在线免疫亲和固相萃取-高效液相色谱法快速测定食品中赭曲霉毒素A
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摘要
目的建立在线免疫亲和固相萃取-高效液相色谱快速测定食品中赭曲霉毒素A(ochratoxin A,OTA)的分析方法。方法谷物、豆类样品采用乙腈-水(80:20,V:V)提取,葡萄酒类样品采用NaHCO_3/NaCl/水溶液(0.4:15:100,m:m:V)提取,3%Tween-20(m:V)溶液稀释后,直接经RIDA~?CREST ICE在线免疫亲和固相萃取系统净化-高效液相色谱荧光检测OTA含量。以乙酸铵-乙腈为流动相洗脱,流速1.0 mL/min,C_(18)色谱柱(150 mm×4.6mm,5μm)分离测定。结果方法检出限分别为0.020μg/kg(豆类、谷物)和0.050μg/kg(葡萄酒类);在OTA的浓度为12.5~500 ng/L的范围内线性关系良好,相关系数r~2为0.9995;日内加标回收率为87.4%~109.8%,相对标准偏差0.50%~4.80%。结论该方法分析速度快,灵敏度高,重现性好,可满足快速准确测定大批量食品样品中OTA含量的需要。
Objective To establish a analytical method for determination of ochratoxin A in food by on-line automatic immunoaffinity solid-phase extraction with high performance liquid chromatography(HPLC). Methods The samples of grain and legumes were extracted with acetonitrile water(80:20, V:V). The wine samples were extracted with NaHCO_3/NaCl/aqueous solution(0.4:15:100, m:m:V), 3% Tween-20(m:V) solution was diluted, and the OTA content was detected directly by the RIDA @CREST ICE on-line immuno affinity solid phase extraction system purification and high performance liquid chromatography fluorescence. The sample solution was eluted with ammonium acetate and acetonitrile as mobile phase at a flow rate of 1.0 mL/min, separated and determined by C_(18) column(150 mm×4.6 mm, 5 μm). Results The detection limits were 0.020 μg/kg(legumes, grains) and 0.050 μg/kg(wine), and the linear relationship was good in the range of 12.5-500 ng/L of OTA, the correlation coefficient r~2 was 0.9995, the recoveries within the day were 87.4%-109.8%, and the relative standard deviations were 0.50%-4.80%. Conclusion The proposed method is rapid, simple, repeatable and sensitive, which is suitable for high throughput determination of OTA in food.
Objective To establish a analytical method for determination of ochratoxin A in food by on-line automatic immunoaffinity solid-phase extraction with high performance liquid chromatography(HPLC). Methods The samples of grain and legumes were extracted with acetonitrile water(80:20, V:V). The wine samples were extracted with NaHCO_3/NaCl/aqueous solution(0.4:15:100, m:m:V), 3% Tween-20(m:V) solution was diluted, and the OTA content was detected directly by the RIDA @CREST ICE on-line immuno affinity solid phase extraction system purification and high performance liquid chromatography fluorescence. The sample solution was eluted with ammonium acetate and acetonitrile as mobile phase at a flow rate of 1.0 mL/min, separated and determined by C_(18) column(150 mm×4.6 mm, 5 μm). Results The detection limits were 0.020 μg/kg(legumes, grains) and 0.050 μg/kg(wine), and the linear relationship was good in the range of 12.5-500 ng/L of OTA, the correlation coefficient r~2 was 0.9995, the recoveries within the day were 87.4%-109.8%, and the relative standard deviations were 0.50%-4.80%. Conclusion The proposed method is rapid, simple, repeatable and sensitive, which is suitable for high throughput determination of OTA in food.
引文
[1]王玉萍,黄昆仑,梁志宏.微生物对赭曲霉毒素A的生物脱毒机理研究进展[J].农业生物技术学报,2017,25(2):316-323.Wang YP,Huang KL,Liang ZH.Research progress of microbial detoxification mechanism of ochratoxin A[J].J Agric Biotechnol,2017,25(2):316-323.
[2]王峻峻,张红印,杨其亚.降解赭曲霉毒素A菌株的筛选及其鉴定[J].食品科学,2014,35(21):154-158.Wang JJ,Zhang HY,Yang QY.Screening and identification of microorganisms for ochratoxin A degradation[J].Food Sci,2014,35(21):154-158.
[3]胡淑荣,应光耀,胡玉莉,等.赭曲霉毒素A快速检测方法研究进展及其在中药中的应用前景[J].中国中药杂志,2017,42(11):2032-2037.Hu SR,Ying GY,Hu YL,et al.Rapid detection of ochratoxin A and its application prospect in traditional Chinese medicine[J].Chin J Tradit Chin Med,2017,42(11):2032-2037.
[4]谢春梅,王华.葡萄与葡萄酒中赭曲霉毒素A检测方法研究进展[J].酿酒科技,2007,(3):92-96.Xie CM,Wang H.Progress in the detection of ochratoxin A in grape and wine[J].Brew Technol,2007,(3):92-96.
[5]庞世琦,刘青,李志勇,等.葡萄酒中赭曲霉毒素A检测方法优化[J].食品科学,2013,34(24):193-196.Pang SQ,Liu Q,Li ZY,et al.Optimization of ochratoxin A detection methods in wine[J].Food Sci,2013,34(24):193-196.
[6]贺亮,郑文,刘玉峰,等.赭曲霉毒素A研究进展[J].中国草食动物科学,2013,37(5):59-61.He L,Zheng W,Liu YF,et al.Ochratoxin A research progress[J].Chin Herbivor Sci,2013,37(5):59-61.
[7]熊科,支慧伟,王晓玲,等.葡萄酒中赭曲霉毒素A的生物降解研究进展[J].中国食品学报,2015,15(10):170-178.Xiong K,Zhi HW,Wang XL,et al.Progress in biodegradation of ochratoxin A in wine[J].J Chin Inst Food Sci Technol,2015,15(10):170-178.
[8]Ochratoxin A.IARC monographs on the evaluation of carcinogenic risk to humans:Some naturally occurring substances;food items and constituents,heterocyclic aromatic amines and mycotoxins[M].Lyon:In IARC,1993.
[9]胡淑荣,应光耀,胡玉莉,等.赭曲霉毒素A快速检测方法研究进展及其在中药中的应用前景[J].中国中药杂志,2017,42(11):2032-2037.Hu SR,Ying GZ,Hu YL,et al.Ochratoxin A rapid detection method research progress and application prospect in traditional Chinese medicine[J].Chin J Tradit Chin Med,2017,42(11):2032-2037.
[10]刘静,张红英,张祥宏.赭曲霉毒素A致癌机制研究进展[J].武警医学,2017,28(1):95-99.Liu J,Zhang HY,Zhang XH.Progress in the carcinogenesis of ochratoxin A(ochratoxin A)[J].Med J Chin Peop Arm Pol For,2017,28(1):95-99.
[11]Bayman P,Baker JL.Ochratoxins:A global perspective[J].Mycopathologia,2006,162(3):215-223
[12]Battilani P,Pietri A.Ochratoxin A in grapes and wine[J].Eur J Plant Pathol,2002,108(7):639-643.
[13]Fazekas B,Kovács ATM.Aflatoxin and ochratoxin a content of spices in hungary[J].Food Addit Contam,2005,22(9):856-863.
[14]Bayman P,Baker JL,Doster MA,et al.Ochratoxin production by the aspergillus ochraceus group and aspergillus alliaceus[J].Appl Environ Microbiol,2002,68(5):2326-2329.
[15]Shotwell OL.Ochratoxin A:Occurrence as natural contaminant of a corn sample[J].Appl Microbiol,1969,17(5):765.
[16]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-Mycotoxins in food limited[S].
[17]Santos EA,Vargas EA.Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee[J].Food Addit Contam,2002,19(5):447-458.
[18]Thirumala-Devi K,Mayo MA,Gopal R,et al.Occurrence of ochratoxin A in black pepper,coriander,ginger and turmeric in India[J].Food Addit Contam,2001,18(9):830-835.
[19]褚庆华,郭德华,王敏,等.香辛料和藤蔓水果干中赭曲霉毒素A的测定[J].化学试剂,2006,28(10):597-600.Zhu QH,Guo DH,Wang M,et al.Determination on ochratoxin A for spices and dried vine fruits[J].Chem Reagent,2006,28(10):597-600.
[20]傅武胜,邱文倩,郑奎城,等.药食两用类食品中赭曲霉毒素A的高效液相色谱-荧光检测方法[J].食品科学,2011,32(14):301-302.Fu WS,Qiu WQ,Zheng KC,et al.High performance liquid chromatography and fluorescence detection for ochratoxin A in medicine and food stuffs[J].Food Sci,2011,32(14):301-302.
[21]梁桂娟.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):136-138.Liang GJ.Determination of ochratoxin A in rice by high performance liquid chromatography with fluorescence detection[J].China Brew,2015,34(8):136-138.
[22]李玉平.高效液相色谱法测定猪肾脏中赭曲霉毒素A[J].食品研究与开发,2017,38(19):152-155.Li YP.Determination of ochratoxin A in pig kidney by high performance liquid chromatography[J].Food Res Dev,2017,38(19):152-155.
[23]莫瑾,龚强,周慧平,等.高效液相色谱-串联质谱法检测茶叶中的赭曲霉毒素A[J].食品安全质量检测学报,2016,7(1):182-187.Mo J,Gong Q,Zhou HP,et al.High performance liquid chromatography tandem mass spectrometry detection of ochratoxin A in tea[J].J Food Saf Qual,2016,7(1):182-187.
[24]孙月.固相萃取-高效液相色谱串联质谱法检测粮食中赭曲霉毒素A[J].江苏农业学报,2016,32(6):1416-1420.Sun Y.Solid phase extraction and high performance liquid chromatography tandem mass spectrometry for the determination of ochratoxin A[J].Jiangsu J Agric Sci,2016,32(6):1416-1420.
[25]康健.葡萄中赭曲霉毒素A的研究[D].天津:天津科技大学,2009.Kang J.A study of ochratoxin A in grapes[D].Tianjin:Tianjin University of Science and Technology,2009.
[26]孟辉.谷物、猪饲料和牛奶中赭曲霉毒素A的检测方法研究[D].北京:中国农业大学,2013.Mong H.Detection of ochratoxin A in grain,pig feed and milk[D].Beijing:China Agricultural University,2013.
[27]褚庆华,郭德华,王敏,等.谷物和酒类中赭曲霉毒素A的测定[J].中国国境卫生检疫杂志,2006,29(2):109-112,117.Zhu QH,Guo D,Wang M,et al.Determination of ochratoxin A in grain and wine[J].Chin J Front Health Quarant,2006,29(2):109-112,117.
[28]GB/T 27404-2008实验室质量控制规范[S].GB/T 27404-2008 Standard of laboratory quality control[S].
[2]王峻峻,张红印,杨其亚.降解赭曲霉毒素A菌株的筛选及其鉴定[J].食品科学,2014,35(21):154-158.Wang JJ,Zhang HY,Yang QY.Screening and identification of microorganisms for ochratoxin A degradation[J].Food Sci,2014,35(21):154-158.
[3]胡淑荣,应光耀,胡玉莉,等.赭曲霉毒素A快速检测方法研究进展及其在中药中的应用前景[J].中国中药杂志,2017,42(11):2032-2037.Hu SR,Ying GY,Hu YL,et al.Rapid detection of ochratoxin A and its application prospect in traditional Chinese medicine[J].Chin J Tradit Chin Med,2017,42(11):2032-2037.
[4]谢春梅,王华.葡萄与葡萄酒中赭曲霉毒素A检测方法研究进展[J].酿酒科技,2007,(3):92-96.Xie CM,Wang H.Progress in the detection of ochratoxin A in grape and wine[J].Brew Technol,2007,(3):92-96.
[5]庞世琦,刘青,李志勇,等.葡萄酒中赭曲霉毒素A检测方法优化[J].食品科学,2013,34(24):193-196.Pang SQ,Liu Q,Li ZY,et al.Optimization of ochratoxin A detection methods in wine[J].Food Sci,2013,34(24):193-196.
[6]贺亮,郑文,刘玉峰,等.赭曲霉毒素A研究进展[J].中国草食动物科学,2013,37(5):59-61.He L,Zheng W,Liu YF,et al.Ochratoxin A research progress[J].Chin Herbivor Sci,2013,37(5):59-61.
[7]熊科,支慧伟,王晓玲,等.葡萄酒中赭曲霉毒素A的生物降解研究进展[J].中国食品学报,2015,15(10):170-178.Xiong K,Zhi HW,Wang XL,et al.Progress in biodegradation of ochratoxin A in wine[J].J Chin Inst Food Sci Technol,2015,15(10):170-178.
[8]Ochratoxin A.IARC monographs on the evaluation of carcinogenic risk to humans:Some naturally occurring substances;food items and constituents,heterocyclic aromatic amines and mycotoxins[M].Lyon:In IARC,1993.
[9]胡淑荣,应光耀,胡玉莉,等.赭曲霉毒素A快速检测方法研究进展及其在中药中的应用前景[J].中国中药杂志,2017,42(11):2032-2037.Hu SR,Ying GZ,Hu YL,et al.Ochratoxin A rapid detection method research progress and application prospect in traditional Chinese medicine[J].Chin J Tradit Chin Med,2017,42(11):2032-2037.
[10]刘静,张红英,张祥宏.赭曲霉毒素A致癌机制研究进展[J].武警医学,2017,28(1):95-99.Liu J,Zhang HY,Zhang XH.Progress in the carcinogenesis of ochratoxin A(ochratoxin A)[J].Med J Chin Peop Arm Pol For,2017,28(1):95-99.
[11]Bayman P,Baker JL.Ochratoxins:A global perspective[J].Mycopathologia,2006,162(3):215-223
[12]Battilani P,Pietri A.Ochratoxin A in grapes and wine[J].Eur J Plant Pathol,2002,108(7):639-643.
[13]Fazekas B,Kovács ATM.Aflatoxin and ochratoxin a content of spices in hungary[J].Food Addit Contam,2005,22(9):856-863.
[14]Bayman P,Baker JL,Doster MA,et al.Ochratoxin production by the aspergillus ochraceus group and aspergillus alliaceus[J].Appl Environ Microbiol,2002,68(5):2326-2329.
[15]Shotwell OL.Ochratoxin A:Occurrence as natural contaminant of a corn sample[J].Appl Microbiol,1969,17(5):765.
[16]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-Mycotoxins in food limited[S].
[17]Santos EA,Vargas EA.Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee[J].Food Addit Contam,2002,19(5):447-458.
[18]Thirumala-Devi K,Mayo MA,Gopal R,et al.Occurrence of ochratoxin A in black pepper,coriander,ginger and turmeric in India[J].Food Addit Contam,2001,18(9):830-835.
[19]褚庆华,郭德华,王敏,等.香辛料和藤蔓水果干中赭曲霉毒素A的测定[J].化学试剂,2006,28(10):597-600.Zhu QH,Guo DH,Wang M,et al.Determination on ochratoxin A for spices and dried vine fruits[J].Chem Reagent,2006,28(10):597-600.
[20]傅武胜,邱文倩,郑奎城,等.药食两用类食品中赭曲霉毒素A的高效液相色谱-荧光检测方法[J].食品科学,2011,32(14):301-302.Fu WS,Qiu WQ,Zheng KC,et al.High performance liquid chromatography and fluorescence detection for ochratoxin A in medicine and food stuffs[J].Food Sci,2011,32(14):301-302.
[21]梁桂娟.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):136-138.Liang GJ.Determination of ochratoxin A in rice by high performance liquid chromatography with fluorescence detection[J].China Brew,2015,34(8):136-138.
[22]李玉平.高效液相色谱法测定猪肾脏中赭曲霉毒素A[J].食品研究与开发,2017,38(19):152-155.Li YP.Determination of ochratoxin A in pig kidney by high performance liquid chromatography[J].Food Res Dev,2017,38(19):152-155.
[23]莫瑾,龚强,周慧平,等.高效液相色谱-串联质谱法检测茶叶中的赭曲霉毒素A[J].食品安全质量检测学报,2016,7(1):182-187.Mo J,Gong Q,Zhou HP,et al.High performance liquid chromatography tandem mass spectrometry detection of ochratoxin A in tea[J].J Food Saf Qual,2016,7(1):182-187.
[24]孙月.固相萃取-高效液相色谱串联质谱法检测粮食中赭曲霉毒素A[J].江苏农业学报,2016,32(6):1416-1420.Sun Y.Solid phase extraction and high performance liquid chromatography tandem mass spectrometry for the determination of ochratoxin A[J].Jiangsu J Agric Sci,2016,32(6):1416-1420.
[25]康健.葡萄中赭曲霉毒素A的研究[D].天津:天津科技大学,2009.Kang J.A study of ochratoxin A in grapes[D].Tianjin:Tianjin University of Science and Technology,2009.
[26]孟辉.谷物、猪饲料和牛奶中赭曲霉毒素A的检测方法研究[D].北京:中国农业大学,2013.Mong H.Detection of ochratoxin A in grain,pig feed and milk[D].Beijing:China Agricultural University,2013.
[27]褚庆华,郭德华,王敏,等.谷物和酒类中赭曲霉毒素A的测定[J].中国国境卫生检疫杂志,2006,29(2):109-112,117.Zhu QH,Guo D,Wang M,et al.Determination of ochratoxin A in grain and wine[J].Chin J Front Health Quarant,2006,29(2):109-112,117.
[28]GB/T 27404-2008实验室质量控制规范[S].GB/T 27404-2008 Standard of laboratory quality control[S].