动物营养学报 >

2021 , Vol. 33 >Issue 8: 4684 - 4693

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.08.047

实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

高效液相色谱同步检测饲料中黄曲霉毒素B1、玉米赤霉烯酮及呕吐毒素的方法

  • 刘颖 ,
  • 金永鹏 ,
  • 罗荪琳 ,
  • 陈义强
展开
  • 中国农业大学动物科技学院, 动物营养学国家重点实验室, 北京 100193
刘颖(1998-),男,湖南邵阳人,硕士研究生,动物营养与饲料科学专业。E-mail:1351662076@qq.com

收稿日期: 2021-01-18

  网络出版日期: 2021-08-11

基金资助

食品安全关键技术研发专项（2018YFC1603002）

收起

A Method for Simultaneous Determination of Aflatoxin B1, Zearalenone and Deoxynivalenol in Feed by High Performance Liquid Chromatography

  • LIU Ying ,
  • JIN Yongpeng ,
  • LUO Sunlin ,
  • CHEN Yiqiang
Expand
  • State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

Received date: 2021-01-18

  Online published: 2021-08-11

Supported by

 

Fold

摘要

本研究旨在建立同步检测饲料中黄曲霉毒素B1、玉米赤霉烯酮及呕吐毒素的高效液相色谱（HPLC）法。样品用乙腈-水提取，经涡旋、高速离心，移取2 mL上清液加入28 mL含1%吐温-20的磷酸盐缓冲液（PBS，pH 7.0），过三合一免疫亲和柱净化，用2 mL甲醇洗脱；洗脱液经50℃浓缩氮气吹干后用50%甲醇-水复溶，采用高效液相色谱仪串联光化学衍生器、荧光检测器和紫外检测器进行检测。结果表明：黄曲霉毒素B1标准品溶液浓度在2.5~50.0 ng/mL、玉米赤霉烯酮标准品溶液浓度在50~2 500 ng/mL、呕吐毒素标准品溶液浓度在250~5 000 ng/mL时线性关系良好，平均加标回收率为81.2%~92.3%，相对标准偏差（RSD）为4.3%~9.5%。采用该方法检测了4种不同饲料样品中的黄曲霉毒素B1、玉米赤霉烯酮及呕吐毒素含量，所有样品均检测出1种以上霉菌毒素，说明饲料中霉菌毒素混合污染较为普遍。由此可见，本试验所建立的方法准确度和精密度高，稳定性好，可作为饲料中黄曲霉毒素B1、玉米赤霉烯酮及呕吐毒素的同步检测方法。

关键词： 黄曲霉毒素B1; 玉米赤霉烯酮; 呕吐毒素; 高效液相色谱

本文引用格式

刘颖 , 金永鹏 , 罗荪琳 , 陈义强 . 高效液相色谱同步检测饲料中黄曲霉毒素B1、玉米赤霉烯酮及呕吐毒素的方法[J]. 动物营养学报, 2021 , 33(8) : 4684 -4693 . DOI: 10.3969/j.issn.1006-267x.2021.08.047

Abstract

A high performance liquid chromatographic (HPLC) method was developed for simultaneous determination of aflatoxin B1, zearalenone and deoxynivalenol in feed. Samples were extracted with acetonitrile-water, after vortexing and high-speed centrifugation, 2 mL of supernatant was transferred and mixed with 28 mL phosphate buffer saline (PBS, pH 7.0) containing 1% tween-20. Then the extracted solution was purified with immune-affinity column and eluted with 2 mL of methanol. The eluent was blow-dried by concentrated nitrogen at 50℃ and then redissolved in 50% methanol-water. Finally, the reconstituted solution was detected by a high performance liquid chromatography coupled with a photochemical derivatizer, a fluorescence detector and an ultraviolet detector. The results showed that the calibration curves of aflatoxin B1 were linear in 2.5 to 50.0 ng/mL, the calibration curves of zearalenone were linear in 50 to 2 500 ng/mL, and the calibration curves of deoxynivalenol were linear in 250 to 5 000 ng/mL. The average recovery ratio was ranged from of 81.2% to 92.3%, and the relative standard deviation (RSD) was ranged from 4.3% to 9.5%. The established method was then used to determine the contents of aflatoxin B1, zearalenone and deoxynivalenol in four kinds of feed samples, it was found that all the detected samples contained more than one mycotoxin, implying that the feed samples were widely contaminated with mycotoxins. In conclusion, the established method has high accuracy, precision and good stability, and it can be used as a simultaneous detection method for aflatoxin B1, zearalenone and deoxynivalenol in feed.

Key words： aflatoxin B1; zearalenone; deoxynivalenol; HPLC

参考文献

[1] 许玲涵.饲料中常见霉菌毒素及防控措施[J].畜牧业环境,2020(3):73. XU L H.Common mycotoxins in feed and their prevention and control measures[J].Animal Industry and Environment,2020(3):73.(in Chinese)
[2] 刘冬梅.霉菌毒素的危害及防治[J].吉林畜牧兽医,2018,39(4):8,11. LIU D M.The harm and prevention of mycotoxins[J].Jilin Animal Husbandry and Veterinary Medicine,2018,39(4):8,11.(in Chinese)
[3] DAI Y Q,HUANG K L,ZHANG B Y,et al.Aflatoxin B1-induced epigenetic alterations:an overview[J].Food and Chemical Toxicology,2017,109:683-689.
[4] ZHOU C,ZHANG Y,YIN S,et al.Biochemical changes and oxidative stress induced by zearalenone in the liver of pregnant rats[J].Human & Experimental Toxicology,2015,34(1):65-73.  
[5] LIU X L,WU R Y,SUN X F,et al.Mycotoxin zearalenone exposure impairs genomic stability of swine follicular granulosa cells in vitro[J].International Journal of Biological Sciences,2018,14(3):294-305.  
[6] BARNES J A,DEROUCHEY J M,TOKACH M D,et al.Effects of vomitoxin concentration in nursery pig diets and the effectiveness of commercial products to mitigate its effects[J].Kansas Agricultural Experiment Station Research Reports,2010(10):79-85.
[7] FROBOSE H L,TOKACH M D,SOLTWEDEL K T,et al.The effects of Biomin product A and vomitoxin on growth performance of nursery pigs[J].Kansas Agricultural Experiment Station Research Reports,2010(10):86-94.
[8] 中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.GB 13078-2017饲料卫生标准[S].北京:中国标准出版社,2017. General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China,Standardization Administration of China. GB 13078-2017 Hygienical standard for feeds[S].Beijing:Standards Press of China,2017.(in Chinese)
[9] SOKOLOVIĆ M,ŠIMPRAGA B.Survey of trichothecene mycotoxins in grains and animal feed in Croatia by thin layer chromatography[J].Food Control,2006,17(9):733-740.  
[10] QU L L,JIA Q,LIU C Y,et al.Thin layer chromatography combined with surface-enhanced raman spectroscopy for rapid sensing aflatoxins[J].Journal of Chromatography A,2018,1579:115-120.
[11] VEGA-HERRERA M,MADARIAGA R,ARANDA M,et al.Confirmation of deoxyniv alenol presence in chilean wheat by high-performance thin-layer chromatography-mass spectrometry[J].Journal of the Chilean Chemical Society,2017,62(2):3435-3437.  
[12] 杜兵耀,马晨,余雄.胶体金免疫层析法检测谷物中的黄曲霉毒素B1[J].饲料工业,2016,37(9):58-60. DU B Y,MA C,YU X.Study on aflatoxin B1 detecting in grain by colloidal gold immunochromatographic assay[J].Feed Industry,2016,37(9):58-60.(in Chinese)
[13] 张文静,吴雨豪,熊勇华.饲料及原料中霉菌毒素的免疫层析快速检测技术[J].中国猪业,2017,12(9):74-77. ZHANG W J,WU Y H,XIONG Y H.Rapid immunochromatography detection of mycotoxins in feed and feed materials[J].China Swine Industry,2017,12(9):74-77.(in Chinese)
[14] REITER E,ZENTEK J,RAZZAZI E.Review on sample preparation strategies and methods used for the analysis of aflatoxins in food and feed[J].Molecular Nutrition and Food Research,2009,53(4):508-524.  
[15] LI P W,ZHOU Q,WANG T,et al.Development of an enzyme-linked immunosorbent assay method specific for the detection of G-group aflatoxins[J].Toxins,2016,8(1):5.
[16] 杨艳,吴鑫,朱应飞,等.酶联免疫吸附法测定植物油中黄曲霉毒素B1[J].食品安全质量检测学报,2018,9(12):3166-3170. YANG Y,WU X,ZHU Y F,et al.Detection of aflatoxin B1 in vegetable oils by enzyme-linked immunosorbent assay[J].Journal of Food Safety and Quality,2018,9(12):3166-3170.(in Chinese)
[17] SKENDI A,IRAKLI M N,PAPAGEORGIOU M D.Optimized and validated high-performance liquid chromatography method for the determination of deoxynivalenol and aflatoxins in cereals[J].Journal of Separation Science,2016,39(8):1425-1432.  
[18] LUCI G,INTORRE L,FERRUZZI G,et al.Determination of ochratoxin A in tissues of wild boar (Sus scrofa L.) by enzymatic digestion (ED) coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD)[J].Mycotoxin Research,2018,34(1):1-8.  
[19] 赵贞,万鹏,李翠枝,等.高效液相色谱法测定饲料中黄曲霉毒素含量的研究[J].畜牧与饲料科学,2019,40(3):27-29,49. ZHAO Z,WAN P,LI C Z,et al.Determination of aflatoxin in feed by high-performance liquid chromatography[J].Animal Husbandry and Feed Science,2019,40(3):27-29,49.(in Chinese)
[20] 魏云计,冯民,朱臻怡,等.高效液相色谱-串联质谱法同时测定饲料中11种霉菌毒素[J].色谱,2017,35(8):891-896. WEI Y J,FENG M,ZHU Z Y,et al.Simultaneous determination of 11 mycotoxins in feeds by high performance liquid chromatography-tandem mass spectrometry[J].Chinese Journal of Chromatography,2017,35(8):891-896.(in Chinese)
[21] DE SANTIS B,DEBEGNACH F,GREGORI E,et al.Development of a LC-MS/MS method for the multi-mycotoxin determination in composite cereal-based samples[J].Toxins,2017,9(5):169.
[22] PARK J,KIM D H,MOON J Y,et al.Distribution analysis of twelve mycotoxins in corn and corn-derived products by LC-MS/MS to evaluate the carry-over ratio during wet-milling[J].Toxins,2018,10(8):319.
[23] GELL R M,CARBONE I.HPLC quantitation of aflatoxin B1 from fungal mycelium culture[J].Journal of Microbiological Methods,2019,158:14-17.
[24] KIM H J,LEE M J,KIM H J,et al.Analytical method development and monitoring of aflatoxin B1,B2,G1,G2 and ochratoxin A in animal feed using HPLC with fluorescence detector and photochemical reaction device[J].Cogent Food & Agriculture,2017,3(1):1419788.
[25] 班晓敏.AFB1、OTA、DON高效液相色谱检测方法的优化和应用[D].硕士学位论文.南京:南京农业大学,2012. BAN X M.Optimization and Application of high performance liquid chromatography detection methods for AFB1,OTA,DON[D].Master's Thesis.Nanjing:Nanjing Agricultural University,2012.(in Chinese)
[26] 何苗,李鑫.高效液相色谱法测定浮小麦中的呕吐毒素[J].海峡药学,2020,32(2):36-38. HE M,LI X.Determination of vomitoxin in light wheat by high performance liquid chromatography[J].Strait Pharmaceutical Journal,2020,32(2):36-38.(in Chinese)
[27] LEE M,SEO D J,JEON S B,et al.Detection of foodborne pathogens and mycotoxins in eggs and chicken feeds from farms to retail markets[J].Korean Journal for Food Science of Animal Resources,2016,36(4):463-468.  
[28] CHEN F F,LUAN C L,WANG L,et al.Simultaneous determination of six mycotoxins in peanut by high-performance liquid chromatography with a fluorescence detector[J].Journal of the Science of Food and Agriculture,2017,97(6):1805-1810.  
[29] 张婷,向仲朝.啤酒中玉米赤霉烯酮的高效液相色谱紫外检测法[J].现代预防医学,2016,43(19):3590-3592. ZHANG T,XIANG Z C.Determination of zearalenone in beer by high-performance liquid chromatography with UV detection[J].Modern Preventive Medicine,2016,43(19):3590-3592.(in Chinese)
[30] 董颖超,李俊,李军国,等.免疫亲和柱-高效液相色谱法检测青贮饲料中的呕吐毒素[J].中国饲料,2010(10):38-39. DONG Y C,LI J,LI J G,et al.Determination of deoxynivalenol in silage feed by immunoaffinity column-high performance liquid chromatography[J].China Feed,2010(10):38-39.(in Chinese)
[31] 关嘉成,苗力,卢杰,等.高效液相色谱法用于小麦中脱氧雪腐镰刀菌烯醇检测与去除的研究[J].广州化工,2020,48(17):68-70. GUAN J C,MIAO L,LU J,et al.Study on the detection and removal of deoxynivalenol in wheat by HPLC[J].Guangzhou Chemical Industry,2020,48(17):68-70.(in Chinese)
[32] 王艳红,胡丹丹,潘雪静.高效液相色谱法测定小麦中黄曲霉毒素B1含量[J].山东化工,2019,48(12):74-75,80. WANG Y H,HU D D,PAN X J.Determination of aflatoxin B1 in wheat by high performance liquid chromatography[J].Shandong Chemical Industry,2019,48(12):74-75,80.(in Chinese)
[33] WU J W,ZHAO R H,CHEN B,et al.Determination of zearalenone in barley by high-performance liquid chromatography coupled with evaporative light scattering detection and natural occurrence of zearalenone in functional food[J].Food Chemistry,2011,126(3):1508-1511.  
[34] IRAKLI M N,SKENDI A,PAPAGEORGIOU M D.HPLC-DAD-FLD method for simultaneous determination of mycotoxins in wheat bran[J].Journal of Chromatographic Science,2017,55(7):690-696.  
[35] 李克,潘丽红,罗小虎,等.高效液相法同时检测谷物中玉米赤霉烯酮和赭曲霉毒素A[J].食品研究与开发,2020,41(6):118-123. LI K,PAN L H,LUO X H,et al.Simultaneous determination of ochratoxin A and zearalenone in cereals by HPLC[J].Food Research and Development,2020,41(6):118-123.(in Chinese)
[36] 李劲秋.饲料霉变的原因、危害及预防措施[J].畜禽业,2016(12):26-27. LI J Q.Causes,harms and preventive measures of feed mildew[J].Livestock and Poultry Industry,2016(12):26-27.(in Chinese)
[37] 龚阿琼,陈晖,全明旭,等.2017年我国饲料原料及饲料毒素检测分析[J].中国饲料,2018(5):91-93. GONG A Q,CHEN H,QUAN M X,et al.Analysis of raw materials and feed toxins in 2017[J].China Feed,2018(5):91-93.(in Chinese)
[38] 史海涛,曹志军,李键,等.中国饲料霉菌毒素污染现状及研究进展[J].西南民族大学学报(自然科学版),2019,45(4):354-366. SHI H T,CAO Z J,LI J,et al.Mycotoxin contamination of feed in China:current status and research advances[J].Journal of Southwest Minzu University (Natural Science Edition),2019,45(4):354-366.(in Chinese)
[39] MA R,ZHANG L,LIU M,et al.Individual and combined occurrence of mycotoxins in feed ingredients and complete feeds in China[J].Toxins,2018,10(3):113.
[40] 张瑞星.饲料中霉菌毒素污染状况调查及霉变饲料对肉鸡氧化损伤和免疫功能的影响[D].硕士学位论文.青岛:青岛农业大学,2016. ZHANG R X.Investigation on contamination of mycotoxin in feed and effect of moldy feed on oxidative damage and immune function of broilers[D].Master's Thesis.Qingdao:Qingdao Agricultural University,2016.(in Chinese)
[41] SHAO M Y,LI L,GU Z L,et al.Mycotoxins in commercial dry pet food in China[J].Food Additives & Contaminants Part B:Surveillance,2018,11(4):237-245.  
[42] LEVKUT M,REVAJOVA V,SLAMINKOVA Z,et al.Lymphocyte subpopulations in blood and duodenal epithelium of broilers fed diets contaminated with deoxynivalenol and zearalenone[J].Animal Feed Science and Technology,2011,165(3/4):210-217.
[43] 杨云斌.玉米赤霉烯酮与黄曲霉毒素B1对HepG2细胞的联合毒性研究[J].食品工业科技,2019,40(18):91-96. YANG Y B.Joint toxicity of zearalenone and aflatoxin B1 on HepG2 cells[J].Science and Technology of Food Industry,2019,40(18):91-96.(in Chinese)
[44] SUN L H,LEI M Y,ZHANG N Y,et al.Individual and combined cytotoxic effects of aflatoxin B1,zearalenone,deoxynivalenol and fumonisin B1 on BRL 3A rat liver cells[J].Toxicon,2015,95:6-12.
Options
文章导航

/

〈 〉

    联系我们

  • 地址：北京市海淀区圆明园西路2号
    　　　中国农业大学动科动医大楼153室
  • 邮编：100193
  • 电话：010－62817823
  • 网址：http://www.chinajan.com
  • E-mail：cjan@chinajan.com
京公网安备 11010802041926号    京ICP备17050989号-2