坚果中黄曲霉毒素的光化学柱后衍生-高效液相色谱法测定
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摘要
目的建立坚果中黄曲霉毒素B1、B2、G1、G2的高效液相色谱荧光检测器测定方法。方法样品以甲醇-水(70:30,v/v)溶液匀质提取,过黄曲霉毒素总量免疫层析亲和柱净化,经LaChrom C18色谱柱分离和光化学柱后衍生反应器衍生后,用带有荧光检测器的高效液相色谱仪测定。采用峰面积外标法定量坚果中黄曲霉毒素B1、B2、G1、G2含量。结果四种黄曲霉毒素在各自的浓度范围内线性关系良好,相关系数均大于0.999,B1、B2、G1、G2的检出限依次为0.10、0.05、0.10、0.05μg/kg。在3个添加水平下回收率为77.5%~109.8%,相对标准偏差为1.43%~2.71%。结论该方法的灵敏度、准确度、精密度均符合黄曲霉毒素的检测技术要求,适用于坚果中黄曲霉毒素B1、B2、G1、G2的日常检测。
Objective To establish a high performance liquid chromatography(HPLC) coupled with post-column photochemical derivatization method for the determination of the aflatoxin B1, B2, G1, and G2 in nuts. Methods The sample was tested by HPLC with fluorescence detector after being extracted from methanol-water(70:30, v/v), purified by immunoaffinity chromatography column of aggregated aflatoxin, separated by LaChrom C18 chromatographic column, and derived from post column photochemical derivative reactor. The contents of aflatoxin B1, B2, G1, and G2 in nuts were measured using peak area external standard method. Results The calibration curves showed a good linearity in each concentration range with correlation coefficient greater than 0.999. The limits of detection(LODs) were 0.10, 0.05, 0.10, and 0.05 μg/kg for the aflatoxins B1, B2, G1, and G2 in nuts, respectively. The recovery rate was within 77.5%~109.8% at three adding levels with the relative standard deviation(RSDs) of 1.43%~2.71%. Conclusion This method is sensitive, accurate and precise which is in accordance with technical standards of the aflatoxins detection. It is proper for routine detection of the aflatoxins B1, B2, G1, and G2 in nuts.
Objective To establish a high performance liquid chromatography(HPLC) coupled with post-column photochemical derivatization method for the determination of the aflatoxin B1, B2, G1, and G2 in nuts. Methods The sample was tested by HPLC with fluorescence detector after being extracted from methanol-water(70:30, v/v), purified by immunoaffinity chromatography column of aggregated aflatoxin, separated by LaChrom C18 chromatographic column, and derived from post column photochemical derivative reactor. The contents of aflatoxin B1, B2, G1, and G2 in nuts were measured using peak area external standard method. Results The calibration curves showed a good linearity in each concentration range with correlation coefficient greater than 0.999. The limits of detection(LODs) were 0.10, 0.05, 0.10, and 0.05 μg/kg for the aflatoxins B1, B2, G1, and G2 in nuts, respectively. The recovery rate was within 77.5%~109.8% at three adding levels with the relative standard deviation(RSDs) of 1.43%~2.71%. Conclusion This method is sensitive, accurate and precise which is in accordance with technical standards of the aflatoxins detection. It is proper for routine detection of the aflatoxins B1, B2, G1, and G2 in nuts.
引文
[1]Deiner UL,Cole RJ,Sanders TH,et al.Epidemiology of aflatoxin formation by Aspergillus flavus[J].Annu Rev Phytopathol,1987,25:240-270.
[2]Kurtzman CP,Horn BW,Hesseltine CW.Aspergillus nomius,a new aflatoxin producing species related to Aspergillus flavus and Aspergillus tamari[J].Antoine van Leeuwenhoek,1987,53:147-158.
[3]Cotty PJ,Bhatnagar D.Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes[J].Appl Environ Microbiol,1994,60:2248-2251.
[4]Chang HL,Devries JW.Rapid high pressure liquid chro-magraphic determination of aflatoxin M1 in milk and nonfat dry milk[J].J Assoc Offic Anal Chem,1982,66(4):913-917.
[5]马志科,昝林森.黄曲霉毒素危害、检测方法及生物降解研究进展[J].动物医学进展,2009,30(9):91-94.Ma ZK,Zan LS.Progress on hazard,detection techniques and biodegradation of aflatoxin[J].Chin J Prog Veter Med,2009,30(9):91-94.
[6]范蓓,李庆鹏,哈益明.关注黄曲霉毒素国内外限量标准,完善食品安全保障措施[J].食品工业科技,2010,(12):26-27.Fan L,Li QP,HA YM.Focus on aflatoxin limited standards at home and abroad,perfect food safety guarantee measures[J].Chin J Sci Technol Food Ind,2010,(12):26-27.
[7]潘崴,庞广昌,张昀.黄曲霉毒素与食品安全[J].食品研究与开发,2004,25(6):11-13.Pan W,Pang GC,Zhang Y.Aflatoxin and food security[J].Chin J Food Res Develop,2004,25(6):11-13.
[8]Parkin DM,Bray F,Ferlay J.Global cancer statistics,2002[J].CA Cancer J Clin,2005,55(2):74-108.
[9]曾运红,谭卫仙.原发性肝癌发病主要危险因素的Meta分析[J].现代预防医学,2004,31(2):172-174.Zeng YH,Tan WX.Meta analysis on the risk factors of primary hepatocellular cancer in Chinese people[J].Chin J Mod Prev Med,2004,31(2):172-174.
[10]GB 2761-2011食品中真菌毒素限量[S].GB 2761-2011 The limited standards of fungal toxin in food[S].
[11]高秀芬,计融,李燕俊,等.高效液相色谱法测定玉米中的黄曲霉毒素[J].中国食品卫生杂志,2007,19(2):105-108.Gao XF,Ji R,Li YJ,et al.Determination of aflatoxins in corns by high performance liquid chromatography[J].Chin J Food Hyg,2007,19(2):105-108.
[12]王娜,潘治利,杨君丽,等.花生中黄曲霉毒素检测方法的研究[J].安徽农业科学,2008,36(24):10305-10306,10310.Wang N,Pan ZL,Yang JL,et al.Study on the method for determination of aflatoxin in peanu[J].Chin J Anhui Agric Sci,2008,36(24):10305-10306,10310.
[13]张浩,杨毅,潘见.LC-MS法测定花生中黄曲霉毒素的研究[J].安徽化工,2007,33(4):68-70.Zhang H,Yang Y,Pan J.The research of aflatoxins in peanut measured by LC-MS method[J].Chin J Anhui Chem Ind,2007,33(4):68-70.
[14]谢刚,王松雪,张艳.超高效液相色谱法快速检测粮食中黄曲霉毒素的含量[J].分析化学,2013,41(2):223-228.Xie G,Wang SX,Zhang Y.Rapid analysis of aflatoxins(B1,B2,G1,G2)in grain by immuno-affinity clear-up column and ultra performance liquid chromatography without derivation[J].Chin J Anal Chem,2013,41(2):223-228.
[15]刘坚,余敦年,熊宁,等.高效液相色谱法对稻谷及稻谷籽粒中黄曲霉毒素的测定研究[J].中国粮油学报,2011,26(4):107-110.Liu J,Yu DN,Xiong N,et al.Study on aflatoxin in paddy and in whole paddy seed by immunoaffinity column-photochemical derivatization and HPLC[J].J Chin Cer Oils Assoc,2011,26(4):107-110.
[16]GB/T 18979-2003食品中黄曲霉毒素的测定免疫亲和层析净化高效液相色谱法和荧光液相法[S].GB/T 18979-2003 Determination aflatoxins content in food—Cleanup by immunoaffinity chromatography and determination by high-performance liquid chromatigraphy and fluoromete[S].
[17]GOBEL R,LUSKY K.Simultaneous determination of aflatoxins,ochratox in A,and zearalenone in grains by new immunoaffinity column/liquid chromatography[J].J AOAC Int,2004,2:411-415.
[18]GB/T 27404-2008实验室质量控制规范食品理化检测[S].GB/T 27404-2008 Criterion on quality control of laboratorieschemical testing of food[S].
[2]Kurtzman CP,Horn BW,Hesseltine CW.Aspergillus nomius,a new aflatoxin producing species related to Aspergillus flavus and Aspergillus tamari[J].Antoine van Leeuwenhoek,1987,53:147-158.
[3]Cotty PJ,Bhatnagar D.Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes[J].Appl Environ Microbiol,1994,60:2248-2251.
[4]Chang HL,Devries JW.Rapid high pressure liquid chro-magraphic determination of aflatoxin M1 in milk and nonfat dry milk[J].J Assoc Offic Anal Chem,1982,66(4):913-917.
[5]马志科,昝林森.黄曲霉毒素危害、检测方法及生物降解研究进展[J].动物医学进展,2009,30(9):91-94.Ma ZK,Zan LS.Progress on hazard,detection techniques and biodegradation of aflatoxin[J].Chin J Prog Veter Med,2009,30(9):91-94.
[6]范蓓,李庆鹏,哈益明.关注黄曲霉毒素国内外限量标准,完善食品安全保障措施[J].食品工业科技,2010,(12):26-27.Fan L,Li QP,HA YM.Focus on aflatoxin limited standards at home and abroad,perfect food safety guarantee measures[J].Chin J Sci Technol Food Ind,2010,(12):26-27.
[7]潘崴,庞广昌,张昀.黄曲霉毒素与食品安全[J].食品研究与开发,2004,25(6):11-13.Pan W,Pang GC,Zhang Y.Aflatoxin and food security[J].Chin J Food Res Develop,2004,25(6):11-13.
[8]Parkin DM,Bray F,Ferlay J.Global cancer statistics,2002[J].CA Cancer J Clin,2005,55(2):74-108.
[9]曾运红,谭卫仙.原发性肝癌发病主要危险因素的Meta分析[J].现代预防医学,2004,31(2):172-174.Zeng YH,Tan WX.Meta analysis on the risk factors of primary hepatocellular cancer in Chinese people[J].Chin J Mod Prev Med,2004,31(2):172-174.
[10]GB 2761-2011食品中真菌毒素限量[S].GB 2761-2011 The limited standards of fungal toxin in food[S].
[11]高秀芬,计融,李燕俊,等.高效液相色谱法测定玉米中的黄曲霉毒素[J].中国食品卫生杂志,2007,19(2):105-108.Gao XF,Ji R,Li YJ,et al.Determination of aflatoxins in corns by high performance liquid chromatography[J].Chin J Food Hyg,2007,19(2):105-108.
[12]王娜,潘治利,杨君丽,等.花生中黄曲霉毒素检测方法的研究[J].安徽农业科学,2008,36(24):10305-10306,10310.Wang N,Pan ZL,Yang JL,et al.Study on the method for determination of aflatoxin in peanu[J].Chin J Anhui Agric Sci,2008,36(24):10305-10306,10310.
[13]张浩,杨毅,潘见.LC-MS法测定花生中黄曲霉毒素的研究[J].安徽化工,2007,33(4):68-70.Zhang H,Yang Y,Pan J.The research of aflatoxins in peanut measured by LC-MS method[J].Chin J Anhui Chem Ind,2007,33(4):68-70.
[14]谢刚,王松雪,张艳.超高效液相色谱法快速检测粮食中黄曲霉毒素的含量[J].分析化学,2013,41(2):223-228.Xie G,Wang SX,Zhang Y.Rapid analysis of aflatoxins(B1,B2,G1,G2)in grain by immuno-affinity clear-up column and ultra performance liquid chromatography without derivation[J].Chin J Anal Chem,2013,41(2):223-228.
[15]刘坚,余敦年,熊宁,等.高效液相色谱法对稻谷及稻谷籽粒中黄曲霉毒素的测定研究[J].中国粮油学报,2011,26(4):107-110.Liu J,Yu DN,Xiong N,et al.Study on aflatoxin in paddy and in whole paddy seed by immunoaffinity column-photochemical derivatization and HPLC[J].J Chin Cer Oils Assoc,2011,26(4):107-110.
[16]GB/T 18979-2003食品中黄曲霉毒素的测定免疫亲和层析净化高效液相色谱法和荧光液相法[S].GB/T 18979-2003 Determination aflatoxins content in food—Cleanup by immunoaffinity chromatography and determination by high-performance liquid chromatigraphy and fluoromete[S].
[17]GOBEL R,LUSKY K.Simultaneous determination of aflatoxins,ochratox in A,and zearalenone in grains by new immunoaffinity column/liquid chromatography[J].J AOAC Int,2004,2:411-415.
[18]GB/T 27404-2008实验室质量控制规范食品理化检测[S].GB/T 27404-2008 Criterion on quality control of laboratorieschemical testing of food[S].
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