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1 5 9 Vol. 5 No Journal of Food Safety and Quality Sep., * 李春丽, 严守雷 ( ) 摘要 : 目的 方法, (P-VBA), (EGDMA),,, 结果 : 5%, 10%, 40%, 0.5 ml/min 5 μg/ml 10 μg/ml %~106.06% 结论,,, 关键词 : ; ; ; Determination of tyramine in soy sauce by molecular imprinting solid phase extraction coupled with high performance liquid chromatography LI Chun-Li, YAN Shou-Lei * (College of Food Science and Technology, Huazhong Agricultural University, Wuhan , China) ABSTRACT: Objective To establish a method for determination of tyramine in soy sauce by molecular imprinting solid phase extraction (MISPE) and high performance liquid chromatography (HPLC). Methods Tyramine molecular imprinted polymer (MIP) was prepared by bulk polymerization using tyramine, p-vinylbenzoic acid and ethylene glycol dimethacrylate as template, functional monomer and cross-linker, respectively. The MISPE column was made using MIP as filler. The extraction conditions were optimized before HPLC determination. Results The optimized conditions were as follows: the loading, washing, eluting solvent were 5% ethanol, 10% methanol and 40% acetonitrile, respectively, and the flow velocity was 0.5 ml/min. The recoveries were in the range of 86.95%~106.06% at the spiked level of 5 μg/ml and 10μg/mL. Conclusion The proposed method allows the efficient extraction of tyramine in soy sauce in pre-treatment and with high sensitivity, which can be applied to trace analysis of tyramine in food. KEY WORDS: tyramine; molecularly imprinted solid phase extraction; high performance liquid chromatography; soy sauce 基金项目 : ( ) Fund: Supported by the National Natural Science Foundation of China( ) * 通讯作者 :,, yanshoulei1225@163.com *Corresponding author: YAN Shou-Lei, Associate Professor, College of Food Science and Technology, Huazhong Agricultural University, Wuhan , China. yanshoulei1225@163.com

2 9, : 引言 (tyramine),,, [1], [2], [3],,,,, 100 mg, 1080 mg [4], 100~800 mg/kg [5] (high performance liquid chromatography, HPLC) [6,7] [8], [9],,,, [10,11], [12],,, (molecularly imprinted technology, MIT), [13], (molecularly imprinted polymer, MIP),,, [14,15],, MIP,,, [16],,,,, 2 材料与方法 2.1 材料与试剂 3, A B C, ; 1 mg/ml 4, (P-VBA) (EGDMA), Sigma-Aldrich ; (AIBN), ;,, ; 2.2 仪器与设备 Milli-Q ( Millipore ); Waters e2695 ( Waters); Agilent ZORBAX XDB-C 18 ( ); HH-8 ( ); US 3120DH ( ); J-E ( Beckman ); 12 CNW ( ) 2.3 实验方法 制备分子印迹固相萃取柱 1.0 mmol 4.0 mmol P-VBA 50 ml, 8.0 ml, 4 30 min, 20.0 mmol EGDMA 0.24 mmol AIBN, 5 min, 10 min, h (NIP),,,, (9:1, v:v) h,,, 50 [17], 100 mg MIP NIP 3 ml,, SPE,, (MISPE NISPE),

3 优化分子印迹固相萃取条件 (1), 5 ml 10 ml MISPE, MIP, MIP [18] (2), (5% 10% 15% 20%, v:v) 10 μg/ml, 1mL, 1 ml/min, HPLC, MISPE (3) 1 ml 5% 10 μg/ml, ( ml/min),, HPLC, (4) 1 ml 10 μg/ml 0.5 ml/min 3 ml (10% 20% 30% 40% 50% 60% 70% 80%, v:v), 0.5 ml/min,, HPLC,, 1 ml 5% 10 μg/ml, 1 ml 10% 0.5 ml/min, 1 ml 10%, 5, HPLC, 1 ml 10 μg/ml, 1 ml40%, 0.5 ml/min,, 5, HPLC, (5) 25 ml 10 μg/ml ( 1 ml) MISPE, 0.5 ml/min,, HPLC (6)MISPE MIPSE, 10 ml - (9:1, v:v),,, 10 μg /ml, 10, HPLC, 高效液相色谱条件 Waters e2695, Agilent ZORBAX XDB C 18 (4.6 mm 250 mm, 5 μm); : A: 0.1% B: C:, ; : 0.6 ml/min; : 225 nm; : 20 μl; : PDA 酱油样品的测定 : (1) GB/T [19], 0.22 μm, HPLC (2) 3, A B C, 10 μg/ml, MISPE,, 0.22 μm, HPLC, (n=3) 3 结果与分析 3.1 分子印迹固相萃取条件的优化 上样溶剂选择, 1 g/95 ml (15 ),,, MISPE,, MISPE 1, MISPE, MISPE 5%, 94%,,,, 5% 上样流速选择 MIP, MIP,, MIP, 2, MIP 0.5 ml/min, MIP 98%, 2 ml/min, MIP 56.3%, 0.5 ml/min 淋洗液和洗脱液选择 3, 10% 10%, ; 10%

4 9, : Fig. 1 Fig. 2 1 Effects of different loading solvents on the binding ration of tyramine 2 Effects of loading flow velocity on the binding ration of tyramine 10%,,, 10% 40% 60% 60% 98%, 96% 76.78%,,, 40%, 40% 3 ml 10% 40% NISPE, 10.73% 5.89%, MISPE NISPE, MISPE, 淋洗液和洗脱液体积选择, 1 ml 10%, 5,, 4 1mL 10%, 2 ml, 15%, 1 ml, 5 ml 40% 5,, 3 ml, 3 ml 40% 萃取容量测定 10 μg /ml, 11 ml,, MISPE, MISPE Fig. 3 3 Effect of washing and elution solvents on the binding ration of tyramine 4 Fig. 4 The relationship of washing and elution volume to cumulative recovery

5 μg,, MISPE 重复使用性能测定 SPE,, MIP, MIPSE 10, 5 MISPE,, 5, 87%,, 5 MISPE Fig. 5 The relationship of cycling times and MISPE column to tyramine recovery 3.2 液相色谱条件的优化 [20], 3, 1,, 3.3 方法的线性相关性及检出限 1 mg/ml μg/ml, 1 HPLC,,, Y=88333X+30526, R 2 =0.9998, 1~500 μg/ml (S/N) 3, (LOD) 0.15 μg/ml 3.4 方法的精密度和回收率 10 μg/ml HPLC, 3, (RSD), RSD<5%,, 5 μg/ml 10 μg/ml, 3,, 2, %~106.06%, RSD, ~ μg/ml 表 1 HPLC 测定酪胺的流动相分离洗脱梯度 Table 1 Mobile phase elution gradient of HPLC for tyramine (min) A(0.1% )/% B( )/% C( )/% 液相色谱图 6, 7.2 min, 6 (C) GB/T , 6 (B) 6 (C), MISPE Table 2 表 2 三种酱油中酪胺的加标回收率 (n=3) Recovery for 3 kinds of soy sauce spiked with tyramine (n=3) 5 μg/ml 10 μg/ml % RSD% A 96.50± ± ± B ± ± ± C ± ± ±

6 9, : A. 10 μg/ml B. MISPE C. GB/T D. Fig. 6 Sample liquid chromatogram A. HPLC chromatogram of 10 μg/ml standard tyramine B. Spiked standard tyramine after purification of MISPE C. Pretreatment by GB/T D. Untreated sample, GB/T ,, 1.5 h, MISPE,,, HPLC,,, 4 结论与讨论 MIP MISPE,, : 5%, 1 ml 10%, 3 ml 40%, 0.5 ml/min, MISPE, (LOD) 0.15 μg/ml 5 μg/ml 10 μg/ml %~106.06%,,, 参考文献 [1],,,. [J]., 2009, 30(10): Cai CG, Zhang H, Wang ZM, et al. Research on food biogenic amines and advances of analytical methods [J]. Food Res Devel, 2009, 30(10): [2] Huang JD, Xing RX, Zhang XM, et al. A molecularly imprinted electrochemical sensor based on multiwalled carbon nanotube-gold nanoparticle composites and chitosan for the detection of tyramine [J]. Food Res Int, 2011, 44: [3]. [J]., 2012, (3): Qu YH. Research progress of biogenic amines in fermented foods [J]. Food Ind, 2012, (3): [4] Shalaby AR. Significance of biogenic amines to food safety and human health [J]. Food Res Int, 1996, 29(7):

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