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1 6 4 Vol. 6 No Journal of Food Safety and Quality Apr., 2015 QuEChERS- B 1 刘 1, 辉 2*, 张燕 1 (1., ; 2.,,, ) 摘要 : 目的 QuEChERS- B 1, 方法 70%, PSA+MgSO 4, 结果 µg/kg, 0.125~0.854 µg/kg, IC ng/ml, 87.66%~97.17%, 4.89%~7.16% (high performance liquid chromatography, HPLC) r 2 =0.9854, 结论, B 1 关键词 : QuEChERS; ; B 1 ; Simultaneous determination for aflatoxin B 1 in tea leaf by QuEChERS -enzyme-linked immunosorbent assay LIU Hui 1, 2*, ZHANG Yan 1 (1. Guangdong Testing Institute of Product Quality Supervision, Foshan , China; 2. Guangdong Provincial Key Laboratory of Food Quality and Safety, Laboratory of Quality and Safety Risk Assessment in Agricultural Products Preservation Ministry of Agriculture,College of Food Science, South China Agriculture University, Guangzhou , China) ABSTRACT: Objective To establish a QuEChERS-enzyme-linked immunosorbent assay (ELISA) method for determining the aflatoxin B 1 (AFB 1 ) in tea leaf rapidly, and optimizing the sample pre-treatment conditions. Methods The samples were extracted with 70% acetonitrile in water, and the supernatant liquid was directly purified with PSA+MgSO 4 for detection of ELISA. Results The limit of detection (LOD) of method was µg/kg, with linear range between and µg/kg, and IC 50 value was ng/ml. Average recovery rate of samples was between 87.66% and 97.17% at 3 adding levels, and the relative standard deviation (RSD) was 4.89%~7.16%. The results showed a good coefficient with high performance liquid chromatography (HPLC) (r 2 =0.9854). Conclusion This method proved to be suitable for the screening of tea leaf samples for the presence of AFB 1, which was easier, faster and more efficient. KEY WORDS: QuEChERS; enzyme-linked immunosorbent assay; aflatoxin B 1 ; tea leaf 基金项目 : (2013B ) Fund: Supported by the Science and Technology Project of Guangdong Province (2013B ) * 通讯作者 :,, lh403@163.com *Corresponding author: LIU Hui, Food Quality Engineer, Guangdong Provincial Product Quality Supervision and Inspection Institute, Foshan , China. lh403@163.com

2 引言 (aflatoxins, AFT) [1], B 1 B 2 G 1 G 2, [2,3], 1993 Ⅰ, AFB 1 [4,5], AFB 1 AFB 1 2 µg/kg, 6 AFB 1 0.5~20 µg/kg, AFB 1, AFB 1 Benford [5] AFB 1,, 11.43%; [6] AFB ; [7] AFB 1,, AFB 1,,, AFB 1 (thin layer chromatography, TLC) [8] (high performance liquid chromatography, HPLC) [9,10] - [11] - [12] (enzyme-linked immunosorbent assay, ELISA) [13],,, ELISA,,, AFB 1,,, Anastassiadas [14] 2003 (quick) (easy) (cheap) (effective) (rugged) (safe) (QuEChERS) AFB 1 ELISA, QuEChERS ELISA AFB 1,, AFB 1,, 2 材料与方法 2.1 材料与仪器 B 1 : ; B 1 ELISA : ; : ; ( Sigma ); (Multiskan MK3); ( Eppendorf ); ( 1200) 2.2 样品来源,, 样品前处理 5.0 g ( ), 25 ml : (7:3), 10 min, 5 ml, (250 mg PSA+450 mg MgSO 4 ) 10 ml, 2 min 10 ml r/min 5 min, 2.4 ELISA 试剂盒的检验步骤,,,,, min,,, 4,, 50 μl,, min, 50 μl, 450 nm A, A 450 nm, Origin Pro 8.5, ELISA, 3 结果与分析 3.1 AFB 1 -ELISA 标准曲线建立 AFB 1, A 450 nm

3 4, : QuEChERS- B ,, 1 IC ng/ml; µg/kg, 0.125~0.854 ng/ml 1 AFB 1 -ELISA (n=5) Fig. 1 Standard curve of AFB 1 by ELISA (n=5) 3.2 样品前处理条件的优化, AFB 1,,,, 提取溶剂的优化 AFB 1 AFB 1,, 1,,, [9,15,16], 76.44%~109.73%,, 60% 70% 80% 90% 100%, AFB 1, 2 2, 70%,, 70% 净化条件的优化,,, QuEChERS,,, -N-(PSA) C 18 (MgSO 4 ), MgSO 4 70%, C 18 +MgSO 4 PSA+MgSO 4,, 3, PSA+MgSO 4, 80%~120%, PSA PSA, 2, 250 mg PSA+450 mg MgSO 4 Table 1 表 1 不同提取剂对 AFB 1 回收率的影响 (n=3) The effects of the extracting solvent on recovery (n=3) /(μg/kg) (%) (%) (%) (%) ± ± ± ±8.82 AFB ± ± ± ± ± ± ± ±6.91 Table 2 表 2 不同比例提取剂对 AFB 1 回收率的影响 (n=3) The effects of different proportions of extracting solvent on recovery (n=3) /(μg/kg) 60% 70% 80% 90% ± ± ± ± ±5.72 AFB ± ± ± ± ± ± ± ± ± ±8.21

1310 6 表 3 不同吸附剂对 AFB 1 回收率的影响 (n=3) Table 3 Recovery data of AFB 1 for different sorbents (n=3) /(μg/kg) C 18 +MgSO 4 /% PSA+MgSO 4 /% 1.0 74.37±8.12 84.81±3.97 AFB 1 2.0 81.93±10.61 94.83±6.16 5.

4 表 3 不同吸附剂对 AFB 1 回收率的影响 (n=3) Table 3 Recovery data of AFB 1 for different sorbents (n=3) /(μg/kg) C 18 +MgSO 4 /% PSA+MgSO 4 /% ± ±3.97 AFB ± ± ± ±7.47,, ELISA,,, [18], 3,, ELISA AFB 1 Fig. 2 2 PSA Comparison of the abilities of different amount of PSA C 18,,, -N-(PSA), -OH -NH -SH, [17], PSA 3.3 方法回收率和精密度, μg/kg 3 AFB 1,,,, 6, 4, 87.66%~97.17%, RSD 4.89%~7.16% 3.4 基质效应,,,,, 3 AFB 1 Fig. 3 Comparison of different normalised standard curve and AFB 1 standard curve by ELISA 3.5 方法验证 AFB 1 ELISA,, HPLC ELISA, 4 5, R 2 >0.95,, ELISA X, HPLC Y,,, 5 Y=1.0433X , R 2 =0.9854, ELISA AFB 1

5 4, : QuEChERS- B 表 4 茶叶中 AFB 1 的加标回收试验结果 (n=6) Table 4 Recovery data of AFB1 in tea leaf (n=6) /(μg/kg) (μg/kg) (%) RSD(%) AFB Table 5 表 5 不同种类样品中 AFB1 添加回收实验结果 (n=3) Recoveries of AFB1 from spiked fish and shrimp by ELISA and HPLC (n=3) HPLC ELISA (ng/g) ± (ng/g) (%) ± (ng/g) (%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Fig. 4 4 (5 ng/ml) Chromatogram data of AFB 1 (5 ng/ml) from tea leaf by HPLC 5 HPLC ELISA AFB 1 Fig. 5 Analytical result of ELISA and HPLC methods

6 实际样品的检测 AFB 1 ELISA HPLC 10, 6 表 6 AFB 1 药物实际样品检测 Table 6 The results of AFB 1 detection from actual samples ELISA (μg/kg) HPLC (μg/kg) ND ND 3 ND ND ND ND 6 ND ND ND ND 9 ND ND : ND 10, 4 AFB 1, HPLC AFB 1 1.6~4.0 μg/kg, ELISA AFB 1 1.8~4.1 μg/kg, AFB 1 4 结论, AFB 1 QuEChERS-ELISA, ELISA, QuEChERS, [19,20], 87.66%~97.17%, RSD 4.89%~7.16%, AFB 1, HPLC,, AFB 1, 参考文献 [1],. [J]., 2009, 30(9): Ma ZK, Zan LS. Progress on hazard, detection techniques and biodegradation of aflatoxin [J]. Prog Vet Med, 2009, 30(9): [2],,. [J]., 2010, 31(24): Zheng Y, Wang YX, Li JJ. Determination of aflatoxins in food by liquid chromatography-tandem mass spectrometry [J]. Food Sci, 2010, 31(24): [3],,,. [J]., 2010, 27(1): Gao RY, Liang MQ, Zhang XC, et al. Determination of aflatoxins content in traditional Chinese medicine using immunoaffiuity column cleanup and by high performance liquid chromatography [J]. J Environ Health, 2010, 27(1): [4] Milita NM, Mihaescu G, Chifiriuc C. Aflatoxins-health risk factors [J]. Bacteriol Virusol Parazitol Epidemiol, 2011, 1(1): [5] Benford D, Leblanc JC, Setzer RW. Application of the margin of exposure (MOE) approach to substances in food that are genotoxic and carcinogenic: example: aflatoxin B 1 (AFB 1 ) [J]. Food Chem Toxicol, 2010, 48(1): [6],,. B 1 [J]., 2014, 24(14): Chen RH, Tan ZX, Zhang QL. Contamination of aflatoxin B 1 in Pu er tea from tea wholesale market of Liwan district [J]. Chin J Health Lab Technol, 2014, 24(14): [7],,,. [J]., 2011, 23(1): Chen JL, Li WX, Yang GY, et al. Biollgical contamination of Pu er tea in a Guangzhou tea market [J]. Carcin Teratogen Mut, 2011, 23(1): [8] Fallah AA. Aflatoxin M 1 contamination in dairy products marketed in Iran during winter and summer [J]. Food Control, 2010, 21(11): [9],,. [J]., 2014, 24(3): He RF, Chen Y, Li YB. Determination of aflatoxins in tea bags by high performance liquid chromatography [J]. Chin J Health Lab Technol, 2014, 24(3): [10] Lee JE, Kwak BM, Ahn JH, et al. Occurrence of aflatoxin M1 in raw milk in South Lorea using an immunoaffinity column and liquid chromatography [J]. Food Control, 2009, 20(2): [11] Abia WA, Warth B, Sulyok M, et al. Determination of multi-mycotoxin occurrence in cereals, nuts and their products in

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7 4, : QuEChERS- B Cameroon by liquid chromatography tandem mass spectrometry (LC -MS/MS) [J]. Food Control, 2013, 31: [12],,,. - B 1,B 2,G 1,G 2 [J]., 2013, 38(2): Yang CL, Li JJ, Hu Q, et al. Determination of four aflatoxins (B 1, B 2, G 1, G 2 ) in fermented condiment by ultra-high performance liquid chromatography-tandem mass spectrometry [J]. China Cond, 2013, 38(2): [13] Guan D, Li PW, Zhang Q, et al. An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M 1 in milk and infant milk products [J]. Food Chem, 2011, 125(4): [14],,,. QuEChERS ELISA [J]., 2015(1): Yang JY, Zhang Y, Zeng DP, et al. Simultaneous determination of fluoroquinolones in seafood by modified QuEChERS and enzyme-linked immunosorbent assay [J]. Sci Technol Food Ind, 2015(1): [15],,,. B 1 [J]., 2014, 5(3): Wu GH, Zhao R, Li N, et al. Determination methods of aflatoxin B 1 in tea [J]. J Food Saf Qual, 2014, 5(3): [16],,,. - B 1 [J]., 2013, 33(3): Zhao HJ, Wang K, Yang WH, et al. Determination of aflatoxins B 1 in tea by high performance liquid chromatographyfluorescence detector with post-column photochemical reaction [J]. J Tea Sci, 2013, 33(3): [17],,,. QuEChERS [J]., 2013, 13(2): YI JH, Duan ZJ, Fang GZ, et al. Study of QuEChERS method for the detection of pesticide and veterinary drug residues in foods [J]. J Chin Inst Food Sci Technol, 2013, 13(2): [18] Tittlemier SA, Gelinas JM, Dufresne G, et al. Development of a direct competitive enzyme-linked immunosorbent assay for the detection of fluoroquinolone residues in shrimp [J]. Food Anal Method, 2008, 1(1): [19],,,. [J]., 2014, 31(8): Wang K, Wei YJ, He Y, et al. Determination of aflatoxins in tea by high performance liquid chromatography tandem mass spectrometry [J]. J Environ Health, 2014, 31 (8): [20],,,. - B 1 [J]., 2014, 20(6): Tan ZX, Jiang JS, Chen RH, et al. Detection of aflatoxin B 1 in the puer tea using affinity column purification-enzyme linked immunosorbent assay [J]. Prev Med Forum, 2014, 20(6): 作者简介 ( 责任编辑 : 杨翠娜 ) 刘辉, 工程师, 主要研究方向为食品技术检测及食品安全风险评估 lh403@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