Microsoft Word 张玉娜-二校 出

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1 7 11 Vol. 7 No Journal of Food Safety and Quality Nov., 张玉娜 1, 丁一 2, 孙欣 3, 万秉纯 3, 王明林 (1., ; 2., ; 3., ) 3* 摘要 : 目的 - ( ) 6 方法, Tween 20,, 50,, 结果 ~2 µg/ml (r ), (S/N=3) 8.27~10.81 µg/l mg/l %~96.8%, 1.24%~6.02%, 结论, 关键词 : ; ; ; Detection of quinolones bactericides in milk by cloud point extraction and high performance liquid chromatography ZHANG Yu-Na 1, DING Yi 2, SUN Xin 3, WAN Bing-Chun 3, WANG Ming-Lin 3* (1. Central Laboratory of Qingdao Agricultural University, Qingdao , China; 2. Shandong Institute for Food and Drug Control, Jinan , China; 3. College of Food Science and Engineering, Shandong Agricultural University, Tai an , China) ABSTRACT: Objective To establish a method for the determination of 6 kinds of quinolones bactericides in milk by cloud point extraction (CPE) and high performance liquid chromatography (HPLC) equipped with ultraviolet detector. Methods The milk samples were demulsified by glacial acetic acid and anhydrous sodium sulfate, then extracted by Tween20. The samples were balanced in 50 water bath for separation after adding concentrated ammonia, n-butyl alcohol, and anhydrous sodium sulfate. Aqueous phase was removed and surfactant phase was detected by HPLC. Results Under the optimum conditions, good linear relationships were obtained in the range of 0.05~2 µg/ml with correlation coefficients (r 2 ) , and the limits of detection (S/N=3) were 8.27~10.81 µg/l. The average recoveries at 3 different spiked levels of 0.05, 0.1 and 0.2 µg/ml were 71.3%~96.8%, with relative standard deviations (RSDs) of 1.24%~6.02%. Conclusion This method is accurate and sensitive, which can meet the requirements of multiple residues determination. KEY WORDS: milk; quinolones bactericides; cloud point extraction; high performance liquid chromatography * 通讯作者 :,, mlwang@sdau.edu.cn *Corresponding author: WANG Ming-Lin, Professor, College of Food Science and Engineering, Shandong Agricultural University, No.61, Daizong Road, Tai an , China. mlwang@sdau.edu.cn

2 11, : 引言 20 60,, [1],,,, [2],, [3] QuEChERS [4] [5],,, (cloud point extraction, CPE) Watanabe 1978, [6],, (high performance liquid chromatography, HPLC) 6, 2 材料与方法 2.1 仪器 试剂与材料 (LC-10AVP, ); (SPD-10A, ); (DS-8510DTH, ); (TGL-16, ) 6 ( 97%, Dr. Ehrenstorfer ); Triton X-100(, ) Triton X-114(, ); Tween 20 Tween 40 PEG 4000 (, ); (, ); (, ); (, ); (, ); (, ) 2.2 标准工作液的配制 6 10 mg 10 ml,, mg/l, 2.3 样品前处理步骤 牛奶破乳过程 10 ml( 0.1 ml), 3 ml 400 g/l Tween 20, 0.95 g Na 2 SO µl,, 35, 15 ml, r/min 10 min 0.45 µm, 浊点萃取步骤 9 ml, 60 µl,, 0.25 g 200 µl,, min, 2 ml, r/min 5 min, 0.22 µm, HPLC 2.4 HPLC 仪器条件 : Inertsil ODS-3(4.6 mm 150 mm, 5 µm); 1; : 0.1% ( 1), : 1 ml/min; : 35 ; : 20 μl 表 1 梯度洗脱程序 Table 1 Program of gradient elution /min /nm % % % % % % % 结果与分析 3.1 HPLC 条件的优化 检测波长的选择 6, 240~290 nm, 280 nm, 250 nm, HPLC, 250 nm; 280 nm 流动相的选择 HPLC,,, 0.1%

3 (A)- (B),, 1 1, 6, 3.2 破乳条件的选择, [7-9],,,, :, 0.15 ml;, 0.95 g; r/min 10 min 3.3 浊点萃取条件的优化 表面活性剂种类的优化 [10,11], Triton X-100 Triton X-114 Tween 20 Tween 80 PEG HLB PEG 4000,, 4 2 6, 2 Tween 20, Tween 表面活性剂浓度的优化, Tween 20, 3 40~90 g/l,,,,,,, 80 g/l 浓氨水体积的优化 ph, 4 : 0~60 µl, 3 ; 3 0~90 µl ph [12], ;, ph, [13,14], 60 µl 90 µl, 90 µl,,, 60 µl 1 6 Fig. 1 Chromatogram of 6 kinds of quinolones : 1. ofloxacin( ); 2.enrofloxacin( ); 3. sarafloxacin( ); 4. oxolinic( ); 5. nalidixic( ); 6. flumequine( )

4 第 11 期 张玉娜, 等: 浊点萃取-高效液相色谱法检测牛奶中的喹诺酮类抗生素 4565 图 2 表面活性剂类型回收率的影响(n=3) Fig. 2 Effect of surfactant on recovery (n=3) 电解质用量的选择 本研究中喹诺酮类物质遇热易分解, 需要添加电解 质降低 Tween 20 的浊点 电解质不仅能够降低浊点萃取温 度, 且能使蛋白质发生盐析, 可以辅助冰乙酸破乳效果, 电解质的存在也增大了溶液离子强度, 使水相密度增大, 而 利 于 相 分 离 [15] 实 验 以 无 水 硫 酸 钠 为 电 解 质, 选 择 0.1~0.3 g 范围内进行添加量的优化, 图 5 为无水硫酸钠添 加量对 6 种奎诺酮类药物回收率的影响 结果显示, 各组 分的回收率在 0.1~0.25 范围内均随着无水硫酸钠用量的增 加而逐渐增大, 因此选择 0.25 g 无水硫酸钠为电解质 Fig. 3 图 3 Tween 20 浓度对回收率的影响(n=3) Effect of concentration of Tween 20 on recovery (n=3) Fig. 5 图 4 浓氨水用量对回收率的影响(n=3) Fig. 4 Effect of the dosage of ammonium hydroxide on recovery (n=3) 图 5 无水硫酸钠的用量对回收率的影响(n=3) Effect of the dosage of sodium sulfate anhydrous on recovery (n=3) 正丁醇添加量的优化 正丁醇能够降低表面活性剂的浊点温度 [16], 且正丁

5 4566 7,, 6, 0.2 ml 6, 0.2 ml Fig. 8 8 (n=3) Effect of equilibration time on recovery (n=3) 3.4 方法学评价 Fig. 6 6 (n=3) Effect of the volume of n-butyl alcohol on recovery (n=3) 平衡温度的优化,, 15~20 [17], 30~70, , 6, 50, 标准曲线 线性范围和检出限 (Y), (X),, (LODs) (LOQs) , 0.05~2 µg/ml, 6 表 2 6 种喹诺酮类标准溶液的线性范围 相关系数 检出限和定量限 Table 2 Linear ranges, correlation coefficients, LODs and LOQs of 6 kinds of quinolones standard solutions LOD (μg/l) LOQ (μg/l) Y=28438X Y=49694X Y=50616X Y=7674X Y=29901X Y=54288X Fig. 7 7 (n=3) Effect of equilibration temperature on recovery (n=3) 平衡时间的优化, 10~50 min ( 8) : 10~30 min, 6, 30 min, 30 min 准确度与精密度 mg/l 3, %~96.8%, 1.24%~6.02%, 实际样品的测定 4 ( : A1, A2, A3, A4),, 4 4, 3

6 11, : Table 3 表 3 6 种喹诺酮类物质的添加回收率及相对标准偏差 (n=3) Average recoveries and relative standard deviations of 6 kinds of quinolones (n=3) 0.05 µg/ml 0.1 µg/ml 0.2 µg/ml (%) RSD(%) (%) RSD(%) (%) RSD(%) 表 4 牛奶实际样品中 6 种喹诺酮药物的检测 (n=3) Table 4 Determination of 6 kinds of quinolones in milk (n=3) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) A1 ND 53.8±0.32 ND ND ND ND A2 ND 22.1±0.68 ND ND ND ND A3 ND ND 37.5±0.72 ND ND ND A4 ND ND ND ND ND ND : ND 4 结论 - ( ) 6,,,, 参考文献 [1]. [J]., 2004, 36(8): Bao HJ. The development of dynamic and rational use of quinolones [J]. Anim Husb Vet Med, 2004, 36(8): [2]. [J]., 2015, 9(21): Zou T. Analysis of rational drug use and adverse reactions of quinolones [J]. Chin J Mod Drug Appl, 2015, 9(21): [3],,,. UPLC-MS [J]., 2016, 35(6) : Li N, Zhang YL, Lin T, et al. Detection of multi-residues of organic phosphorus in jam by GC [J]. J Instrum Anal, 2016, 35(6) : [4],,,. - - [J]., 2012, 30(6): Gao F, Zhao Y, Shao B, et al. Determination of residues of pesticides and veterinary drugs in milk by ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry [J]. Chin J Chromatogr, 2012, 30(6): [5],,, [J]., 2011, 39(2): Wang L, Li YQ, Wang HB, et al. Simultaneous Determination of Twenty Veterinary Drug Residues in Milk and Meat Using Matrix Solid Phase Dispersion-Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry [J]. Chin J Anal Chem, 2011, 39(2): [6] Watanabe H, Tanaka H. A non-ionic surfactant as a new solvent for liquid-liquid extraction of zinc(ii)with 1-(2-pyridylazo)-2-naphthol [J]. Talanta, 1978, 25(10): [7] Wu C, Liu Y, Wu Q, et al. Combined use of liquid liquid microextraction and carbon nanotube reinforced hollow fiber microporous membrane solid-phase microextraction for the determination of triazine herbicides in water and milk samples by high-performance liquid chromatography [J]. Food Anal Method, 2012, 5(3): [8],,,. - [J]., 2007, 25(6): Wang J, Cui YM, Liu W, et al. Determination of six pesticides in milk using cloud point extraction-high performance liquid chromatography [J]. Chin J Chromatogr, 2007, 25(6): [9],,,. HPLC [J] , 22(6): Dai J, Chen SW, Gu XH, et al. Detection of chloramphenicol residues in shrimp and milk by HPLC [J]. J Wuxi Univ Light Ind, 2003, 22(6):

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