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1 7 7 Vol. 7 No Journal of Food Safety and Quality Jul., 2016 姚敬 *, 黄伟雄, 李敏, 陈明, 王静 (, ) 摘要 : 目的 -, 方法 HLB ;, 15 N 13 2 C-SEM-NBA 结果 0.5~100 μg/kg, , 0.5 μg/kg, 85.1%~114.6%, 3.2%~6.9% %, 结论, 关键词 : ; ; ; - Study on the determination of semicarbazide in flour products and transformation law between azodicarbazide and semicarbazide YAO Jing *, HUANG Wei-Xiong, LI Min, CHEN Ming, WANG Jing (Guangdong Provincial Center for Disease Control and Prevention, Guangzhou , China) ABSTRACT: Objective To establish a method for the determination of semicarbazide(sem) residues in flour products by ultra high performance liquid chromatography-tandam mass spectrometry, and also study the effect of azodicarbazide(ada) adding levels on the semicarbazide content in the bread and their transform condition. Methods Samples were hydrolyzed with hydrochloric acid, derivated by nitrobenzaldehyde(2-nba), cleaned by HLB column, and extracted with ethyl acetate. Mass spectrometer was used by electrospray ionization source positive iron mode for scanning monitoring, and then the target of semicarbazide derivatives was quantified with isotope internal standard of 15 N 13 2 C-SEM-NBA. Results SEM had a good linear relationship in the range of 0.5~100 μg/kg with the correlation coefficient of , the limit of detection was 0.5 μg/kg. The recoveries was 85.1%~114.6% and RSD was 3.2%~6.9%. The positive percentage of semicarbazide in the ten commercial bread was 41.7%. ADA can transform to SEM under the hot and damp condition, and the SEM content of surface was higher than that of inside in the same bread. Conclusion The method is rapid, precise and sensitive, which is suitable for the quantitative analysis of semicarbazide in flour product. 基金项目 : (A ) Fund: Supported by Medical Research Fund Of Guangdong Province (A ) * 通讯作者 :,, yiuking@21cn.com *Corresponding author: YAO Jing, Associate Chief Technician, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou , China. yiuking@21cn.com

2 KEY WORDS: flour product; azodicarbazide; semicarbazide; ultra high performance liquid chromatography-tandam mass spectrometry 1 引言 (semicarbazide, SEM), (median lethal dose, LD50) 225mg/kg, 2003,, [1] (azodicarbazide, ADA),,, [2], - [3-5] ( 1) ADA [6] ADA, GB [7] ADA 0.45mg/kg ADA, ADA [8] ,, - [6-9],,, ADA, 2 材料与方法 2.1 仪器与试剂 - : LC-20 AD XR () ABI 4000 ( AB Sciex ); Waters BEH C 18 (50 mm 2.1mm, 1.7 μm, ); VORTEX-5 ( ); phb3ph ( ) (SEM HCl, CAS : , Sigma-Aldrich ) SCA- 13 C- 15 N 2 (CAS : , Sigma-Aldrich ); (>97%,Chem Service ) (, Sigma ); (, Merck ); (, ); ( 18MΩ cm) 2.2 实验方法 仪器条件 (1) : Waters Acquity TM UPLC BEH C 18 (2.1 mm 50 mm, 1.7 μm); : 40 ; 15 ; : 50μL; 0.2mL/min; A 0.1%, B, 1 Table 1 表 1 流动相梯度洗脱条件 Gradient elution condition of mobile phase (min) (ml/min) A(%) B(%) Fig. 1 1 [3-5] Schematic diagram of the conversion of azodicarbonamide and semicarbazide

3 7, : 2875 (2) : (electrospray ionization,esi); : ; : (multi-reaction monitoring,mrm) 2 表 2 质谱参数 Table 2 Massspectrometer parameters (m/z) (V) (m/z) (V) SEM * / SEM- 15 N 2 13 C * / ( : * ) Fig. 2 2 Signal values of standard and internal standard 样品前处理 (1) 2 g,, 50 ml, 0.10 μg/ml 50 μl, 0.2 mol/l 10 ml,, 0.1 mol/l2-nba 100 μl, 30 min, h (2), 0.3 mol/l 3 ml ph 4~ r/min 10 min, HLB 3 ml 3 ml, 10 ml, 6 ml, 3 ml 40, 1 ml0.1%, 0.22 μm, 标准溶液配制 0.02 μg/ml μl, 10 ml, 0.10 μg/ml 50 μl, 0.1 mol/l 2-NBA100 μl,, h, 3 SEM-NBA Fig. 3 SEM-NBA fragment ion peak : 209/166(m/z); : 209/192(m/z) Above: 209/166(m/z); Below: 209/192(m/z) 3.2 衍生化酸度条件选择 37, 10μg/L SEM, ph 2.3~10.2 4, ph4~6,, 3 结果与分析 3.1 仪器条件优化 0.1%,,,, min, 15 N 13 2 C-SEM- NBA 2.87 min, 2, 2, 209/ /192, 212/168, 209/166(m/z) ( 3) Fig. 4 4 ph The effect of ph on the efficience of derivatization

4 线性试验与检出限,, 0.5~100 μg/kg, , Y=352X μg/kg h, ;,, μg/kg 3.4 加标回收率与精密度, 4.0 μg/kg 85.1%~114.6% 6, 3.2%~6.9%,, 3 4, [9], ADA,,, 考察偶氮甲酰胺添加水平对面包中氨基脲含量的影响 mg/kg,, ( 280 g, 150 ml, 8.4 g, 2.8 g, 12.5 g, 11 g, 3.4 g) 3 h, min,, 小麦粉制品中氨基脲的检出结果 60, : 4, 21.1~35.9 μg/kg; 16, 72.3~255.6 μg/kg, 75%; 4, 1.83~10.45 μg/kg; 1, 1.86 μg/kg %, 1.83~255.6μg/kg,,,, 3.6 偶氮甲酰胺添加水平对面包中氨基脲含量的影响 考察面制品中氨基脲的形成条件, 4, 1, ; 2 50 μg/kg,, ( 5A); 3 50 μg/kg, 60~70 ( 5B); 4 50 μg/kg, 4, 5 ( 5C), 5 Fig.5 Chromatograms of semicarbazide content in flour samples after different treatment A. ; B. ; C. A. Blank experiment anduntreated spiked samples; B. flour samples treated by heating; C. flour samples treated by moistening Table 3 表 3 偶氮甲酰胺添加水平对面包中氨基脲含量的影响 (n=3) Effects of azodicarbonamide addition on theyield of semicarbazide in flour(n=3) (μg/kg) : 10.0 mg/kg : 20.0 mg/kg : 40.0 mg/kg 81.2±1.3% 374±1.0% 675±1.4% 37.8±2.6% 136±1.7% 366±2.2% 89.5±1.9% 407±2.3% 702±1.6%

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