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

Home , Difloxacin, Orbifloxacin, Sarafloxacin, Spiramycin, Sulfadimethoxine, Sulfadimidine

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 (99.1%), doxycycline (98.2%), and tiamulin Bohemia, NY). (99.9%) were from Sigma-Aldrich (St. Louis, MO). Spiramycin (e) Ultrasonic machine.—B5510J-DTH (Branson, Danbury, (96.0%), tilmicosin (98.5%), and tylosin (98.0%) were from Dr. CT). Ehrenstorfer GmbH (Augsburg, Germany). Pirlimycin (86.6%) (f) Centrifuge.—AX-320 (Tomy Seiko Co., Tokyo, Japan). was from Pfizer Japan Inc. (Tokyo, Japan). Mirosamicin (g) Microcentrifuge.—5415R (Eppendorf Co. Ltd, Hamburg, (97.7%) was from Kyoritsu Pharmaceutical Co. (Tokyo, Japan). Germany). (i) Internal standard (IS).—Demeclocycline (92.3%) was (h) Polypropylene centrifuge tubes.—15 mL and 50 mL from Hayashi Pure Medical Industry. (Corning Inc., Corning, NY). (i) Glass volumetric flasks.—50 and 100 mL (SIBATA Preparation of Standard Solutions and Calibration Scientific Technology Ltd, Saitama, Japan). Standards (j) Polymethylpentene and opaque volumetric flasks.—10 mL (VITLAB GmbH, Grossostheim, Germany). (a) Stock standard solutions of 33 individual compounds (k) SPE manifold system.—Vacuum manifold system (GL except TCs (100 µg/mL).—Stock standard solutions were Sciences Inc., Tokyo, Japan). prepared individually. The suitable quantity of standard taking (l) SPE polymer cartridges for the cleanup procedure.— into account the substance purity was weighed in a 50 mL glass InertSepTM PLS-3 cartridge, 20 cc/200 mg (GL Sciences Inc.). volumetric flask. Clopidol was dissolved in 5 mL acetonitrile, 232 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table 1. log P values of veterinary drugs and made up to 50 mL with methanol. Sulfadimidine and oxolinic acid were dissolved in acetonitrile, and made up to Analytes logP 50 mL with acetonitrile. The rest of compounds were dissolved Quinolones in methanol, and made up to 50 mL with methanol. Stock Marbofloxacin –0.5 standard solutions were kept in amber glass vials in the dark at 4°C, under which conditions, they were stable for one year. Norfloxacin –1.0 (b) Mixed standard solutions except TCs (1 µg/mL).—An Ofloxacin –0.4 aliquot (500 µL) of each stock standard solution shown in (a) Enrofloxacin –0.2 was transferred and mixed together in a 50 mL glass volumetric Ciprofloxacin –1.1 flask, and made up to 50 mL with methanol. This mixed standard solution was kept in an amber glass vial in the dark at 4°C, Danofloxacin –0.3 under which conditions this was stable for 3 months. Orbifloxacin 0.9 (c) Stock standard solutions of 4 TCs (1000 µg/mL).— Sarafloxacin 0.3 Stock standard solutions of TCs (oxytetracycline, tetracycline, Difloxacin 1.6 chlortetracycline and doxycycline) were prepared individually. The suitable quantity of standard taking into account Oxolinic acid 1.7 the substance purity was weighed in a 10 mL opaque Nalidixic acid 1.4 polymethylpentene volumetric flask (light-shielding). TCs were Flumequine 2.9 dissolved in methanol and made up to 10 mL with methanol. Tetracyclines The stock standard solutions were kept in polypropylene vials in the dark at –20°C, under which conditions they were stable Oxytetracycline –1.6 for 1 month. Tetracycline –2.0 (d) Mixed oxytetracycline and tetracycline standard solution Chlortetracycline –1.3 (1 µg/mL).—An aliquot (100 µL) of each stock standard solution Doxycycline –0.7 of oxytetracycline and tetracycline shown in (c) was transferred

a and mixed together in a 10 mL opaque polymethylpentene Demeclocycline 0.7 volumetric flask, and made up to 10 mL with acetonitrile Macrolides containing 0.1% formic acid (ACN/FA) immediately before Spiramycin 2.1 use. This solution was diluted 10 times with ACN/FA. Tilmicosin 3.6 (e) Working standard solutions for 35 veterinary drugs (except for chlortetracycline and doxycycline) (from 0.001 Mirosamicin 2.0 to 0.1 µg/mL).—Working standard solutions were prepared Oleandomycin 2.6 immediately before use by serial dilution of each mixed standard Erythromycin A 2.7 solution shown in (b) and (d) with ACN/FA. (f) Matrix-matched standard solutions for 35 veterinary Tylosin 1.0 drugs (from 0.025 to 50 ng/mL).—Calibration curves for Josamycin 2.9 35 veterinary drugs (except chlortetracycline and doxycycline) Lincosamides were obtained from matrix-matched calibration samples. Blank Lincomycin A 0.2 samples were prepared as described in the Sample Preparation section. Matrix-matched standard solutions were prepared by Pirlimycin 1.7 mixing an aliquot (500 µL) of blank solution and the appropriate Sulfonamides volume of working standard solutions shown in (e), and then Sulfadiazine –0.1 made up to 1 mL with ACN/FA, e.g., a 0.025 ng/mL solution Sulfathiazole 0.1 was made by mixing an aliquot (500 µL) of blank solution and the working standard solution (0.001 µg/mL, 25 µL), and then Sulfamerazine 0.1 made up to 1 mL. Sulfadimidine 0.3 (g) Mixed chlortetracycline and doxycycline standard Sulfamonomethoxine 0.8 solution (1 µg/mL).—An aliquot (100 µL) of each stock Sulfamethoxazole 0.9 standard solution of chlortetracycline and doxycycline shown in (c) was transferred and mixed together in a 10 mL opaque Sulfaquinoxaline 1.7 polymethylpentene volumetric flask, and made up to 10 mL Sulfadimethoxine 1.6 with ACN/FA immediately before use. This solution was diluted Others 10 times with ACN/FA. Thiabendazole 2.5 (h) Working standard solutions for chlortetracycline and doxycycline (from 0.001 to 0.1 µg/mL).—Working standard 5-hydroxythiabendazole 2.1 solutions were prepared immediately before use by serial Clopidol 2.6 dilution of the mixed standard solution shown in (g) with Tiamulin 5.6 ACN/FA. a The internal standard material for the quantification of chlortetracycline (i) IS.—Demeclocycline was the IS for the quantification of and doxycycline. chlortetracycline and doxycycline. Demeclocycline (10.9 mg) was accurately weighed in a 10 mL opaque polymethylpentene Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 233 volumetric flask, dissolved in methanol, and made up to 10 mL 50 mL polypropylene centrifuge tubes containing 1 mL of 25% with methanol. The stock IS solution (1000 µg/mL) was kept NaCl solution (B). The tube (B) was vortexed for 1 min., and in polypropylene vials in the dark at –20°C, under which then centrifuged at 9600 × g for 10 min at 4°C. The supernatant conditions the solution was stable for 1 month. Working IS was loaded to the conditioned PLS-3 cartridge at approximately solutions (from 0.01 to 1 µg/mL) were prepared immediately 1 mL/min. The target compounds were retained on the cartridge, before use by serial dilution of the stock IS solution with while the solution containing the matrix of food was passed ACN/FA. through the cartridge. The cartridge was washed with 5 mL of (j) IS calibration standard solutions for chlortetracycline water, and then vacuum-dried for 3 min at a pressure of 10 mm Hg. and doxycycline (from 0.025 to 50 ng/mL).—Calibration In addition, the second extraction from the remaining substance curves for chlortetracycline and doxycycline were obtained in tube (A) was performed as shown in the “Second extraction” from IS calibration samples. IS calibration standard solutions stage of Figure 1. The characteristics of the remaining matrixes were prepared by mixing the working IS solution shown in (i) were varied and depended on the different type of samples, as (0.01 µg/mL, 100 µL) and the appropriate volume of working well as the pellets or the insoluble matrix floating on the top solutions shown in (h), and made up to 1 mL with ACN/FA, of the hexane layer. The following procedure was used for all e.g., a 0.025 ng/mL solution was made by mixing the working sample types. Water (2 mL) was added to the tube (A) and then IS standard solution (0.01 µg/mL, 100 µL) together with the (A) was vortexed. Subsequently, 10 mL ACN/FA was added. working standard solution for chlortetracycline and doxycycline The tube (A) was vortexed again for 1 min, and ultrasonicated (0.001 µg/mL, 25 µL), and then brought to 1 mL volume. for 10 min. Magnesium sulfate was added for dehydration (3 g each for bovine muscle, swine muscle, chicken muscle, prawn, LC Separation Conditions milk, and honey). A 4 g amount of magnesium sulfate was added for salmon, red sea bream, and flounder; 5g was added for egg. (a) Mobile phase.—The 0.05% formic acid solution was Then tube (A) was vigorously shaken for 1 min, and centrifuged prepared by mixing 0.5 mL of formic acid with 1 L water. (A) at 1800 × g for 10 at 4°C. The 0.05% formic acid solution and (B) acetonitrile were mixed As shown as the black arrow in Figure 1, the organic phase using the pump in gradient mode as follows: 5% B (3 min); was used as the elution solution for the PLS-3 cartridge 5–90% B (12 min); 90% B (5 min); 90-5% B (0.1 min); and previously loaded with Na2EDTA–McIlvaine’s buffer layer. 5% B (5 min). The eluate from the cartridge was collected into an opaque (b) Flow rate.—0.3 mL/min. polymethylpentene volumetric flask. (c) Column temperature.—40°C. The eluate was made up to 10 mL with ACN/FA. An aliquot (d) Injection volume.—2 µL. (1 mL) was transferred to a microtube, diluted to 2-fold with ACN/FA, and centrifuged at 16 000 × g for 5 min at 4°C. The MS/MS Conditions supernatant was transferred into an amber polypropylene vial tube. The resultant solution was analyzed by LC-MS/MS twice (a) Ionization mode.—Positive-ion ESI. on the same day. Each quantitative value was taken as a mean (b) Ion spray voltage.—5500 V. of two measurements. (c) Source temperature.—650°C. (d) Entrance potential.—10 V. Single-Laboratory Validation Tests with Spiked Samples (e) Curtain gas pressure.—20 psi (nitrogen). (f) Collision gas pressure.—7 psi (nitrogen). The method was validated according to the guidelines of the (g) Ion source gas pressure 1.—80 psi (nitrogen). Japanese Ministry of Health, Labour, and Welfare. Selectivity (h) Ion source gas pressure 2.—40 psi (nitrogen). was confirmed by analyzing blank samples. Trueness, (i) Acquisition function.—Selected reaction monitoring repeatability (RSDr), and within-run reproducibility (RSDWR) (SRM); the SRM program is shown in Table 2. were determined by means of the recoveries using samples spiked with 37 veterinary drugs and demeclocycline at levels Sample Preparation of 10 or 100 µg/kg, performed with two samples per day over five different days. LOQs and LODs were estimated from the The schematic procedure of sample preparation is shown in repeatability data of the blank samples spiked with 0.1, 0.25, Figure 1. For the sample preparation, glass vessels were not 0.5, 1, 2.5, and 5 µg/kg for each of the 37 veterinary drugs used, because silica in the glass could make an interference examined. LOQs were calculated as 10 times the SD, and LODs signal during LC-MS/MS analysis of TCs. were calculated as 3 times the SD using the Analyst software Thoroughly minced sample (5.0 g) was poured in 50 mL (AB Sciex). polypropylene centrifuge tubes (A). IS was spiked at a level of 10 µg/kg. Na2EDTA–McIlvaine’s buffer (pH 7.0, 20 mL) was Results and Discussion added. The tube (A) was vortexed for 1 min. A 5 mL amount of hexane was added. The tube (A) was vortexed again for 1 min, LC-MS/MS Parameters ultrasonicated for 10 min, and then centrifuged at 9 600 × g for 20 min at 4°C. The hexane layer was discarded by pipetting. The MS scans of the 37 veterinary drugs revealed that the Hexane washing was used at all sample types to ease the most abundant molecular ion was the protonated molecule operations. [M+H]+. As each [M+H]+ is a precursor ion, a further As shown in “First extraction” of Figure 1, the MS/MS scan was performed after the collision energy was Na2EDTA–McIlvaine’s buffer layer was transferred into new increased. Two fragment ions (corresponding to quantitative 234 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table 2. SRM parameters

Retention time, Declustering Collision Collision cell exit Analytes Transition, m/z min potential, V energy, eV potential, V Ion ratio, %c

Quinolones

Marbofloxacin 363.0→320.1a 5.9 81 21 20 52.2 363.0→121.9 79 10

Norfloxacin 320.0→276.2a 6.0 96 23 12 34.7 320.0→230.9 53 12 Ofloxacin 362.0→318.0a 6.0 46 29 16 84.2 362.0→261.0 41 16 Ciprofloxacin 332.0→288.1a 6.1 81 25 24 110 332.0→231.1 57 18 Danofloxacin 358.0→82.0a 6.2 81 70 23 57.9 358.0→255.0 65 23 Enrofloxacin 360.2→316.0a 6.3 116 29 10 66.5 360.2→245.0 36 8 Orbifloxacin 396.0→295.1a 6.4 116 35 18 41.4 396.0→351.9 27 18 Sarafloxacin 385.9→341.9a 6.6 116 27 22 86.7 385.9→298.8 39 22 Difloxacin 400.0→356.0a 6.6 116 29 20 85.0 400.0→299.1 45 16 Oxolinic acid 261.9→243.9a 7.7 61 27 18 16.9 261.9→215.9 41 18 Nalidixic acid 232.9→215.0a 8.5 56 21 16 96.6 232.9→186.9 35 14 Flumequine 262.0→201.9a 8.6 66 41 14 42.4 262.0→126.1 67 20 Tetracyclines

Oxytetracycline 461.1→426.1a 6.1 50 27 26 59.5 461.1→443.1 19 24 Tetracycline 445.1→410.1a 6.3 36 29 24 15.8 445.1→225.9 77 16 Chlortetracycline 479.1→443.9a 6.9 91 29 28 74.4 479.1→462.0 23 26 Doxycycline 445.1→428.1a 7.0 50 25 24 9.6 445.1→154.0 37 12 Demeclocyclineb 465.1→429.9a 6.6 76 31 22 56.7 465.1→288.9 45 18 Macrolides

Spiramycin 843.4→174.0a 6.5 6 45 22 28.6 843.4→142.2 45 10 Tilmicosin 869.4→174.0a 7.0 1 57 40 20.0 869.4→696.4 57 16 Mirosamicin 728.3→158.0a 7.3 6 37 12 16.2 728.3→116.0 61 12 Oleandomycin 688.4→158.2a 7.4 116 35 10 52.2 688.4→544.3 23 36 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 235

Table 2. (continued)

Retention time, Declustering Collision Collision cell exit Analytes Transition, m/z min potential, V energy, eV potential, V Ion ratio, %c

Erythromycin A 734.3→158.1a 7.6 86 39 10 80.3 734.3→576.3 27 34

Tylosin 916.4→174.0a 7.7 1 51 14 10.3 916.4→101.0 85 12 Josamycin 828.3→109.2a 8.6 11 67 14 68.8 828.3→174.0 43 16 Lincomycins

Lincomycin A 407.1→126.2a 5.6 121 37 12 12.0 407.1→359.1 27 22 Pirlimycin 411.0→112.1a 6.6 101 31 8 18.9 411.0→363.2 25 18 Sulfonamides

Sulfadiazine 251.0→155.9a 5.8 81 21 14 73.7 251.0→107.9 33 18 Sulfathiazole 255.9→155.9a 6.0 56 21 14 52.3 255.9→107.9 33 16 Sulfamerazine 264.9→155.9a 6.4 101 25 14 88.2 264.9→107.9 37 18 Sulfadimidine 278.9→155.9a 6.8 56 23 12 58.3 278.9→107.9 25 16 Sulfamonomethoxine 281.0→107.9a 7.0 191 35 10 42.9 281.0→155.9 17 12 Sulfamethoxazole 253.9→155.9a 7.4 76 23 12 69.0 253.9→107.9 33 16 Sulfaquinoxaline 300.9→155.9a 7.9 66 23 14 52.4 300.9→107.9 39 10 Sulfadimethoxine 310.9→155.9a 7.9 81 29 14 39.3 310.9→107.9 39 10 Others

5-hydroxythiabendazole 217.8→190.9a 5.5 51 37 14 38.5 217.8→146.8 47 22 Clopidol 191.9→100.9a 5.7 171 37 16 25.6 192.9→155.9 33 14 Thiabendazole 201.9→174.9a 6.0 161 37 14 70.8 201.9→131.0 45 12 Tiamulin 494.3→192.2a 8.1 86 29 14 63.3

494.3→119.0 61 12

a Ion used for quantification. b The internal standard material for the quantification of chlortetracycline and doxycycline. c The relative ion abundance ratio of the selected product ions for the standard solution, 10 ng/mL of each compound. 236 Kanda et al.: Journal of aoaC InternatIonal Vol. 98, no. 1, 2015

Highly polar veterinary drugs (A) (A) (A) medium polar veterinary drugs

Vortex (1 min) Vortex (1 min) Hexane Discard by pipetting Ultrasonicate (10 min) Centrifuge (9600 xg,20 min, 4oC) Sample (5g) +Hexane (5 mL) +I.S.

Na2EDTA–McIlvain’s buffer (pH 7, 20 mL)

Load to PLS-3 (200 mg, 20 mL) First extraction

(B) (B)

Vortex (1 min) Wash with water (5 mL) Aqueous phase Centrifuge (9600 xg,10 min, 4oC) Vaccum-dry (3 min) Discard +25% NaCl sol. (1 mL) the passed through solution Collect Second extraction the eluate solution

Remaining matrix

(A) (A) (A) (A) Supernatant

Vortex (1 min) Vortex (1 min) Violently shake (1 min) Ultrasonicate (10 min) Centrifuge Using the second (1800 xg,10 min, 4oC) extracted solution as the elution solution for PLS-3 +Water (2 mL) +Acetonitrile containing + MgSO4 0.1% formic acid (10 mL) (3, 4 or 5 g)

Analyse Aliquot Supernatant by LC/MS/MS

Make up to 10 mL Transfer to Dilute by 2-fold Pour into a microtube an amber Centrifuge polypropylene (16000 xg, 5 min , 4oC) vial tube

Figure 1. Schematic representation of the sample preparation procedure for the analysis of 37 veterinary drugs in livestock and fi shery products. and confi rmative ions) were monitored for each of the 37 metal ion impurities remaining in the C18 column, which made veterinary drugs (Table 2). Several MS parameters including their peaks broad. The novel organic hybrid silica base column ion-spray voltage, source temperature, declustering potential, (YMC-Triart) has been reported to reduce metal ion impurities entrance potential, and four gas pressures were systematically and achieve good chromatographic retention and separation of varied according to the manual of fl ow injection analysis, and metal chelating and hydrophilic compounds. Using the column, we selected the conditions that yielded the best sensitivity, as the peak shapes of TCs and QLs were better, and the sensitivities listed in the Experimental section. In particular, we noted the were improved by a factor of 3. Thiabendazole, sulfathiazole, curtain gas, ion source gas 1 and 2 conditions that measured sulfamerazine, and clopidol diluted with an organic solvent macrolides with high sensitivity. Because MS scans of some of were poorly retained on the column because the organic solvent penicillins showed that the most abundant molecular ion was may act as a part of the mobile phase. We minimized the drug the deprotonated molecule [M-H]–, it was excluded from the injection volume to 2 µL, which resulted in the peak widths at analytes in this study. half height ranging from 0.1 to 0.44 min and the tailing factors ranging from 0.85 to 1.21. LC Conditions Extraction and Cleanup Procedure LC conditions to determine multi-class veterinary drugs in livestock and fi shery products were previously reported The extraction and cleanup procedure was developed using by our laboratory (9), in which a gradient mixture of 0.1% 11 veterinary drugs, norfl oxacin, ciprofl oxacin, chlortetracycline, formic acid in 10 mM ammonium acetate and acetonitrile as doxycycline, 5-hydroxythiabendazole, clopidol, erythromycin the mobile phase and a C18 column were used. However, the A, spiramycin, lincomycin A, oxolinic acid, and sulfadimidine. sensitivities of TCs and QLs were low under these conditions. Among these drugs, norfl oxacin, ciprofl oxacin, chlortetracycline, Because the ionization mode of these drugs was positive-ion doxycycline, 5-hydroxythiabendazole, and clopidol did ESI, the ammonium ion which lowered the sensitivity of not achieve acceptable values following the guidelines of [M+H]+ was excluded from the mobile phase. The peak shapes Japanese Ministry of Health, Labour, and Welfare (10, 11) by of FQs and thiabendazole were split. The peak shapes of TCs, using our reported QuEChERS methods (8, 9), because these sulfathiazole, sulfamerazine, clopidol, and oxolinic acid were compounds were soluble in the aqueous phase. Erythromycin poor. The tailing factors of these drugs were 0.3–0.6. TCs and A and spiramycin represent the macrolides class. Lincomycin QLs which are strong metal chelating compounds interact with A represents the lincosamides. Oxolinic acid and sulfadimidine Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 237

Acetonitrile containing 0.1% formic acid Na2EDTA-MacIlvain Buffer 120% (a) 100%

80%

60%

40%

a 20%

0% First extraction Second extraction using Na2EDTA-MacIlvain Buffer using Acetonitrile containing 0.1% formic acid 120% (b) Extracted Ratio (%) 100% 80% 60% 40% 20% 0%

clopidol tylosin norfloxacin doxycycline spiramycin Lincomycin ciprofloxacin oxolinic acid sulfadimidine chlortetracycline

5-hydroxythiabendazole Figure 2. Effect on the extracted ratios of 11 veterinary drugs from swine muscle, twice extraction by the same solvent (a), by the different solvents (b). Mean of 5 replications. had higher accuracy than other drugs on LC-MS/MS. These follows. Eleven drugs spiked into a swine muscle were extracted compounds served as indicators, showing that the LC-MS/MS with Na2EDTA-McIlvaine’s buffer. The extraction solution was measurements are stable. After spiking 50 µL of a 1 µg/mL loaded onto the PLS-3 cartridge, and was eluted with ACN/FA. standard solution of these drugs into a minced swine muscle, the This eluate was analyzed by LC-MS/MS. The recovery rates (a) following studies were performed. At this time, the drugs were were calculated. Na2EDTA-McIlvaine’s buffer spiked with 11 quantified by using matrix-matched calibration standard curves. drugs was loaded onto the PLS-3 cartridge. The recovery rates Veterinary drugs were extracted from the sample using from the PLS-3 cartridge (b) were calculated. The extraction an ultrasonic machine (33–35). This procedure allowed the rates by Na2EDTA-McIlvaine’s buffer were corrected (a) using simultaneous handling of many samples and lowered the risk of (b). The pH of the buffer was set as 7.0 because the retention contamination. The sufficient extraction ability was confirmed of drugs was better than at pH 4. Na2EDTA was added to the using the incurred swine muscle containing chlortetracycline. buffer because the extraction of TCs and QLs were better with As extraction solvents, we compared ACN/FA used on our buffer containing Na2EDTA which had the ability to chelate modified QuEChERS method (9) and Na2EDTA-McIlvaine’s divalent cations (8, 13, 16, 19, 22). As shown in Figure 2a, the buffer used on our antibiotic extraction (5–7). The extracted rates extraction rate of each drug, i.e., norfloxacin, ciprofloxacin, of 11 drugs by Na2EDTA-McIlvaine’s buffer were calculated as chlortetracycline, doxycycline, and lincomycin A was better with

Non-combination of the eluted solution Combination of the eluted solution 120% (a) a 100%

80%

60% 40%

Recovery Recovery Ratio (%) 20%

15 14 (b)

a 13 12 11 10 9 8 7 6 5 4

Rate of matrix effect 3 2 1 0

clopidol tylosin norfloxacin doxycycline spiramycin Lincomycin ciprofloxacin oxolinic acid sulfadimidine chlortetracycline

5-hydroxythiabendazole Figure 3. Effect of the two conditions eluting from the SPE polymer cartridge on the recovery rate of 11 veterinary drugs (a), the rate of matrix effect (b). Mean of 5 replications. 238 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

(a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle (a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle

4 3 5 5x10 1x10 5x10 1x104 Marbofloxacin Oxolinic acid

0 0 0 0 5x104 1x103 Norfloxacin 5x105 1x104 Nalidixic acid

0 0 15x104 1x103 0 0 3 Ofloxacin 5x104 1x10 Flumequine

0 0 4 5x10 1x103 0 0 Ciprofloxacin 5x104 1x103 Oxytetracycline

0 0 1x104 1x103 Danofloxacin 0 0 5x104 1x103

Intensity Tetracycline Intensity

0 0 4 10x10 1x103 Enrofloxacin 0 0 1x104 1x103 Chlortetracycline

0 0 10x104 1x103 Orbifloxacin 0 0 5x104 1x103 Doxycycline

0 0 5x104 1x103 Sarafloxacin 0 0 1x104 1x103 Demeclocycline 0 0 3 10x104 1x10 Difloxacin

0 0 5 6 7 8 9 5 6 7 8 9 Retention time (min) 0 0 5 6 7 8 9 5 6 7 8 9 Retention time (min)

(a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle (a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle 3 3 4x10 1x10 2x105 2x103 Spiramycin Sulfadiazine

0 0 0 0 4x103 1x103 Tilmicosin 2x105 1x103 Sulfathiazole

0 0

4 5x10 1x103 0 0 Mirosamaycin 3 1x105 1x10 Sulfamerazine

0 0 3 3x104 1x10 Oleandomycin 0 0

3 1x105 1x10 Sulfadimidine

0 0

3 3x104 1x10 Erythromycin A 0 0 3 2x104 1x10 Sulfamonomethoxine Intensity Intensity

0 0 3 1x104 1x10 Tylosin 0 0

1x105 1x103 Sulfamethoxazole 0 0

5x104 1x103 Josamycin

0 0

1x105 1x103 0 0 Sulfaquinoxaline

5x105 1x104 Lincomycin A

0 0

5 3 0 3x10 1x10 0 Sulfadimethoxine 1x105 1x103 Pirlimycin

0 0 0 0 5 6 7 8 9 5 6 7 8 9 5 6 7 8 9 5 6 7 8 9 Retention time (min) Retention time (min)

Figure 4. Chromatograms obtained in the MRM mode (quantification transition) for swine muscle spiked with 10 mg/kg of 37 veterinary drugs (a), and for corresponding blank swine muscle (b). Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 239

(a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle (2) The second extracted solution was re-used as the elution 5-hydroxythiabendazole solution. The eluate was diluted by 2-fold with ACN/FA, and analyzed by LC-MS/MS.

1x105 2x103 On (1) and (2) conditions, the recovery rates of 11 veterinary Clopidol drugs were the same (Figure 3a). However, the matrix effects were dramatically different. The matrix effect was defined as 0 0 the ratio of the slope of the matrix-matched calibration curve 3x105 1x103 Thiabendazole Intensity and the standard solution calibration curve. On the condition of (1), strong matrix enhancements were found for norfloxacin, 0 0 ciprofloxacin, chlortetracycline, doxycycline, spiramycin, and 3x105 1x103 Tiamulin lincomycin A. In contrast, the matrix enhancements were not observed under (2) conditions. Because the pork fatty acids and

0 0 5 6 7 8 9 5 6 7 8 9 phospholipids were reported to be retained by the SPE polymer Retention time (min) cartridge (36, 37), the interfering matrix was considered to be Figure 4. (continued) Chromatograms obtained in the MRM mode cleaned-up when the second extraction solution was passed (quantification transition) for swine muscle spiked with 10 mg/kg through the SPE polymer cartridge (Figure 3b). Finally, we of 37 veterinary drugs (a), and for corresponding blank swine muscle (b). chose the extraction and cleanup procedure shown in Figure 1.

Na2EDTA-McIlvaine’s buffer than with ACN/FA. The second Instrument Performance extraction using Na2EDTA-McIlvaine’s buffer did not improve recovery rates. The first extraction using Na2EDTA-McIlvaine’s Figure 4a shows the SRM chromatograms obtained from buffer and second extraction step using ACN/FA improved swine muscle spiked with 10 µg/kg of 37 veterinary drugs and recovery rates to over 70%, except for chlortetracycline and demeclocycline. No matrix effect was observed on peak shape doxycycline, which were unstable in solution. Therefore, in all samples. The retention time determined for the spiked polar veterinary drugs were extracted with two different polar samples was not significantly different from that determined for solvents, Na EDTA-McIlvaine’s buffer (pH 7.0) and ACN/FA. 2 the standards. The relative ion abundance ratios of the selected Subsequently, we evaluated the two conditions to elute the compounds from the SPE polymer cartridge which retained the product ions for each compound are shown in Table 2 together with those of the standard solutions. All of the relative ion compounds first-extracted by Na2EDTA-McIlvaine’s buffer. (1) A new ACN/FA (10 mL) was used as the elution solution. abundance ratios of the spiked samples were within 20% of those The resultant eluate and the second extracted solution were of the standard solutions, which satisfied the permitted tolerance mixed and analyzed by LC-MS/MS. required in the EU guidelines (38). These results indicated that

swine muscle chicken muscle bovine muscle prawn salmon traut red sea bream flounder milk egg honey

2.2

2.0

1.8

1.6

a 1.4

1.2

The slope slope The ratio 1.0

0.8

0.6

0.4

0.2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Analytes

Figure 5. Slope ratio between matrix-matched and solvent calibrations. The compliance interval covering the range between 0.8 and 1.2 for the tolerable matrix effect was plotted. Veterinary drug code: (1) marbofloxacin; (2) norfloxacin; (3) ofloxacin; (4) ciprofloxacin; (5) danofloxacin; (6) enrofloxacin; (7) orbifloxacin; (8) sarafloxacin; (9) difloxacin; (10) oxolinic acid;1) (1 nalidixic acid; (12) flumequine; (13) oxytetracycline; (14) tetracycline; (15) chlortetracycline; (16) doxycycline; (17) demeclocycline (the internal standard material for the quantification of chlortetracycline and doxycycline); (18) spiramycin; (19) tilmicosin; (20) mirosamycin; (21) oleandomycin; (22) erythromycin A (23) tylosin; (24) josamycin; (25) lincomycin A; (26) pirlimycin; (27) sulfadiazine; (28) sulfathiazole; (29) sulfamerazine; (30) sulfadimidine; (31) sulfamonomethoxine; (32) sulfamethoxazole; (33) sulfaquinoxaline; (34) sulfadimethoxine; (35) 5-hydroxythiabendazole; (36) clopidol; (37) thiabendazole; (38) tiamulin. 240 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table 3. Validation results of veterinary drugs Swine muscle Chicken muscle

Nalidixic acid 93 (6; 9) 101 (4; 6) 0.5 10c 89 (3; 3) 95 (3; 4) 0.5 10c Flumequine 91 (5; 5) 97 (3; 5) 0.2 500 88 (5; 5) 96 (3; 4) 0.2 500 Tetracyclines Oxytetracycline 79 (7;10) 77 (4; 8) 1 72 (3; 9) 75 (4; 6) 1

Tetracycline 79 (11; 9) 80 (4; 8) 1 200e 78 (4; 8) 72 (4; 5) 1 200e Chlortetracycline 92 (9;12) 93 (4; 7) 2 101 (10;13) 95 (6;12) 2 Doxycycline 83 (9; 9) 81 (2; 6) 0.5 50 96 (9; 9) 88 (5;10) 1 50 Demeclocyclinef 76 (12;10) 69 (3;11) 0.5 59 (9;14) 75 (5; 8) 1 Macrolides Spiramycin 85 (8; 9) 87 (13;10) 1 200 94 (15;12) 83 (9;13) 1 200 Tilmicosin 92 (10;11) 94 (4; 5) 1 100 82 (8;14) 100 (5; 8) 1 70 Mirosamycin 87 (7; 10) 96 (2; 9) 0.2 50 83 (6; 10) 96 (3; 3) 0.2 40 Oleandomycin 94 (6; 6) 98 (3; 3) 0.5 100 87 (7; 6) 100 (8; 7) 0.5 200 Erythromycin A 100 (7; 8) 98 (4; 4) 0.5 50 91 (4; 3) 98 (2; 5) 0.5 50 Tylosin 82 (6; 6) 91 (4; 7) 0.2 50 73 (9;10) 80 (8; 9) 0.2 50 Josamycin 88 (3; 3) 90 (4; 4) 0.2 40 82 (4; 6) 91 (3; 6) 0.2 40 Lincosamides Lincomycin A 92 (4; 4) 92 (3; 8) 0.5 200 85 (5; 5) 94 (4; 4) 0.5 200

Pirlimycin 78 (6; 5) 77 (6; 7) 0.2 10c 75 (6; 7) 79 (5; 6) 0.2 10c Sulfonamides Sulfadiazine 98 (4; 7) 102 (5; 8) 0.2 100 96 (4; 5) 109 (5; 6) 0.2 100 Sulfathiazole 96 (4; 7) 109 (6;10) 0.5 100 94 (6; 6) 108 (6;10) 0.5 100 Sulfamerazine 96 (8; 8) 109 (7; 8) 0.5 100 89 (8;10) 101 (10; 7) 0.2 10c Sulfadimidine 97 (7; 7) 104 (5; 6) 0.5 100 91 (5; 5) 105 (4; 5) 0.2 100 Sulfamonomethoxine 95 (10;11) 108 (3; 4) 2 20 91 (6; 6) 101 (4; 4) 2 100 Sulfamethoxazole 95 (4; 4) 102 (3; 4) 0.5 20 92 (4; 5) 98 (5; 4) 0.5 20 Sulfaquinoxaline 92 (7; 10) 97 (3; 9) 1 10 c 86 (6; 7) 95 (4; 5) 1 50 Sulfadimethoxine 84 (5; 4) 96 (3; 7) 0.2 200 89 (4; 6) 99 (2; 2) 0.2 50 Others Thiabendazole 84 (7; 6) 96 (2; 4) 0.2 83 (5; 5) 98 (7; 6) 0.2 100g 50g 5-hydroxythiabendazole 77 (5; 6) 86 (2; 5) 0.1 81 (3; 6) 93 (3; 3) 0.1 Clopidol 89 (4; 6) 99 (4; 4) 1 200 89 (3; 3) 99 (4; 3) 0.5 5000 Tiamulin 84 (6; 6) 87 (4; 7) 0.2 40 79 (7; 5) 86 (4; 5) 0.2 100 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 241

Table 3. (continued) Bovine muscle Prawn

Nalidixic acid 88 (5; 8) 96 (2; 3) 0.5 10c 91 (3; 7) 96 (1; 3) 0.5 10c Flumequine 86 (4; 7) 97 (2; 6) 0.2 500 90 (5; 9) 96 (2; 4) 0.2 10c Tetracyclines Oxytetracycline 70 (5; 6) 73 (3; 6) 1 77 (9;14) 78 (3; 6) 2 200

c Tetracycline 71 (6; 7) 71 (5; 6) 1 200e 75 (7; 6) 78 (5; 8) 1 10 Chlortetracycline 106 (5; 9) 96 (2;11) 2 92 (7; 8) 84 (5; 7) 2 10c Doxycycline 90 (5; 7) 90 (4;10) 1 100 81 (7;10) 77 (3; 6) 1 10c Demeclocyclinef 61 (9;11) 69 (3;12) 1 64 (6; 7) 75 (5; 9) 1 Macrolides Spiramycin 87 (9; 8) 85 (8;10) 1 200 86 (7; 8) 90 (5;10) 1 200 Tilmicosin 91 (9; 8) 99 (2; 3) 0.2 100 93 (5;10) 96 (3; 7) 0.2 10c Mirosamycin 86 (4; 8) 97 (4; 5) 0.2 10c 82 (4; 6) 91 (2; 7) 0.2 10c Oleandomycin 91 (5; 7) 103 (3; 5) 1 50 95 (2; 5) 101 (1; 3) 0.2 10c Erythromycin A 93 (5; 7) 102 (3; 5) 1 50 99 (5; 5) 102 (2; 2) 1 200 Tylosin 81 (5; 7) 90 (1; 6) 1 50 82 (6; 9) 92 (4; 5) 0.1 100 Josamycin 85 (5; 7) 96 (2; 4) 1 10c 91 (4; 7) 94 (3; 4) 0.5 10c Lincosamides Lincomycin A 94 (15;12) 83 (9;13) 2 200 92 (2; 5) 98 (2; 5) 0.5 100 Pirlimycin 71 (3; 3) 76 (3; 4) 0.2 100 82 (4; 9) 87 (2; 4) 0.2 10c

Sulfonamides Sulfadiazine 103 (4; 5) 118 (4; 4) 1 100 95 (4; 6) 101 (4; 4) 0.2 10c Sulfathiazole 100 (5; 6) 114 (3; 5) 1 100 91 (4; 6) 101 (2; 8) 1 10c Sulfamerazine 93 (6; 6) 102 (2; 5) 0.5 100 93 (3; 5) 99 (2; 4) 0.2 10c Sulfadimidine 92 (3; 4) 104 (1; 4) 0.5 100 93 (4; 5) 100 (2; 2) 0.2 10c Sulfamonomethoxine 92 (4; 6) 99 (2; 2) 2 10 96 (6; 6) 100 (3; 3) 1 10c Sulfamethoxazole 89 (5; 8) 99 (2; 3) 0.5 10c 96 (3; 5) 97 (2; 2) 0.5 10c Sulfaquinoxaline 82 (5; 8) 94 (3; 4) 0.5 100 91 (5; 10) 92 (2; 4) 0.2 10c Sulfadimethoxine 85 (5; 7) 98 (3; 3) 0.2 50 94 (4; 7) 96 (2; 5) 0.2 10c Others Thiabendazole 83 (7; 6) 93 (4; 8) 0.2 85 (5; 7) 90 (5; 7) 0.2 100g 20g 5-hydroxythiabendazole 71 (3; 3) 79 (4; 7) 0.1 88 (4; 6) 95 (3; 5) 0.1 Clopidol 92 (3; 6) 105 (2; 8) 0.5 200 93 (4; 8) 100 (4; 3) 0.2 10c Tiamulin 76 (4; 6) 90 (3; 8) 0.2 10c 80 (4; 7) 83 (3; 5) 0.1 10c 242 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table 3. (continued) Salmon trout Red sea bream

Nalidixic acid 89 (6; 5) 93 (3; 4) 0.5 10c 92 (3; 4) 97 (2; 2) 0.5 10c Flumequine 92 (5; 8) 94 (4; 5) 0.2 500 92 (3; 4) 97 (1; 5) 0.2 40 Tetracyclines Oxytetracycline 76 (7; 9) 79 (6; 7) 1 200 78 (5; 9) 79 (4; 9) 2 200 Tetracycline 77 (7; 8) 76 (4; 5) 1 10c 74 (6;11) 76 (4; 8) 1 10c Chlortetracycline 103 (3; 5) 97 (4; 4) 2 10c 106 (7;10) 98 (3; 4) 1 10c Doxycycline 93 (6; 5) 87 (2; 5) 0.5 10c 113 (9; 8) 105 (5; 9) 0.5 50 Demeclocyclinef 64 (8;15) 71 (4; 6) 1 54 (10;12) 67 (3; 7) 1 Macrolides Spiramycin 93 (8; 9) 102 (5; 6) 1 200 90 (7;10) 91 (7;11) 1 200 Tilmicosin 90 (5; 8) 96 (3; 3) 0.2 10c 90 (6; 8) 96 (2; 9) 0.5 10c Mirosamycin 87 (6; 6) 90 (4; 4) 0.2 10c 94 (3; 3) 103 (3; 7) 0.2 10c Oleandomycin 93 (5; 5) 99 (4; 3) 1 10c 99 (3; 4) 100 (3; 9) 0.2 50 Erythromycin A 97 (4; 5) 99 (4; 8) 0.2 200 102 (4; 5) 103 (4;10) 0.2 60 Tylosin 89 (4; 4) 95 (4; 6) 0.2 100 90 (4; 6) 96 (2; 5) 0.2 100 Josamycin 86 (4; 4) 92 (3; 6) 0.5 10c 92 (4; 5) 94 (1; 5) 0.5 50 Lincosamides Lincomycin A 90 (4; 3) 94 (2; 4) 2 100 96 (3; 8) 99 (2; 4) 0.2 50

Pirlimycin 81 (5; 5) 84 (4; 4) 0.2 10c 80 (5; 6) 86 (4; 8) 0.2 10c Sulfonamides Sulfadiazine 89 (7; 9) 92 (4; 7) 0.2 100 96 (5; 5) 101 (2; 3) 0.1 10c Sulfathiazole 93 (5; 7) 97 (4; 5) 0.5 10c 82 (4; 8) 87 (2; 5) 0.5 10c Sulfamerazine 93 (3; 8) 95 (3; 4) 0.1 10c 97 (4; 6) 99 (3; 8) 0.2 10c Sulfadimidine 81 (5;11) 90 (5; 5) 0.2 10c 96 (2; 5) 97 (3; 5) 0.2 10c Sulfamonomethoxine 92 (3; 7) 96 (2; 5) 0.2 100 81 (5; 8) 87 (4;12) 2 100 Sulfamethoxazole 78 (6; 9) 81 (4; 4) 0.5 10c 95 (4; 4) 101 (1; 6) 0.5 10c Sulfaquinoxaline 78 (6; 9) 86 (4; 6) 0.5 10c 74 (4; 6) 77 (1; 6) 0.2 10c Sulfadimethoxine 82 (5; 5) 90 (3; 4) 0.2 100 83 (3; 4) 90 (1; 5) 0.2 10c Others Thiabendazole 75 (5; 6) 79 (5; 7) 0.2 86 (6; 7) 94 (1; 3) 0.2 20g 20g 5-hydroxythiabendazole 82 (5; 5) 90 (3; 4) 0.1 85 (4; 6) 89 (2; 5) 0.1 Clopidol 94 (6; 5) 97 (4; 5) 0.2 10c 95 (4; 7) 101 (2; 5) 2 10c Tiamulin 82 (5; 5) 87 (1; 5) 1 10c 77 (4; 6) 85 (2; 6) 0.1 10c Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 243

Table 3. (continued) Flounder Milk

Nalidixic acid 90 (5; 4) 100 (1; 4) 0.5 10c 91 (3; 6) 98 (2; 4) 0.2 10c Flumequine 89 (5; 5) 97 (2; 2) 0.2 600 89 (2; 9) 94 (4; 5) 0.2 100 Tetracyclines Oxytetracycline 72 (6; 7) 77 (4; 4) 1 200 94 (5; 8) 93 (3; 3) 2

c Tetracycline 73 (5; 6) 76 (4; 6) 2 10 89 (4; 4) 93 (3; 8) 1 100e Chlortetracycline 102 (4; 9) 95 (2; 2) 1 10c 98 (4; 6) 92 (2; 4) 2 Doxycycline 112 (6;10) 103 (3; 4) 1 10c 98 (4; 5) 94 (4; 5) 1 10c Demeclocyclinef 63 (9; 9) 74 (3; 7) 1 88 (9;10) 93 (6; 6) 1 Macrolides Spiramycin 84 (7;10) 95 (5; 5) 1 200 89 (6; 6) 89 (3; 4) 1 200 Tilmicosin 89 (7; 8) 93 (5; 9) 0.2 10c 88 (5;10) 90 (3; 7) 0.5 50 Mirosamycin 89 (6; 6) 95 (4; 5) 0.2 10c 91 (4; 7) 91 (3; 4) 0.1 10c Oleandomycin 89 (6; 6) 95 (4; 3) 0.2 10c 94 (5; 7) 93 (1; 6) 0.2 50 Erythromycin A 97 (3; 3) 102 (3; 4) 0.2 200 96 (3; 4) 93 (3; 4) 0.2 40 Tylosin 87 (5; 6) 95 (4; 3) 0.2 100 88 (7; 7) 88 (3; 3) 0.2 50 Josamycin 86 (4; 4) 95 (3; 3) 0.5 10c 86 (2; 4) 87 (3; 7) 0.5 10c Lincosamides Lincomycin A 86 (5; 5) 96 (3; 5) 0.5 100 88 (3; 4) 91 (2; 3) 0.5 150

Pirlimycin 75 (5; 6) 82 (3; 3) 0.2 10c 93 (5; 8) 92 (5; 8) 0.2 300 Sulfonamides Sulfadiazine 92 (7; 7) 102 (4; 5) 0.2 10c 92 (3; 7) 98 (3; 4) 0.2 70 Sulfathiazole 74 (7; 7) 89 (4; 5) 0.5 10c 95 (4; 8) 97 (3; 7) 0.5 90 Sulfamerazine 93 (7; 7) 99 (5; 5) 0.2 10c 92 (4; 9) 91 (5; 7) 0.2 10c Sulfadimidine 90 (6; 5) 97 (3; 5) 0.2 10 c 93 (5; 5) 96 (5; 5) 0.2 25 Sulfamonomethoxine 74 (7; 5) 85 (3;12) 2 100 89 (4; 6) 98 (4; 5) 1 10c Sulfamethoxazole 86 (4; 7) 98 (2; 4) 0.5 10c 96 (4; 5) 93 (3; 3) 0.2 10c Sulfaquinoxaline 64 (6; 8) h 75 (2; 3) 0.2 10c 91 (4; 8) 95 (4; 5) 0.5 10 Sulfadimethoxine 72 (5; 5) 84 (3; 5) 0.2 100 93 (4; 5) 94 (3; 3) 0.1 20 Others Thiabendazole 82 (5; 5) 93 (3; 5) 0.2 87 (4; 5) 92 (3; 3) 0.2 20g 100g 5-hydroxythiabendazole 81 (4; 4) 94 (3; 4) 0.1 81 (4; 9) 89 (5; 9) 0.1 Clopidol 91 (5; 5) 100 (2; 2) 2 10c 90 (4; 4) 93 (4; 4) 1 20 Tiamulin 75 (6; 5) 85 (2; 5) 0.1 10c 77 (5; 5) 80 (5;11) 0.1 10c 244 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table 3. (continued) Egg Honey

a b a b Trueness, % (RSDr , %; RSDWR , %) Trueness, % (RSDr , %; RSDWR , %) Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg Quinolones Marbofloxacin 79 (4; 6) 75 (4; 9) 0.5 10c 96 (5; 5) 101 (2; 2) 1 10c Norfloxacin 80 (4; 5) 81 (4; 6) 2 10c 92 (6; 6) 95 (1; 4) 1 10c Ofloxacin 88 (4; 5) 91 (3; 5) 0.2 10c 98 (4; 5) 102 (2; 3) 1 10c Enrofloxacin 90 (4; 5) 91 (5; 7) 0.5 92 (5; 6) 98 (4; 4) 2 10c,d 10c,d Ciprofloxacin 81 (7; 7) 82 (2; 7) 1 95 (6; 4) 100 (2; 4) 5 Danofloxacin 89 (5; 9) 87 (6; 7) 5 10c 96 (5; 4) 102 (3; 4) 1 10c Orbifloxacin 86 (5; 5) 85 (4;12) 0.5 10c 92 (3; 6) 100 (4; 4) 0.5 10c Sarafloxacin 83 (3; 3) 88 (3; 4) 1 10c 91 (5; 7) 98 (3; 3) 2 10c Difloxacin 90 (4; 7) 94 (3; 4) 0.5 10c 96 (3; 3) 100 (3; 4) 2 10c Oxolinic acid 90 (3; 5) 96 (2; 2) 2 10c 97 (3; 5) 100 (2; 4) 1 10c Nalidixic acid 90 (3; 2) 93 (2; 6) 0.2 10c 96 (3; 3) 100 (3; 3) 0.2 10c Flumequine 76 (3; 5) 84 (3; 4) 0.2 10c 95 (3; 3) 100 (3; 4) 0.2 10c Tetracyclines Oxytetracycline 76 (3; 4) 75 (2; 4) 1 95 (6; 5) 96 (2; 2) 1 Tetracycline 84 (6; 7) 79 (4; 7) 1 400e 92 (5; 5) 92 (2; 6) 2 300e Chlortetracycline 99 (3; 7) 90 (3; 5) 1 85 (6;10) 83 (4; 4) 2 Doxycycline 113 (5; 8) 112 (4; 7) 0.5 10c 103 (9; 9) 101 (3; 4) 0.5 10c Demeclocyclinef 60 (7; 7) 57 (4;10) 2 88 (6; 9) 97 (3;10) 2 Macrolides Spiramycin 94 (7;10) 90 (4; 6) 1 10c 96 (7; 7) 98 (5; 5) 1 10c Tilmicosin 92 (5; 7) 96 (7; 3) 1 10c 94 (4; 6) 101 (4; 6) 1 10c Mirosamycin 92 (4; 4) 96 (1; 3) 0.1 10c 96 (3; 4) 103 (4; 4) 0.1 50 Oleandomycin 93 (2; 6) 98 (2; 3) 0.5 10c 94 (4; 5) 102 (4; 4) 0.2 10c Erythromycin A 98 (2; 4) 98 (2; 4) 0.2 90 101 (3; 5) 98 (2; 5) 0.2 10c Tylosin 91 (4; 6) 91 (3; 5) 0.2 200 97 (4; 4) 102 (4; 3) 0.2 10c Josamycin 84 (2; 3) 89 (1; 3) 0.5 10c 90 (6; 6) 95 (2; 4) 0.5 10c Lincosamides Lincomycin A 81 (4; 8) 78 (4; 7) 0.2 100 96 (3; 5) 99 (2; 3) 0.5 10c Pirlimycin 81 (4; 7) 82 (4; 8) 0.2 10c 95 (3; 3) 96 (4; 7) 0.2 10c Sulfonamides Sulfadiazine 72 (9;16) 57 (12;24) 0.2 20 97 (4; 6) 99 (2; 3) 0.2 10c Sulfathiazole 97 (4; 3) 100 (3; 4) 1 10c 92 (6; 6) 98 (5; 5) 0.5 10c Sulfamerazine 91 (4; 5) 90 (3; 6) 0.2 10c 92 (6; 4) 100 (3; 3) 0.2 10c Sulfadimidine 94 (3; 6) 96 (4; 3) 0.2 10 92 (4; 5) 98 (4; 4) 0.2 10c Sulfamonomethoxine 92 (3; 5) 93 (2; 5) 0.5 10c 95 (4; 6) 100 (3; 4) 2 10c Sulfamethoxazole 91 (2; 4) 94 (2; 4) 0.2 10c 92 (5; 5) 100 (2; 3) 0.2 10c Sulfaquinoxaline 90 (3; 4) 90 (2; 3) 0.2 10 90 (3; 5) 98 (2; 3) 0.2 10c Sulfadimethoxine 90 (2; 3) 91 (1; 3) 0.1 1000 92 (4; 4) 100 (2; 3) 0.1 10c Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 245

Table 3. (continued) Egg Honey

a b a b Trueness, % (RSDr , %; RSDWR , %) Trueness, % (RSDr , %; RSDWR , %) Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg Others Thiabendazole 89 (5; 4) 95 (3; 3) 0.2 93 (6; 6) 99 (2; 3) 0.1 100g 20g 5-hydroxythiabendazole 88 (4; 3) 90 (3; 3) 0.1 97 (4; 6) 100 (2; 3) 2 Clopidol 29 (12; 30) h 25 (26; 36) h 2 10c 93 (4; 7) 99 (3; 3) 0.2 10c Tiamulin 77 (4; 5) 84 (2; 3) 0.1 1000 76 (5; 6) 82 (3; 8) 0.1 10c a RSDr of repeatability. b RSDWR of within-run reproducibility. c The 10 mg/kg default regulatory limit; MRLs for some analytes in livestock and fishery products have not been defined. d MRL is the sum of enrofloxacin and ciprofloxacin. e MRL is the sum of oxytetracycline, tetracycline, and chlortetracycline. f The internal standard material for the quantification of chlortetracycline and doxycycline. g MRL is the sum of thiabendazole and 5-hydroxythiabendazole. h Did not satisfy the criteria of the Japanese guideline. the matrix did not significantly affect the fragmentation patterns muscle and honey; sulfathiazole in bovine muscle, prawn, of each precursor ion of 37 veterinary drugs to two product ions. flounder, and honey; sulfamerazine in honey; sulfadimidine in honey; sulfamonomethoxine in bovine muscle and honey; Linearity of Calibration sulfaquinoxaline in salmon trout, sulfadimethoxine in salmon trout; 5-hydroxythiabendazole in prawn; thiabendazole in The matrix-matched calibration curves of 35 veterinary bovine muscle and prawn. Therefore, we consider that the drugs, except for chlortetracycline and doxycycline, were matrix-matched standard calibration curves were adequate for obtained for a series of standard solutions containing each the quantification of each of the 37 veterinary drugs in livestock matrix at five concentrations by plotting the peak area against and fishery products. the concentration. Chlortetracycline and doxycycline were unstable in the resultant solution. Therefore demeclocycline Method Validation was used as an IS to more accurately measure the concentrations of both chlortetracycline and doxycycline. Chlortetracycline Validation was carried out following the guidelines of the and doxycycline calibration curves were obtained for a series Japanese Ministry of Health, Labour, and Welfare (5, 6). The of standard solutions at five concentrations by plotting the developed method in this study was validated by means of peak area against the concentration, corrected by 0.01 µg/mL recovery tests using swine muscle, chicken muscle, bovine demeclocycline. All of the correlation coefficient (r) values muscle, prawn, salmon trout, red sea bream, flounder, milk, egg, were over 0.999, and deviations in individual points from and honey samples which were spiked with 50 µL of working the calibration curves were lower than 20%. Accordingly, standard solutions (1 µg/mL or 10 µg/mL) in two replicates for satisfactory linearity was obtained in the range examined for 5 separate days. As shown in Table 3, the overall recovery of each compound. the 37 drugs ranged from 25 to 118%. The RSDr ranged from 1 To evaluate the matrix effect, slopes derived from the to 26%. The RSDWR ranged from 2 to 36%. In this method, the standard solution and matrix-matched calibration curves numbers of analytes that satisfied the guidelines criteria were derived from each livestock and fishery product were compared 37 out of 37 in swine muscle, chicken muscle, bovine muscle, at the same range as described above. The slope ratios of the prawn, salmon trout, red sea bream, milk, and honey samples, matrix-matched/standard solution calibration curves were 35 out of 37 in egg, and 34 out of 37 in flounder. Only two obtained for each of the 37 veterinary drugs (Figure 5). The slope analytes (sulfadiazine and clopidol) in egg and three analytes ratios ranging from 0.8 to 1.2 were considered to be tolerable, (norfloxacin, danofloxacin, and sulfaquinoxaline) in flounder whereas the ratio higher than 1.2 or lower than 0.8 implied a were not sufficiently recovered. Selectivity was confirmed strong matrix effect (39). A significant matrix effect was noticed by analyzing blank samples, and no interfering peaks were on marbofloxacin in bovine muscle and prawn; danofloxacin observed at the same retention times of the target analytes. in chicken muscle, prawn, flounder, and honey; orbifloxacin Figure 4b shows the SRM chromatograms obtained from the in prawn, difloxacin in bovine muscle; tetracycline in swine blank swine muscle. muscle and prawn; chlortetracycline in prawn; demeclocycline in prawn; spiramycin in swine muscle, chicken muscle, bovine LODs and LOQs muscle, prawn, salmon trout, flounder, and honey; tilmicosin in swine muscle, chicken muscle, bovine muscle, red sea bream, As shown in Table 3, LOQs for the 37 veterinary drugs ranged flounder, and honey; pirlimycin in honey; sulfadiazine in bovine from 0.1 to 5 µg/kg, which was less than the 10 µg/kg default 246 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015 regulatory limit, set by the positive list system for agricultural TCs and QLs were measured with good sensitivity and chemical residues in foods in Japan. LODs ranged from 0.03 to excellent peak shapes using the novel hybrid column, a mobile 2 µg/kg. phase consisting of a mixture of 0.05% formic acid and acetonitrile, and a minimum injection volume. By preparing the Survey of Livestock and Fishery Products gas pressure on MS/MS parameters, macrolides were measured with high sensitivity. LOQs of the 37 veterinary drugs were To demonstrate the applicability of the developed method in lower than the MRLs. this study for the determination of 37 veterinary drug residues, Using this method, the numbers of analytes that were 110 samples (20 swine muscle, 15 chicken muscle, 13 bovine validated in accordance with the Japanese Ministry of Health, muscle, 10 prawn, 10 salmon trout, 7 red sea bream, 10 flounder, Labour, and Welfare guideline were 37 analytes out of 37 in 5 milk, 10 egg, and 10 honey), purchased from retail outlets in swine muscle, chicken muscle, bovine muscle, prawn, salmon Japan, were tested. When the peak was detected, the ion ratios trout, red sea bream, milk, and honey, 35 in egg, and 34 in were compared with those of the standard solutions at comparable flounder. FQs, TCs, and 5-hydroxythiabendazole, which could concentrations. Because the relative ion abundance ratios were not be determined using previously reported methods, were within 20% recommended by EU guidelines (38), the identity of successfully analyzed using our novel method. residual drugs was accurate. No analyte was detected in bovine This method was successfully applied on 110 commercially muscle, prawn, red sea bream, milk, or egg. In swine muscle, available livestock and fishery products. Veterinary drug TCs were detected in ten samples. In four samples, more than one residues were found in 24 samples. It is necessary to continue TC was found; oxytetracycline (2.5 µg/kg) and chlortetracycline monitoring for the residues of 37 veterinary drugs in livestock (3.9 µg/kg), oxytetracycline (1.3 µg/kg) and doxycycline and fishery products using this method. The method developed (1.4 µg/kg), tetracycline (1.7 µg/kg) and chlortetracycline in this study provides high-quality performance and ease of (10.9 µg/kg), chlortetracycline (18.7 µg/kg) and doxycycline implementation for the routine monitoring of 37 polar veterinary (4.0 µg/kg). On the other hand, oxytetracycline was found in drugs in livestock and fishery products. four samples (1.3, 3.6, 4.5, and 9.5 µg/kg), tetracycline in one sample (6.4 µg/kg), and doxycycline in one sample (0.8 µg/kg). References In chicken muscle, three veterinary drugs were detected in four samples. Clopidol (3.4 µg/kg), oxytetracycline (8.5 µg/kg), (1) T urnidge, J. (2004) Antimicrob. 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