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1 5 7 Vol. 5 No Journal of Food Safety and Quality Jul., AFB 1 周 * 闯, 班晓敏, 刘胜利, 张海彬 (, ) 摘要 : 目的 AFB 1 HPLC 方法 AFB 1, HPLC 结果 AFB 1 0.5~80 μg/ml, Y= X , R 2 = , 0.60 ng/ml, 86.40%~99.06% 43, AFB % 11.63% 结论 HPLC AFB 1, 关键词 : AFB 1 ; ; ; Determination of AFB 1 in corns by high performance liquid chromatography without derivations ZHOU Chuang, BAN Xiao-Min, LIU Sheng-Li, ZHANG Hai-Bin * (College of Veterinary Medicine, Nanjing Agricultural University, Nanjing , China) ABSTRACT: Objective To develop a method for the determination of AFB 1 in the corns by high performance liquid chromatography (HPLC) without any derivations to make the determination process simpler. Methods The extraction, purification and detection conditions were optimized. Finally, an HPLC method without derivations was developed. Results When the concentrations of AFB 1 were at 0.5~80 μg/ml, a good linearity between the concentration and peak area ratio was obtained. The linear regression equation was Y= X with R 2 = The limit of detection was 0.60 ng/ml and recovery rates were between 86.40% and 99.06%. Relevance rate and the rate of exceeding the allowed standards of China in the 43 corn samples was 39.53% and 11.63%, respectively. Conclusion The HPLC without derivations could be used to detect the content of AFB 1 in the corns, and it had simpler process, better operability and more practical application value. KEY WORDS: AFB 1 ; corns; high performance liquid chromatography; determination 1 引言 (aflatoxins) [1],, B 1 B 2 G 1 G 2 [2-3], AFB 1 [4], AFB 1, [5] AFB 1, AFB 1 5 μg/kg [6-7] * 通讯作者 :,, haibinzh@njau.edu.cn *Corresponding author: ZHANG Hai-Bin, Professor, College Of Veterinary Medicine Nanjing Agricultural University, No.1, Weigang, Xuanwu District, Nanjing , China. haibinzh@njau.edu.cn

2 7, : - AFB AFB 1, AFB 1 (enzyme-linked immunosorbent assay, ELISA) [8] (HPLC) [9-10] (LC-MS/MS) [11-12], ELISA, LC-MS/MS, HPLC (FLD) [8-12] AFB 1 HPLC-FLD,,,, HPLC [11-13] AFB 1 (TFA) ( ) (PBPB) [13],,, [14] HPLC-FLD, AFB 1,, 2 材料与方法 2.1 材料与试剂, 43 AFB 1 ( 98%, Sigma ); (); ( ); Whatman No.4 GF/C ( ); ( ); ; (0.45 μm)( ); ( ) 2.2 仪器设备 (LC-20AT, ); (RF-10AXL, ); (Inertsil ODS-3V, 150 mm 4.6 mm, 5 μm, ); (WH-1 ); (XL-04A, ) 2.3 AFB 1 高效液相色谱检测条件的优化 AFB 1 标准品工作液的制备 AFB 1,, 1 mg/ml μg/ml 高效液相检测条件的筛选 AFB 1, 30, 0.8 ml/min (20:80, v/v); (25:75, v/v); (30:70, v/v); : (35:65, v/v); (30:70, v/v) E X : 360~370 nm, E M : 410~430 nm AFB 1 20 μl 2.4 AFB 1 免疫亲和柱柱容的确定 AFB 1 标准品工作液的制备 AFB 1,, 1 mg/ml 10 μg/ml, μg/ml 实验步骤 (1) 1 ml, 1 /s ( ) AFB 1 (2), 20 ml 2, 10 ml, 5 ml/min, (3) 1 ml ( ) AFB 1,, 1 /s,,, AFB 1 1 ml, 2 ml (4), μg/ml (5) HPLC 2.5 样品中 AFB 1 的提取样品的预处理 1 kg,, 25 g, 5 g (80:20, v/v) (70:30, v/v) (60:40, v/v) (50:50, v/v) (40:60, v/v) 125 ml 30 min

3 Whatman No.4, 15 ml 30 ml,, 5 AFB 1, 20 μg/kg,, 3, (RSD) 样品通过免疫亲和柱前的准备,,,, HPLC, AFB 1, AFB 1,,,, 样品的免疫亲和柱净化 AFB 1 AFB 1,, : (1) 15 ml, 1 /s ( ) AFB 1 (2), 20 ml 2, 10 ml, 5 ml/min, (3) 1 ml ( ) AFB 1,, 1 /s,,, AFB 1 1 ml, 2 ml 样品的 HPLC 检测 (1) μg/ml,, 3,,,,, 0.45 μm, HPLC, (2),,, HPLC 3:1, 10:1 (3) AFB 1, μg/kg,, 3, (RSD) 样品的检测结果分析 43 AFB 1, EXCEL 3 结果与分析 3.1 AFB 1 高效液相检测条件的优化结果, : : =30:70(v/v); : 0.8 ml/min; : Ex=365 nm, Em=418 nm; : 30 ; : 20 μl; : Inertsil ODS-3V, 150 mm 4.6 mm, 5 μm HPLC μg/ml,,, 17.5 min, AFB 1 0.5~80 μg/ml Y= X , R 2 = ng/ml(1.2 μg/kg), 1, 免疫亲和柱柱容的测定结果 μg/ml, HPLC 5,, ( AFB 1 Fig. 1 AFB 1 standard curve

4 7, : - AFB (P>0.05), (P<0.01) : (70:30, v/v),, 91%, (70:30, v/v) %±0.94%, (RSD) 1.04% 3.4 样品中 AFB 1 高效液相色谱检测方法的验证结果 2 AFB 1 (a) (b) (c) Fig. 2 AFB 1 chromatogram of standards (a), extracting solution(b) and labeling(c) μg/ml), μg/ml ( μg/ml) ; 3.0 μg/ml, ; μg/ml 2.0~3.0 μg 3.3 提取液的优化结果 5 -(80:20, v/v) (70:30, v/v) (60:40, v/v) (50:50, v/v) (40:60, v/v), 1 1, 5 3, 1 : (80:20, v/v) : (60:40, v/v); 2 : (70:30, v/v); 3 : (50:50, v/v) (40:60, v/v) 3, 10, AFB 1,, μg/kg,, , 3, 10 (RSD) 3.29% 2.33% 3.50%, 3 (P>0.05) 3, 3 10, 3 (RSD) 2.36% 2.76% 1.48%, 3 (P>0.05) 3.5 送检玉米样品的分析结果 4, AFB μg/kg,, μg/kg 10 [7], B 1, 4 讨论与结论 HPLC AFB 1 [9-10],,, [15], 3 Table 1 80:20 表 1 提取液体积比对提取效果影响 (n=3) Effect of extraction by different extracting solution (n=3) 70:30 5 % 60:40 50:50 40: %± % 84.10± ± ± ± ±1.45

5 表 2 加标回收实验 (n=3) Table 2 Recoveries of experiments (n=3) 10 % 5 μg/kg 10 μg/kg 50 μg/kg ± 93.68± ± ± 表 3 精密度的测定结果 Table 3 Results of determined variation 10 % 5 μg/kg 10 μg/kg 50 μg/kg ± 93.73± ± ±1.37 表 4 Table 4 ( ) ( ) 被检玉米样品中 AFB 1 的检测结果 The test results of AFB 1 in corn samples (%) ( ) (%) (μg/kg) (μg/kg) AFB 1, HPLC, 86.40%~99.06%, AFB 1,,, AFB 1, 1.2 μg/kg 5 μg/kg, AFB 1 [7], AFB 1,,, [16] AFB 1 HPLC, AFB 1,, [10,17],,,, [18], HPLC AFB 1 参考文献 [1] Magan N, Olsen M. Mycotoxins in food: Detection and control [M]. Cambridge: Woodhead Publishing, [2] Goryacheva IY, Rusanova TY, Pankin KE. Fluorescent properties of aflatoxins in organized media based on surfactants, cyclodextrins, and calixresorcinarenes [J]. J Anal Chem, 2008, 63(8): [3],,,. 4 [J]., 2011, 40(1): Gao XF, Yin SA, Zhang HY, et al. Aflatoxin contamination of corn samples collected from six regions of China[J]. J Hyg Res, 2011, 40(1): [4] Eaton DL, Groopman JD. The toxicology of aflatoxins: human health, veterinary, and agricultural significance[m]. New York: Academic Press, [5],,. [J]., 2013, (010): Gao XF, Yang DJ, Ji R. Correlationship of four aflatoxins in corns[j]. Chin Prev Med, 2013 (010): [6] Commission Regulation (EC) No. 466/2001 of 8 March 2001 setting maximum levels for certain contaminants in foodstuffs, Off J Eur Communities L77/1, [7] GB [S]. GB Maximum levels of mycotoxins in foods [S]. [8],,,. B 1 [J]., 2004, 29(10): Ye XZ, Wang XL, Zhao YS, et al. Analysis of Determination Methods of AFB 1 [J]. Food Ferment Ind, 2004, 29(10): [9] Maragos C. Analysis of mycotoxins with capillary electrophoresis [J]. Semin Food Anal, 1998, 3: [10] Beaver RW. Determination of aflatoxins in corn and peanuts

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Food Anal Methods, 2014, 7(3): 690 696. [12] Rahmani A, Jinap S, Soleimany F, et al. Sample preparation optimization for the simultaneous determination of mycotoxins in cereals[j].

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6 7, : - AFB using high performance liquid chromatography[j]. Arch Environ Contam Toxicol, 1989, 18(3): [11] Xue XF, Selvaraj JN, Zhao L, et al. Simultaneous Determination of Aflatoxins and Ochratoxin A in Bee Pollen by Low-Temperature Fat Precipitation and Immunoaffinity Column Cleanup Coupled with LC-MS/MS[J]. Food Anal Methods, 2014, 7(3): [12] Rahmani A, Jinap S, Soleimany F, et al. Sample preparation optimization for the simultaneous determination of mycotoxins in cereals[j]. Eur Food Res Technol, 2011, 232(4): [13]. B 1 [D]. :, Ma L. Study on Detection Technology for Determination of Aflatoxin B 1 With high sensitivity[d].wuhan: Chinese Academy of Agriculture Sciences, [14],,,. B 1 [J]., 2005, 27(2): Li PW, Ma L, Yang JE, et al. A review on analytical methods for aflatoxin B 1 in grains and oilseeds products[j]. Chin J Oil Crop Sci, 2005, 27(2): , Duarte D, Liu RN, Wang JX. The Mycotoxin Blue Book [M]. Beijing: China Agricultural Science and Technology Press, [17] Jansen H, Jansen R, Brinkman UAT, et al. Fluorescence enhancement for aflatoxins in HPLC by post-column split-flow iodine addition from a solid-phase iodine reservoir[j]. Chromatographia, 1987, 24(1): [18],,,. β- B 1 [J]., 2012, 33(15): Zhang M, Guo T, Liu X, et al. Mechanisms Underlying Fluorescence Enhancement of Aflatoxin B 1 by β-cyclodextrin and Its Derivatives[J]. Food Chem, 2012, 33(15): 作者简介 ( 责任编辑 : 赵静 ) 周闯, 硕士, 主要从事真菌毒素的检测方法的应用与研究 zhouchuang1989@126.com [15]. AFB 1 OTA DON [D]. :, Ban XM. Optimization and Application of High Performance Liquid Chromatography Detection Methods for AFB 1, OTA, DON[D]. Nanjing: Nanjing Agricultural University, 张海彬, 教授, 主要研究方向为真菌毒素与畜产品安全 haibinzh@njau.edu.cn [16] Duarte D,,. [M]. :

Microsoft Word - 3098-3102 20150611007-蔡伟江-二校

Microsoft Word - 3098-3102 20150611007-蔡伟江-二校 6 8 Vol. 6 No. 8 2015 8 Journal of Food Safety and Quality Aug., 2015 蔡伟江 *, 邓鹏飞, 刘光兰, 李丹 (, 519040) 摘要 : 目的, 方法 (4.6 mm 250 mm, 5.0 μm) ; : : : =70: 16: 14(V:V:V),, : 1.0 ml/min; : 40 ; : 10