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1 4 6 Vol. 4 No Journal of Food Safety and Quality Dec., 2013 孙佳 1, 毛燕 2 1,, 李刚 2*, 张丽 1 (1., ; 2., ) 摘要 : 目的方法,, 1 ml/min, Venusil HILIC (250 mm 4.6 mm, 5 µm, 100 Å), 结果 0.6 µg/ml, 2 µg/ml, R 30%, RSD 7% 结论, 关键词 : ; ; ; HILIC Application of hydrophilic interaction liquid chromatography to determine hydrogen peroxide in food SUN Jia 1, MAO Yan 2, LI Gang 1, 2*, ZHANG Li 1 (1. College of Food Science and Technology, Jilin Agricultural University, Changchun , China; 2. Jilin Province Product Quality Supervision Test Institute, Changchun , China) ABSTRACT: Objective To establish a high pressure liquid chromatography (HPLC) method for determination of the hydrogen peroxide content in food. Methods The food sample was directly extracted with water, and the residual hydrogen perioxide (H 2 O 2 ) was determined without derivatization via hydrophilic interaction chromatography (HILIC) subsequently. The separation was carried out through a Venusil HILIC column (250 mm 4.6 mm, 5 µm, 100 Å) with acetonitrile as mobile phase at a flow rate of 1 ml/min. Results The detection limit and quantitation limit of H 2 O 2 were 0.6 µg/ml and 2 µg/ml, respectively. The average recovery of food sample was higher than 30% with relative standard deviation (RSD) of lower than 7%. Conclusion This method has a high sensitivity, a good precision and accuracy, which can be applied to determine the H 2 O 2 in food. KEY WORDS: hydrogen peroxide; food; high pressure liquid chromatography; HILIC column 1 引言 (hydrogen peroxide), [1] GB [2],, [3],, 基金项目 : (2012-ZJK10) Fund: Supported by the Jilin Province Quality and Technical Supervision Science Projects (2012-ZJK10) * 通讯作者 :,, lg56ky@163.com *Corresponding author: LI Gang, Professor, Jilin Province Product Quality Supervision Test Institute, No.1088, Nanhu Road, Changchun , China. lg56ky@163.com

2 1874 4,,,, : [4] [5-6] [7-9] [10-11] [12-13] [14-20] [10, 21] GB/T [22],,, (high pressure liquid chromatography, HPLC), HPLC, [20],,,,, Venusil HILIC,,,,,, 2 材料与方法 2.1 仪器与材料 Agilent1100 ( VWD DAD ); AE240 ( - ); ( ); HPD-25 ( ); ( ); KQ-250DE ( ); () ( Tedia, ); (, ); 30%(, ); ( ) 2.2 方法标准溶液的配制 : 0.66 g g 200 ml,, 1 mg/ml,, 10, 30, 50, 100, 200 μg/ml, 样品的处理 : 100 g,, 10 g( 0.01 g) 250 ml, 50 ml, 3 min,, 10 min,,, 4000 r/min 5 min ml C 18 (, C 18 4 ml 20 ml ),,, 20 ml,,, 20 ml 10 ml, 50 ml, 0.45 μm 2 ml, : 10 g( 0.01 g) 50 ml,,,, 4000 r/min 5 min ml 2.3 色谱条件 Venusil HILIC (250 mm 4.6 mm, 5 μm, 100 Å),, 40 ; 220 nm, 1.0 ml/min, 5 μl 3 结果与讨论 3.1 色谱条件的优化色谱柱的选择, H 2 O 2, SCX SAX, ; C 18,, H 2 O 2, H 2 O 2 HILIC,, 检测波长的选择

3 6, : 1875, 0.1 mg/ml H 2 O 2, DAD H 2 O 2 ( 1),, H 2 O 2, nm,,,, 195 nm 225 nm H 2 O 2, nm, H 2 O 2,, 220 nm 1 Fig. 1 UV spectra of H 2 O 2 H 2 O 2 HILIC,, H 2 O 2, , H 2 O 2 :, 220 nm, H 2 O 2 ;, H 2 O 2, ; H 2 O 2, 100% H 2 O 2 试液含水量对色谱峰影响, H 2 O 2,, -(v/v) 0/100 20/80 40/60 60/40 80/20 100/0 1 mg/ml H 2 O 2,, 2 2,, H 2 O 2,,, 3.2 HPLC 色谱图 H 2 O 2 HPLC 3 4, 3 4 H 2 O 流动相的选择 100 % 80 % 60 % 40 % 20 % 0 % Fig. 2 2 The effect of water contents in samples on chromatographic peak form

4 4 6 Vol. 4 No Journal of Food Safety and Quality Dec., 标准曲线的绘制及相关性 H 2 O 2, 20 μl,, ( 5),, : H 2 O 2 : Y=8.986X (R=0.9996) 5 Fig. 3 1: ; 2:. 1: acetonitrile; 2: hydrogen peroxide. 3 Chromatogram of H 2 O 2 standard solution Fig. 5 5 Standard curve of hydrogen peroxide, H 2 O μg/ml,, 3.4 检出限和定量限 1: ; 2:. 1: acetonitrile; 2: hydrogen peroxide. 4 Fig. 4 Liquid chromatogram of sample contained H 2 O 2, 1, H 2 O 2, H 2 O 2 10 μg/ml,, 8, 20 μl,, 220 nm,,, DAD 0.6 μg/ml, 2 μg/ml 3.5 精密度实验 50 μg/ml, 5,, RSD 1 基金项目 : (2012-ZJK10) Fund: Supported by the Jilin Province Quality and Technical Supervision Science Projects (2012-ZJK10) * 通讯作者 :,, lg56ky@163.com *Corresponding author: LI Gang, Professor, Jilin Province Product Quality Supervision Test Institute, No.1088, Nanhu Road, Changchun , China. lg56ky@163.com

5 6, : 样品前处理条件的研究 H 2 O 2,,, H 2 O 2, 表 1 精密度实验结果 Table 1 Results of precision test average RSD% 表 2 提取次数的影响 Table 2 The effect of extraction times 表 3 过氧化氢的加样回收率 (n=5) Table 3 The average recovery of H 2 O 2 (n=5) (μg/g) (%) RSD% H 2 O 2,,,, H 2 O 2,,, H 2 O 2, 3 H 2 O 2, 2 50 ml 3 H 2 O 2, : 15 mg/kg 3.7 回收率实验 : 6, 5, 5 g, 1000 μg/ml ml, 2.2.2, 20 μl, 3 3, 6 30%, 7%,

6 H 2 O 2 4 结论 (HILIC),,, C 18,,,, 参考文献 [1]. [D]., Li SP. New Methods for Determination of Hydrogen peroxide [D]. University of South China, [2] GB [S]. GB Hygiene standards for use of food additives [S]. [3],,,. [J]., 2005, 26(9): Zhou JH, Li H, Song SC, et al. Determine the effect of hydrogen peroxide on bone mesenchymal stem cells (BMSC) [J]. Guangdong Med J, 2005, 26(9): [4],. [J]., 2001, 17(1): 1 5. Zhang GF, Cheng HZ. Determination of hydrogen peroxide by micelle electrochemiluminescence with flow injection analysis [J]. J Anal Sci, 2001, 17(1): 1 5. [5],,,. / H 2 O 2 [J]., 2006, 31(5): Dong ZQ, Yuan R, Chai YQ, et al. A biosensor for hydrogen peroxide based on horseradish peroxidase immobilized in ZrO2/Au So-l gel Matrix [J]. J Southwest China Normal Univ, 2006, 31(5): [6]. [D]., Liu HF. The development of the biosensor method for rapidly detecting hydrogen peroxide residue in food [D]. Shanghai Jiaotong Univ, [7],,. [J]., 2008, 35(8): Zhang WW, Yang Y, Cheng PC. Determination of hydrogen peroxide in water-soaked foodstuffs by ammonium molybdate tetrahydrate spectrophotometry [J]. Mod Preventive Med, 2008, 35(8): [8],. - [J]., 2003, 20(4): Sheng Y, Chai SF. Indirect determination of trace amount of hydrogen peroxide in food by the i2-ccl4extraction spectrophotometry [J]. Chin J Spectrosc Lab, 2003, 20(4): [9],,. [J]., 2008, 28(11): Zhang YJ, Ma J, Zhang L. Spectrophotometric determination of hydrogen peroxide in acidic dye system with titanium oxalate [J]. Ind Water Treat, 2008, 28(11): [10] Arnous A, Petrakis C, Makris DP, et al. A peroxyoxalate chemilumine- scence-basedassay for the evaluation of hydrogen peroxide scavenging activity employing 9, 10-diphenylanthracene as the fluorophore [J]. J Pharmacol Toxicol Methods, 2002, 48: [11],,. [J]., 2008, 27(4): Li XX, Sheng HL, Qi HL. Determination of hydrogen peroxide on a peroxidase-immobilized capillary column by flow-injection chemiluminescence [J]. J Instrum Anal, 2002, 48: [12],. [J]., 2009, 20(6): Zhou Y, Zhang DX. Determination of hydrogen peroxide in water-soaked foodstuffs by fluorescence analysis [J]. J Publ Health Preventive Med, 2009, 20(6): [13],,. T [J]., 2008, 25(6): Sheng L, Zhu JL, Han XQ. Determination of hydrogen peroxide based on fluorescence quenching with safraninet system [J]. Chin J Spectrosc Lab, 2008, 25(6): [14] Kobayashi K, Kawai S. Enzymatic determination of hydrogen peroxide using gas chromatography [J]. J Chromatogr A, 1982, 245(3): [15] Effkemann S, Pinkernell U, Karst U. Peroxide analysis in laundry detergents using liquid chromatography [J]. Anal Chim Acta, 1998, 363(1): [16],. - [J]., 2005, 23(4): Xu JR, Cheng ZM. Determination of peroxides in environmental samples by high performance liquid chromatography with fluorescence detection [J]. Chin J Chromatogr, 2005, 23(4): [17],,,. [J]., 2003, 21(5): Guo ZA, Zhao JC, Dang GC. Determination of peroxyacetic acid

7 6, : 1879 and hydrogen peroxide by ion suppression chromatography in disinfectants [J]. Chin J Chromatogr, 2003, 21(5): [18],,. [J]., 2003, 13(5): Hu JM, Shi WP, Lin SB. Study on method for determination of H 2 O 2 in cosmetics by HPLC [J]. Chin J Health Lab Technol, 2003, 13(5): [19],,. [J]., 2003, 31(5): Wang KX, Xu LJ, Yao M. Determinationof hydrogen peroxide using flow injection analysis-conduetivity method with ion chromatography [J]. Anal Chem, 2003, 31(5): [20],,. - [J]., 2009, 26(2): Li YH, Liu Y, Zhou JJ. Determination of peroxides in food samples by high performance liquid chromatography with variable wavelength detector [J]. Chin J Spectrosc Lab, 2009, 26(2): [21],,. [J]., 2008, 27(4): Li XX, Sheng HL, Qi HL.Determination of hydrogen peroxide on a peroxidase-immobilized capillary column by flow-injection chemiluminescence [J]. J Instrum Anal, 2008, 27(4): [22] GB/T [S]. GB/T The determination methods of hydrogen peroxide in the food [S]. 作者简介 ( 责任编辑 : 张宏梁 ) 孙佳, 硕士研究生, 主要研究方向为农产品加工及贮藏工程 sunjiasunpeng@163.com 李刚, 教授, 主要研究方向为食品分析检测 lg56ky@163.com

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.