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1 6 12 Vol. 6 No Journal of Food Safety and Quality Dec., 2015 ELISA 2,4-1, 魏茂琼, 刘宏程 2* 1,, 林涛, 林昕, 兰珊珊, 沙凌杰 2, 王丽 (1. (), ; 2., ) 2 摘要 : 目的 2,4-(2,4-dichlorophenoxyacetic acid, 2,4-D) 方法 ELISA 2,4-D, ; - (ultra performance liquid chromatography-mass spectrometry, UPLC-MS) 结果, ELISA UPLC-MS µg/kg 3 µg/kg, 80.19%~117.37% 80.04%~112.5%; 4 8 结论 UPLC-MS, ELISA,,,, ELISA 2,4-D 关键词 : 2,4-; ; ; - ; Detection of 2,4-dichlorophenoxyacetic acid in vegetables by direct competitive ELISA method WEI Mao-Qiong 1, 2, LIU Hong-Cheng 1, 2*, LIN Tao 1, 2, LIN Xin 1, 2, LAN Shan-Shan 1, 2, SHA Ling-Jie 1, 2, WANG Li 2 (1. Laboratory of Quality & Safety Risk Assessment for Agro-Product, Ministry of Agriculture (Kunming), Kunming , China; 2. Agri-Food Quality Standard & Testing Technology Institute, Yunnan Academy of Agricultural Sciences, Kunming , China) ABSTRACT: Objective To study the method for the rapid determination of 2,4- dichlorophenoxyacetic acid (2,4-D) in vegetables by direct competitive enzyme-linked immunosorbent assay (ELISA). Methods The samples were pretreated by acetonitrile, then the residual of 2,4-D in vegetables was detected by ELISA kit, and the sensitivity, recovery and stability of the method were analyzed. The sensitivity and stability of the method were compared with ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Results By sample preparation, the limit of detection (LOD) of direct competitive ELISA and UPLC-MS method could reach to µg/kg and 3 µg/kg respectively. The recovery rates were 80.19%~117.37% and 80.04%~112.5%. The stability result of the kit indicated that it could be preserved at 4 for more than eight months. 基金项目 : (2012CA005) ()(2014DA001) (GJFP ) Fund: Supported by Social Development in Yunnan Province Science and Technology Plan (2012CA005), Science and Technology Innovation Platform Construction Plan (Public Service of Science and Technology, 2014DA001), National Agricultural Product Quality Safety Risk Assessment Project (GJFP ) * 通讯作者 :,, liuorg@163.com *Corresponding author: LIU Hong-Cheng, Researcher, Laboratory of Quality and Safety Risk Assessment for Agro-products (Kunming), Ministry of Agriculture, Institute of Quality Standards and Testing Technology, Yunnan Academy of Agricultural Sciences, No.9 Xueyun Road, Kunming , China. liuorg@163.com

2 Conclusion Compared with UPLC-MS method, direct competitive ELISA method is simple, efficient, fast, sensitive and suitable for screening of the mass samples. Therefore, the ELISA method is more suitable for rapid detection of 2,4-D in vegetables. KEY WORDS: 2,4-dichlorophenoxyacetic acid; vegetables; direct competitive enzyme-linked immunosorbent assay; ultra performance liquid chromatography-mass spectrometry; rapid detection 1 引言 2,4-2,4-D,,, 2,4-D,, [1] 2,4-D, 2,4-D, 2,4-D,, [2] 2,4-D, ,4-D 0.03 mg/l [3] GB ,4-D mg/kg, 2,4-D 2,4-D [4], (GC-MS) (HPLC) [5] [6] GC-MS 2,4-D 2,4-D -,, (immunoassay, IA), [7,8] (ELISA) 2,4-D,, 2,4-D -(UPLC-MS) ELISA 2,4-D,, ELISA 2,4-D 2 材料与方法 2.1 材料与试剂, : 2,4-D (Abraxis, USA, NO.54003A): ; ( ) (, ); 2,4-D: 100 µg/ml: 2.2 仪器 (Bio-Rad680, ); (HY-5, ); (AE-100, Mettler Toledo ); (50~200 µl); ; (OA-SYS, Organomation ), ; (Agilent1290, Agilent ); (Agilent6460, Agilent ); (IKA T18 ULTRA-TURRAX, IKA ) ,4-D 标准溶液的配制 0.2 µg/ml : 0.1 ml 2,4-D, 50 ml ng/ml 2.4 方法 ELISA 法测定 2,4-D (1), [9], 10 g 50 ml, 20 ml,, 10

3 12, : ELISA 2, min 4000 r/min 10 min, 1 ml,, 20 s (2) 1 50 µl 2 50 µl, 3 50 µl 2,4-D,, 37 1 h 4,, 4~5, 5: 100 µl,, min 6: 100 µl, 450 nm (OD) UPLC-MS 法测定 [10,11],, 15.0 g 250 ml, 30 ml 1 min 5.0~7.0 g 100 ml, 1 min, 1 h, 10.0 ml 150 ml,,, : 1 µl 3 结果与分析 3.1 酶联免疫试剂盒测定方法标准工作曲线制作 ng/ml, 50 μl/, 3 2,4-D,,, : %=B/B 0 =( )/ 100% 0~80 ng/ml, : Y= X , r 2 = 方法最低检测限 UPLC-MS 2,4-D 6 6, [12] (LOD)= ELISA +3 2,4-D µg/kg µg/kg( 1) 2,4-D 0.5 mg/kg; [13] ELISA 2,4-D, 50 ng/ml; [14] (HPLC) 2,4-D, 2,4-D 0.74 mg/kg, 2 mg/kg,, Fig ,4-D Standard curve of 2,4-D solution 表 1 番茄与黄瓜空白样品的最低检测限 Table 1 The LOD of tomatoes and cucumbers blank samples /µg kg -1 /µg kg -1 (MEAN, n=6) (SD) (LOD) 样品添加回收实验 2,4-D µg/l, %~117.37%, 2.73%~10.0% 稳定性试验 ELISA 4 8, 2,4-D : (80%~120%), 4.34%~10.59%, UPLC-MS 法测定标准工作曲线 2,4-D 0.01~10 µg/ml,, r 2 =0.99

4 Table 2 表 2 ELISA 法样品添加回收试验 Recovery of 2,4-D from tomatoes and cucumbers (n=6) /µg kg -1 /% CV/% Table 3 表 3 UPLC-MS 法样品添加回收试验 (n=6) Recovery of 2,4-D from tomatoes and cucumbers (n=6) /µg kg -1 /µg kg -1 /% 结果计算 [10], 2,4-D, (1) 2,4-D : A V1 V3 (1) As V2 m : ω 2,4-D, mg/kg ρ 2,4-D, mg/l A 2,4-D, mau s A s 2,4-D, mau s V 1, ml V 2, ml V 3, ml m, g 检出限 2,4-, 2.4, 6, 3 µg/kg 回收率验证试验, 2,4-D 2,4-D, 3, µg/kg, 80.04%~112.5% 4 结论,,

12, : ELISA 2,4-4965 2 h ELISA, 1.739 µg/kg 1.727 µg/kg, 5~50 µg/kg, 80.19%~117.37%, 2.73%~10.0%, 1.739 µg/kg, 3 µg/kg, ELISA, 2,4-D, 2,4-D, 2,4-D 参考文献 [1] Salman JM, Hameed BH.

Enhanced removal of pentachlorophenol and 2,4-D from aqueous solution by an aminated biosorbent [J]. J Hazard Mater, 2009, 165: 408 414. [3] Guidelines for drinking-water quality.

J Henan Agric Sci, 2011, 40(8): 40 43. [5] Sarikaya R, Yilmaz M.

Chemosphere, 2003, 52(1): 195 201. [6],,,. 2,4-D [J]., 2013, 35(1): 55 57. Hou ZG, Liu JG, Zhao XF, et al. Determination of 2,4-D-ethylhexyl Residue in Corn by GC-MS [J].

5 12, : ELISA 2, h ELISA, µg/kg µg/kg, 5~50 µg/kg, 80.19%~117.37%, 2.73%~10.0%, µg/kg, 3 µg/kg, ELISA, 2,4-D, 2,4-D, 2,4-D 参考文献 [1] Salman JM, Hameed BH. Adsorption of 2,4-dichlorophenoxy acetic acid and carbofuran pesticides onto granular activated carbon [J]. Desalination, 2010, 256: [2] Deng SB, Ma R, Yu Q, et al. Enhanced removal of pentachlorophenol and 2,4-D from aqueous solution by an aminated biosorbent [J]. J Hazard Mater, 2009, 165: [3] Guidelines for drinking-water quality. Addendum to recomm endations [S]. WHO, 2003: [4],,,. 2,4-D [J]., 2011, 40(8): Gong F, Xi J, Zhi AM, et al. A review on the toxicity and residues determination of 2,4-Dichlorophenoxy [J]. J Henan Agric Sci, 2011, 40(8): [5] Sarikaya R, Yilmaz M. Investigation of acute toxicity and the effect of 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide on the behavior of the common carp (Cyprinus carpio L., 1758; Pisces, Cyprinidae) [J]. Chemosphere, 2003, 52(1): [6],,,. 2,4-D [J]., 2013, 35(1): Hou ZG, Liu JG, Zhao XF, et al. Determination of 2,4-D-ethylhexyl Residue in Corn by GC-MS [J]. J Jilin Agric Univ, 2013, 35(1): [7] Ramón-Azcón J. Detection of pesticide residues using an immunodevice based on negative dielectrophoresis [J]. Biosens Bioelectron, 2009, 24(6): [8] Lacasaña M. Association between organophosphate pesticides exposure and thyroid hormones in floriculture workers[j]. Toxicol Appl Pharmacol, 2010, 243(1): [9],,,. [J]., 2014, 30(4): Liu HC, Lin T, Shao JL, et al. Determination of plant growth regulator residues in fruits by quick extraction and high performance liquid chromatography [J]. J Anal Sci, 2014, 30(4): [10],,, 8 [J]. 2011, 27(7): Shao JL, Li QW, Liu HC, et al. Determination of Eight Carbamate Pesticides in Vegetables by High Performance Liquid Chromatography [J]. Mod Food Sci Technol, 2011, 27(7): [11],,,. [J]., 2011,27(2): Ji SJ, Yu JC, Gao X, et al. Determination of carbendazim residues in cabbage by HPLC [J]. Food Mach, 2011, 27(2): [12],,,. ELISA [J]., 2013, 13(11): Xi X, Zhang MZ, Li MJ, et al. Development of dcelisa method for rapid detection of streptomycin residue in milk and honey [J]. J Chin Inst Food Sci Technol, 2013, 13(11): [13],,,. 2,4-D [J]., 2006, 9(3): Li HN, Cao YY, Sun YQ, et al. The preparation of polyclone antibody for 2,4-D residue [J]. J Agric Univ Hebei, 2006, 9(3): [14],. - 2,4-D [J]., 2014, 9: Zheng ZF, Guo JL. Solid phase extraction coupled with HPLC for the determination of 2,4-D residues in citrus [J]. Chem Eng Eq, 2014, 9: 作者简介 ( 责任编辑 : 白洪健 ) 魏茂琼, 研究实习员, 主要研究方向为食品安全 weimaobsyz@163.com 刘宏程, 研究员, 主要研究方向为农产品安全 liuorg@163.com

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0.

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0. 118 217 5 38 1 DOI 1.3969/j.issn.5-6521.217.1.28 Food Research And Development 1 2 1 2 1 2 1 2 1 2 1 2 1. 731 2. 731 - UPLC-MS/MS 5. 1 mol/l ProElut PXC 5 % - Thermo Hypersil Gold mm 2.1 mm 1.9 μm ESI