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1 5 5 Vol. 5 No Journal of Food Safety and Quality May., 吴玉銮 1, 冼燕萍 1, 郭新东 1*, 罗海英 1, 王莉 1, 韩婉清 1, 陈立伟 1, 李核 (1., ; 2., ) 2 摘要 : 目的 (dspe- UPLC-MS/MS) 方法,,,, MgSO 4 NaAC, N- (PSA)/ (MWNTs), UPLC BEH HILIC (50 mm 2.1 mm, 1.7 µm ), -10 mmol/l ( 0.1%, v/v),,, 结果 μg/kg; 1.0~100 μg/l, 0.999; 3, (n=6) 89.6%~117.2%, 4.8%~12.9%, (n=5) 15% 结论, 2-4- 关键词 : 2- ; 4- ; - ; ; Quantitative determination of 2-and 4-methylimidazole in soy sauce using dispersive solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry WU Yu-Luan 1, XIAN Yan-Ping 1, GUO Xin-Dong 1*, LUO Hai-Ying 1, WANG Li 1, HAN Wan-Qing 1, CHEN Li-Wei 1, LI He 2 (1. Guangzhou Quality Supervision and Testing Institute, Guangzhou , China; 2. School of Chemistry and Environment, South China Normal University, Guangzhou , China) ABSTRACT: Objective To establish a simple and rapid method for determination of 2-methylimidazole (2-MID) and 4-methylimidazole (4-MID) in soy sauce using dispersive solid phase extraction (dspe) clean-up and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods The isotope internal standard was added into the samples, which then was diluted with water and adjusted to alkaline, processed by liquid-liquid extraction with acetonitrile, followed by adding anhydrous MgSO 4 and anhydrous NaAC. Then the extract of dark soy sauce was purified by primary secondary amine (PSA) sorbent plus multi-walled carbon nanotubes (MWNTs). The ultimate solution was separation with UPLC BEH HILIC (50 mm 2.1 mm, 1.7 µm), elution with acetonitrile and 10 mmol/l ammonium acetate (containing 基金项目 : (2013DFH30070) (2010B ) Fund: Supported by the International S&T Cooperation Program of China (ISTCP) (2013DFH30070) and Guangdong Province Science and Technology Project (2010B ) * 通讯作者 :,, gdone@21cn.com *Corresponding author: GUO Xin-Dong, Professor, Guangzhou Quality Supervision and Testing Institute, No.38, Baqier Road, Guangzhou , China. gdone@21cn.com

2 % formic acid, v/v), and detection with tandem mass spectrometry under ESI at positive and multiple reaction monitoring (MRM) mode. The targets were quantified by isotope internal standard. Results The limit of detection (LOD) of the MIDs were both 1.5 μg/kg, and the linear range were 1.0~100 μg/l with the correlation coefficients larger than The average recoveries ranged from 89.6% to 117.2%, and within-day precisions were in a range of 4.8%~12.9% when three levels of standard were spiked into light and dark soy sauce. The inter-day precisions were less than 15%. Conclusion The developed method is simple, rapid, accurate and sensitive for the detection of 2-MID and 4-MID in soy souse. KEY WORDS: 2-methylimidazole; 4-methylimidazole; ultra-high performance liquid chromatography-tandem mass spectrometry; dispersive solid phase extraction; soy souse 1 引言,,,,,,, 2- (2-methylimidazole, 2-MID) 4- (4-methylimidazole, 4-MID) [1] ( 1),, [2], 2-MID 4-MID [3], (IARC) (Group 2A) [4], 2-MID 4-MID, 2 2-MID 4-MID (UV) [5,6] (GC) [2] (LC) [7,8] - (GC-MS) [9,10] - (GC-MS/MS) [11] - (LC-MS/MS) [12-15] 2,,, UV, ; GC GC-MS ; LC, ; - /,, GC-MS/MS LC-MS/MS, LC-MS/MS,,,,,,, [14], 2,, (UPLC) MS/MS MRM,, 2-MID 4-MID,, 2-MID 4-MID Fig. 1 Structures of 2-MID and 4-MID 2 材料与方法 2.1 主要仪器与试剂 ACQUITY TM Waters Xevo TM TQ MS ( Waters ); MS3 basic ( IKA ); KQ-500E ( ); 3K30 ( Sigma ); Turbo LV ( Biotage ); Milli-Q ( Millipore )

3 5, : MID ( 97%, ); 4-MID ( 99%, Dr. Ehrenstorfer ); : 4-MID-d 6 ( 98%, CDN ISOTOPES, ); ( HPLC, CNW Technologies GmbH ); ( 25%,, ); (18.2 MΩ); QuEChERS ( Agilent, : ), 6 g MgSO g ; N- (PSA, µm, 6 nm, CNW Technologies GmbH ); (MWNTs, nm, 99%, ) : 5 5, 2.2 标准溶液的配制 4-MID 2-MID, 100 mg/l, 0.2% - (v/v) 0.5 mg/l 4-MID-d 6, 20 mg/l, 0.2% - (v/v) 0.5 mg/l 2.3 样品前处理 0.5~1.0 g( g) 15 ml, 100 μl, 3 ml 0.2 ml,, 5 ml, 2 min, 2.5 g MgSO 4 NaAC (QuEChERS ), 2 min, 4500 r/min 5 min, 1.0 ml 10 ml, 40, 1.0 ml 0.5% - (v/v), 1 min, 0.22 μm UPLC-MS/MS ;, 20 mg PSA 10 mg MWNTs 1.5 ml, 1 min, 30 s, r/min 3 min, 0.22 μm UPLC- MS/MS 2.4 UPLC-MS/MS 检测条件 : Waters ACQUITY UPLC BEH HILIC (50 mm 2.1 mm, 1.7 µm); : 10 mmol/l ( 0.1%, v/v)+ =(7:93, v/v); : 0.3 ml/min; : 30 ; : 2 μl : (ESI), (MRM) ;, 0.2 ml/min; (m/z) 1, 0.1 s 3 结果与讨论 3.1 脱水剂用量对回收率的影响,,,, (XTR),,,,, ph 5,,,,,,, ;,,,,,,,,, ;,,, 表 1 2 种待测物及同位素内标物的质谱分析条件 Table 1 MS parameters for the analysis of studied compounds (m/z) (m/z) (V) (ev) (min) 1 4-MID 83 56*/ / MID 83 42*/ / IS 4-MID-d : *

4 AOAC , MgSO 4 NaAC [16], Agilent QuEChERS g MgSO 4 NaAC,, 2, 2, 2.5 g ( MgSO 4 NaAC 2 g 0.5 g), 2, 78%~90%,,, TIC 3 4,, ( 3B 4C), ( 3A 4B),, μg/kg( 2 ), [10] [11] ( 7.27 μg/kg 5 μg/kg),, 2.3, 3 TIC Fig. 3 Comparison of TIC of light soya sauce before (B) and after (A) dehydration (1. 4-MID and 4-MID-d 6 ; 2. 2-MID) 3.2 吸附剂种类和用量对回收率的影响, MgSO 4 NaAC,, 4B, PSA MWNTs PSA Fig. 2 2 Effect of the amount of water remover on the recoveries of MIDs 4 TIC Fig. 4 Comparison of TIC of dark soya sauce before (C) and after (B) dehydration, and purified with PSA+MWNTs (A) (1. 4-MID and 4-MID-d 6 ; 2. 2-MID)

5 5, : ; MWNTs,, PSA [17,18] PSA ( mg) MWNTs ( mg), PSA,,, 2-MID ; MWNTs,,, PSA MWNTs 20 mg 10 mg ( 5 ), 2, 20 mg PSA 10 mg MWNTs, TIC ( 4A), 3.3 仪器条件的优化 2, ESI [M+H] + (m/z 83), m/z 68( -CH 3 ) m/z 56( -C 2 H 3 ) m/z 42( -NC 2 H 3 ),,, m/z 68 m/z 56 4-MID, m/z 56 m/z 42 2-MID 1 2,, Wang [14] C30, C30, : mm 150 mm ACQUITY UPLC BEH C18 ; 50 mm 100 mm ACQUITY UPLC BEH HILIC 2, 50 mm ACQUITY UPLC BEH HILIC HILIC,, 5% ( ),, ( ), mmol/l -10 mmol/l -10 mmol/l ( 0.1%, )3 90%, 2 ( 6B); 93%,,, 3 6, 2-10 mmol/l Fig. 5 5 Effect of the type and amount of sorbent on the recoveries of MIDs

6 1362 5,,, +10 mmol/l ( 0.1%, v/v)=93: 7(v/v), (10.0 μg/l) 线性关系和检出限,,, 1.0~100 μg/l, 4-MID-d μg/l, 6 (1. 4-MID 4-MID-d 6 ; 2. 2-MID) Fig. 6 Comparison of analytes in different mobile phase systems (1. 4-MID and 4-MID-d 6 ; 2. 2-MID) A. acetonitrile +10 mmol/l ammonium acetate (containing 0.1% formic acid, volume fraction) = 93+7 (v/v); B. acetonitrile +10 mmol/l ammonium acetate (containing 0.1% formic acid, volume fraction) = 90+10(v/v); C. acetonitrile +5 mmol/l ammonium acetate = 93+7(v/v); D. acetonitrile +10 mmol/l ammonium acetate = 93+7(v/v). ( 0.1%, ) ( 6A), 2 4-MID-d 6,, 2,, 0.999, 1.0~100 μg/l ; 3 (S/N) 10 (S/N) ( 5 ), 2 (LODs) 1.5 µg/kg, (LOQs) 5.0 µg/kg, 3.5 方法学评价和实际样品检测 3 (n=6); 5, 3, %~117.2%, 4.8%~12.9%, Fig. 7 7 (10.0 μg/l) Selective ion chromatogram of standard (10.0 μg/l) 表 2 2 种目标物的线性范围 线性方程 相关系数 检出限和定量限 Table 2 Linear ranges, regression equations, LODs and LOQs for 2 analytes (μg/l) R 2 (µg/kg) (µg/kg) 4-MID 1.0~100 Y= X MID 1.0~100 Y= 0.727X

7 5, : Table 3 表 3 样品的添加回收和精密度实验数据 Recoveries and repeatabilities for 2 analytes in samples (μg/kg) (%, n=6) (%, n=5) (μg/kg) (%) RSD (%, n=6) (μg/kg) RSD (%, n=5) 4-MID 11 10, 20, , 108.3, , 9.6, MID N.D. 5, 10, , 89.6, , 6.2, MID , 600, , 106.5, , 11.0, MID 17 15, 30, , 95.7, , 8.2, %~14.1%, 5 5, 10 4-MID, 11~1621 μg/kg; MID, 5.9~51.2 μg/kg 4 结论 - 4-MID 2-MID, BEH HILIC, 4-MID 2-MID ;, MgSO 4 NaAC, ;, PSA MWNTs, ;, 4-MID 2-MID 参考文献 [1] Moon JK, Shibamoto T. Formation of carcinogenic 4(5)-methylimidazole in Maillard reaction systems [J]. J Agric Food Chem, 2011, 59(2): [2],,, [J]., 2012, 37 (8): Peng XQ, Feng W, Shi HY, et al. Determination of 2-methylimidazole and 4-methylimidazole in caramel pigment by capillary gas chromatography [J]. China Condi, 2012, 37 (8): [3] Chan P, Mahler J, Travlos G, et al. Induction of thyroid lesions in 14-week toxicity studies of 2- and 4-methylimidazole in Fischer 344/N rats and B6C3F1 mice [J]. Arch Toxicol, 2006, 80 (3): [4] Grosse Y, Baan R, Secretan-Lauby B, et al. Carcinogenicity of chemicals in industrial and consumer products, food contaminants and flavourings, and water chlorination byproducts [J]. Lancet Onc, 2011, 12(4): [5],,,. 4- [J]., 2008, 25(2): Li JM, Wang YJ, Zou N, et al. Detemination of 4-methyliminidazol in soy sauce by ultraviolet-visible spectrometry [J]. Chin J Spectrosc Lab, 2008, 25(2): [6],,,. 4- [J]., 2010, (7): Zhao JP, Gao LH, Jin WW, et al. Detemination of 4-methyliminidazol in soy sauce [J]. China Brewing, 2010, (7): [7],,,. 4- [J]., 2007, 33 (12): Wang MF, Wang YH, Guo XD, et al. Determination of pre-column dansylation of 4-methylimidazole by capillary HPLC [J]. Food Ferment Ind, 2007, 33 (12): [8] Moretton C, Cretier G, Nigay H, et al. 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(8): [9] Casal S, Fernandes JO, Oliveira MBPP, et al. Gas chromatographic mass spectrometric quantification of 4-(5-) methylimidazole in roasted coffee after ion-pair extraction[j]. J Chromatogr A, 2002, 976(1-2): [10] 珺,, [J]., 2012, 28(5): , 578 Xiong J, Chen ML, Lai YD. Determination of 4-methylimidazole in liquid seasoning by solid phase extraction combined with gas chromatography/selected ion mass spectrometry [J]. Mod Food Sci Technol, 2012, 28(5): , 578.

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