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1 6 3 Vol. 6 No Journal of Food Safety and Quality Mar., 2015 王丽英 1, 任贝贝 1 1,, 杨立新 2, 路杨 1, 常凤启 1, 刘印平 (1., ; 2., ) 1* 摘要 : 目的 4- (4-methylimidazole, 4-MeI) - 方法 MCX, - ( 0.05% ), MRM 4-MeI, 结果 10~1000 µg/l, R , 90.5%~101.6%, (RSD)<6.6% 4-MeI, 4-MeI 23.3~ µg/l, µg/l; 4-MeI 111.2~ µg/l, µg/l; 4-MeI 10.8~307.1 µg/l, 94.8 µg/l 结论, 4-MeI 关键词 : - ; 4- ; ; ; Determination of 4-methylimidazole in soy sauce, vinegar and beverage by ultra high-performance liquid chromatography-tandem mass spectrometry WANG Li-Ying 1, REN Bei-Bei 1, YANG Li-Xin 1, 2, LU Yang 1, CHANG Feng-Qi 1, LIU Yin-Ping 1* (1. Hebei Provincial Center for Disease Control and Prevention, Shijiazhuang , China; 2. College of Environmental Sciences and Engineering, Peking University, Beijing , China) ABSTRACT: Objective To establish a method to determine the 4-methylimidazole (4-MeI) concentrations in soy sauce, vinegar and beverage by ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Methods Soy sauce, vinegar and beverage samples were purified by MCX solid phase extraction column. Then acetonitrile-water (containing 0.05% ammonia) was used as chromatographic separation conditions, the 4-MeI signal was collected in MRM mode and quantified by isotope internal standard. Results The linear relationship was good from 10 µg/l to 1000 µg/l and the correlation coefficient was The recovery of low, medium and high levels was 90.5%~101.6% and the relative standard deviation was less than 6.6%. 4-MeI was contained in all samples. The concentration level of 4-MeI ranged from 23.3~ mg/kg with a mean of µg/l, 111.2~ µg/l with a mean of µg/l and 10.8~307.1 µg/l with a mean of 94.8 µg/l in soy sauce, vinegar and beverage samples, respectively. Conclusion This method is simple, rapid and of high sensitivity, which is suitable for detection of 4-MeI in soy sauce, vinegar and beverages. KEY WORDS: ultra high-performance liquid chromatography-tandem mass spectrometry; 4-methylimidazole; * 通讯作者 :,, liuyinping0930@163.com *Corresponding author: LIU Yin-Ping, Chief Technician, Hebei Provincial Center for Disease Control and Prevention, Shijiazhuang , China. liuyinping0930@163.com

2~2077.8 µg/l, 622.5 µg/l; 4-MeI 10.8~307.1 µg/l, 94.

2 3, : soy sauce; vinegar; beverage 1 引言,,,, [1,2],, [3,4],,, ( ph ), 4- (4-methylimidazole, 4-MeI) [5] 2012, (CSPI), 4-MeI 4-MeI, [6] GB ( ), 4-MeI 0.02%, 4-MeI 4-MeI (gas chromatography, GC) [7,8], - (gas chromatography-mass spectrometry, GC-MS) [9], (liquid chromatography, LC) [10], - (liquid chromatography-mass spectrometry, LC-MS) [ 11-15] GC, GC-MS,, LC-MS/MS,,, (solid phase extraction, SPE), (ultra high-performance liquid chromatography-tandem mass spectrometry, UHPLC-MS/MS), 4-MeI,, 4-MeI 2 材料与方法 2.1 材料试剂与仪器, (, ); (, J.T.Baker ); 4-MEI(C 4 H 6 N 2 ) ( 99.5%, Sigma ); D 5-4-MEI (C/D/N Isotopes, ) ( ) TSQ Quantum Access MAX (Thermo Scientific, Sunnyvale, CA); ( ); ( Organomation ); Waters Oasis MCX 3 cc(60 mg) ( Waters ) 2.2 实验方法液相色谱条件 Polaris C 18 -A (1.8 µm, 2.1 mm 100 mm); 0.3 ml/min; 10 µl, 30 ; : A ( 0.05% ), B, : 5% B, 3 min, 3 min 5 min 40%, 5.1 min 5%, 6.5 min 质谱条件 : ; : ; : 3.0 kv; : 35 Arb; : 7 Arb; : 300, : MeI m/z=83.1>42.6 m/z=83.1>56.5, D 5-4-MeI m/z=88.0>60.5 m/z=88.0>61.5,

GC-MS,, LC-MS/MS,,, (solid phase extraction, SPE), (ultra high-performance liquid chromatography-tandem mass spectrometry, UHPLC-MS/MS), 4-MeI,, 4-MeI 2 材料与方法 2.1 材料试剂与仪器, (, ); (, J.T.

3 964 6 ESI 标准溶液配制 4-MeI : 4-MeL D5-4-MeI g( g), 10 ml, 4-MeL D 5-4-MeI 1.0 mg/ml 4 20 µl 1 mg/ml 4-MeL 10 ml,, 2.0 µg/ml 10 µl 1 mg/ml D 5-4-MeI 10 ml,, 1.0 µg/ml : 4-MeL 1.5 ml, 0.1 ml (1.0 µg/ml), 0.05% / (v:v, 95:5) 1.0 ml 4-MeI ng/ml 样品前处理 (1) : 0.4 ml 1.5 ml, 0.8 ml, 0.1 ml 1 µg/ml D 5-4-MeI, 10 min, r/min 3 min 10 ml, 200 µl 0.02 mol/l, 1 ml,, SPE : 1.0 ml 1.5 ml, r/min 3 min, 0.5 ml 10 ml, 100 µl 0.02 mol/l, 1.3 ml, 0.1 ml 1 µg/ml D 5-4-MeI,, SPE, :, 10 min 0.5 ml 10 ml, 100 µl 0.02 mol/l, 1.3 ml, 0.1 ml 1 µg/ml D 5-4-MeI,, SPE (2) 3 ml, 3 ml Waters Oasis MCX 3 cc(60 mg), 2 ml, 2 ml 2 ml,, 2 ml 5% / (v:v), 45,, 0.05% / (95:5) 1.0 ml,, 0.22 µm, 1.5 ml, UHPLC-MS/MS 3 结果与讨论 3.1 前处理方法的优化,,,, 1 ml r/min 3 min,,,,,,,,,,,,, MCX 1 1,, 3.2 UHPLC-MS/MS 色谱分离条件和质谱条件的确定,, ( 0.05% ) 100 ng/ml, 4-MeI, ESI+,,, 4-MeI (2002/657/EC),, MRM 4-MeI(m/z=83.1>42.6 m/z=83.1>56.5) D 5-4-MeI (m/z=88.0>60.5 m/z=88.0>61.5), MeI D 5-4-MeI MeI (m/z=83.1>56.5) D 5-4-MeI(m/z=88.0>60.5), 1.98 min 1.94 min

1 ml 1 µg/ml D 5-4-MeI, 10 min, 10000 r/min 3 min 10 ml, 200 µl 0.02 mol/l, 1 ml,, SPE : 1.0 ml 1.5 ml, 10000 r/min 3 min, 0.5 ml 10 ml, 100 µl 0.02 mol/l, 1.3 ml, 0.

4 3, : Fig. 1 Chromatogram of soy sauce a: ; b: a: Extract with acetonitrile; b: Extract without acetonitrile Y X(10~1000 µg/l),, R ; 1.5 µg/l ( S/N=3) 方法的回收率和精密度,, 6, (RSD) 1, 90.5%~ 101.6%, (RSD) 3.1%~6.6% 3.4 样品中 4-MeI 含量的测定 2 4-MeI D 5-4-MeI Fig. 2 The chromatograms of 4-MeI and D5-4-MeI 3.3 方法学的考察线性关系 2.2.3, UHPLC-MS/MS 4-MeI, 4-MeI, 4-MeI MeI, 4-MeI 4-MeI 2 4-MeI 3 表 1 4-MeI 在酱油醋饮料样品中添加水平回收率及 RSD (n=6) Table 1 The spiked levels recovery and RSD of 4-MeI in soy sauce, vinegar and beverage samples (n=6) (μg/l) /% RSD/% /% RSD/% /% RSD/%

5 966 6 Table 2 表 2 酱油醋饮料样品中 4-MeI 检测结果 The detection results of 4-MeI in soy sauce, vinegar, and beverage samples (µg/l) (%) (µg/l) (%) (µg/l) (%) < ~ < ~ ~ ~ ~ ~ ~ > > > MeI,,, 4-MeI 3 4-MeI Fig. 3 The concentration distribution of4-mei in soy sauce, vinegar and beverage samples A: ; B: ; C: A: soy sauce; B: vinegar; C: beverage 4-MeI 23.3~ µg/l, µg/l Yamaguchi [16], [12],,,,, 4-MeI ;, 4-MeI,, 4-MeI 4-MeI 111.2~ µg/l, µg/l 4-MeI 4,, 4-MeI ;, 4 Fig. 4 The chromatograms of vinegar sample 4-MeI 10.8~ µg/l, 94.8 µg/l, 4-MeI,,,, 4-MeI, 4 结论与讨论, UHPLC-MS/MS 4-MeI, 4-MeI 4-MeI

3 The concentration distribution of4-mei in soy sauce, vinegar and beverage samples A: ; B: ; C: A: soy sauce; B: vinegar; C: beverage 4-MeI 23.3~4130.8 µg/l, 823.

6 3, : ,, 4-MeI,, 4-MeI,, 4-MeI 参考文献 [1] Yoshikawa S, Fujiwara M. Determination of 4(5)-methylimidazole in food by thin-layer chromatography [J]. Shokuhin Eiseigaku Zasshi, 1981, 22: [2] Moon JK, Shibamoto T. Formation of carcinogenic 4(5)-methylimidazole in Maillard reaction systems [J]. J Agric Food Chem, 2011, 59: [3] Lee KG., Jang HW, Shibamoto T. Formation of carcinogenic 4(5)-methylimidazole in caramel model systems: a role of sulfite [J]. Food Chem, 2012, 136: [4] Hengel M, Shibamoto T. Carcinogenic 4(5)-Methylimidazole Found in Beverages, Sauces, and Caramel Colors: Chemical Properties, Analysis, and Biological Activities [J]. J Agric Food Chem, 2013, 61: [5] Cunha SC, Barrado AI, Faria MA. Assessment of 4-(5-)methylimidazole in soft drinks and dark beer [J]. J. Food Compos Anal, 2011, 24: [6] Chan P, Mahler J, Travlos G. Induction of thyroid lesions in 14-week toxicity studies of 2 and 4-methylimidazole in Fisher 344/N rats and B6C3F1 mice [J]. Arch Toxicol, 2006, 80 (3): [7] Fuchs G, Sundell S. Quantitative determination of 4-methylimidazole as 1-acetyl derivative in caramel color by gas liquid chromatography [J]. J Agric Food Chem, 1975, 23: [8] Wilks RA, Shingler AJ, Thurman LS. The isolation of 4-methylimidazole from caramel color and its determination by thin-layer and gas-liquid chromatography [J]. J Chromatogr A, 1973, 87: [9] Fernandes JO, Ferreira MA. Gas chromatographic mass spectrometric determination of 4-(5)methylimidazole in ammonia caramel colour using ion-pair extraction and derivatization with isobutylchloroformate [J]. J Chromatogr A, 1997, 786: [10] Moretton C, Crétier G, Nigay H. Quantification of 4-methylimidazole in class III and IV caramel colors: validation of a new method based on heart-cutting two-dimensional liquid chromatography (LC-LC) [J]. J Agric Food Chem, 2011, 59: [11],,,. UPLC-MS-MS 4-2- [J]., 2014, 35(08): Zhang L, Kong XH, He Q, et al. Determination of 4-(5-)-methylimidazole and its isomer 2-methylimidazole in beverage by ultra performance liquid chromatography tandem mass spectrometry [J]. Food Sci, 2014, 35(08): [12],,, [J]., 2014, 5(5): Wu YL, Xian YP, Guo XD, et al. Quantitative determination of 2-and 4-methylimidazole in soy sauce using dispersive solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry [J]. J Food Saf Qual, 2014, 5(5): [13] Klejdus B, Moravcová J, Kubán V. Reversed-phase high-performance liquid chromatographic/mass spectrometric method for separation of 4-methylimidazole and 2-acetyl-4- (1,2,3,4-tetrahydroxybutyl)imidazole at pg levels [J]. Anal Chim Acta, 2003, 477: [14] Klejdus B, Moravcová J, Lojková L. Solid-phase extraction of 4(5)-methylimidazole (4MeI) and 2-acetyl- 4(5)-(1,2,3,4- tetrahydroxybutyl)-imidazole (THI) from foods and beverages with subsequent liquid chromatographic_electrospray mass spectrometric quantification [J]. J Sep Sci, 2006, 29: [15] Lojková L, Klejdus B, Moravcová J. Supercritical fluid extraction (SFE) of 4(5)-methylimidazole (4-MeI) and 2-acetyl- 4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI) from ground-coffee with high-performance liquid chromatographicelectrospray mass spectrometric quantification (HPLC/ESI-MS) [J]. Food Addit Contam, 2006, 23: [16] Yamaguchi H, Masuda T. Determination of 4(5)-methylimidazole in soy sauce and other foods by LC-MS/MS after solid-phase extraction [J]. J Agric Food Chem, 2011, 59: 作者简介 ( 责任编辑 : 杨翠娜 ) 王丽英, 博士, 中级技师, 主要研究方向为食品安全检测 wangliying2011@163.com 刘印平, 硕士, 中级技师, 主要研究方向为食品安全检测 liuyinping0930@163.com

Formation of carcinogenic 4(5)-methylimidazole in caramel model systems: a role of sulfite [J]. Food Chem, 2012, 136: 1165 1168. [4] Hengel M, Shibamoto T.

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