysis is desirable to speed up large namely, microbiological screening (5–7) and screening using amounts of sample inspections. LC-MS/MS (8, 9). However, the sensitivity of microbiological (2) Polar veterinary drugs must be simultaneously extracted screening was insufficient to detect residual levels of multi-class from livestock and fishery products. We attempted to use aqueous veterinary drugs. Moreover, when positive results were found solvent on the first extraction and then organic solvent on the second extraction. Different pretreatment procedures, such as Received August 13, 2013. Accepted by JB May 26, 2014. Corresponding author’s e-mail: [email protected]. quick, easy, cheap, effective, rugged, and safe (QuEChERS) tokyo.jp methods (8, 9 12–18, 26) or pressurized liquid extraction (PLE) DOI: 10.5740/jaoacint.13-272 were used recently. By using acetonitrile in the QuEChERS KANDA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 98, NO. 1, 2015 231 method, extraction of TCs, MLs, and FQs was insufficient Before use, the PLS-3 cartridges were conditioned with 5 mL (8, 9, 12, 14, 15, 18, 26). By using other extraction solutions acetonitrile, and then 5 mL Na2EDTA-McIlvaine’s buffer i.e., acidified acetonitrile (13–16, 26), methanol (12, 18), or solution (pH 7.0). methanol-acetonitrile (10, 17), extraction of these drugs was (m) Microtubes.—1.5 mL (Eppendorf Co. Ltd). insufficient as well. As shown in Table 1, log P of these drugs (n) Polypropylene and amber vial tubes.—300 µL (GL was negative, which means that these drugs were soluble in Sciences Inc.). the aqueous phase. Actually, a mixture of water and organic solvent was used (19, 20, 21, 23, 32). Using water at PLE was Reagents significantly more effective for the extraction of QLs, PC V, and SDs (25–29). (a) Water.—Obtained using a Milli-Q system (Millipore (3) During the measurement by LC-MS/MS, the matrix Corp., Billerica, MA). interferes with the ionization of the target compounds, which (b) Solvent.—Acetonitrile (LC grade), hexane (for pesticide precludes the quantification. The matrix interference from residue and polychlorinated biphenyl analysis grade) and livestock and fishery products is removed by a cleanup using methanol (LC grade; Wako Pure Chemical Industries Ltd, the SPE polymer cartridge. Osaka, Japan). (4) To increase the sensitivity, LC conditions (mobile phase, (c) Formic acid (99%).—LC-MS grade (Wako Pure column, and injection volume) and MS/MS parameters were Chemical Industries Ltd). modified. (d) Citric acid monohydrate, Na2EDTA, sodium chloride, (5) The analytical method developed in this study was and anhydrous magnesium sulfate.—Analytical grade (Wako validated in 10 livestock and fishery products: swine muscle, Pure Chemical Industries Ltd). chicken muscle, bovine muscle, prawn, salmon trout, red sea (e) Disodium hydrogen phosphate dihydrate.—Analytical bream, flounder, milk, egg, and honey in accordance with the grade (Merck KGaA, Darmstadt, Germany). Japanese guidelines. (f) Polar extraction solution 1; Na2EDTA-McIlvaine’s buffer solution (pH 7.0).—Prepared by dissolving 30.92 g disodium Experimental hydrogen phosphate dihydrate, 2.73 g citric acid monohydrate, and 37.13 g Na2EDTA in water and diluting to 1 L. Samples (g) Polar extraction solution 2; Acetonitrile containing 0.1% formic acid.—Freshly prepared by mixing 0.1 mL of formic Livestock and fishery products (swine muscle, chicken acid with 100 mL of acetonitrile. muscle, bovine muscle, prawn, salmon trout, red sea bream, (h) Standard (purity grade).—Marbofloxacin (98.0%), flounder, milk, egg, and honey) were purchased from local norfloxacin (98.0%), ciprofloxacin (98.0%), difloxacin (98.0%), supermarkets in Japan and were confirmed to be free of the flumequine (98.0%), oxytetracycline (99.0%), erythromycin targeted analytes in this study. The tissues were minced with an A (98.0%), sulfadiazine (99.0%), sulfathiazole (98.0%), electric household food processor and stored at –20°C. sulfamonomethoxine (99.0%), sulfamethoxazole (99.0%), sulfadimethoxine (99.0%), 5-hydroxythiabendazole (98.0%), Apparatus clopidol (98.0%), and thiabendazole (99.0%) were purchased from Wako Pure Chemical Industries Ltd Ofloxacin (97.7%), (a) LC system.—LC-20A series (Shimadzu Corp., Kyoto, orbifloxacin (99.6%), and lincomycin A (98.0%) were from Japan). Hayashi Pure Medical Industry (Osaka, Japan). Danofloxacin (b) MS system.—API 5500 Qtrap mass spectrometer with (100.0%), enrofloxacin (99.8%), oxolinic acid (98.8%), an electrospray ionization (ESI) interface and Analyst (Version nalidixic acid (99.8%), oleandomycin (96.5%), josamycin 1.4.2) software (AB Sciex, Framingham, MA). (86.8%), sulfamerazine (99.5%), sulfadimidine (99.4%), and (c) LC column.—Triart C18 column (150 × 2.0 mm, 5 µm sulfaquinoxaline (99.6%) were from Kanto Chemical Co. particle size) (YMC Co. Ltd, Kyoto, Japan). (Tokyo, Japan). Sarafloxacin (97.3%), tetracycline (97.7%), (d) Mixer.—Vortex-Genie 2 (Scientific Industries Inc., chlortetracycline
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