6 4 Vol. 6 No. 4 2015 4 Journal of Food Safety and Quality Apr., 2015 朱 珍 1, 朱 俊 霖 1, 庄 松 娟 2, 李 振 兴 1* (1., 266003; 2., 266071) 摘 要 : 目 的 方 法 0.1%,,,,,,, Aglient ZORBAX Eclipse-C 18, -0.1%, 275 nm, 305 nm, 0.8 ml/min 结 果 50 200 500 µg/kg 72.20%~85.68%, (RSD) 1.716%~9.926% 结 论, > > 关 键 词 : ; ; ; Detection of dissociative o-tyrosine in electron beam irradiated seafood by high performance liquid chromatography ZHU Zhen 1, ZHU Jun-Lin 1, ZHUANG Song-Juan 2, LI Zhen-Xing 1* (1.Food Safety Laboratory, Ocean University of China, Qingdao 266003, China; 2. Qingdao Institute for Food and Drug Control, Qingdao 266071, China ) ABSTRACT: Objective To establish a method for the determination of o-tyrosine in irradiated seafood by high performance liquid chromatography (HPLC). Methods The sample was homogenized with 0.1% formic acid, the ultrasonic bath and centrifugation were followed. Then the resulting supernatants were treated with acetone at 20 for 2 h and centrifuged again. After evaporation of acetone from supernatants, the remaining solution was dried freeze. Finally, the powder was collected, reconstituted in formic acid and detected by HPLC analysis with external reference method. For HPLC separation, Aglient ZORBAX Eclipse-C 18 column was used with a flow rate at 0.8 ml/min, mobile phase: methanol-0.1% formic acid in Milli-Q water, and the fluorescence detector was set at λex275 nm and λem305 nm. Results The recoveries of o-tyrosine at the spiked amounts of 50, 200, and 500 μg/kg were 72.20%~85.68% with RSD value between 1.716% to 9.926%. Conclusion This method can be applied to detect o-tyrosine in electron beam irradiated seafood. The content of dissociative o-tyrosine in some seafood was metapenaeusensis>lateolabrax japonicas> scallop. KEY WORDS: electron beam irradiation; o-tyrosine; high performance liquid chromatography; seafood 基 金 项 目 : (2013GGB01192) Fund: Supported by Science and Technology Development Plan Project of Shandong Province (2013GGB01192) * 通 讯 作 者 :,,, E-mail: lizhenxing@ouc.edu.cn *Corresponding author: LI Zhen-Xing, Associate Professor, Food Science and Engineering College, No.5, Yushan Road, Qingdao 266003, China. E-mail: lizhenxing@ouc.edu.cn
4, : 1447 1 引 言,, 60 Co 137 Cs gamma 5MeV X [1], [2,3] [4], [5],, 2013 6172 [6], 39.5%, DNA [7,8], gamma,,, - ( ),, [9],, : ( ) ( ),, [10,11],,, 2 材 料 与 方 法 2.1 材 料 与 试 剂 DL- ( 98%, Sigma ); (, TEDIA ); (, );, (, ); Milli-Q ; (1 ml); ( 66, 0.22 μm); (2 ml,,,, Agilent ) ( ) 2.2 实 验 仪 器 与 设 备 (Agilent 1260, Aglient ); (Hei-Vap, Heidolph ); (DS-1, ); (3K-15, Sigma ); (KQ5200B, ); ( 0.1 g, TE601-2, ); ( 0.0001g, BS-224-S, ); (Milli-Q, Millipore ); Aglient ZORBAX Eclipse-C 18 (4.6 mm 250 mm, 5.0 µm, Aglient ) 2.3 标 准 溶 液 的 配 制 : 5 mg, 0.1% 5 ml, 4 : 0.1%, 4 2.4 样 品 处 理 2.4.1 游 离 邻 酪 氨 酸 的 提 取 Chen [10], 0.8 g ( 0.01 g) 50 ml, 3.5 ml 0.1%, 18000 r/min 2 min, 15 min, 8000 r/min, 4 20 min,, 8 ml, -40 2 h, 8000 r/min, 4 15 min, 45,, 0.8 ml 0.1%, 0.22 µm, 2.4.2 结 合 邻 酪 氨 酸 的 提 取 [11], 0.200 g ( 0.001 g) 50 ml, 20 ml - (3:1),, 8000 r/min, 4 10 min,,,,,, 6 ml, 6 mol/l, 110,,
1448 6 2.5 色 谱 条 件 及 参 数 2.5.1 非 衍 生 高 效 液 相 色 谱 : AglientZORBAXEclipse-C 18 (4.6 mm 250 mm, 5.0 µm); : 0.1% (A)- (B); : 0~14 min, 92%(A); 14~14.2 min, 92%(A)~20%(A); 14.2~21 min, 20%(A); 21~21.2 min, 20%(A)~92%(A); 21.2~30 min, 92%(A); : 20 µl; : 30 ; 275 nm, 305 nm 2.5.2 柱 前 衍 生 - 高 效 液 相 色 谱 OPA : 0.100 g, 0.100 g, 0.200 g, 50% 10 ml, 0.22 µm, 4 : =1:40 (v:v), 1 min 20 µl : AglientZORBAXEclipse-C 18 (4.6 mm 250 mm, 5.0 µm); : 0.02 mol/l (A)- (B); : 0~10 min, 80%~65%(A), 10~15 min, 65%(A), 15~15.01 min,65%~80%(a), 15.01~20 min, 80%(A); : 30 ; 330 nm, 450 nm 3 结 果 与 分 析 3.1 方 法 的 线 性 范 围 检 出 限 定 量 限 精 密 度 -, 0.1~9500 µg/l, 0.8 µg/l, 5 µg/l, ( 1),,,,, : 1 10 50 100 500 1000 3000 5000 10000 µg/l, (Y) (X, µg/l), 1~10000 µg/l, Y=0.0503X+0.9417, 0.9999, 2 (S/N=3) 2.7 µg/l, (S/N=10) 8 µg/l A: OPA - A: Chromatogram of o-tyrosine standards by pre-column derivatization HPLC with OPA B: B: Chromatogram of o-tyrosine standards by HPLC without derivatization Y: Y: orth o-tyrosine 1 Fig. 1 Liquid chromatogram of o-tyrosine standards
4, : 1449 2 Fig. 2 Standard curve of o-tyrosine, 6, 1 1,,, (RSD) 10% 3.2 方 法 的 加 标 回 收 率, 50 200 500 µg/kg, 3, 2.4 2.5, 2, 72.20%~85.68%, 1.716%~9.926% 3.3 辐 照 样 品 的 测 定 ( 10 kw, 10 MeV) 10 kgy 2.4 2.5,, 3: 表 1 鲈 鱼 基 围 虾 扇 贝 中 精 密 度 实 验 结 果 (n=6) Table 1 Results of precision test (n=6) o-tyrosine /ng 1 2 3 4 5 6 Average/ng RSD/% Lateolabraxjaponicus 114.589 106.731 116.554 114.589 112.625 114.589 113.280 3.141 Metapenaeusensis 41.898 43.862 41.898 39.933 41.898 39.933 41.570 1.350 Scallop 58.514 65.558 65.262 60.182 61.387 62.345 62.208 2.549 表 2 鲈 鱼 基 围 虾 扇 贝 中 邻 酪 氨 酸 的 加 标 回 收 率 和 相 对 标 准 偏 差 (n=3) Table 2 Recoveries and RSDs of o-tyrosine in real samples by LC-FLD (n=3) (%) (%) (%) (%) (%) (%) HPLC-FLD 50 µg/kg 200 µg/kg 500 µg/kg Lateolabraxjaponicus 72.20 6.573 76.63 1.716 79.31 3.072 Metapenaeusensis 82.08 9.926 82.56 3.430 84.40 9.072 Scallop 85.29 3.803 80.99 5.782 85.68 9.214 表 3 Table 3 辐 照 水 产 品 中 的 邻 酪 氨 酸 含 量 (n=3) Content of o-tyrosine in seafood (n=3) µg/kg µg/kg Lateolabraxjaponicus 53.514 65.558 65.262 61.445 5.609 Metapenaeusensis 128.342 114.589 98.872 113.935 14.746 Scallop 30.110 41.898 43.862 38.623 7.438 : 10 kgy,
1450 6,, 10 kgy 4 讨 论 4.1 实 验 原 理 初 探 : -, OPA( ), [11] ; [12],,,,, :, 110, HPLC [13] ;,,,,,, HPLC [14] -,,, ;,, [8], 4.2 实 验 结 果 讨 论, -,,, ;, 80%, Chen [10] 61.445 113.935 38.623 µg/kg,,, [15], 767 764 454 mg/100 g, 10-6 ~10-5 [16] HPLC-MS-MS 10 kgy, 0.539 0.598 0.537 0.619 0.614 mg/kg,,, ; [8] 10 kgy, 178.5/288 µg/kg,,, ; Wolfgang [17] 0 2 4 6 8 10 kgy,, 10 kgy 45 17 8 3 µg/g, 5 结 论 与 展 望,,,,, > >,,,,, 参 考 文 献 [1]. [J]., 2013, (15): 109 112. Geng JN. Food irradiation and its application in food processing [J]. Food Res Devel, 2013, (15): 109 112. [2],,,. [J]., 2007, (01): 61 64. Ha YM, Shi HD, Wang F, et al. Application and characteristics of food irradiation by electron beam [J]. J Nucl Agric Sci, 2007, (01): 61 64. [3] Peter B, Roberts. Food irradiation is safe:half a century of studies[j].radiat Phys Chem, 2014, (105): 78 82. [4],,,.
4, : 1451 [J]., 2012, 2(4): 11 14. Li ZX, Zhang LM, Gu KF, et al.comparative study and suggestion on relevant standards and regulations fordomestic and international aquatic irradiation [J].Chin Fish Qual Stand, 2012, 2(4): 11 14. [5],,. [J]., 2010, 4: 75 78. Yu L, Bu Y, Wei ZP. The application study of irradiation on aquatic products [J]. Meat Res, 2010, 4: 75 78. [6]. [M]. :, 2014 National Bureau of Statistics of the People's Republic of China. China statistical yearbook [M]. Beijing: Chinese Statistics Press, 2014. [7],,,. [J]., 2009, (5): 142 144. Wang F, Wang ZD, Zhou HJ, et al. The methods and standards of detection techniques for irradiated foods [J]. Food Mech, 2009, (5): 142 144. [8],. [J]., 2005, (06): 260 265. Ha YM, Zhou HJ. The reviewon analytical detection methodsfor irradiated foods [J]. Food Sci, 2005, (06): 260 265. [9],,,, [J]., 2011, 30(10): 1143 1147. Wu M, Li JX, Zhang ZG, et al. Identification of irradiated protein-rich food by dissociative tyrosine [J].J Instrum Anal. 2011, 30(10): 1143 1147. [10] Chen S, Fan LH, Song J, et al. Identification of irradiated meats by determining o- and m-tyrosine as markers [J]. Meat Sci, 2013, 93(2): 226 232. [11],,,. γ- Lin L, Zhang K, Qian H. Determination of o-tyrosine in irradiated chicken products by HPLC [J]. Food Sci, 2010, (10): 188 191. [13],,,. [J]., 2011, (01): 28 32. Zheng R, Ding ZP, Yan LM, et al. Study pre-treatment of HPLC determinatethe o-tyrosine in irradiation food [J].Shanghai Meas Test, 2011, (01): 28 32. [14] Ibe FI, Grinter R, Massey R, et al. Detection of o-tyrosine in irradiated chicken by reverse-phase HPLC and fluorescence detection [J]. Food Add Contam, 1991, (8): 787 792. [15], ( ) [M]. :, 2009. Wang YD, China Food Composition (The Second Edition) [M].Beijing: Peking University Press,2009. [16],,,. HPLC-MS/MS [J]., 2011, 30(10): 1187 1190. Wang M, Zhang XQ, Yang ZY, et al. Determination of o-tyrosine in γ-ray irradiation protein-rich food by HPLC-MS/MS [J]. J Instrum Anal, 2011, 30(10): 1187 1190. [17] Wolfgang G, Hein TJ, Simat HS. Determination of non-protein bound o-tyrosine as a marker for the detection of irradiated shrimps [J]. Eur Food Res Technol, 2000, (210): 299 304. 作 者 简 介 ( 责 任 编 辑 : 李 振 飞 ) 朱 珍, 硕 士 研 究 生, 主 要 研 究 方 向 为 辐 照 食 品 的 检 测 E-mail: 15275251431@139.com [J]., 2009, (10):236 238. Wang DH, Wang M, Han L, et al.determination of o-tyrosine in γ-ray irradiated food by HPLC [J]. Food Sci, 2009, (10): 236 238. [12],,. 李 振 兴, 博 士, 副 教 授, 主 要 研 究 方 向 为 水 产 品 的 安 全 与 质 量 控 制 E-mail: lizhenxing@ouc.edu.cn [J]., 2010, (10): 188 191.