高效液相色谱-串联质谱法测定杂粮豆类中11种真菌毒素
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摘要
利用高效液相色谱-串联质谱建立杂粮豆类中11种真菌毒素(包括黄曲霉毒素B_1、B_2、G_1、G_2、赭曲霉毒素A、T-2毒素、脱氧雪腐镰刀菌烯醇、伏马毒素B1、B2和B3、玉米赤霉烯酮)同时检测的方法。实验对提取溶剂、提取时间和净化方式等进行优化,建立双体系联合提取方法,首先采用甲醇-水(70∶30,V/V)体系提取,再以乙腈-水(84∶16,V/V)体系二次提取,提取时间3 min,在此基础上采用C18吸附剂进行净化处理。结果表明,11种毒素的线性范围良好,相关系数大于0.999,添加回收率为70.0%～108%,相对标准偏差为1.3%～8.6%。所建方法简单、快速、灵敏度高、重复性好,可满足杂粮豆类中11种真菌毒素的同时检测需要。
A method was developed for the simultaneous determination of 11 mycotoxins in minor food legumes, including a?atoxins B_1, B_2, G_1, G_2, ochratoxins A, T-2, deoxynivalenol, fumonisins B1, B2 and B3 and zearalenone. Sample extraction and purification conditions were optimized. Samples were extracted with methanol-water(70:30, V/V) and acetonitrilewater(84:16, V/V) consecutively(the extraction process took 3 min), and then the extract was puri?ed using C18 sorbent.The results showed that the calibration curves for all mycotoxins were linear with correlation coef?cients above 0.999. The average recoveries of spiked samples ranged from 70.0% to 108%, with relative standard deviations(RSDs) from 1.3%to 8.6%. The method was simple and rapid with high sensitivity and repeatability, and it was suitable for simultaneous detection of 11 mycotoxins in minor food legumes.
A method was developed for the simultaneous determination of 11 mycotoxins in minor food legumes, including a?atoxins B_1, B_2, G_1, G_2, ochratoxins A, T-2, deoxynivalenol, fumonisins B1, B2 and B3 and zearalenone. Sample extraction and purification conditions were optimized. Samples were extracted with methanol-water(70:30, V/V) and acetonitrilewater(84:16, V/V) consecutively(the extraction process took 3 min), and then the extract was puri?ed using C18 sorbent.The results showed that the calibration curves for all mycotoxins were linear with correlation coef?cients above 0.999. The average recoveries of spiked samples ranged from 70.0% to 108%, with relative standard deviations(RSDs) from 1.3%to 8.6%. The method was simple and rapid with high sensitivity and repeatability, and it was suitable for simultaneous detection of 11 mycotoxins in minor food legumes.
引文
[1]ROCHA M E B,FREIRE F C O,MAIA F E F,et al.Mycotoxins and their effects on human and animal health[J].Food Control,2014,36:159-165.DOI:10.1016/j.foodcont.2013.08.021.
[2]郑翠梅,张艳,王雪松,等.液相色谱-质谱联用同时检测粮食中多种真菌毒素的应用进展[J].粮食科技与经济,2012,37(1):45-49.DOI:10.16465/j.gste.2012.01.001.
[3]FERRE F S.Worldwide occurrence of mycotoxins in rice[J].Food Control,2016,62:291-298.DOI:/10.1016/j.foodcont.2015.10.051.
[4]李志霞,聂继云,闫震,等.果品主要真菌毒素污染检测、风险评估与控制研究进展[J].中国农业科学,2017,50(2):332-347.DOI:10.3864/j.issn.0578-1752.2017.02.012.
[5]MARIN S,RAMOS A J,CANO-SANCHO G,et al.Mycotoxins:occurrence,toxicology and exposure assessment[J].Food and Chemical Toxicology,2013,60:218-237.DOI:10.1016/j.fct.2013.07.047.
[6]HUSSEIN H S,BRASEL J M.Toxicity,metabolism,and impact of mycotoxins on humans and animals[J].Toxicology,2001,167(2):101-134.DOI:10.1016/S0300-483X(01)00471-1.
[7]谭斌,翟小童.杂豆质量安全问题及对策[J].食品科学技术学报,2016,34(6):7-11.DOI:10.3969/j.issn.2095-6002.2016.06.002.
[8]刘尚文.在农业结构调整中提升杂粮豆类地位[J].粮食加工,2004(3):3-5.DOI:10.3969/j.issn.1007-6395.2004.03.001.
[9]杨辉,李绥峰,张巍.小杂粮主要病虫害发生规律及防治对策[J].现代园艺,2010(5):49-50.DOI:10.14051/j.cnki.xdyy.2010.05.003.
[10]SCOTT P M,WALBEEK W,KENNEDY B,et al.Mycotoxins(ochratoxin A,citrinin,and sterigmatocystin)and toxigenic fungi in grains and other agricultual products[J].Journal of Agricultural and Food Chemistry,1972,20(6):1103-1109.DOI:10.1021/jf60184a010.
[11]JIN P G,HAN Z,CAI Z X,et al.Simultaneous determination of 10mycotoxins in grain by ultra-high-performance liquid chromatographytandem mass spectrometry using 13C15-deoxynivalenol as internal standard[J].Food Additives and Contaminants,2010,27(12):1701-1713.DOI:10.1080/19440049.2010.517222.
[12]马皎洁,邵兵,林肖惠,等.我国部分地区2010年产谷物及其制品中多组分真菌毒素污染状况研究[J].中国食品卫生杂志,2011,23(6):481-488.DOI:10.13590/j.cjfh.2011.06.023.
[13]曹娅,孙利,王明林,等.粮谷中8种痕量真菌毒素的定量分析方法[J].分析测试学报,2013,32(2):150-155.DOI:10.3969/j.issn.1004-4957.2013.02.002.
[14]陈慧菲,朱天仪,陈凤香,等.QuEChERS-超高效液相色谱串联质谱法测定谷物中的8种真菌毒素[J].粮食与油脂,2016,29(5):67-70.DOI:10.3969/j.issn.1008-9578.2016.05.019.
[15]OVIEDO M S,RAMIREZ M L,BARROS G G,et al.Influence of water activity and temperature on growth and mycotoxin production by Alternaria alternata on irradiated soya beans[J].International Journal of Food Microbiology,2011,149(2):127-132.DOI:10.1016/j.ijfoodmicro.2011.06.007.
[16]杜英秋,张瑞英,杨焕春,等.国内外真菌毒素检测标准现状及分析[J].粮食与油脂,2014,27(7):60-66.DOI:10.3969/j.issn.1008-9578.2014.07.017.
[17]张亮.T-2毒素与黄曲霉毒素特异性抗体的制备及应用[D].北京:中国农业科学院,2015:1-4.
[18]CASTILLO M D,GONZáLEZ H H,MARTíNEZ E J,et al.Mycoflora and potential for mycotoxin production of freshly harvested black bean from the Argentinean main production area[J].Mycopathologia,2004,158(1):107-112.DOI:10.1023/B:MYCO.0000038426.05215.89.
[19]TSENG T C,TU J C,TZEAN S S.Mycoflora and mycotoxins in dry bean(Phaseolus vulgaris)produced in Taiwan and in Ontario,Canada[J].Botanical Bulletin of Academia Sinica,1995,36(4):229-234.
[20]TSENG T C,TU J C.Mycoflora and mycotoxins in adzuki and mung beans produced in Ontario,Canada[J].Microbios,1997,90:87-95.
[21]IHA M H,TRUCKSESS M W,TOURNAS V H.Effect of processing on ochratoxin A content in dried beans[J].Food Additives&Contaminants:Part A,2009,26:1389-1395.DOI:10.1080/02652030903013286.
[22]马俊,邱富娜,刘金娥,等.ELISA和TLC检测饲料中AFB1含量的比较研究[J].畜牧与饲料科学,2013,34(2):19-23.DOI:10.16003/j.cnki.issn1672-5190.2013.02.023.
[23]KINANI S,BOUCHONNET S,BOURCIER S,et al.Study of the chemical derivatization of zearalenone and it metabolites for gas chromatography-mass spectrometry analysis of environmental samples[J].Journal of Chromatography A,2008,1190(1/2):307-315.DOI:10.1002/rcm.3771.
[24]AMELIN V G,KARASEVA N M,TRETYAKOV A V.Combination of the QuEChERS method with dispersive liquid-liquid microextraction and derivatization in the determination of mycotoxins ingrain and mixed feed by gas-liquid chromatography with an electroncapture detector[J].Journal of Analytical Chemistry,2013,68(6):552-557.DOI:10.1134/S1061934813060026.
[25]PEI S C,LEE W J,ZHANG G P,et al.Development of antizearalenone monoclonal antibody and detection of zearalenone in corn products from China by ELISA[J].Food Control,2013,31:65-70.DOI:10.1016/j.foodcont.2012.09.006.
[26]BOWERS E,HELLMICH R,MUNKVOLD G.Comparison of fumonisin contamination using HPLC and ELISA methods in bt and near-isogenic maize hybrids infested with European corn borer or western bean cutworm[J].Journal of Agriculture and Food Chemistry,2014,62:6463-6472.DOI:10.1021/jf5011897.
[27]黎睿,谢刚,王松雪.高效液相色谱法同时检测粮食中常见8种真菌毒素的含量[J].食品科学,2015,36(6):206-210.DON:10.7506/spkx1002-6630-201506039.
[28]JUAN C,COVARELLI L,BECCARI G,et al.Simultaneous analysis of twenty-six mycotoxins in durum wheat grain from Italy[J].Food Control,2016,62:322-329.DOI:10.1016/j.foodcont.2015.10.032.
[29]DESMARCHELIER A,TESSIOT S,BESSAIRE T,et al.Combining the quick,easy,cheap,effective,rugged and safe approach and cleanup by immunoaffinity column for the analysis of 15 mycotoxins by isotope dilution liquid chromatography tandem mass spectrometry[J].Journal of Chromatography A,2014,1337:75-84.DOI:10.1016/j.chroma.2014.02.025.
[30]BERTHILLER F,BRERA C,IHA M H,et al.Developments in mycotoxin analysis:an update for 2015-2016[J].World Mycotoxin Journal,2017,10(1):5-29.DOI:10.3920/WMJ2016.2138.
[31]国家卫生和计划生育委员会.食品中黄曲霉毒素B组和G组的测定:GB 5009.22-2016[S].北京:中国标准出版社,2016.
[32]国家卫生和计划生育委员会.食品中赭曲霉毒素A的测定:GB5009.96-2016[S].北京:中国标准出版社,2016.
[33]国家卫生和计划生育委员会.食品中脱氧雪腐镰刀菌烯醇及其乙酰化衍生物的测定:GB 5009.111-2016[S].北京:中国标准出版社,2016.
[34]国家卫生和计划生育委员会.食品中玉米赤霉烯酮的测定:GB5009.209-2016[S].北京:中国标准出版社,2016.
[35]高蓓,姜德铭,杨永坛.同位素内标-液相色谱-串联质谱法同时测定粮食及其制品中的5种真菌毒素[J].色谱,2017,35(6):601-607.DOI:10.3724/SP.J.1123.2016.12028.
[36]刘丹,韩小敏,李凤琴,等.花生油和玉米油中多组分真菌毒素高效液相色谱-串联质谱检测方法的建立[J].食品科学,2017,38(10):297-304.DOI:10.7506/spkx1002-6630-201710048.
[37]SUN J,LI W X,ZHANG Y.QuEChERS purification combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for simultaneous quantification of 25 mycotoxins in cereals[J].Toxins,2016,8(12):375.DOI:10.3390/toxins8120375.
[38]刘青,邹志飞,余炀炀,等.食品中真菌毒素法规限量标准概述[J].中国酿造,2017,36(1):12-18.DOI:10.11882/j.issn.0254-5071.2017.01.003.
[2]郑翠梅,张艳,王雪松,等.液相色谱-质谱联用同时检测粮食中多种真菌毒素的应用进展[J].粮食科技与经济,2012,37(1):45-49.DOI:10.16465/j.gste.2012.01.001.
[3]FERRE F S.Worldwide occurrence of mycotoxins in rice[J].Food Control,2016,62:291-298.DOI:/10.1016/j.foodcont.2015.10.051.
[4]李志霞,聂继云,闫震,等.果品主要真菌毒素污染检测、风险评估与控制研究进展[J].中国农业科学,2017,50(2):332-347.DOI:10.3864/j.issn.0578-1752.2017.02.012.
[5]MARIN S,RAMOS A J,CANO-SANCHO G,et al.Mycotoxins:occurrence,toxicology and exposure assessment[J].Food and Chemical Toxicology,2013,60:218-237.DOI:10.1016/j.fct.2013.07.047.
[6]HUSSEIN H S,BRASEL J M.Toxicity,metabolism,and impact of mycotoxins on humans and animals[J].Toxicology,2001,167(2):101-134.DOI:10.1016/S0300-483X(01)00471-1.
[7]谭斌,翟小童.杂豆质量安全问题及对策[J].食品科学技术学报,2016,34(6):7-11.DOI:10.3969/j.issn.2095-6002.2016.06.002.
[8]刘尚文.在农业结构调整中提升杂粮豆类地位[J].粮食加工,2004(3):3-5.DOI:10.3969/j.issn.1007-6395.2004.03.001.
[9]杨辉,李绥峰,张巍.小杂粮主要病虫害发生规律及防治对策[J].现代园艺,2010(5):49-50.DOI:10.14051/j.cnki.xdyy.2010.05.003.
[10]SCOTT P M,WALBEEK W,KENNEDY B,et al.Mycotoxins(ochratoxin A,citrinin,and sterigmatocystin)and toxigenic fungi in grains and other agricultual products[J].Journal of Agricultural and Food Chemistry,1972,20(6):1103-1109.DOI:10.1021/jf60184a010.
[11]JIN P G,HAN Z,CAI Z X,et al.Simultaneous determination of 10mycotoxins in grain by ultra-high-performance liquid chromatographytandem mass spectrometry using 13C15-deoxynivalenol as internal standard[J].Food Additives and Contaminants,2010,27(12):1701-1713.DOI:10.1080/19440049.2010.517222.
[12]马皎洁,邵兵,林肖惠,等.我国部分地区2010年产谷物及其制品中多组分真菌毒素污染状况研究[J].中国食品卫生杂志,2011,23(6):481-488.DOI:10.13590/j.cjfh.2011.06.023.
[13]曹娅,孙利,王明林,等.粮谷中8种痕量真菌毒素的定量分析方法[J].分析测试学报,2013,32(2):150-155.DOI:10.3969/j.issn.1004-4957.2013.02.002.
[14]陈慧菲,朱天仪,陈凤香,等.QuEChERS-超高效液相色谱串联质谱法测定谷物中的8种真菌毒素[J].粮食与油脂,2016,29(5):67-70.DOI:10.3969/j.issn.1008-9578.2016.05.019.
[15]OVIEDO M S,RAMIREZ M L,BARROS G G,et al.Influence of water activity and temperature on growth and mycotoxin production by Alternaria alternata on irradiated soya beans[J].International Journal of Food Microbiology,2011,149(2):127-132.DOI:10.1016/j.ijfoodmicro.2011.06.007.
[16]杜英秋,张瑞英,杨焕春,等.国内外真菌毒素检测标准现状及分析[J].粮食与油脂,2014,27(7):60-66.DOI:10.3969/j.issn.1008-9578.2014.07.017.
[17]张亮.T-2毒素与黄曲霉毒素特异性抗体的制备及应用[D].北京:中国农业科学院,2015:1-4.
[18]CASTILLO M D,GONZáLEZ H H,MARTíNEZ E J,et al.Mycoflora and potential for mycotoxin production of freshly harvested black bean from the Argentinean main production area[J].Mycopathologia,2004,158(1):107-112.DOI:10.1023/B:MYCO.0000038426.05215.89.
[19]TSENG T C,TU J C,TZEAN S S.Mycoflora and mycotoxins in dry bean(Phaseolus vulgaris)produced in Taiwan and in Ontario,Canada[J].Botanical Bulletin of Academia Sinica,1995,36(4):229-234.
[20]TSENG T C,TU J C.Mycoflora and mycotoxins in adzuki and mung beans produced in Ontario,Canada[J].Microbios,1997,90:87-95.
[21]IHA M H,TRUCKSESS M W,TOURNAS V H.Effect of processing on ochratoxin A content in dried beans[J].Food Additives&Contaminants:Part A,2009,26:1389-1395.DOI:10.1080/02652030903013286.
[22]马俊,邱富娜,刘金娥,等.ELISA和TLC检测饲料中AFB1含量的比较研究[J].畜牧与饲料科学,2013,34(2):19-23.DOI:10.16003/j.cnki.issn1672-5190.2013.02.023.
[23]KINANI S,BOUCHONNET S,BOURCIER S,et al.Study of the chemical derivatization of zearalenone and it metabolites for gas chromatography-mass spectrometry analysis of environmental samples[J].Journal of Chromatography A,2008,1190(1/2):307-315.DOI:10.1002/rcm.3771.
[24]AMELIN V G,KARASEVA N M,TRETYAKOV A V.Combination of the QuEChERS method with dispersive liquid-liquid microextraction and derivatization in the determination of mycotoxins ingrain and mixed feed by gas-liquid chromatography with an electroncapture detector[J].Journal of Analytical Chemistry,2013,68(6):552-557.DOI:10.1134/S1061934813060026.
[25]PEI S C,LEE W J,ZHANG G P,et al.Development of antizearalenone monoclonal antibody and detection of zearalenone in corn products from China by ELISA[J].Food Control,2013,31:65-70.DOI:10.1016/j.foodcont.2012.09.006.
[26]BOWERS E,HELLMICH R,MUNKVOLD G.Comparison of fumonisin contamination using HPLC and ELISA methods in bt and near-isogenic maize hybrids infested with European corn borer or western bean cutworm[J].Journal of Agriculture and Food Chemistry,2014,62:6463-6472.DOI:10.1021/jf5011897.
[27]黎睿,谢刚,王松雪.高效液相色谱法同时检测粮食中常见8种真菌毒素的含量[J].食品科学,2015,36(6):206-210.DON:10.7506/spkx1002-6630-201506039.
[28]JUAN C,COVARELLI L,BECCARI G,et al.Simultaneous analysis of twenty-six mycotoxins in durum wheat grain from Italy[J].Food Control,2016,62:322-329.DOI:10.1016/j.foodcont.2015.10.032.
[29]DESMARCHELIER A,TESSIOT S,BESSAIRE T,et al.Combining the quick,easy,cheap,effective,rugged and safe approach and cleanup by immunoaffinity column for the analysis of 15 mycotoxins by isotope dilution liquid chromatography tandem mass spectrometry[J].Journal of Chromatography A,2014,1337:75-84.DOI:10.1016/j.chroma.2014.02.025.
[30]BERTHILLER F,BRERA C,IHA M H,et al.Developments in mycotoxin analysis:an update for 2015-2016[J].World Mycotoxin Journal,2017,10(1):5-29.DOI:10.3920/WMJ2016.2138.
[31]国家卫生和计划生育委员会.食品中黄曲霉毒素B组和G组的测定:GB 5009.22-2016[S].北京:中国标准出版社,2016.
[32]国家卫生和计划生育委员会.食品中赭曲霉毒素A的测定:GB5009.96-2016[S].北京:中国标准出版社,2016.
[33]国家卫生和计划生育委员会.食品中脱氧雪腐镰刀菌烯醇及其乙酰化衍生物的测定:GB 5009.111-2016[S].北京:中国标准出版社,2016.
[34]国家卫生和计划生育委员会.食品中玉米赤霉烯酮的测定:GB5009.209-2016[S].北京:中国标准出版社,2016.
[35]高蓓,姜德铭,杨永坛.同位素内标-液相色谱-串联质谱法同时测定粮食及其制品中的5种真菌毒素[J].色谱,2017,35(6):601-607.DOI:10.3724/SP.J.1123.2016.12028.
[36]刘丹,韩小敏,李凤琴,等.花生油和玉米油中多组分真菌毒素高效液相色谱-串联质谱检测方法的建立[J].食品科学,2017,38(10):297-304.DOI:10.7506/spkx1002-6630-201710048.
[37]SUN J,LI W X,ZHANG Y.QuEChERS purification combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for simultaneous quantification of 25 mycotoxins in cereals[J].Toxins,2016,8(12):375.DOI:10.3390/toxins8120375.
[38]刘青,邹志飞,余炀炀,等.食品中真菌毒素法规限量标准概述[J].中国酿造,2017,36(1):12-18.DOI:10.11882/j.issn.0254-5071.2017.01.003.