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

23 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 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 169-73, Japan Residues of 37 polar veterinary drugs belonging to six families (quinolones, tetracyclines, macrolides, lincosamides, sulfonamides, and others) in livestock and fishery products were determined using a validated LC-MS/MS method. There were two key points in sample preparation. First, extraction was performed with two solutions of different polarity. Highly polar compounds were initially extracted with Na 2 EDTA-McIlvaine s buffer (ph 7.). Medium polar compounds were then extracted from the same samples with acetonitrile containing.1% formic acid. Secondly, cleanup was performed using a single SPE polymer cartridge. The first extracted solution was applied to the cartridge. Highly polar compounds were retained on the cartridge. Then, the second extracted solution was applied to the same cartridge. Both highly and medium polar compounds were eluted from the cartridge. This method satisfied the guideline criteria for 37 out of 37 drugs in swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, milk, and honey; 35 out of 37 in egg; and 34 out of 37 in flounder. The LOQ ranged from.1 to 5 µg/kg. Residues were detected in 24 out of 11 samples and analyzed using the validated method. Veterinary drugs are widely used on farms to treat and prevent diseases. However, over-dosing and noncompliance with the withdrawal period may cause drug residues to remain in animal tissues (1, 2). Drug-contaminated livestock and fishery products may have a potential risk for the consumer s health because they can provoke drug-resistant pathogenic strains of bacteria, allergic reactions, and toxicity (3, 4). Therefore, it is necessary to monitor livestock and fishery products for the residual veterinary drugs using accurate analysis. We have used two major analytical strategies to measure residual substances, namely, microbiological screening (5 7) and screening using LC-MS/MS (8, 9). However, the sensitivity of microbiological screening was insufficient to detect residual levels of multi-class veterinary drugs. Moreover, when positive results were found Received August 13, 213. Accepted by JB May 26, 214. Corresponding author s e-mail: Maki_1_Kanda@member.metro. tokyo.jp DOI: 1.574/jaoacint.13-272 with microbiological methods, specific chromatographic analyses were needed to identify the antibiotics. The identifying process was so complicated that it was difficult to identify each residual drug. The accuracy of analysis for the residual drugs has been required worldwide in recent years. In Japan, the analytic methodologies used by inspection institutes had to be validated until December 13, 213 according to the notice issued by the Japanese Ministry of Health, Labour, and Welfare (1, 11). On the other hand, the simultaneous analysis methodologies for multi-class veterinary drug residues using LC-MS/MS have already been reported (8, 9, 12 29). However, the trueness and precision of reported analysis using LC-MS/MS (8, 9, 12 23, 25, 27) for fluoroquinolones (FQs), tetracyclines (TCs), penicillins (PCs), 5-hydroxythiabendazole, and clopidol did not achieve acceptable values according to the Guidelines for the Validation of Analytical Methods for Residual Agricultural Chemicals in Food. Furthermore, the sensitivity of some analysis was insufficient to detect residual levels of multi-class veterinary drugs (12, 17 19, 22, 23, 25, 27). On the Japanese positive list system, veterinary drugs of which no established maximum residues limits (MRLs) were given the default regulatory limit (uniform limit of level) at 1 µg/kg. Therefore, the analysis of multi-class drugs needs the LOQ for each drug to be less than 1 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; 3, 31). TC residues were found in swine muscle, fish, and honey. The residues of enrofloxacin were found in shrimp from Asia. Therefore, we need analytical methods to accurately measure the residue concentrations of these drugs. The aim of this study was to determine residues of 37 polar veterinary drugs belonging to six families [quinolones (QLs), TCs, macrolides (MLs), lincosamides, sulfonamides (SDs), and others] in livestock and fishery products using a validated LC- MS/MS method. By addressing the following five points, we improved pretreatment procedures and LC-MS/MS conditions: (1) Simple and rapid analysis is desirable to speed up large amounts of sample inspections. (2) Polar veterinary drugs must be simultaneously extracted from livestock and fishery products. We attempted to use aqueous solvent on the first extraction and then organic solvent on the second extraction. Different pretreatment procedures, such as quick, easy, cheap, effective, rugged, and safe (QuEChERS) methods (8, 9 12 18, 26) or pressurized liquid extraction (PLE) were used recently. By using acetonitrile in the QuEChERS

Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 231 method, extraction of TCs, MLs, and FQs was insufficient (8, 9, 12, 14, 15, 18, 26). By using other extraction solutions i.e., acidified acetonitrile (13 16, 26), methanol (12, 18), or methanol-acetonitrile (1, 17), extraction of these drugs was insufficient as well. As shown in Table 1, log P of these drugs was negative, which means that these drugs were soluble in the aqueous phase. Actually, a mixture of water and organic solvent was used (19, 2, 21, 23, 32). Using water at PLE was significantly more effective for the extraction of QLs, PC V, and SDs (25 29). (3) During the measurement by LC-MS/MS, the matrix interferes with the ionization of the target compounds, which precludes the quantification. The matrix interference from livestock and fishery products is removed by a cleanup using the SPE polymer cartridge. (4) To increase the sensitivity, LC conditions (mobile phase, column, and injection volume) and MS/MS parameters were modified. (5) The analytical method developed in this study was validated in 1 livestock and fishery products: swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, flounder, milk, egg, and honey in accordance with the Japanese guidelines. Experimental Samples Livestock and fishery products (swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, flounder, milk, egg, and honey) were purchased from local supermarkets in Japan and were confirmed to be free of the targeted analytes in this study. The tissues were minced with an electric household food processor and stored at 2 C. Apparatus (a) LC system. LC-2A series (Shimadzu Corp., Kyoto, Japan). (b) MS system. API 55 Qtrap mass spectrometer with an electrospray ionization (ESI) interface and Analyst (Version 1.4.2) software (AB Sciex, Framingham, MA). (c) LC column. Triart C18 column (15 2. mm, 5 µm particle size) (YMC Co. Ltd, Kyoto, Japan). (d) Mixer. Vortex-Genie 2 (Scientific Industries Inc., Bohemia, NY). (e) Ultrasonic machine. B551J-DTH (Branson, Danbury, CT). (f) Centrifuge. AX-32 (Tomy Seiko Co., Tokyo, Japan). (g) Microcentrifuge. 5415R (Eppendorf Co. Ltd, Hamburg, Germany). (h) Polypropylene centrifuge tubes. 15 ml and 5 ml (Corning Inc., Corning, NY). (i) Glass volumetric flasks. 5 and 1 ml (SIBATA Scientific Technology Ltd, Saitama, Japan). (j) Polymethylpentene and opaque volumetric flasks. 1 ml (VITLAB GmbH, Grossostheim, Germany). (k) SPE manifold system. Vacuum manifold system (GL Sciences Inc., Tokyo, Japan). (l) SPE polymer cartridges for the cleanup procedure. InertSep TM PLS-3 cartridge, 2 cc/2 mg (GL Sciences Inc.). Before use, the PLS-3 cartridges were conditioned with 5 ml acetonitrile, and then 5 ml Na 2 EDTA-McIlvaine s buffer solution (ph 7.). (m) Microtubes. 1.5 ml (Eppendorf Co. Ltd). (n) Polypropylene and amber vial tubes. 3 µl (GL Sciences Inc.). Reagents (a) Water. Obtained using a Milli-Q system (Millipore Corp., Billerica, MA). (b) Solvent. Acetonitrile (LC grade), hexane (for pesticide residue and polychlorinated biphenyl analysis grade) and methanol (LC grade; Wako Pure Chemical Industries Ltd, Osaka, Japan). (c) Formic acid (99%). LC-MS grade (Wako Pure Chemical Industries Ltd). (d) Citric acid monohydrate, Na 2 EDTA, sodium chloride, and anhydrous magnesium sulfate. Analytical grade (Wako Pure Chemical Industries Ltd). (e) Disodium hydrogen phosphate dihydrate. Analytical grade (Merck KGaA, Darmstadt, Germany). (f) Polar extraction solution 1; Na 2 EDTA-McIlvaine s buffer solution (ph 7.). Prepared by dissolving 3.92 g disodium hydrogen phosphate dihydrate, 2.73 g citric acid monohydrate, and 37.13 g Na 2 EDTA in water and diluting to 1 L. (g) Polar extraction solution 2; Acetonitrile containing.1% formic acid. Freshly prepared by mixing.1 ml of formic acid with 1 ml of acetonitrile. (h) Standard (purity grade). Marbofloxacin (98.%), norfloxacin (98.%), ciprofloxacin (98.%), difloxacin (98.%), flumequine (98.%), oxytetracycline (99.%), erythromycin A (98.%), sulfadiazine (99.%), sulfathiazole (98.%), sulfamonomethoxine (99.%), sulfamethoxazole (99.%), sulfadimethoxine (99.%), 5-hydroxythiabendazole (98.%), clopidol (98.%), and thiabendazole (99.%) were purchased from Wako Pure Chemical Industries Ltd Ofloxacin (97.7%), orbifloxacin (99.6%), and lincomycin A (98.%) were from Hayashi Pure Medical Industry (Osaka, Japan). Danofloxacin (1.%), enrofloxacin (99.8%), oxolinic acid (98.8%), nalidixic acid (99.8%), oleandomycin (96.5%), josamycin (86.8%), sulfamerazine (99.5%), sulfadimidine (99.4%), and sulfaquinoxaline (99.6%) were from Kanto Chemical Co. (Tokyo, Japan). Sarafloxacin (97.3%), tetracycline (97.7%), chlortetracycline (99.1%), doxycycline (98.2%), and tiamulin (99.9%) were from Sigma-Aldrich (St. Louis, MO). Spiramycin (96.%), tilmicosin (98.5%), and tylosin (98.%) were from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Pirlimycin (86.6%) was from Pfizer Japan Inc. (Tokyo, Japan). Mirosamicin (97.7%) was from Kyoritsu Pharmaceutical Co. (Tokyo, Japan). (i) Internal standard (IS). Demeclocycline (92.3%) was from Hayashi Pure Medical Industry. Preparation of Standard Solutions and Calibration Standards (a) Stock standard solutions of 33 individual compounds except TCs (1 µg/ml). Stock standard solutions were prepared individually. The suitable quantity of standard taking into account the substance purity was weighed in a 5 ml glass volumetric flask. Clopidol was dissolved in 5 ml acetonitrile,

232 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 Table 1. log P values of veterinary drugs Analytes Quinolones logp Marbofloxacin.5 Norfloxacin 1. Ofloxacin.4 Enrofloxacin.2 Ciprofloxacin 1.1 Danofloxacin.3 Orbifloxacin.9 Sarafloxacin.3 Difloxacin 1.6 Oxolinic acid 1.7 Nalidixic acid 1.4 Flumequine 2.9 Tetracyclines Oxytetracycline 1.6 Tetracycline 2. Chlortetracycline 1.3 Doxycycline.7 Demeclocycline a.7 Macrolides Spiramycin 2.1 Tilmicosin 3.6 Mirosamicin 2. Oleandomycin 2.6 Erythromycin A 2.7 Tylosin 1. Josamycin 2.9 Lincosamides Lincomycin A.2 Pirlimycin 1.7 Sulfonamides Sulfadiazine.1 Sulfathiazole.1 Sulfamerazine.1 Sulfadimidine.3 Sulfamonomethoxine.8 Sulfamethoxazole.9 Sulfaquinoxaline 1.7 Sulfadimethoxine 1.6 Others Thiabendazole 2.5 5-hydroxythiabendazole 2.1 Clopidol 2.6 Tiamulin 5.6 a The internal standard material for the quantification of chlortetracycline and doxycycline. and made up to 5 ml with methanol. Sulfadimidine and oxolinic acid were dissolved in acetonitrile, and made up to 5 ml with acetonitrile. The rest of compounds were dissolved in methanol, and made up to 5 ml with methanol. Stock standard solutions were kept in amber glass vials in the dark at 4 C, under which conditions, they were stable for one year. (b) Mixed standard solutions except TCs (1 µg/ml). An aliquot (5 µl) of each stock standard solution shown in (a) was transferred and mixed together in a 5 ml glass volumetric flask, and made up to 5 ml with methanol. This mixed standard solution was kept in an amber glass vial in the dark at 4 C, under which conditions this was stable for 3 months. (c) Stock standard solutions of 4 TCs (1 µg/ml). Stock standard solutions of TCs (oxytetracycline, tetracycline, chlortetracycline and doxycycline) were prepared individually. The suitable quantity of standard taking into account the substance purity was weighed in a 1 ml opaque polymethylpentene volumetric flask (light-shielding). TCs were dissolved in methanol and made up to 1 ml with methanol. The stock standard solutions were kept in polypropylene vials in the dark at 2 C, under which conditions they were stable for 1 month. (d) Mixed oxytetracycline and tetracycline standard solution (1 µg/ml). An aliquot (1 µl) of each stock standard solution of oxytetracycline and tetracycline shown in (c) was transferred and mixed together in a 1 ml opaque polymethylpentene volumetric flask, and made up to 1 ml with acetonitrile containing.1% formic acid (ACN/FA) immediately before use. This solution was diluted 1 times with ACN/FA. (e) Working standard solutions for 35 veterinary drugs (except for chlortetracycline and doxycycline) (from.1 to.1 µg/ml). Working standard solutions were prepared immediately before use by serial dilution of each mixed standard solution shown in (b) and (d) with ACN/FA. (f) Matrix-matched standard solutions for 35 veterinary drugs (from.25 to 5 ng/ml). Calibration curves for 35 veterinary drugs (except chlortetracycline and doxycycline) were obtained from matrix-matched calibration samples. Blank samples were prepared as described in the Sample Preparation section. Matrix-matched standard solutions were prepared by mixing an aliquot (5 µl) of blank solution and the appropriate volume of working standard solutions shown in (e), and then made up to 1 ml with ACN/FA, e.g., a.25 ng/ml solution was made by mixing an aliquot (5 µl) of blank solution and the working standard solution (.1 µg/ml, 25 µl), and then made up to 1 ml. (g) Mixed chlortetracycline and doxycycline standard solution (1 µg/ml). An aliquot (1 µl) of each stock standard solution of chlortetracycline and doxycycline shown in (c) was transferred and mixed together in a 1 ml opaque polymethylpentene volumetric flask, and made up to 1 ml with ACN/FA immediately before use. This solution was diluted 1 times with ACN/FA. (h) Working standard solutions for chlortetracycline and doxycycline (from.1 to.1 µg/ml). Working standard solutions were prepared immediately before use by serial dilution of the mixed standard solution shown in (g) with ACN/FA. (i) IS. Demeclocycline was the IS for the quantification of chlortetracycline and doxycycline. Demeclocycline (1.9 mg) was accurately weighed in a 1 ml opaque polymethylpentene

Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 233 volumetric flask, dissolved in methanol, and made up to 1 ml with methanol. The stock IS solution (1 µg/ml) was kept in polypropylene vials in the dark at 2 C, under which conditions the solution was stable for 1 month. Working IS solutions (from.1 to 1 µg/ml) were prepared immediately before use by serial dilution of the stock IS solution with ACN/FA. (j) IS calibration standard solutions for chlortetracycline and doxycycline (from.25 to 5 ng/ml). Calibration curves for chlortetracycline and doxycycline were obtained from IS calibration samples. IS calibration standard solutions were prepared by mixing the working IS solution shown in (i) (.1 µg/ml, 1 µl) and the appropriate volume of working solutions shown in (h), and made up to 1 ml with ACN/FA, e.g., a.25 ng/ml solution was made by mixing the working IS standard solution (.1 µg/ml, 1 µl) together with the working standard solution for chlortetracycline and doxycycline (.1 µg/ml, 25 µl), and then brought to 1 ml volume. LC Separation Conditions (a) Mobile phase. The.5% formic acid solution was prepared by mixing.5 ml of formic acid with 1 L water. (A) The.5% formic acid solution and (B) acetonitrile were mixed using the pump in gradient mode as follows: 5% B (3 min); 5 9% B (12 min); 9% B (5 min); 9-5% B (.1 min); and 5% B (5 min). (b) Flow rate..3 ml/min. (c) Column temperature. 4 C. (d) Injection volume. 2 µl. MS/MS Conditions (a) Ionization mode. Positive-ion ESI. (b) Ion spray voltage. 55 V. (c) Source temperature. 65 C. (d) Entrance potential. 1 V. (e) Curtain gas pressure. 2 psi (nitrogen). (f) Collision gas pressure. 7 psi (nitrogen). (g) Ion source gas pressure 1. 8 psi (nitrogen). (h) Ion source gas pressure 2. 4 psi (nitrogen). (i) Acquisition function. Selected reaction monitoring (SRM); the SRM program is shown in Table 2. Sample Preparation The schematic procedure of sample preparation is shown in Figure 1. For the sample preparation, glass vessels were not used, because silica in the glass could make an interference signal during LC-MS/MS analysis of TCs. Thoroughly minced sample (5. g) was poured in 5 ml polypropylene centrifuge tubes (A). IS was spiked at a level of 1 µg/kg. Na 2 EDTA McIlvaine s buffer (ph 7., 2 ml) was 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, ultrasonicated for 1 min, and then centrifuged at 9 6 g for 2 min at 4 C. The hexane layer was discarded by pipetting. Hexane washing was used at all sample types to ease the operations. As shown in First extraction of Figure 1, the Na 2 EDTA McIlvaine s buffer layer was transferred into new 5 ml polypropylene centrifuge tubes containing 1 ml of 25% NaCl solution (B). The tube (B) was vortexed for 1 min., and then centrifuged at 96 g for 1 min at 4 C. The supernatant was loaded to the conditioned PLS-3 cartridge at approximately 1 ml/min. The target compounds were retained on the cartridge, while the solution containing the matrix of food was passed through the cartridge. The cartridge was washed with 5 ml of water, and then vacuum-dried for 3 min at a pressure of 1 mm Hg. In addition, the second extraction from the remaining substance in tube (A) was performed as shown in the Second extraction stage of Figure 1. The characteristics of the remaining matrixes were varied and depended on the different type of samples, as well as the pellets or the insoluble matrix floating on the top of the hexane layer. The following procedure was used for all sample types. Water (2 ml) was added to the tube (A) and then (A) was vortexed. Subsequently, 1 ml ACN/FA was added. The tube (A) was vortexed again for 1 min, and ultrasonicated for 1 min. Magnesium sulfate was added for dehydration (3 g each for bovine muscle, swine muscle, chicken muscle, prawn, milk, and honey). A 4 g amount of magnesium sulfate was added for salmon, red sea bream, and flounder; 5g was added for egg. Then tube (A) was vigorously shaken for 1 min, and centrifuged at 18 g for 1 at 4 C. As shown as the black arrow in Figure 1, the organic phase was used as the elution solution for the PLS-3 cartridge previously loaded with Na 2 EDTA McIlvaine s buffer layer. The eluate from the cartridge was collected into an opaque polymethylpentene volumetric flask. The eluate was made up to 1 ml with ACN/FA. An aliquot (1 ml) was transferred to a microtube, diluted to 2-fold with ACN/FA, and centrifuged at 16 g for 5 min at 4 C. The supernatant was transferred into an amber polypropylene vial tube. The resultant solution was analyzed by LC-MS/MS twice on the same day. Each quantitative value was taken as a mean of two measurements. Single-Laboratory Validation Tests with Spiked Samples The method was validated according to the guidelines of the Japanese Ministry of Health, Labour, and Welfare. Selectivity was confirmed by analyzing blank samples. Trueness, repeatability (RSDr), and within-run reproducibility (RSD WR ) were determined by means of the recoveries using samples spiked with 37 veterinary drugs and demeclocycline at levels of 1 or 1 µg/kg, performed with two samples per day over five different days. LOQs and LODs were estimated from the repeatability data of the blank samples spiked with.1,.25,.5, 1, 2.5, and 5 µg/kg for each of the 37 veterinary drugs examined. LOQs were calculated as 1 times the SD, and LODs were calculated as 3 times the SD using the Analyst software (AB Sciex). Results and Discussion LC-MS/MS Parameters The MS scans of the 37 veterinary drugs revealed that the most abundant molecular ion was the protonated molecule [M+H] +. As each [M+H] + is a precursor ion, a further MS/MS scan was performed after the collision energy was increased. Two fragment ions (corresponding to quantitative

234 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 Table 2. SRM parameters Analytes Transition, m/z Retention time, min Declustering potential, V Collision energy, ev Collision cell exit potential, V Ion ratio, % c Quinolones Marbofloxacin 363. 32.1 a 5.9 81 21 2 52.2 363. 121.9 79 1 Norfloxacin 32. 276.2 a 6. 96 23 12 34.7 32. 23.9 53 12 Ofloxacin 362. 318. a 6. 46 29 16 84.2 362. 261. 41 16 Ciprofloxacin 332. 288.1 a 6.1 81 25 24 11 332. 231.1 57 18 Danofloxacin 358. 82. a 6.2 81 7 23 57.9 358. 255. 65 23 Enrofloxacin 36.2 316. a 6.3 116 29 1 66.5 36.2 245. 36 8 Orbifloxacin 396. 295.1 a 6.4 116 35 18 41.4 396. 351.9 27 18 Sarafloxacin 385.9 341.9 a 6.6 116 27 22 86.7 385.9 298.8 39 22 Difloxacin 4. 356. a 6.6 116 29 2 85. 4. 299.1 45 16 Oxolinic acid 261.9 243.9 a 7.7 61 27 18 16.9 261.9 215.9 41 18 Nalidixic acid 232.9 215. a 8.5 56 21 16 96.6 232.9 186.9 35 14 Flumequine 262. 21.9 a 8.6 66 41 14 42.4 262. 126.1 67 2 Tetracyclines Oxytetracycline 461.1 426.1 a 6.1 5 27 26 59.5 461.1 443.1 19 24 Tetracycline 445.1 41.1 a 6.3 36 29 24 15.8 445.1 225.9 77 16 Chlortetracycline 479.1 443.9 a 6.9 91 29 28 74.4 479.1 462. 23 26 Doxycycline 445.1 428.1 a 7. 5 25 24 9.6 445.1 154. 37 12 Demeclocycline b 465.1 429.9 a 6.6 76 31 22 56.7 465.1 288.9 45 18 Macrolides Spiramycin 843.4 174. a 6.5 6 45 22 28.6 843.4 142.2 45 1 Tilmicosin 869.4 174. a 7. 1 57 4 2. 869.4 696.4 57 16 Mirosamicin 728.3 158. a 7.3 6 37 12 16.2 728.3 116. 61 12 Oleandomycin 688.4 158.2 a 7.4 116 35 1 52.2 688.4 544.3 23 36

Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 235 Table 2. (continued) Analytes Transition, m/z Retention time, min Declustering potential, V Collision energy, ev Collision cell exit potential, V Ion ratio, % c Erythromycin A 734.3 158.1 a 7.6 86 39 1 8.3 734.3 576.3 27 34 Tylosin 916.4 174. a 7.7 1 51 14 1.3 916.4 11. 85 12 Josamycin 828.3 19.2 a 8.6 11 67 14 68.8 828.3 174. 43 16 Lincomycins Lincomycin A 47.1 126.2 a 5.6 121 37 12 12. 47.1 359.1 27 22 Pirlimycin 411. 112.1 a 6.6 11 31 8 18.9 411. 363.2 25 18 Sulfonamides Sulfadiazine 251. 155.9 a 5.8 81 21 14 73.7 251. 17.9 33 18 Sulfathiazole 255.9 155.9 a 6. 56 21 14 52.3 255.9 17.9 33 16 Sulfamerazine 264.9 155.9 a 6.4 11 25 14 88.2 264.9 17.9 37 18 Sulfadimidine 278.9 155.9 a 6.8 56 23 12 58.3 278.9 17.9 25 16 Sulfamonomethoxine 281. 17.9 a 7. 191 35 1 42.9 281. 155.9 17 12 Sulfamethoxazole 253.9 155.9 a 7.4 76 23 12 69. 253.9 17.9 33 16 Sulfaquinoxaline 3.9 155.9 a 7.9 66 23 14 52.4 3.9 17.9 39 1 Sulfadimethoxine 31.9 155.9 a 7.9 81 29 14 39.3 31.9 17.9 39 1 Others 5-hydroxythiabendazole 217.8 19.9 a 5.5 51 37 14 38.5 217.8 146.8 47 22 Clopidol 191.9 1.9 a 5.7 171 37 16 25.6 192.9 155.9 33 14 Thiabendazole 21.9 174.9 a 6. 161 37 14 7.8 21.9 131. 45 12 Tiamulin 494.3 192.2 a 8.1 86 29 14 63.3 494.3 119. 61 12 a b c Ion used for quantification. The internal standard material for the quantification of chlortetracycline and doxycycline. The relative ion abundance ratio of the selected product ions for the standard solution, 1 ng/ml of each compound.

236 Kanda et al.: Journal of aoac InternatIonal Vol. 98, no. 1, 215 (A) (A) (A) Highly polar veterinary drugs medium polar veterinary drugs Vortex (1 min) Vortex (1 min) Ultrasonicate (1 min) Hexane Discard by pipetting Sample (5g) +I.S. Na 2 EDTA McIlvain s buffer (ph 7, 2 ml) Centrifuge (96 xg,2 min, 4 o C) +Hexane (5 ml) Load to First extraction PLS-3 (2 mg, 2 ml) (B) (B) Aqueous phase Vortex (1 min) Centrifuge (96 xg,1 min, 4 o C) Wash with water (5 ml) Vaccum-dry (3 min) +25% NaCl sol. (1 ml) Discard the passed through solution Second extraction Collect the eluate solution Remaining matrix (A) (A) (A) (A) Supernatant Vortex (1 min) Vortex (1 min) Violently shake (1 min) Ultrasonicate (1 min) Centrifuge (18 xg,1 min, 4 o C) +Water (2 ml) +Acetonitrile containing + MgSO 4.1% formic acid (1 ml) (3, 4 or 5 g) Using the second extracted solution as the elution solution for PLS-3 Aliquot Supernatant Analyse by LC/MS/MS Make up to 1 ml Transfer to a microtube Dilute by 2-fold Pour into an amber Centrifuge polypropylene (16 xg, 5 min, 4 o C) vial tube Figure 1. Schematic representation of the sample preparation procedure for the analysis of 37 veterinary drugs in livestock and fishery products. and confirmative ions) were monitored for each of the 37 veterinary drugs (Table 2). Several MS parameters including ion-spray voltage, source temperature, declustering potential, entrance potential, and four gas pressures were systematically varied according to the manual of flow injection analysis, and we selected the conditions that yielded the best sensitivity, as listed in the Experimental section. In particular, we noted the curtain gas, ion source gas 1 and 2 conditions that measured macrolides with high sensitivity. Because MS scans of some of penicillins showed that the most abundant molecular ion was the deprotonated molecule [M-H], it was excluded from the analytes in this study. LC Conditions LC conditions to determine multi-class veterinary drugs in livestock and fishery products were previously reported by our laboratory (9), in which a gradient mixture of.1% formic acid in 1 mm ammonium acetate and acetonitrile as the mobile phase and a C18 column were used. However, the sensitivities of TCs and QLs were low under these conditions. Because the ionization mode of these drugs was positive-ion ESI, the ammonium ion which lowered the sensitivity of [M+H] + was excluded from the mobile phase. The peak shapes of FQs and thiabendazole were split. The peak shapes of TCs, sulfathiazole, sulfamerazine, clopidol, and oxolinic acid were poor. The tailing factors of these drugs were.3.6. TCs and QLs which are strong metal chelating compounds interact with metal ion impurities remaining in the C18 column, which made their peaks broad. The novel organic hybrid silica base column (YMC-Triart) has been reported to reduce metal ion impurities and achieve good chromatographic retention and separation of metal chelating and hydrophilic compounds. Using the column, the peak shapes of TCs and QLs were better, and the sensitivities were improved by a factor of 3. Thiabendazole, sulfathiazole, sulfamerazine, and clopidol diluted with an organic solvent were poorly retained on the column because the organic solvent may act as a part of the mobile phase. We minimized the drug injection volume to 2 µl, which resulted in the peak widths at half height ranging from.1 to.44 min and the tailing factors ranging from.85 to 1.21. Extraction and Cleanup Procedure The extraction and cleanup procedure was developed using 11 veterinary drugs, norfloxacin, ciprofloxacin, chlortetracycline, doxycycline, 5-hydroxythiabendazole, clopidol, erythromycin A, spiramycin, lincomycin A, oxolinic acid, and sulfadimidine. Among these drugs, norfloxacin, ciprofloxacin, chlortetracycline, doxycycline, 5-hydroxythiabendazole, and clopidol did not achieve acceptable values following the guidelines of Japanese Ministry of Health, Labour, and Welfare (1, 11) by using our reported QuEChERS methods (8, 9), because these compounds were soluble in the aqueous phase. Erythromycin A and spiramycin represent the macrolides class. Lincomycin A represents the lincosamides. Oxolinic acid and sulfadimidine

Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 237 Acetonitrile containing.1% formic acid Na 2 EDTA-MacIlvain Buffer 12% 1% (a) 8% 6% 4% Extracted Ratio (%) a 2% % 12% 1% 8% 6% 4% 2% % norfloxacin First extraction using Na 2 EDTA-MacIlvain Buffer ciprofloxacin 5-hydroxythiabendazole clopidol chlortetracycline Second extraction using Acetonitrile containing.1% formic acid 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. doxycycline spiramycin tylosin Lincomycin oxolinic acid sulfadimidine (b) had higher accuracy than other drugs on LC-MS/MS. These compounds served as indicators, showing that the LC-MS/MS measurements are stable. After spiking 5 µl of a 1 µg/ml standard solution of these drugs into a minced swine muscle, the following studies were performed. At this time, the drugs were quantified by using matrix-matched calibration standard curves. Veterinary drugs were extracted from the sample using an ultrasonic machine (33 35). This procedure allowed the simultaneous handling of many samples and lowered the risk of contamination. The sufficient extraction ability was confirmed using the incurred swine muscle containing chlortetracycline. As extraction solvents, we compared ACN/FA used on our modified QuEChERS method (9) and Na 2 EDTA-McIlvaine s buffer used on our antibiotic extraction (5 7). The extracted rates of 11 drugs by Na 2 EDTA-McIlvaine s buffer were calculated as follows. Eleven drugs spiked into a swine muscle were extracted with Na 2 EDTA-McIlvaine s buffer. The extraction solution was loaded onto the PLS-3 cartridge, and was eluted with ACN/FA. This eluate was analyzed by LC-MS/MS. The recovery rates (a) were calculated. Na 2 EDTA-McIlvaine s buffer spiked with 11 drugs was loaded onto the PLS-3 cartridge. The recovery rates from the PLS-3 cartridge (b) were calculated. The extraction rates by Na 2 EDTA-McIlvaine s buffer were corrected (a) using (b). The ph of the buffer was set as 7. because the retention of drugs was better than at ph 4. Na 2 EDTA was added to the buffer because the extraction of TCs and QLs were better with buffer containing Na 2 EDTA which had the ability to chelate divalent cations (8, 13, 16, 19, 22). As shown in Figure 2a, the extraction rate of each drug, i.e., norfloxacin, ciprofloxacin, chlortetracycline, doxycycline, and lincomycin A was better with Recovery Ratio (%) a Non-combination of the eluted solution 12% 1% 8% 6% 4% 2% Combination of the eluted solution (a) Rate of matrix effect a % 15 14 13 12 11 1 9 8 7 6 5 4 3 2 1 norfloxacin ciprofloxacin 5-hydroxythiabendazole clopidol chlortetracycline 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. doxycycline spiramycin tylosin Lincomycin oxolinic acid sulfadimidine (b)

238 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 5x1 4 Marbofloxacin 1x1 3 5x1 5 Oxolinic acid 1x1 4 5x1 4 Norfloxacin 1x1 3 5x1 5 Nalidixic acid 1x1 4 15x1 4 Ofloxacin 1x1 3 5x1 4 Flumequine 1x1 3 5x1 4 Ciprofloxacin 1x1 3 5x1 4 Oxytetracycline 1x1 3 Intensity 1x1 4 Danofloxacin 1x1 3 Intensity 5x1 4 Tetracycline 1x1 3 1x1 4 Enrofloxacin 1x1 3 1x1 4 Chlortetracycline 1x1 3 1x1 4 Orbifloxacin 1x1 3 5x1 4 Doxycycline 1x1 3 5x1 4 Sarafloxacin 1x1 3 1x1 4 Demeclocycline 1x1 3 1x1 4 Difloxacin 1x1 3 5 6 7 8 9 5 6 7 8 9 Retention time (min) 5 5 6 7 8 9 6 7 8 9 Retention time (min) 4x1 3 Spiramycin 1x1 3 2x1 5 Sulfadiazine 2x1 3 4x1 3 Tilmicosin 1x1 3 2x1 5 Sulfathiazole 1x1 3 5x1 4 Mirosamaycin 1x1 3 1x1 5 Sulfamerazine 1x1 3 3x1 4 Oleandomycin 1x1 3 1x1 5 Sulfadimidine 1x1 3 Intensity 3x1 4 Erythromycin A 1x1 3 Intensity 2x1 4 Sulfamonomethoxine 1x1 3 1x1 4 Tylosin 1x1 3 1x1 5 Sulfamethoxazole 1x1 3 5x1 4 Josamycin 1x1 3 1x1 5 Sulfaquinoxaline 1x1 3 5x1 5 Lincomycin A 1x1 4 1x1 5 Pirlimycin 1x1 3 3x1 5 Sulfadimethoxine 1x1 3 5 6 7 8 9 5 6 7 8 9 Retention time (min) 5 6 7 8 9 5 6 7 8 9 Retention time (min) Figure 4. Chromatograms obtained in the MRM mode (quantification transition) for swine muscle spiked with 1 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, 215 239 Intensity 1x1 5 3x1 5 3x1 5 5-hydroxythiabendazole Clopidol Thiabendazole Tiamulin 5 6 7 8 9 5 6 7 8 9 Retention time (min) Figure 4. (continued) Chromatograms obtained in the MRM mode (quantification transition) for swine muscle spiked with 1 mg/kg of 37 veterinary drugs (a), and for corresponding blank swine muscle (b). Na 2 EDTA-McIlvaine s buffer than with ACN/FA. The second extraction using Na 2 EDTA-McIlvaine s buffer did not improve recovery rates. The first extraction using Na 2 EDTA-McIlvaine s buffer and second extraction step using ACN/FA improved recovery rates to over 7%, except for chlortetracycline and doxycycline, which were unstable in solution. Therefore, polar veterinary drugs were extracted with two different polar solvents, Na 2 EDTA-McIlvaine s buffer (ph 7.) and ACN/FA. Subsequently, we evaluated the two conditions to elute the compounds from the SPE polymer cartridge which retained the compounds first-extracted by Na 2 EDTA-McIlvaine s buffer. (1) A new ACN/FA (1 ml) was used as the elution solution. The resultant eluate and the second extracted solution were mixed and analyzed by LC-MS/MS. 2x1 3 1x1 3 1x1 3 (2) The second extracted solution was re-used as the elution solution. The eluate was diluted by 2-fold with ACN/FA, and analyzed by LC-MS/MS. On (1) and (2) conditions, the recovery rates of 11 veterinary drugs were the same (Figure 3a). However, the matrix effects were dramatically different. The matrix effect was defined as the ratio of the slope of the matrix-matched calibration curve and the standard solution calibration curve. On the condition of (1), strong matrix enhancements were found for norfloxacin, ciprofloxacin, chlortetracycline, doxycycline, spiramycin, and lincomycin A. In contrast, the matrix enhancements were not observed under (2) conditions. Because the pork fatty acids and phospholipids were reported to be retained by the SPE polymer cartridge (36, 37), the interfering matrix was considered to be cleaned-up when the second extraction solution was passed through the SPE polymer cartridge (Figure 3b). Finally, we chose the extraction and cleanup procedure shown in Figure 1. Instrument Performance Figure 4a shows the SRM chromatograms obtained from swine muscle spiked with 1 µg/kg of 37 veterinary drugs and demeclocycline. No matrix effect was observed on peak shape in all samples. The retention time determined for the spiked samples was not significantly different from that determined for the standards. The relative ion abundance ratios of the selected product ions for each compound are shown in Table 2 together with those of the standard solutions. All of the relative ion abundance ratios of the spiked samples were within 2% of those of the standard solutions, which satisfied the permitted tolerance 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. 1.8 1.6 The slope ratio a 1.4 1.2 1..8.6.4.2 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 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.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; (1) oxolinic acid; (11) 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; (2) mirosamycin; (21) oleandomycin; (22) erythromycin A (23) tylosin; (24) josamycin; (25) lincomycin A; (26) pirlimycin; (27) sulfadiazine; (28) sulfathiazole; (29) sulfamerazine; (3) sulfadimidine; (31) sulfamonomethoxine; (32) sulfamethoxazole; (33) sulfaquinoxaline; (34) sulfadimethoxine; (35) 5-hydroxythiabendazole; (36) clopidol; (37) thiabendazole; (38) tiamulin.

24 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 Table 3. Validation results of veterinary drugs Swine muscle Chicken muscle Trueness, % (RSD a r, %; RSDW b R, %) Trueness, % (RSD a r, %; RSDW b R, %) Analytes 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg Quinolones Marbofloxacin 82 (6; 6) 89 (4; 7).5 5 8 (5; 5) 86 (6; 6).5 1 c Norfloxacin 74 (6; 7) 77 (4; 7) 2 2 74 (6; 9) 76 (1;1) 2 2 Ofloxacin 82 (6; 6) 93 (3; 7).2 1 c 82 (6; 7) 91 (5; 1).2 5 Enrofloxacin 87 (7; 7) 92 (4; 6).5 82 (6; 6) 87 (7;1).2 5 d 5 d Ciprofloxacin 72 (7; 6) 81 (3; 6) 1 73 (7; 5) 76 (7; 6) 1 Danofloxacin 85 (9; 9) 84 (5; 7) 2 1 77 (1;13) 76 (4;11) 2 2 Orbifloxacin 89 (8; 9) 98 (7; 8).5 2 87 (6; 5) 92 (8; 8).5 1 c Sarafloxacin 8 (9; 9) 88 (4; 7).5 1 c 81 (6; 8) 89 (3; 8).5 1 Difloxacin 88 (5; 9) 96 (5; 5) 1 2 8 (9; 9) 92 (7; 6).5 1 c Oxolinic acid 94 (5; 7) 11 (3; 4).5 2 89 (4; 6) 1 (5; 5).5 3 Nalidixic acid 93 (6; 9) 11 (4; 6).5 1 c 89 (3; 3) 95 (3; 4).5 1 c Flumequine 91 (5; 5) 97 (3; 5).2 5 88 (5; 5) 96 (3; 4).2 5 Tetracyclines Oxytetracycline 79 (7;1) 77 (4; 8) 1 72 (3; 9) 75 (4; 6) 1 Tetracycline 79 (11; 9) 8 (4; 8) 1 2 e 78 (4; 8) 72 (4; 5) 1 2 e Chlortetracycline 92 (9;12) 93 (4; 7) 2 11 (1;13) 95 (6;12) 2 Doxycycline 83 (9; 9) 81 (2; 6).5 5 96 (9; 9) 88 (5;1) 1 5 Demeclocycline f 76 (12;1) 69 (3;11).5 59 (9;14) 75 (5; 8) 1 Macrolides Spiramycin 85 (8; 9) 87 (13;1) 1 2 94 (15;12) 83 (9;13) 1 2 Tilmicosin 92 (1;11) 94 (4; 5) 1 1 82 (8;14) 1 (5; 8) 1 7 Mirosamycin 87 (7; 1) 96 (2; 9).2 5 83 (6; 1) 96 (3; 3).2 4 Oleandomycin 94 (6; 6) 98 (3; 3).5 1 87 (7; 6) 1 (8; 7).5 2 Erythromycin A 1 (7; 8) 98 (4; 4).5 5 91 (4; 3) 98 (2; 5).5 5 Tylosin 82 (6; 6) 91 (4; 7).2 5 73 (9;1) 8 (8; 9).2 5 Josamycin 88 (3; 3) 9 (4; 4).2 4 82 (4; 6) 91 (3; 6).2 4 Lincosamides Lincomycin A 92 (4; 4) 92 (3; 8).5 2 85 (5; 5) 94 (4; 4).5 2 Pirlimycin 78 (6; 5) 77 (6; 7).2 1 c 75 (6; 7) 79 (5; 6).2 1 c Sulfonamides Sulfadiazine 98 (4; 7) 12 (5; 8).2 1 96 (4; 5) 19 (5; 6).2 1 Sulfathiazole 96 (4; 7) 19 (6;1).5 1 94 (6; 6) 18 (6;1).5 1 Sulfamerazine 96 (8; 8) 19 (7; 8).5 1 89 (8;1) 11 (1; 7).2 1 c Sulfadimidine 97 (7; 7) 14 (5; 6).5 1 91 (5; 5) 15 (4; 5).2 1 Sulfamonomethoxine 95 (1;11) 18 (3; 4) 2 2 91 (6; 6) 11 (4; 4) 2 1 Sulfamethoxazole 95 (4; 4) 12 (3; 4).5 2 92 (4; 5) 98 (5; 4).5 2 Sulfaquinoxaline 92 (7; 1) 97 (3; 9) 1 1 c 86 (6; 7) 95 (4; 5) 1 5 Sulfadimethoxine 84 (5; 4) 96 (3; 7).2 2 89 (4; 6) 99 (2; 2).2 5 Others Thiabendazole 84 (7; 6) 96 (2; 4).2 1 g 83 (5; 5) 98 (7; 6).2 5 g 5-hydroxythiabendazole 77 (5; 6) 86 (2; 5).1 81 (3; 6) 93 (3; 3).1 Clopidol 89 (4; 6) 99 (4; 4) 1 2 89 (3; 3) 99 (4; 3).5 5 Tiamulin 84 (6; 6) 87 (4; 7).2 4 79 (7; 5) 86 (4; 5).2 1

Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 241 Table 3. (continued) Bovine muscle Prawn Trueness, % (RSD a r, %; RSDW b R, %) Trueness, % (RSD a r, %; RSDW b R, %) Analytes 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg Quinolones Marbofloxacin 85 (4; 8) 11 (4; 4) 1 1 79 (6; 9) 89 (3; 5) 1 1 c Norfloxacin 73 (6; 6) 81 (4; 6) 2 1 c 74 (6; 5) 85 (3; 6) 1 1 c Ofloxacin 84 (8; 1) 95 (5; 7).2 1 c 8 (7; 7) 9 (5; 8).2 1 c Enrofloxacin 84 (3; 5) 95 (2; 6) 1 86 (4; 6) 99 (3; 3).5 5 d 1 d Ciprofloxacin 71 (6; 6) 8 (2; 3) 1 74 (7; 6) 87 (3; 4) 2 Danofloxacin 79 (8; 7) 83 (4; 6) 5 2 78 (4; 8) 85 (1; 7) 2 1 Orbifloxacin 88 (5; 8) 11 (3; 5).5 2 9 (2; 4) 97 (3; 4).5 1 c Sarafloxacin 81 (4; 5) 89 (4; 5) 1 1 c 86 (6; 8) 91 (4; 8) 1 1 c Difloxacin 88 (4; 3) 97 (3; 5) 1 1 c 92 (4; 8) 98 (4; 5).5 1 c Oxolinic acid 89 (5; 7) 1 (2; 3).5 1 92 (5; 8) 98 (2; 3).5 3 Nalidixic acid 88 (5; 8) 96 (2; 3).5 1 c 91 (3; 7) 96 (1; 3).5 1 c Flumequine 86 (4; 7) 97 (2; 6).2 5 9 (5; 9) 96 (2; 4).2 1 c Tetracyclines Oxytetracycline 7 (5; 6) 73 (3; 6) 1 77 (9;14) 78 (3; 6) 2 2 Tetracycline 71 (6; 7) 71 (5; 6) 1 2 e 75 (7; 6) 78 (5; 8) 1 1 c Chlortetracycline 16 (5; 9) 96 (2;11) 2 92 (7; 8) 84 (5; 7) 2 1 c Doxycycline 9 (5; 7) 9 (4;1) 1 1 81 (7;1) 77 (3; 6) 1 1 c Demeclocycline f 61 (9;11) 69 (3;12) 1 64 (6; 7) 75 (5; 9) 1 Macrolides Spiramycin 87 (9; 8) 85 (8;1) 1 2 86 (7; 8) 9 (5;1) 1 2 Tilmicosin 91 (9; 8) 99 (2; 3).2 1 93 (5;1) 96 (3; 7).2 1 c Mirosamycin 86 (4; 8) 97 (4; 5).2 1 c 82 (4; 6) 91 (2; 7).2 1 c Oleandomycin 91 (5; 7) 13 (3; 5) 1 5 95 (2; 5) 11 (1; 3).2 1 c Erythromycin A 93 (5; 7) 12 (3; 5) 1 5 99 (5; 5) 12 (2; 2) 1 2 Tylosin 81 (5; 7) 9 (1; 6) 1 5 82 (6; 9) 92 (4; 5).1 1 Josamycin 85 (5; 7) 96 (2; 4) 1 1 c 91 (4; 7) 94 (3; 4).5 1 c Lincosamides Lincomycin A 94 (15;12) 83 (9;13) 2 2 92 (2; 5) 98 (2; 5).5 1 Pirlimycin 71 (3; 3) 76 (3; 4).2 1 82 (4; 9) 87 (2; 4).2 1 c Sulfonamides Sulfadiazine 13 (4; 5) 118 (4; 4) 1 1 95 (4; 6) 11 (4; 4).2 1 c Sulfathiazole 1 (5; 6) 114 (3; 5) 1 1 91 (4; 6) 11 (2; 8) 1 1 c Sulfamerazine 93 (6; 6) 12 (2; 5).5 1 93 (3; 5) 99 (2; 4).2 1 c Sulfadimidine 92 (3; 4) 14 (1; 4).5 1 93 (4; 5) 1 (2; 2).2 1 c Sulfamonomethoxine 92 (4; 6) 99 (2; 2) 2 1 96 (6; 6) 1 (3; 3) 1 1 c Sulfamethoxazole 89 (5; 8) 99 (2; 3).5 1 c 96 (3; 5) 97 (2; 2).5 1 c Sulfaquinoxaline 82 (5; 8) 94 (3; 4).5 1 91 (5; 1) 92 (2; 4).2 1 c Sulfadimethoxine 85 (5; 7) 98 (3; 3).2 5 94 (4; 7) 96 (2; 5).2 1 c Others Thiabendazole 83 (7; 6) 93 (4; 8).2 1 g 85 (5; 7) 9 (5; 7).2 2 g 5-hydroxythiabendazole 71 (3; 3) 79 (4; 7).1 88 (4; 6) 95 (3; 5).1 Clopidol 92 (3; 6) 15 (2; 8).5 2 93 (4; 8) 1 (4; 3).2 1 c Tiamulin 76 (4; 6) 9 (3; 8).2 1 c 8 (4; 7) 83 (3; 5).1 1 c

242 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 Table 3. (continued) Salmon trout Red sea bream Trueness, % (RSD a r, %; RSDW b R, %) Trueness, % (RSD a r, %; RSDW b R, %) Analytes 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg Quinolones Marbofloxacin 82 (5; 5) 87 (1; 5) 1 1 c 85 (4;1) 92 (2; 6) 1 1 c Norfloxacin 72 (5; 6) 75 (3; 3) 2 1 c 77 (5;11) 81 (3; 6) 1 1 c Ofloxacin 87 (5; 6) 96 (3; 5) 1 1 c 88 (3; 8) 95 (3; 6).5 1 c Enrofloxacin 89 (5; 7) 92 (5; 8) 1 88 (4; 6) 99 (3; 7).5 1 c,d Ciprofloxacin 75 (7; 9) 8 (2; 6) 5 75 (5; 6) 88 (4; 6) 2 Danofloxacin 84 (8;11) 84 (4; 5) 2 1 84 (9; 7) 93 (4;14) 5 1 Orbifloxacin 92 (6; 5) 93 (2; 5).2 1 c 93 (4; 5) 98 (1; 5).2 1 c Sarafloxacin 82 (4; 6) 89 (3; 4) 1 3 86 (5; 5) 94 (2; 5) 1 1 c Difloxacin 92 (5; 5) 94 (3; 3).5 1 c 92 (5; 4) 95 (3; 5) 1 1 c Oxolinic acid 92 (4; 8) 95 (4; 4).5 1 95 (2; 3) 98 (1; 2).2 6 Nalidixic acid 89 (6; 5) 93 (3; 4).5 1 c 92 (3; 4) 97 (2; 2).5 1 c Flumequine 92 (5; 8) 94 (4; 5).2 5 92 (3; 4) 97 (1; 5).2 4 Tetracyclines Oxytetracycline 76 (7; 9) 79 (6; 7) 1 2 78 (5; 9) 79 (4; 9) 2 2 Tetracycline 77 (7; 8) 76 (4; 5) 1 1 c 74 (6;11) 76 (4; 8) 1 1 c Chlortetracycline 13 (3; 5) 97 (4; 4) 2 1 c 16 (7;1) 98 (3; 4) 1 1 c Doxycycline 93 (6; 5) 87 (2; 5).5 1 c 113 (9; 8) 15 (5; 9).5 5 Demeclocycline f 64 (8;15) 71 (4; 6) 1 54 (1;12) 67 (3; 7) 1 Macrolides Spiramycin 93 (8; 9) 12 (5; 6) 1 2 9 (7;1) 91 (7;11) 1 2 Tilmicosin 9 (5; 8) 96 (3; 3).2 1 c 9 (6; 8) 96 (2; 9).5 1 c Mirosamycin 87 (6; 6) 9 (4; 4).2 1 c 94 (3; 3) 13 (3; 7).2 1 c Oleandomycin 93 (5; 5) 99 (4; 3) 1 1 c 99 (3; 4) 1 (3; 9).2 5 Erythromycin A 97 (4; 5) 99 (4; 8).2 2 12 (4; 5) 13 (4;1).2 6 Tylosin 89 (4; 4) 95 (4; 6).2 1 9 (4; 6) 96 (2; 5).2 1 Josamycin 86 (4; 4) 92 (3; 6).5 1 c 92 (4; 5) 94 (1; 5).5 5 Lincosamides Lincomycin A 9 (4; 3) 94 (2; 4) 2 1 96 (3; 8) 99 (2; 4).2 5 Pirlimycin 81 (5; 5) 84 (4; 4).2 1 c 8 (5; 6) 86 (4; 8).2 1 c Sulfonamides Sulfadiazine 89 (7; 9) 92 (4; 7).2 1 96 (5; 5) 11 (2; 3).1 1 c Sulfathiazole 93 (5; 7) 97 (4; 5).5 1 c 82 (4; 8) 87 (2; 5).5 1 c Sulfamerazine 93 (3; 8) 95 (3; 4).1 1 c 97 (4; 6) 99 (3; 8).2 1 c Sulfadimidine 81 (5;11) 9 (5; 5).2 1 c 96 (2; 5) 97 (3; 5).2 1 c Sulfamonomethoxine 92 (3; 7) 96 (2; 5).2 1 81 (5; 8) 87 (4;12) 2 1 Sulfamethoxazole 78 (6; 9) 81 (4; 4).5 1 c 95 (4; 4) 11 (1; 6).5 1 c Sulfaquinoxaline 78 (6; 9) 86 (4; 6).5 1 c 74 (4; 6) 77 (1; 6).2 1 c Sulfadimethoxine 82 (5; 5) 9 (3; 4).2 1 83 (3; 4) 9 (1; 5).2 1 c Others Thiabendazole 75 (5; 6) 79 (5; 7).2 2 g 86 (6; 7) 94 (1; 3).2 2 g 5-hydroxythiabendazole 82 (5; 5) 9 (3; 4).1 85 (4; 6) 89 (2; 5).1 Clopidol 94 (6; 5) 97 (4; 5).2 1 c 95 (4; 7) 11 (2; 5) 2 1 c Tiamulin 82 (5; 5) 87 (1; 5) 1 1 c 77 (4; 6) 85 (2; 6).1 1 c 1 c,d

Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 243 Table 3. (continued) Flounder Milk Trueness, % (RSD a r, %; RSDW b R, %) Trueness, % (RSD a r, %; RSDW b R, %) Analytes 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg Quinolones Marbofloxacin 78 (8; 7) 88 (4; 3) 1 1 c 91 (2; 4) 92 (4; 9) 1 75 Norfloxacin 67 (7; 7) h 78 (5; 4) 2 1 c 84 (4; 5) 89 (2; 5) 2 1 c Ofloxacin 78 (5; 5) 9 (5; 4).5 1 c 93 (3; 5) 93 (5; 6).5 1 c Enrofloxacin 83 (6; 6) 91 (4; 5).5 93 (4; 5) 91 (4; 5) 1 1 c,d 5 d Ciprofloxacin 71 (5; 6) 8 (4; 4) 1 87 (5;1) 86 (2; 7) 2 Danofloxacin 31 (17; 27) h 57 (6; 7) h 5 1 86 (8; 9) 85 (4; 9) 2 5 Orbifloxacin 85 (3; 5) 96 (4; 3).2 1 c 89 (6; 6) 93 (4; 4).5 2 Sarafloxacin 8 (4; 4) 89 (3; 4) 1 1 c 88 (6; 6) 92 (2; 9) 5 1 c Difloxacin 86 (5; 5) 96 (4; 5).5 1 c 94 (3; 3) 92 (5; 7) 1 1 c Oxolinic acid 89 (5; 6) 1 (2; 5).5 5 95 (2; 7) 97 (3; 2).5 1 c Nalidixic acid 9 (5; 4) 1 (1; 4).5 1 c 91 (3; 6) 98 (2; 4).2 1 c Flumequine 89 (5; 5) 97 (2; 2).2 6 89 (2; 9) 94 (4; 5).2 1 Tetracyclines Oxytetracycline 72 (6; 7) 77 (4; 4) 1 2 94 (5; 8) 93 (3; 3) 2 Tetracycline 73 (5; 6) 76 (4; 6) 2 1 c 89 (4; 4) 93 (3; 8) 1 1 e Chlortetracycline 12 (4; 9) 95 (2; 2) 1 1 c 98 (4; 6) 92 (2; 4) 2 Doxycycline 112 (6;1) 13 (3; 4) 1 1 c 98 (4; 5) 94 (4; 5) 1 1 c Demeclocycline f 63 (9; 9) 74 (3; 7) 1 88 (9;1) 93 (6; 6) 1 Macrolides Spiramycin 84 (7;1) 95 (5; 5) 1 2 89 (6; 6) 89 (3; 4) 1 2 Tilmicosin 89 (7; 8) 93 (5; 9).2 1 c 88 (5;1) 9 (3; 7).5 5 Mirosamycin 89 (6; 6) 95 (4; 5).2 1 c 91 (4; 7) 91 (3; 4).1 1 c Oleandomycin 89 (6; 6) 95 (4; 3).2 1 c 94 (5; 7) 93 (1; 6).2 5 Erythromycin A 97 (3; 3) 12 (3; 4).2 2 96 (3; 4) 93 (3; 4).2 4 Tylosin 87 (5; 6) 95 (4; 3).2 1 88 (7; 7) 88 (3; 3).2 5 Josamycin 86 (4; 4) 95 (3; 3).5 1 c 86 (2; 4) 87 (3; 7).5 1 c Lincosamides Lincomycin A 86 (5; 5) 96 (3; 5).5 1 88 (3; 4) 91 (2; 3).5 15 Pirlimycin 75 (5; 6) 82 (3; 3).2 1 c 93 (5; 8) 92 (5; 8).2 3 Sulfonamides Sulfadiazine 92 (7; 7) 12 (4; 5).2 1 c 92 (3; 7) 98 (3; 4).2 7 Sulfathiazole 74 (7; 7) 89 (4; 5).5 1 c 95 (4; 8) 97 (3; 7).5 9 Sulfamerazine 93 (7; 7) 99 (5; 5).2 1 c 92 (4; 9) 91 (5; 7).2 1 c Sulfadimidine 9 (6; 5) 97 (3; 5).2 1 c 93 (5; 5) 96 (5; 5).2 25 Sulfamonomethoxine 74 (7; 5) 85 (3;12) 2 1 89 (4; 6) 98 (4; 5) 1 1 c Sulfamethoxazole 86 (4; 7) 98 (2; 4).5 1 c 96 (4; 5) 93 (3; 3).2 1 c Sulfaquinoxaline 64 (6; 8) h 75 (2; 3).2 1 c 91 (4; 8) 95 (4; 5).5 1 Sulfadimethoxine 72 (5; 5) 84 (3; 5).2 1 93 (4; 5) 94 (3; 3).1 2 Others Thiabendazole 82 (5; 5) 93 (3; 5).2 2 g 87 (4; 5) 92 (3; 3).2 1 g 5-hydroxythiabendazole 81 (4; 4) 94 (3; 4).1 81 (4; 9) 89 (5; 9).1 Clopidol 91 (5; 5) 1 (2; 2) 2 1 c 9 (4; 4) 93 (4; 4) 1 2 Tiamulin 75 (6; 5) 85 (2; 5).1 1 c 77 (5; 5) 8 (5;11).1 1 c

244 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 215 Table 3. (continued) Egg Honey Trueness, % (RSD a r, %; RSDW b R, %) Trueness, % (RSD a r, %; RSDW b R, %) Analytes 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg 1 μg/kg 1 μg/kg LOQ, μg/kg MRL, μg/kg Quinolones Marbofloxacin 79 (4; 6) 75 (4; 9).5 1 c 96 (5; 5) 11 (2; 2) 1 1 c Norfloxacin 8 (4; 5) 81 (4; 6) 2 1 c 92 (6; 6) 95 (1; 4) 1 1 c Ofloxacin 88 (4; 5) 91 (3; 5).2 1 c 98 (4; 5) 12 (2; 3) 1 1 c Enrofloxacin 9 (4; 5) 91 (5; 7).5 92 (5; 6) 98 (4; 4) 2 1 c,d Ciprofloxacin 81 (7; 7) 82 (2; 7) 1 95 (6; 4) 1 (2; 4) 5 95 (6; 5) 96 (2; 2) 1 Danofloxacin 89 (5; 9) 87 (6; 7) 5 1 c 96 (5; 4) 12 (3; 4) 1 1 c Orbifloxacin 86 (5; 5) 85 (4;12).5 1 c 92 (3; 6) 1 (4; 4).5 1 c Sarafloxacin 83 (3; 3) 88 (3; 4) 1 1 c 91 (5; 7) 98 (3; 3) 2 1 c Difloxacin 9 (4; 7) 94 (3; 4).5 1 c 96 (3; 3) 1 (3; 4) 2 1 c Oxolinic acid 9 (3; 5) 96 (2; 2) 2 1 c 97 (3; 5) 1 (2; 4) 1 1 c Nalidixic acid 9 (3; 2) 93 (2; 6).2 1 c 96 (3; 3) 1 (3; 3).2 1 c Flumequine 76 (3; 5) 84 (3; 4).2 1 c 95 (3; 3) 1 (3; 4).2 1 c Tetracyclines Oxytetracycline 76 (3; 4) 75 (2; 4) 1 Tetracycline 84 (6; 7) 79 (4; 7) 1 4 e 92 (5; 5) 92 (2; 6) 2 3 e Chlortetracycline 99 (3; 7) 9 (3; 5) 1 85 (6;1) 83 (4; 4) 2 Doxycycline 113 (5; 8) 112 (4; 7).5 1 c 13 (9; 9) 11 (3; 4).5 1 c Demeclocycline f 6 (7; 7) 57 (4;1) 2 88 (6; 9) 97 (3;1) 2 Macrolides Spiramycin 94 (7;1) 9 (4; 6) 1 1 c 96 (7; 7) 98 (5; 5) 1 1 c Tilmicosin 92 (5; 7) 96 (7; 3) 1 1 c 94 (4; 6) 11 (4; 6) 1 1 c Mirosamycin 92 (4; 4) 96 (1; 3).1 1 c 96 (3; 4) 13 (4; 4).1 5 Oleandomycin 93 (2; 6) 98 (2; 3).5 1 c 94 (4; 5) 12 (4; 4).2 1 c Erythromycin A 98 (2; 4) 98 (2; 4).2 9 11 (3; 5) 98 (2; 5).2 1 c Tylosin 91 (4; 6) 91 (3; 5).2 2 97 (4; 4) 12 (4; 3).2 1 c Josamycin 84 (2; 3) 89 (1; 3).5 1 c 9 (6; 6) 95 (2; 4).5 1 c Lincosamides Lincomycin A 81 (4; 8) 78 (4; 7).2 1 96 (3; 5) 99 (2; 3).5 1 c Pirlimycin 81 (4; 7) 82 (4; 8).2 1 c 95 (3; 3) 96 (4; 7).2 1 c Sulfonamides Sulfadiazine 72 (9;16) 57 (12;24).2 2 97 (4; 6) 99 (2; 3).2 1 c Sulfathiazole 97 (4; 3) 1 (3; 4) 1 1 c 92 (6; 6) 98 (5; 5).5 1 c Sulfamerazine 91 (4; 5) 9 (3; 6).2 1 c 92 (6; 4) 1 (3; 3).2 1 c Sulfadimidine 94 (3; 6) 96 (4; 3).2 1 92 (4; 5) 98 (4; 4).2 1 c Sulfamonomethoxine 92 (3; 5) 93 (2; 5).5 1 c 95 (4; 6) 1 (3; 4) 2 1 c Sulfamethoxazole 91 (2; 4) 94 (2; 4).2 1 c 92 (5; 5) 1 (2; 3).2 1 c Sulfaquinoxaline 9 (3; 4) 9 (2; 3).2 1 9 (3; 5) 98 (2; 3).2 1 c Sulfadimethoxine 9 (2; 3) 91 (1; 3).1 1 92 (4; 4) 1 (2; 3).1 1 c 1 c,d