免疫亲和柱净化-高效液相色谱柱后光化学衍生-荧光检测器检测食品中黄曲霉毒素
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摘要
目的建立一种免疫亲和固相萃取柱净化-高效液相色谱柱后光化学衍生-荧光检测器同时测定食品中黄曲霉毒素B_1、B_2、G_1和G_2的方法。方法以甲醇-水(70:30,V:V)为提取溶剂,采用高速均质提取,并经过黄曲霉毒素免疫亲和柱净化。结果经月旭公司Welch Ultimate~XB-C_(18)色谱柱(250 mm×4.6 mm,5μm)分离后使用光化学衍生器进行柱后衍生,并采用带荧光检测器的高效液相色谱仪检测,流动相为甲醇/水(45:55,V:V)。黄曲霉毒素B_1、B_2、G_1和G_2线性范围在0.3~50.0μg/L之间,线性相关系数均大于0.999,B_1、B_2、G_1和G_2检出限分别为0.15μg/kg、0.05μg/kg、0.15μg/kg、0.05μg/kg。在3个加标浓度下大米、花生和瓜子等试样的回收率在80.7%~92.6%之间;相对标准偏差(RSD)在2.04~3.87%之间。结论该方法的灵敏度、准确度和精密度均符合黄曲霉毒素的检测技术要求,且简便快速,适用于食品中黄曲霉毒素B_1,B_2,G_1和G_2的准确测定。
Objective To establish an immunoaffinity chromatography-high performance liquid chromatography(HPLC) fluorescence detector coupled with post-column photochemical derivatization method for determination of the aflatoxin B_1, B_2, G_1, and G_2 in food.Methods The sample was tested by HPLC with fluorescence detector after being homogenizer extracted from methanol-water(70:30, V:V), purified by immunoaffinity chromatography column of aggregated aflatoxin, separated by Welch Ultimate~XB-C_(18) HPLC column, eluted with a mobile phase consisting of methanol and waters(45:55, V:V) and derived from post column photochemical derivative reactor.The content of aflatoxin B_1, B_2, G_1, and G_2 in food was 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 within linear range 0.3~50.0 μg/L.The limits of detection(LODs) were 0.15, 0.05, 0.15 and 0.05 μg/kg for the aflatoxins B_1, B_2, G_1, and G_2 in food, respectively.The recovery rate was within 80.7 %~92.6 % at 3 adding levels with the relative standard deviation(RSDs) of 2.04~3.87%.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 B_1, B_2, G_1, and G_2 in food.
Objective To establish an immunoaffinity chromatography-high performance liquid chromatography(HPLC) fluorescence detector coupled with post-column photochemical derivatization method for determination of the aflatoxin B_1, B_2, G_1, and G_2 in food.Methods The sample was tested by HPLC with fluorescence detector after being homogenizer extracted from methanol-water(70:30, V:V), purified by immunoaffinity chromatography column of aggregated aflatoxin, separated by Welch Ultimate~XB-C_(18) HPLC column, eluted with a mobile phase consisting of methanol and waters(45:55, V:V) and derived from post column photochemical derivative reactor.The content of aflatoxin B_1, B_2, G_1, and G_2 in food was 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 within linear range 0.3~50.0 μg/L.The limits of detection(LODs) were 0.15, 0.05, 0.15 and 0.05 μg/kg for the aflatoxins B_1, B_2, G_1, and G_2 in food, respectively.The recovery rate was within 80.7 %~92.6 % at 3 adding levels with the relative standard deviation(RSDs) of 2.04~3.87%.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 B_1, B_2, G_1, and G_2 in food.
引文
[1]张春艳,周孝治,陈菊芳,等.光化学衍生-高效液相色谱法测定黄曲霉毒素含量[J].湖南农业大学学报(自然科学版),2010,36(5):569?573.Zhang CY,Zhou XZ,Chen JF,et al.Determination of aflatoxins by high performance liquid chromatography with post-column photochemical derivatization[J].JHunan Agric Univer,2010,36(5):569?573.
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[3]陈新,王雄,雷达.免疫亲和-光化学柱后衍生高效液相色谱荧光法测定花生粕中黄曲霉毒素的研究[J].中国饲料,2007,24:30?34.Chen X,Wang X,Lei D.Determination of aflatoxin B1,B2,G1and G2 by using photochemical derivatization-HPLCfluorescence detector[J].Chin Feed,2007,24:30?34.
[4]Hasan A,Abdurrahman A,Sahan S.Determination of aflatoxin levels in some dairy and food products which consumed in ankara[J].Food Control,2005,16(3):263?266.
[5]计成,赵丽红.黄曲霉毒素生物降解的研究及前景展望[J].动物营养学报,2010,22(2):241?245.Ji C,Zhao LH.Research and prospects of aflatoxin biological detoxification[J].Chin J Animal Nutrition,2010,22(2):241?245.
[6]Lin LM,Zhang J,Wang P.Thin-layer chromatography of mycotoxins and comparison with other chromatography methods[J].J Chromatogr A,1998,815(1):3?20.
[7]Guan D,Li P,Zhang W,et al.An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products[J].Food Chem,2011,125(4):1359?1364.
[8]谢体波,刘红,陆苇,等.间接竞争ELISA检测试剂盒测定粮油食品中的黄曲霉毒素B1[J].食品安全质量检测学报,2015,6(7):2834?2839.Xie TB,Liu H,Lu W,et al.Determination of aflatoxin B1 in cooking oil,peanut,and grain by using directcompetitive ELISA kit[J].J Food Saf Qual,2015,6(7):2834?2839.
[9]李平,谢体波,易重任,等.黄曲霉毒素M1 ELISA试剂盒的检测效果研究[J].食品安全质量检测学报,2015,6(6):2297?2302.Li P,Xie TB,Yi ZR,et al.Research on detection result of aflatoxin M1 enzyme-linked immune sorbent assay detection kit[J].J Food Saf Qual,2015,6(6):2297?2302.
[10]朱鹏飞,刘文卫,凌霞,等.柱前衍生高效液相色谱荧光法测定粮谷类食物中黄曲霉毒素B1、B2、G1、G2[J].中国卫生检验杂志,2015,25(6):807?815.Zhu PF,Liu WW,Ling X,et al.Determination of aflatoxin B1,B2,G1,G2 in cereal food by high performance liquid chromatography with fluorescence detector and pre-column derivatization[J].Chin J Health Lab Tec,2015,25(6):807?815
[11]GB/T 18979-2003食品中黄曲霉毒素的测定免疫亲和层析柱高效液相色谱法和荧光光度法[S].GB/T 18979-2003 Determination aflatoxins content in food-clean up by immunoaffinity chromatographyand determination byhigh-performance liquid chromatigraphy and fluorometer[S].
[12]Nonaka Y,Saito K,Hanioka N,et al.Determination of aflatoxins in food samples by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry[J].J Chromatogr A,2009,1216(20):4416?4422.
[13]Tanaka H,Takino M,Sugita-Konishi Y,et al.Development of a liquid chromatography/time-of-flight mass spectrometric method for the simultaneous determination of trichothecenes,zearalenone and aflatoxins in food stuffs[J].Rapid Commun Mass Sp,2006,20(9):1422?1428.
[14]Andri PB,Joerg S,Elke A.Comparison of two post-column derivation system,ultraviolet irradiation and electrochemical determination for the liquid chromatographic determination of aflatoxin in food[J].J Chromatogr A,2002,85(2):411?416.
[15]王君,刘秀梅.食品中黄曲霉毒素B1、B2、G1、G2的高效液相色谱测定方法[J].中国食品卫生杂志,2005,17(6):498?500.Wang J,Liu X M.High performance liquid chromatographic method for determination of Aflatoxin B1,B2,G1 and G2 in foods[J].Chin J Food Hyg,2005,17(6):498?500.
[16]范海东,王玲锋,赵冬吉.自制光化学衍生反应器高效液相色谱法测定大豆油中黄曲霉毒素[J].理化检验-化学分册,2013,49(8):999?1001.Fan HD,Wang LF,Zhao D.Determination of aflatoxins in soybean oil by Homemade photochemical reactor-high performance liquid chromatography[J].Phy Test Chem Anal Part B:Chem Anal,2013,49(8):999.
[2]冯靓,蔡增轩,谭莹,等.HPLC同时测定食品中黄曲霉毒素B1、B2、G1、G2[J].中国卫生检验杂志,2007,17(3):511?513.Feng L,Cai ZX,Tan Y,et al.Simultaneous detection of aflatoxin B1,B2,G1,G2 in foods by HPLC[J].Chin J Health Lab tec,2007,17(3):511?513.
[3]陈新,王雄,雷达.免疫亲和-光化学柱后衍生高效液相色谱荧光法测定花生粕中黄曲霉毒素的研究[J].中国饲料,2007,24:30?34.Chen X,Wang X,Lei D.Determination of aflatoxin B1,B2,G1and G2 by using photochemical derivatization-HPLCfluorescence detector[J].Chin Feed,2007,24:30?34.
[4]Hasan A,Abdurrahman A,Sahan S.Determination of aflatoxin levels in some dairy and food products which consumed in ankara[J].Food Control,2005,16(3):263?266.
[5]计成,赵丽红.黄曲霉毒素生物降解的研究及前景展望[J].动物营养学报,2010,22(2):241?245.Ji C,Zhao LH.Research and prospects of aflatoxin biological detoxification[J].Chin J Animal Nutrition,2010,22(2):241?245.
[6]Lin LM,Zhang J,Wang P.Thin-layer chromatography of mycotoxins and comparison with other chromatography methods[J].J Chromatogr A,1998,815(1):3?20.
[7]Guan D,Li P,Zhang W,et al.An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products[J].Food Chem,2011,125(4):1359?1364.
[8]谢体波,刘红,陆苇,等.间接竞争ELISA检测试剂盒测定粮油食品中的黄曲霉毒素B1[J].食品安全质量检测学报,2015,6(7):2834?2839.Xie TB,Liu H,Lu W,et al.Determination of aflatoxin B1 in cooking oil,peanut,and grain by using directcompetitive ELISA kit[J].J Food Saf Qual,2015,6(7):2834?2839.
[9]李平,谢体波,易重任,等.黄曲霉毒素M1 ELISA试剂盒的检测效果研究[J].食品安全质量检测学报,2015,6(6):2297?2302.Li P,Xie TB,Yi ZR,et al.Research on detection result of aflatoxin M1 enzyme-linked immune sorbent assay detection kit[J].J Food Saf Qual,2015,6(6):2297?2302.
[10]朱鹏飞,刘文卫,凌霞,等.柱前衍生高效液相色谱荧光法测定粮谷类食物中黄曲霉毒素B1、B2、G1、G2[J].中国卫生检验杂志,2015,25(6):807?815.Zhu PF,Liu WW,Ling X,et al.Determination of aflatoxin B1,B2,G1,G2 in cereal food by high performance liquid chromatography with fluorescence detector and pre-column derivatization[J].Chin J Health Lab Tec,2015,25(6):807?815
[11]GB/T 18979-2003食品中黄曲霉毒素的测定免疫亲和层析柱高效液相色谱法和荧光光度法[S].GB/T 18979-2003 Determination aflatoxins content in food-clean up by immunoaffinity chromatographyand determination byhigh-performance liquid chromatigraphy and fluorometer[S].
[12]Nonaka Y,Saito K,Hanioka N,et al.Determination of aflatoxins in food samples by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry[J].J Chromatogr A,2009,1216(20):4416?4422.
[13]Tanaka H,Takino M,Sugita-Konishi Y,et al.Development of a liquid chromatography/time-of-flight mass spectrometric method for the simultaneous determination of trichothecenes,zearalenone and aflatoxins in food stuffs[J].Rapid Commun Mass Sp,2006,20(9):1422?1428.
[14]Andri PB,Joerg S,Elke A.Comparison of two post-column derivation system,ultraviolet irradiation and electrochemical determination for the liquid chromatographic determination of aflatoxin in food[J].J Chromatogr A,2002,85(2):411?416.
[15]王君,刘秀梅.食品中黄曲霉毒素B1、B2、G1、G2的高效液相色谱测定方法[J].中国食品卫生杂志,2005,17(6):498?500.Wang J,Liu X M.High performance liquid chromatographic method for determination of Aflatoxin B1,B2,G1 and G2 in foods[J].Chin J Food Hyg,2005,17(6):498?500.
[16]范海东,王玲锋,赵冬吉.自制光化学衍生反应器高效液相色谱法测定大豆油中黄曲霉毒素[J].理化检验-化学分册,2013,49(8):999?1001.Fan HD,Wang LF,Zhao D.Determination of aflatoxins in soybean oil by Homemade photochemical reactor-high performance liquid chromatography[J].Phy Test Chem Anal Part B:Chem Anal,2013,49(8):999.