5 12 Vol. 5 No. 12 2014 12 Journal of Food Safety and Quality Dec., 2014 王培锋 *, 王凤美, 张鸿伟, 徐彪, 邱芳, 何京澄, 周维政 (, 266002) 摘要 : 目的 Q-Exactive 方法, QuEChERS,, ACQUITY BEH C 18, 0.1% -, Q-Exactive, 结果 31 5 10 6 0.99, (limit of detection, LOD) 10 μg/kg 结论, 关键词 : ; ; Q-Exative Screening of antibiotics in foodstuffs of animal origin by high performance liquid chromatography-quadrurpole/electrostatic field orbitrap high resolution mass spectrometry WANG Pei-Feng *, WANG Feng-Mei, ZHANG Hong-Wei, XU Biao, QIU Fang, HE Jing-Cheng, ZHOU Wei-Zheng (Technical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao 266002, China) ABSTRACT: Objective To develop a method of high performance liquid chromatography (HPLC) coupled with linear ion trap/orbitrap high resolution mass spectrometry (QuEChERS) to detect antibiotics quickly in food of animal origin. Methods Antibiotics in samples were extracted using a solvent of acidic acetonitrile, following by purification with QuEChERS, dilution with acetonitrile and H 2 O. The separation was performed on an ACQUITY BEH C 18 column using gradient elution with aqueous solution (containing 0.1% formic acid)/acetonitrile as a mobile phase. The accurate mass of quasi-molecular ion were acquired by full scanning of electrostatic field orbitrap and applied for rapid screening and analysis, further confirmation was accomplished according to the retention time and qualitative fragments obtained by data dependent scan mode. Results Under the optimal conditions, 31 antibiotics were routinely detected with mass accuracy error below 5 10 6, and good linearity were provided in their respective linear ranges with correlation coefficients all higher than 0.99, the limit of detection (LOD) for all the 31 antibiotics were 10 μg/kg. Conclusion The method was applied to fish, milk, and honey and satisfying screening results proved that it is an efficient path 基金项目 : (2013IK176)(12-1-3-80-jh) (201510036-01) Fund: Supported by the Scientific and Technological Project of the General Administration of Quality Supervision, Inspection and Quarantine of the People s Republic of China (2013IK176), Science and Technology Support Plan of Qingdao (12-1-3-80-jh) and Scientific Research Projects by Quality Inspection Service Industry (201510036-01) * 通讯作者 :,, Email: 33642759@qq.com *Corresponding author: WANG Pei-Feng, Engineer, Technical Center of Shandong Entry-Exit Inspection & Quarantine Bureau, No.70, Qutangxia Road, Shinan District, Qingdao 266002, Shandong Province, China. E-mail: 33642759@gmail.com
3770 5 for rapid analysis of antibiotics in food of animal origin. KEY WORDS: antibiotics, food of animal origin, Q-exactive high resolution mass spectrometry 1 引言, (fluoroquinolones, FQs) 3, 6, 7, FQs, FQs,, FQs,,, (sulfonamides, SAs),,,,,, FQs SAs [1-3] [4-7] - [8-10], -,,,,, /,,,,, / [11] [12] [13-15] /, 2 材料和方法 2.1 仪器与试剂 Thermo Q-Exactvie ( Thermo Fisher ); Himac CR22GII ( Hitachi ); MS1Minishaker ( IKA ); FA1104 ( ); Milli-Q (18.2 MΩ cm) ( Millipore ) (, Fluka ); (, Merck ); (, Mallinckrodt Bake ) -6 1-5-, Dr. Ehrenstorfer, 99% 2.2 标准品溶液的配制, 100 μg/ml, 18, 2.3 实验部分 2 g( 0.01 g) 50 ml, 4 ml, 1 min, 10 ml1%, 1 min, Quchers 5980-0032, 10 min, 4 10000 r/min 5 min, 30 min,
12, : - 3771 6 ml Quchers 5980-4950, 1 min, 10000 r/min 5 min, 4 ml, 40, :0.1% (1:9), 1 min, 15000 r/min 10 min,, 2.4 色谱 - 质谱条件 2.4.1 色谱条件 : Waters ACQUITY UPLC BEH C 18 (2.1 mm 100 mm, 1.7 μm, Waters ); : 300 μl/min; : 10 μl; : 30 ; A 0.1%, B ; 2.4.2 质谱条件 : Q-Exactive; : (electron spray ionization, ESI); : 350 ; : 3.0 kv; : 30 L/min; : 3.3 L/min; ; (m/z): 100~1000 R=70000 31 Q-Exactive MS 1 Table 1 1 31 Analysis parameters of the 31 antibiotics No. Compounds Retention time(min) Ion formation Theoretical (m/z) Measured (m/z) Mass accuracy error/10 6 (ppm) 1 2.03 [M+H] + 251.05972 251.05956 0.64 2 2.09 [M+H] + 311.08085 311.08120 1.12 3 3.28 [M+H] + 311.08085 311.08129 1.41 4 2.08 [M+H] + 291.14517 291.14510 0.24 5 2.43 [M+H] + 279.09102 279.09088 0.50 6 1.95 [M+H] + 215.04849 215.04840 0.42 7 2.16 [M+H] + 250.06447 250.06510 2.52 8 2.10 [M+H] + 256.02089 256.02064 0.97 9 2.27 [M+H] + 265.07537 265.07630 0.26 10 4.08 [M+H] + 279.09102 279.09113 1.27 11 2.83 [M+H] + 281.07029 281.06959 2.49 12 3.27 [M+H] + 281.07029 281.07064 1.64 13 3.27 [M+H] + 285.02075 285.02053 1.12 14 2.98 [M+H] + 254.05939 254.05953 2.39 15 3.27 [M+H] + 301.07537 301.07529 0.93 16 3.34 [M+H] + 315.09102 315.09071 0.98 17 2.48 [M+H] + 271.03179 271.03175 0.15 18 5 2.83 [M+H] + 281.07029 281.07036 0.25 19 2.83 [M+H] + 285.02075 285.02073 0.07 20 2.06 [M+H] + 320.14050 320.14064 0.44 21 2.23 [M+H] + 360.17180 360.17176 0.11 22 2.16 [M+H] + 358.15615 358.15618 0.11 23 2.43 [M+H] + 386.13107 386.13105 0.08 24 2.09 [M+H] + 334.15165 334.15156 0.21 25 3.70 [M+H] + 262.08740 262.08729 0.80 26 2.10 [M+H] + 332.14050 332.14062 0.66 27 2.08 [M+H] + 362.15106 362.15083 0.91 28 2.46 [M+H] + 400.14672 400.14665 0.17 29 2.02 [M+H] + 363.14631 363.14635 0.14 30 3.62 [M+H] + 232.08424 232.08431 0.30 31 3.07 [M+H] + 262.07100 262.07077 0.87
3772 5 3 结果与讨论 3.1 样品的提取,,, ( 0.1% 0.1%), 0.1%,, 3.2 色谱 / 质谱条件的优化 Waters ACQUITY UPLC BEH C 18, 31 ACQUITY UPLC BEH C 18 1.7 μm,,, ( -0.1% -0.1% ), 0.1%,, -0.1%, 0.1% - 31,,,, ESI + ESI - 31,, 31 ESI,, [M+H] +, 31,, 5 10-6 9 1~3 3.3 回收率 3, 1 (limit of quantity, LOQ) 2 (2LOQ) 5 (5LOQ) 3, 6, (relative standard deviation, RSD), 31 60.39%~110.90%,RSD 1.56%~9.83%( 2) Fig. 1 1 9 Extracted ion chromatograms of the 9 kinds of representative antibiotics in fish muscle
第 12 期 王培锋, 等: 高效液相色谱-四极杆轨道阱高分辨质谱对动物源食品中抗生素类成分的快速筛查 Fig. 2 Fig. 3 图 2 蜂蜜添加回收中 9 种代表抗生素提取离子色谱图 Extracted ion chromatograms of the 9 kinds of representative antibiotics in honey 图 3 牛奶添加回收中 9 种代表抗生素提取离子色谱图 Extracted ion chromatograms of the 9 kinds of representative antibiotics in milk 3773
3774 5 4 31 Fig. 4 TIC of 31 kinds of antibiotics in standard solution Compounds 表 2 鱼肉, 牛奶, 蜂蜜中 31 种抗生素化合物的添加回收率 Table 2 Recoveries of the 31 antibiotics spiked in cosmetic samples (n=6) Add Level (μg/l) Average(%) RSD(%) Average(%) RSD(%) Average(%) RSD(%) 10 100.03 4.23 100.08 6.92 100.28 2.85 20 99.05 4.50 98.05 8.19 98.06 3.42 50 95.08 4.98 98.064 7.38 99.29 4.62 10 102.36 513 90.35 3.58 93.37 2.84 20 100.35 3.85 92.35 3.20 107.23 3.57 50 105.97 5.46 110.90 3.49 102.93 4.02 10 98.05 4.09 96.35 5.24 95.63 6.89 20 96.05 4.10 98.35 3.53 90.36 5.22 50 99.02 3.00 99.94 3.24 101.44 6.50 10 90.20 5.53 98.22 5.55 94.10 4.02 20 97.66 4.25 96.44 4.17 97.96 3.50 50 95.97 3.63 95.67 5.04 96.81 4.51 10 84.92 5.45 94.52 3.63 75.33 2.38 20 84.84 3.83 94.46 5.59 76.06 3.36 50 90.32 5.14 100.51 2.04 89.87 4.91 10 64.30 6.50 74.85 5.05 80.79 4.09 20 79.18 5.73 89.29 6.03 83.62 4.14 50 81.79 3.14 94.38 3.58 93.19 3.84 10 93.65 4.55 93.08 3.87 94.94 3.25 20 94.05 3.80 92.45 4.68 94.11 3.41 50 92.33 5.59 91.68 6.65 98.67 5.09 10 85.35 3.69 95.01 4.16 68.33 9.45 20 74.97 4.88 83.44 4.68 87.61 6.36 50 77.83 6.55 86.67 7.69 99.71 2.78 10 70.12 7.24 78.02 6.85 100.35 5.40 20 85.65 5.31 95.35 5.74 89.25 6.77 50 86.89 6.55 96.70 6.43 99.23 4.50
12, : - 3775 续表 2 Compounds Add Level (μg/l) Average(%) RSD(%) Average(%) RSD(%) Average(%) RSD(%) 10 91.37 2.49 79.49 3.62 95.72 5.12 20 93.34 5.98 81.21 6.69 93.53 3.97 50 103.75 2.61 90.29 4.60 79.46 7.94 10 96.03 4.35 83.04 2.87 82.81 1.61 20 82.21 5.39 91.52 5.71 73.36 4.78 50 74.61 3.43 106.83 3.43 93.55 5.49 10 92.57 2.70 80.76 2.22 74.45 3.99 20 95.46 3.21 95.13 3.63 97.03 1.57 50 94.15 2.65 97.27 2.49 99.18 6.48 10 95.11 7.70 92.42 6.49 91.32 5.28 20 99.72 8.37 99.73 8.39 94.44 5.22 50 97.62 6.45 95.24 6.07 190.96 3.60 10 75.45 2.98 83.97 2.42 74.26 5.94 20 93.37 1.41 103.95 2.50 96.16 3.74 50 78.77 3.31 87.68 3.50 100.49 5.65 10 99.79 3.55 101.08 2.84 98.34 6.08 20 93.54 4.56 104.12 3.85 95.58 7.93 50 97.38 5.52 94.99 5.31 93.12 3.86 10 63.11 6.75 82.43 4.93 77.58 1.66 20 67.67 5.56 87.55 3.90 79.09 2.60 50 67.67 5.20 84.17 3.12 82.50 4.76 10 89.54 3.50 88.57 4.69 90.53 4.91 20 90.59 3.59 94.14 3.30 99.77 5.19 50 99.27 4.39 97.08 3.46 94.21 4.78 10 95.30 2.85 93.81 2.87 97.93 5.49 20 90.46 2.96 100.69 3.22 98.96 4.60 50 98.25 8.22 95.95 8.01 105.53 6.92 10 92.70 6.35 92.04 6.18 103.15 3.56 20 99.55 5.50 99.72 6.49 106.61 3.08 50 98.51 3.40 98.59 5.51 112.41 4.96 10 75.80 5.33 84.34 4.50 98.43 9.83 20 70.97 6.67 78.95 5.72 93.12 3.18 50 71.36 4.94 79.45 5.91 95.94 4.45 10 96.23 4.76 96.02 5.82 92.42 9.78 20 99.09 2.88 99.20 4.28 94.17 6.43 50 91.31 6.82 99.40 7.56 98.28 9.79 10 99.08 2.94 96.87 1.92 94.98 6.71 20 91.58 3.08 101.92 2.72 99.61 2.04 50 97.09 6.26 94.62 4.54 91.28 2.58 10 72.76 5.32 80.97 4.80 68.67 5.81 20 80.86 6.73 90.03 7.41 67.68 3.68 50 60.39 10.68 67.30 9.48 80.03 9.33
3776 5 续表 2 Compounds 5 Add Level (μg/l) Average(%) RSD(%) Average(%) RSD(%) Average(%) RSD(%) 10 72.18 5.76 80.40 7.34 58.50 7.30 20 69.16 7.20 77.04 8.43 83.15 2.07 50 78.24 7.85 87.12 8.59 103.64 8.06 10 97.21 7.64 85.90 6.87 84.56 4.35 20 91.51 8.25 99.67 7.61 82.40 2.31 50 98.95 3.61 87.85 2.78 92.96 4.25 10 86.42 6.30 96.18 6.24 91.76 3.48 20 90.42 5.01 100.66 5.47 88.72 2.98 50 66.62 6.92 94.08 5.21 92.13 3.69 10 60.34 3.50 67.16 3.78 76.40 9.87 20 86.84 7.71 76.54 5.45 74.16 2.44 50 65.37 9.97 61.67 9.42 86.27 1.56 10 73.65 4.94 81.93 3.60 80.05 8.80 20 78.23 4.49 87.04 3.59 75.37 1.77 50 60.30 3.62 67.15 5.09 108.90 3.17 10 82.82 7.05 72.09 5.24 87.70 1.29 20 78.64 2.82 80.90 2.01 88.24 6.48 50 75.33 7.48 83.80 6.65 81.68 5.87 10 95.68 6.31 84.18 6.08 92.57 6.21 20 98.30 5.46 109.36 5.50 98.39 4.51 50 94.16 3.97 92.54 3.26 103.65 6.79 10 64.23 5.39 73.68 3.37 76.26 8.57 20 76.53 11.85 75.10 7.68 86.46 4.30 50 67.10 4.75 74.67 4.07 83.63 4.83 3.4 实际样品的检测 Q-Exactvie,, 200, 4 结论 Q-Exactvie, 12 min 31,, 参考文献 [1]. [M]. :, 2007. Pang GF. Modern analytical technology for pesticide residue and veterinary drug residue [M]. Beijing: Science Press, 2007. [2] Regulation (EC) No 1223/2009 of The European Parliament and of the Council of 30 November 2009 on cosmetic products [Z]. [3]. [M]. :, 1994. Zhu BL. Animal drug residues in food [M]. Shanghai: Shanghai Science and Technology Press, 1994 [4] Wu ZJ, Peng Y. Analysis of penicillin antibiotics in cosmetics using ultra performance liquid chromatography [J]. Adv Mater Res, 2011, 233-235: 87 90. [5] Jin W, Yang YJ, Wang WY, et al. Simultaneous determination of antibiotics in anti-acne cosmetics by rapid LC with DAD [J]. Chromatographia, 2009, 69: 1221-1226. [6],,. 12 [J]., 2007, 25(2): 238 240. Zheng HH, Wang P, Li J. Determination of 12 sulfonamides in cosmetics by ultra performance liquid chromatography [J]. Chin
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