全自动免疫亲和在线净化—高效液相色谱法快速测定粮食中赭曲霉毒素A
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摘要
建立了全自动免疫亲和在线净化-高效液相色谱法快速测定粮食中赭曲霉毒素A(Ochtatoxin A,OTA)的方法。玉米、小麦样品经乙腈-水(60:40,V:V)提取,3%Tween-20(m/V)水溶液10倍稀释后,用自动进样器注入RIDA~? REST在线固相萃取系统,经赭曲霉毒素A免疫亲和小柱净化后,以乙腈-2%乙酸水(50:50,V:V)为流动相,流速为1.0 mL/min,C18色谱柱(150 mm×3.5 mm,3.5μm)分离,荧光检测器测定。根据3倍信噪比的峰响应值,确定赭曲霉毒素A的检出限为0.24μg/kg,在0.012 5～0.5μg/L范围内呈线性相关,R~2值为0.999 8;在玉米、小麦样品中加标回收率为80.1%～106.9%,变异系数为2.4%～8.2%。本方法一次装柱可检测60个样品,24 h可检测100个样品,满足谷物中赭曲霉毒素A快速准确定量检测的需要。
A rapid analytical method using the on-line full-automatic immunoaffinity column clean-up and high-performance liquid chromatography for the determination of ochtatoxin A in cereal was developed.Samples were extracted by acetonitrile-water solution(60:40).The extraction solution were diluted 10 times with 3%Tween-20; water solution,then injected into the RIDA~? CREST on-line immunoaffinity column clean-up system,and washed out by acetonitrile:2% acetic acid solution(50:50)as mobile phase at the flow rate of 1.0 mL/min.Ochtatoxin A was analyzed by HPLC-FLD.The detection limit of OTA was 0.24μg/kg.The linear ranges of OTA was 0.012 5 ～0.5μg/kg with R~2 of 0.999 8.The recovery rates in maize and wheat samples spiked with OTA were in the range of 80.1%～106.9%,with the relative standard deviation of 2.4%～8.2%. Each on-line immunoaffinity column could be used for 60 samples.100 samples could be detected per day.This method could satisfy the demands for ochratoxin A in cereal.
A rapid analytical method using the on-line full-automatic immunoaffinity column clean-up and high-performance liquid chromatography for the determination of ochtatoxin A in cereal was developed.Samples were extracted by acetonitrile-water solution(60:40).The extraction solution were diluted 10 times with 3%Tween-20; water solution,then injected into the RIDA~? CREST on-line immunoaffinity column clean-up system,and washed out by acetonitrile:2% acetic acid solution(50:50)as mobile phase at the flow rate of 1.0 mL/min.Ochtatoxin A was analyzed by HPLC-FLD.The detection limit of OTA was 0.24μg/kg.The linear ranges of OTA was 0.012 5 ～0.5μg/kg with R~2 of 0.999 8.The recovery rates in maize and wheat samples spiked with OTA were in the range of 80.1%～106.9%,with the relative standard deviation of 2.4%～8.2%. Each on-line immunoaffinity column could be used for 60 samples.100 samples could be detected per day.This method could satisfy the demands for ochratoxin A in cereal.
引文
[1]Commission Regulation(EC)No 1881/2006,setting maximum levels for certain contaminants in food stuffs[S]
[2]CLARK H A,SNEDEKER S M.Ochratoxin A:its cancer risk and potential for exposure[J]. J Toxicol Environ Health B,2006,9(3):265-296
[3]OLSEN M,JONSSON N,MAGEN N,et al.Prevention of ochratoxin A in cereals in Europe[J]. Adv Food Mycol,2006,571:317-348
[4]GB 2761—2017食品安全国家标准食品中真菌毒素限量[S]GB 2761—2017 National food safety standard Maximum lev-els of mycotoxins in food[S]
[5]赖卫华,许杨,熊勇华,等.赭曲霉毒素A无毒体系胶体金试纸条的研制及与传统胶体金试纸条的比较[J].食品科学,2008,29(9):365-368LAI W H,XU Y,XIONG Y H,et al.Development of colloidal gold strip with Mimotope peptide for rapid detection of ochratoxin A and comparison between it and traditional strip[J].Food Science,2008,29(9):365-368
[6]李鑫,李培武,张奇,等.农产品中赭曲霉毒素A免疫层析试纸条快速检测技术研究[J].中国油料作物学报,2014,36(5):648-652LI X,LI P W,ZHANG Q,et al.Development of an immunochromatographic assay for ochratoxin A in agro-products[J].Chinese Journal of Oil Crop Sciences 2014,36(5):648-652
[7]周耀锋,熊斯诚,江湖,等.量子点荧光微球免疫层析试纸条定量检测玉米中赭曲霉毒素A[J].分析化学,2015,43(12):1837-1843ZHOU Y F,XIONG S C,JIANG H,et al.Immunochromatographic assay for quantitative detection of ochratoxin A in maize by quantum dot beads[J].Chinese Journal of Analytical Chemistry,2015,43(12):1837-1843
[8]ZHENG Z,HANNEKEN J,HOUCHINS D,et al.Validation of an ELISAtest kit for the detection of ochratoxin A in several food commodities by comparison with HPLC[J].Mycopathologia,2005,159:265-272
[9]ZAMFIR LG,GEANA I,BOURIGUA S,et al.Highly sensitive label-freeimmunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection[J].Sensors Actuators B Chem,2011,159(1):178-184
[10]黄飚,陶文沂,张莲芬,等.赭曲霉毒素A的高灵敏时间分辨荧光免疫分析[J].生物化学与生物物理进展,2005,32(7):662-666HUANG B,TAO W Y,ZHANG L F,et al.Ultrasensitive time-resolved fluoroimmunoassay of ochratoxin A[J].Progress in Biochemistry and Biophysics,2005,32(7):662-666
[11]谢刚,王松雪,张艳,等.粮食中赭曲霉毒素A的快速定量检测-超高效液相色谱法[J].食品安全质量检测学报,2014,5(3):783-790XIE G,WANG S X,ZHANG Y,et al.Rapid analysis of ochratoxin A in grain by ultra performance liquid chromatography[J].Journal of Food Safety and Quality,2014,5(3):783-790
[12]梁桂娟,张琼,杨波.高效液相色谱-荧光检测法检测大米中的赭曲霉毒素A[J].中国酿造,2015,34(8):136-LIANG G J,ZHANG Q,YANG B.Determination of ochratoxin A in rice by HPLC-FLD[J].China Brewing,2015,34(8):136-138
[13]辛媛媛,张艳,王松雪,等.UPLC-MS/MS法测定玉米中13种真菌毒素[J].中国粮油学报,2015,30(12):126-130XIN Y Y,ZHANG Y,WANG S X,et al.Determination of13 mycotoxins in corn by UPLC-MS/MS[J].Journal of the Chinese Cereals and Oils Association,2015,30(12):126-130
[14]WILFRED A A,BENEDIKT W,MICHAEL S,et al.Determination of multi-mycotoxin occurrence in cereals,nuts and their products in Cameroon by liquid chromatography tandem mass spectrometry(LC-MS/MS)[J].Food Control,2013,31(2):438-453
[15]JOSEP R,CARLA S,JORDI M.Evaluation of matrix solid-phase dispersion(MSPD)extraction for multi-mycotoxin determination in different flours using LC-MS/MS[J].Talanta,2011,85:206-215
[16]SHARMAN M,MACDONALD S,GILBERT,et al.Automated liquid chromatographic determination of ochratoxin A in cereals and animal products using immunoaffinity column clean-up[J].Journal of Chromatography A,1992,603(1-2):285-289
[17]ESKOLA M,KOKKONEN M,RIZZO A.Application of manual and automated systems for purification of ochratoxin A and zearalenone in cereals with immunoaffinity columns[J].Agric.J Agric Food Chem,2002,50(1):41-47
[18]SENYUVA H Z.Immunoaffinity column clean-up techniques in food analysis:a review[J].Journal of Chromatography B,2010,878(2):115-132
[19]JINAP S,RIJK T,ARZANDEH S.Aflatoxin determination using in-line immunoaffinity chromatography in foods[J].Food Control.2012,26(1):42-48
[20]RIA R,MONIKA P,ELAINE M.Automated aflatoxin analysis using inline reusable immunoaffnity column cleanup and LC-fluorescence detection[J].Journal of AOAC International,2015,98(6):1585-1590
[21]李丽,黎睿,谢刚,等.全自动免疫亲和在线净化-高效液相色谱法快速高通量测定饲料中黄曲霉毒素[J].分析测试学报,2017,36(6):800-804LI L,LI R,XIE G,et al.Rapid and high throughput analysis of aflatoxins in feed matrix using on-line immunoaffinity column clean-up and high Performance liquid chromatography[J].Journal of Instrumental Analysis,2017,36(6):800-804
[22]INTERNATIONAL STANDARD ISO 15141-1-1998.Foodstuffs—Determination of ochratoxin A in cereals andcereal products—Part 1:High performance liquid chromatographic method with silica gel clean up[S]
[23]GHALI R,HMAISSIA K,GHORBEL H,et al.HPLC determination of ochratoxin A in high consumption Tunisian foods[J].Food Control,2009,2018:716-720
[24]GILBERT J,SLACK P T,MANN P J,et al.Liquid chromatographic method with immunoaffinity column cleanup for determination of ochratoxin A in barley:collaborative study[J].Journal of AOAC International,2000,83(6):1377-1383
[25]GB 5009.22-2016食品安全国家标准食品中黄曲霉毒素B族和G族的测定[S]GB 5009.22—2016 National food safety standard Determination of aflatoxins B and G in foods[S]
[26]GB/T 27417-2017合格评定化学分析方法确认和验证指南[S]GB/T 27417—2017 Comformity assessment-Guidance on validation and verification of chemical analytical methods[S]
[27]No 519/2014.Amending Regulation(EC)No 401/2006 as regards methods of sampling of large lots,spices and food supplements,performance criteria for T-2,HT-2 toxin and citrinin and screening methods of analysis[S]
[28]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 laboratories-chemical testing of food[S].
[2]CLARK H A,SNEDEKER S M.Ochratoxin A:its cancer risk and potential for exposure[J]. J Toxicol Environ Health B,2006,9(3):265-296
[3]OLSEN M,JONSSON N,MAGEN N,et al.Prevention of ochratoxin A in cereals in Europe[J]. Adv Food Mycol,2006,571:317-348
[4]GB 2761—2017食品安全国家标准食品中真菌毒素限量[S]GB 2761—2017 National food safety standard Maximum lev-els of mycotoxins in food[S]
[5]赖卫华,许杨,熊勇华,等.赭曲霉毒素A无毒体系胶体金试纸条的研制及与传统胶体金试纸条的比较[J].食品科学,2008,29(9):365-368LAI W H,XU Y,XIONG Y H,et al.Development of colloidal gold strip with Mimotope peptide for rapid detection of ochratoxin A and comparison between it and traditional strip[J].Food Science,2008,29(9):365-368
[6]李鑫,李培武,张奇,等.农产品中赭曲霉毒素A免疫层析试纸条快速检测技术研究[J].中国油料作物学报,2014,36(5):648-652LI X,LI P W,ZHANG Q,et al.Development of an immunochromatographic assay for ochratoxin A in agro-products[J].Chinese Journal of Oil Crop Sciences 2014,36(5):648-652
[7]周耀锋,熊斯诚,江湖,等.量子点荧光微球免疫层析试纸条定量检测玉米中赭曲霉毒素A[J].分析化学,2015,43(12):1837-1843ZHOU Y F,XIONG S C,JIANG H,et al.Immunochromatographic assay for quantitative detection of ochratoxin A in maize by quantum dot beads[J].Chinese Journal of Analytical Chemistry,2015,43(12):1837-1843
[8]ZHENG Z,HANNEKEN J,HOUCHINS D,et al.Validation of an ELISAtest kit for the detection of ochratoxin A in several food commodities by comparison with HPLC[J].Mycopathologia,2005,159:265-272
[9]ZAMFIR LG,GEANA I,BOURIGUA S,et al.Highly sensitive label-freeimmunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection[J].Sensors Actuators B Chem,2011,159(1):178-184
[10]黄飚,陶文沂,张莲芬,等.赭曲霉毒素A的高灵敏时间分辨荧光免疫分析[J].生物化学与生物物理进展,2005,32(7):662-666HUANG B,TAO W Y,ZHANG L F,et al.Ultrasensitive time-resolved fluoroimmunoassay of ochratoxin A[J].Progress in Biochemistry and Biophysics,2005,32(7):662-666
[11]谢刚,王松雪,张艳,等.粮食中赭曲霉毒素A的快速定量检测-超高效液相色谱法[J].食品安全质量检测学报,2014,5(3):783-790XIE G,WANG S X,ZHANG Y,et al.Rapid analysis of ochratoxin A in grain by ultra performance liquid chromatography[J].Journal of Food Safety and Quality,2014,5(3):783-790
[12]梁桂娟,张琼,杨波.高效液相色谱-荧光检测法检测大米中的赭曲霉毒素A[J].中国酿造,2015,34(8):136-LIANG G J,ZHANG Q,YANG B.Determination of ochratoxin A in rice by HPLC-FLD[J].China Brewing,2015,34(8):136-138
[13]辛媛媛,张艳,王松雪,等.UPLC-MS/MS法测定玉米中13种真菌毒素[J].中国粮油学报,2015,30(12):126-130XIN Y Y,ZHANG Y,WANG S X,et al.Determination of13 mycotoxins in corn by UPLC-MS/MS[J].Journal of the Chinese Cereals and Oils Association,2015,30(12):126-130
[14]WILFRED A A,BENEDIKT W,MICHAEL S,et al.Determination of multi-mycotoxin occurrence in cereals,nuts and their products in Cameroon by liquid chromatography tandem mass spectrometry(LC-MS/MS)[J].Food Control,2013,31(2):438-453
[15]JOSEP R,CARLA S,JORDI M.Evaluation of matrix solid-phase dispersion(MSPD)extraction for multi-mycotoxin determination in different flours using LC-MS/MS[J].Talanta,2011,85:206-215
[16]SHARMAN M,MACDONALD S,GILBERT,et al.Automated liquid chromatographic determination of ochratoxin A in cereals and animal products using immunoaffinity column clean-up[J].Journal of Chromatography A,1992,603(1-2):285-289
[17]ESKOLA M,KOKKONEN M,RIZZO A.Application of manual and automated systems for purification of ochratoxin A and zearalenone in cereals with immunoaffinity columns[J].Agric.J Agric Food Chem,2002,50(1):41-47
[18]SENYUVA H Z.Immunoaffinity column clean-up techniques in food analysis:a review[J].Journal of Chromatography B,2010,878(2):115-132
[19]JINAP S,RIJK T,ARZANDEH S.Aflatoxin determination using in-line immunoaffinity chromatography in foods[J].Food Control.2012,26(1):42-48
[20]RIA R,MONIKA P,ELAINE M.Automated aflatoxin analysis using inline reusable immunoaffnity column cleanup and LC-fluorescence detection[J].Journal of AOAC International,2015,98(6):1585-1590
[21]李丽,黎睿,谢刚,等.全自动免疫亲和在线净化-高效液相色谱法快速高通量测定饲料中黄曲霉毒素[J].分析测试学报,2017,36(6):800-804LI L,LI R,XIE G,et al.Rapid and high throughput analysis of aflatoxins in feed matrix using on-line immunoaffinity column clean-up and high Performance liquid chromatography[J].Journal of Instrumental Analysis,2017,36(6):800-804
[22]INTERNATIONAL STANDARD ISO 15141-1-1998.Foodstuffs—Determination of ochratoxin A in cereals andcereal products—Part 1:High performance liquid chromatographic method with silica gel clean up[S]
[23]GHALI R,HMAISSIA K,GHORBEL H,et al.HPLC determination of ochratoxin A in high consumption Tunisian foods[J].Food Control,2009,2018:716-720
[24]GILBERT J,SLACK P T,MANN P J,et al.Liquid chromatographic method with immunoaffinity column cleanup for determination of ochratoxin A in barley:collaborative study[J].Journal of AOAC International,2000,83(6):1377-1383
[25]GB 5009.22-2016食品安全国家标准食品中黄曲霉毒素B族和G族的测定[S]GB 5009.22—2016 National food safety standard Determination of aflatoxins B and G in foods[S]
[26]GB/T 27417-2017合格评定化学分析方法确认和验证指南[S]GB/T 27417—2017 Comformity assessment-Guidance on validation and verification of chemical analytical methods[S]
[27]No 519/2014.Amending Regulation(EC)No 401/2006 as regards methods of sampling of large lots,spices and food supplements,performance criteria for T-2,HT-2 toxin and citrinin and screening methods of analysis[S]
[28]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 laboratories-chemical testing of food[S].