液相色谱-串联质谱法测定蜂王浆中双甲脒及其代谢物的残留量
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摘要
采用固相萃取-液相色谱-串联质谱法(SPE-LC-MS/MS)测定蜂王浆中双甲脒、单甲脒、2,4-二甲基苯基甲酰胺和2,4-二甲基苯胺的残留量。样品经氨水稀释、氨化乙腈沉淀蛋白并提取,通过中性Al_2O_3固相萃取柱净化,以液相色谱-串联质谱多反应监测正离子模式检测,同位素稀释内标法和外标法定量。结果表明,双甲脒、单甲脒、2,4-二甲基苯基甲酰胺和2,4-二甲基苯胺的定量限分别为0.05、0.5、5.0和0.5μg/kg,在空白蜂王浆基质溶液0～300μg/kg范围内绘制线性工作曲线,线性相关系数大于0.997,对空白蜂王浆进行添加浓度5.0、10、100、200μg/kg的实验,总体回收率为50.5%～110%,相对标准偏差为0.8%～15.0%。该方法简便、快捷,定量限能够满足目前国内外残留限量要求,可为蜂王浆中双甲脒及其代谢物残留量的测定提供参考。
Royal jelly is a popular nutritional supplement, its quality directly affects consumer's health. In this work, solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry(SPE-LC-MS/MS) was developed for determination of amitraz, N-2,4-dimethyl phenyl-N-methylformamidine(DMPF), N-(2,4-dimethylphenyl) formamide(DMF) and 2,4-dimethylaniline(DMA) in royal jelly. The protein of royal jelly was precipitated, and the extracts were dissolved and distilled with ammonia solution and ammonia of acetonitrile solution. The supernatant solution was cleaned up with solid phase extraction column of N-Al_2O_3 cartridges and eluted with ammonia of acetonitrile solution into a glass culture tube, the eluate evaporated to dryness and reconstituted in acetonitrile solution, and then filtered through nylon membrane into a glass LC vial for LC-MS/MS analysis. The solution was separated by Agilent Eclipse XDB-C18 column(150 mm×4.6 mm×5 μm) and the quantitative detection was performed on LC-MS/MS by multiple reaction monitoring(MRM) mode under positive electrospray ionization(ESI~+). Isotopes dilution internal standard method or external standard method were used to determine the residue contents in sample. The limits of quantification(LOQs, S/N=10) of amitraz, DMPF, DMF and DMA are 0.05, 0.5, 5.0 and 0.5 μg/kg, respectively. Single-laboratory method validation data were determined, the calibration standard curves concentration are 0, 5.0, 10, 100, 200, 300 μg/kg and correlation coefficients are more than 0.997. Appropriate amounts of the standard target compounds were spiked into the "blank" royal jelly and fixed at four final concentration levels of 5.0, 10, 100 and 200 μg/kg, the recoveries and relative standard deviations(RSDs) using spiked matrix calibration standard curves are 50.5%-110% and 0.8%-15.0%, respectively. This method is convenient, rapid and effective, it is suitable for determination and confirmation of amitraz and its metabolites in royal jelly sample, and in line with the regulations of European Union(EU).
Royal jelly is a popular nutritional supplement, its quality directly affects consumer's health. In this work, solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry(SPE-LC-MS/MS) was developed for determination of amitraz, N-2,4-dimethyl phenyl-N-methylformamidine(DMPF), N-(2,4-dimethylphenyl) formamide(DMF) and 2,4-dimethylaniline(DMA) in royal jelly. The protein of royal jelly was precipitated, and the extracts were dissolved and distilled with ammonia solution and ammonia of acetonitrile solution. The supernatant solution was cleaned up with solid phase extraction column of N-Al_2O_3 cartridges and eluted with ammonia of acetonitrile solution into a glass culture tube, the eluate evaporated to dryness and reconstituted in acetonitrile solution, and then filtered through nylon membrane into a glass LC vial for LC-MS/MS analysis. The solution was separated by Agilent Eclipse XDB-C18 column(150 mm×4.6 mm×5 μm) and the quantitative detection was performed on LC-MS/MS by multiple reaction monitoring(MRM) mode under positive electrospray ionization(ESI~+). Isotopes dilution internal standard method or external standard method were used to determine the residue contents in sample. The limits of quantification(LOQs, S/N=10) of amitraz, DMPF, DMF and DMA are 0.05, 0.5, 5.0 and 0.5 μg/kg, respectively. Single-laboratory method validation data were determined, the calibration standard curves concentration are 0, 5.0, 10, 100, 200, 300 μg/kg and correlation coefficients are more than 0.997. Appropriate amounts of the standard target compounds were spiked into the "blank" royal jelly and fixed at four final concentration levels of 5.0, 10, 100 and 200 μg/kg, the recoveries and relative standard deviations(RSDs) using spiked matrix calibration standard curves are 50.5%-110% and 0.8%-15.0%, respectively. This method is convenient, rapid and effective, it is suitable for determination and confirmation of amitraz and its metabolites in royal jelly sample, and in line with the regulations of European Union(EU).
引文
[1] 刘喜生,李春雁,靳藜. 蜂王浆的药理和生理作用[J]. 中国蜂业,2015,66(5):48-49. LIU Xisheng, LI Chunyan, JIN Li. The pharmacological and physiological effects of royal jelly[J]. Apiculture of China, 2015, 66(5): 48-49(in Chinese).
[2] 中国医药保健品进出口商会. 2012年蜂王浆产品出口额创十年最高[J]. 中国蜂业,2013,64:45-46. China Chamber of Commerce for Import & Export of Medicines & Health Products. In 2012, the highest level for export of royal jelly products in last ten years[J]. Apiculture of China, 2013, 64: 45-46(in Chinese).
[3] 王少成,姜元振. 双甲脒(螨克)防治果、菜、棉植食性螨、蚜、木虱等的药效评价[J]. 农药科学与管理,1992,(2):6-7. WANG Shaocheng, JIANG Yuanzhen. The efficacy evaluation of amitraz to control fruit, vegetables, cotton for mite, aphid and psylla[J]. The Pesticide Science and Management, 1992, (2): 6-7(in Chinese).
[4] 代平礼,王强,孙继虎,等. 4种农药对意大利蜜蜂的毒力测定[J]. 农药,2007,46(8):546-547. DAI Pingli, WANG Qiang, SUN Jihu, et al. Bioassay of four pesticides on Apis mellifera[J]. Agrochemicals, 2007, 46(8): 546-547(in Chinese).
[5] GERARDO P S, GABRIEL O C, DAVID M S, et al. Comparing effects of three acaricides on Varroajacobsoni (Acari: Varroidae) and Apismellifera (Hymenoptera: Apidae) using two application techniques[J]. Florida Entomologist, 2000, 83(4): 468-476.
[6] OSANOA O, ADMIRAALA W, KLAMERC H J C, et al. Comparative toxic and genotoxic effects of chloroacetanilides, formamidines and their degradation products on vibrio fischeri and chironomusriparius[J]. Environmental Pollution, 2002, 119(2): 195-202.
[7] RIAL-OTERO R, GASPAR E M, MOURA I, et al. Chromatographic-based methods for pesticide determination in honey: an overview[J]. Talanta, 2007, 71(2): 503-514.
[8] The European Union. Council Regulation(EU)No 2377/90/EEC/1990[S]. 1990.
[9] Japan. Positive list system for agricultural chemical residues in foods[EB/OL]. http://www.mhlw.go.jp/english/topics/foodsafety/positivelist060228/2017/11/01.
[10] The United States of America. Code of federal regulations (40 CFR Part 180,180.287)[S]. 2006.
[11] 郑用权,姚建仁,邵向东,等. 蜂蜜中双甲脒残留量检测方法[J]. 农药科学与管理,2000,21(3):14-16. ZHENG Yongquan, YAO Jianren, SHAO Xiang-dong, et al. Residue analysis method of amitraz in honey by GC[J]. Pesticide Science and Administration, 2000, 21(3): 14-16(in Chinese).
[12] AMOLI J S, HASAN J, HEJAZY M. Determination of amitraz residue by headspace gas chromatography in honey and beeswax samples from Iran[J]. American Journal of Food Technology, 2009, 4(1): 56-59.
[13] SHAMSIPUR M, HASSAN J, SALAR-AMOLI J, et al. Headspace solvent microextraction-gas chromatographic thermionic specific detector determination of amitraz in honey after hydrolysis to 2,4-dimethylaniline[J]. Journal of Food Composition and Analysis, 2008, (21): 264-270.
[14] JIMéNEZ J J, BERNAL J L, NOZAL M J D, et al. Extraction and clean-up methods for the determination of amitraz total residues in beeswax by gas chromatography with electron capture detection[J]. Analytica Chimica Acta, 2004, 524(1/2): 271-278.
[15] 伊雄海,杨惠琴,郭德华,等. 气相色谱质谱法测定鳗鱼中双甲脒及其代谢物残留量[J]. 上海交通大学学报:农业科学版,2009,27(5):524-527. YI Xionghai, YANG Huiqin, GUO Dehua, et al. Determination of acaricides amitraz and its metabolite residues in Eel-GC-MS method[J]. Journal of Shang Hai Jiao Tong University: Agricultural Science, 2009, 27(5): 524-527(in Chinese).
[16] YU H, TAO Y F, LE T, et al. Simultaneous determination of amitraz and its metabolite residue in food animal tissues by gas chromatography-electron capture detector and gas chromatography-mass spectrometry with accelerated solvent extraction[J]. Journal of Chromatography B, 2010, 878(21): 1 746-1 752.
[17] 李阿丹,王晶,崔宗岩,等. 固相微萃取-气相色谱-串联质谱法测定蜂蜜中双甲脒及其代谢产物残留[J]. 燕山大学学报,2016,40(3):236-240. LI Adan, WANG Jing, CUI Zongyan, et al. Simutaneous determination of amitraz and its metabolite 2, 4-dimethylaniline in honey by solid-phase microextraction coupled with gas chromatography-tandem mass spectrometry[J]. Journal of Yanshan University, 2016, 40(3): 236-240(in Chinese).
[18] 薛晓锋,赵静,邱静,等. 气相色谱-质谱法同时测定蜂蜜中的双甲脒及其代谢物残留[J]. 现代科学仪器,2005,(1):65-67. XUE Xiaofeng, ZHAO Jing, QIU Jing, et al. Simultaneous determination of amitraz and its metabolite (2,4-xylidine) residues in honey by gas chromatography-mass spectrometry[J]. Modern Scientific Instruments, 2005, (1): 65-67(in Chinese).
[19] 周召千,丁慧瑛,吴娟,等. 动物源产品中双甲脒及其代谢产物残留量的气相色谱-质谱法测定[J]. 农药研究与应用,2009,13(4):23-26. ZHOU Zhaoqian, DING Huiying, WU Juan, et al. Determination of amitraz and its metabolites residues in foods of aminal origin by gas chromatography-mass spectrometry[J]. Agrochemicals Research & Application, 2009, 13(4): 23-26(in Chinese).
[20] YAMINI Y, FARAJI M, GHAMBARIAN M. Hollow-fiber liquid-phase microextraction followed by gas chromatography glame ionization detection for the determination of amitraz in honey and water samples[J]. Food Analytical Methods, 2015, 8(3): 758-766.
[21] BASHIRI JUYBARI M, MEHDINIA A, JABBARI A, et al. Determination of amitraz in the honey samples by dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection[J]. American Journal of Analytical Chemistry, 2011, 2(5): 632-637.
[22] GB 23200.103—2016 食品安全国家标准蜂王浆中双甲脒及其代谢产物残留量的测定气相色谱-质谱法[S]. 北京:中国标准出版社,2016.
[23] 赵增运,吴斌,沈崇钰,等. 高效液相色谱法测定蜂蜜中的双甲脒残留[J]. 中国养蜂,2005,56(5):4-6. ZHAO Zengyun, WU Bin, SHEN Chongyu, et al. Determination of amitraz residue in honey by HPLC with UV detector[J]. Apiculture of China, 2005, 56(5): 4-6(in Chinese).
[24] GB/T 21169—2007 蜂蜜中双甲脒及其代谢物残留量测定-液相色谱法[S]. 北京:中国标准出版社,2007.
[25] TOKMANA N, SOLERA C, FARRé M, et al. Determination of amitraz and its transformation products in pears by ethyl acetate extraction and liquid chromatography-tandem mass spectrometry[J]. Journal of Chromatography A, 2009, 1 216(15): 3 138-3 146.
[26] XU J Z, MIAO J J, LIN H, et al. Determination of amitraz and 2,4-dimethylaniline residues in honey by using LC with UV detection and MS/MS[J]. Journal of Separation Science, 2009, 32(23/24): 4 020-4 024.
[27] 郭浩,郭东东,李恒,等. 固相萃取/液相色谱-串联质谱法测定鱼塘水中双甲脒及其代谢产物[J]. 分析测试学报,2014,33(12):1 416-1 420. GUO Hao, GUO Dongdong, LI Heng, et al. Detection of amitraz and its metabolites in pond-water using SPE/LC-MS/MS[J]. Journal of Instrumental Analysis, 2014, 33(12): 1 416-1 420(in Chinese).
[28] GUO H, ZHANG P, WANG J W, et al. Determination of amitraz and its metabolites in whole blood using solid-phase extraction and liquid chromatography-tandem mass spectrometry[J]. Journal of Chromatography B, 2014, 951/952(1): 89-95.
[29] KORTA E, BAKKALI A, BERRUETA L A, et al. Study of acaricide stability in honey. Characterization of amitraz degradation products in honey and beeswax[J]. Journal of Agricultural and Food Chemistry, 2001, 49(12): 5 835-5 842.
[30] STAHNKE H, KITTLAUS S, KEMPE G N, et al. Reduction of matrix effects in liquid chromatography-electrospray ionization-mass spectrometry by dilution of the sample extracts: how much dilution is needed?[J]. Analytical Chemistry, 2012, 84(3): 1 474-1 482.
[31] FERRER C, LOZANO A, AGUERA A, et al. Overcoming matrix effects using the dilution approach in multiresidue methods for fruits and vegetables[J]. Journal of Chromatography A, 2011, 1 218(42): 7 634-7 639.
[2] 中国医药保健品进出口商会. 2012年蜂王浆产品出口额创十年最高[J]. 中国蜂业,2013,64:45-46. China Chamber of Commerce for Import & Export of Medicines & Health Products. In 2012, the highest level for export of royal jelly products in last ten years[J]. Apiculture of China, 2013, 64: 45-46(in Chinese).
[3] 王少成,姜元振. 双甲脒(螨克)防治果、菜、棉植食性螨、蚜、木虱等的药效评价[J]. 农药科学与管理,1992,(2):6-7. WANG Shaocheng, JIANG Yuanzhen. The efficacy evaluation of amitraz to control fruit, vegetables, cotton for mite, aphid and psylla[J]. The Pesticide Science and Management, 1992, (2): 6-7(in Chinese).
[4] 代平礼,王强,孙继虎,等. 4种农药对意大利蜜蜂的毒力测定[J]. 农药,2007,46(8):546-547. DAI Pingli, WANG Qiang, SUN Jihu, et al. Bioassay of four pesticides on Apis mellifera[J]. Agrochemicals, 2007, 46(8): 546-547(in Chinese).
[5] GERARDO P S, GABRIEL O C, DAVID M S, et al. Comparing effects of three acaricides on Varroajacobsoni (Acari: Varroidae) and Apismellifera (Hymenoptera: Apidae) using two application techniques[J]. Florida Entomologist, 2000, 83(4): 468-476.
[6] OSANOA O, ADMIRAALA W, KLAMERC H J C, et al. Comparative toxic and genotoxic effects of chloroacetanilides, formamidines and their degradation products on vibrio fischeri and chironomusriparius[J]. Environmental Pollution, 2002, 119(2): 195-202.
[7] RIAL-OTERO R, GASPAR E M, MOURA I, et al. Chromatographic-based methods for pesticide determination in honey: an overview[J]. Talanta, 2007, 71(2): 503-514.
[8] The European Union. Council Regulation(EU)No 2377/90/EEC/1990[S]. 1990.
[9] Japan. Positive list system for agricultural chemical residues in foods[EB/OL]. http://www.mhlw.go.jp/english/topics/foodsafety/positivelist060228/2017/11/01.
[10] The United States of America. Code of federal regulations (40 CFR Part 180,180.287)[S]. 2006.
[11] 郑用权,姚建仁,邵向东,等. 蜂蜜中双甲脒残留量检测方法[J]. 农药科学与管理,2000,21(3):14-16. ZHENG Yongquan, YAO Jianren, SHAO Xiang-dong, et al. Residue analysis method of amitraz in honey by GC[J]. Pesticide Science and Administration, 2000, 21(3): 14-16(in Chinese).
[12] AMOLI J S, HASAN J, HEJAZY M. Determination of amitraz residue by headspace gas chromatography in honey and beeswax samples from Iran[J]. American Journal of Food Technology, 2009, 4(1): 56-59.
[13] SHAMSIPUR M, HASSAN J, SALAR-AMOLI J, et al. Headspace solvent microextraction-gas chromatographic thermionic specific detector determination of amitraz in honey after hydrolysis to 2,4-dimethylaniline[J]. Journal of Food Composition and Analysis, 2008, (21): 264-270.
[14] JIMéNEZ J J, BERNAL J L, NOZAL M J D, et al. Extraction and clean-up methods for the determination of amitraz total residues in beeswax by gas chromatography with electron capture detection[J]. Analytica Chimica Acta, 2004, 524(1/2): 271-278.
[15] 伊雄海,杨惠琴,郭德华,等. 气相色谱质谱法测定鳗鱼中双甲脒及其代谢物残留量[J]. 上海交通大学学报:农业科学版,2009,27(5):524-527. YI Xionghai, YANG Huiqin, GUO Dehua, et al. Determination of acaricides amitraz and its metabolite residues in Eel-GC-MS method[J]. Journal of Shang Hai Jiao Tong University: Agricultural Science, 2009, 27(5): 524-527(in Chinese).
[16] YU H, TAO Y F, LE T, et al. Simultaneous determination of amitraz and its metabolite residue in food animal tissues by gas chromatography-electron capture detector and gas chromatography-mass spectrometry with accelerated solvent extraction[J]. Journal of Chromatography B, 2010, 878(21): 1 746-1 752.
[17] 李阿丹,王晶,崔宗岩,等. 固相微萃取-气相色谱-串联质谱法测定蜂蜜中双甲脒及其代谢产物残留[J]. 燕山大学学报,2016,40(3):236-240. LI Adan, WANG Jing, CUI Zongyan, et al. Simutaneous determination of amitraz and its metabolite 2, 4-dimethylaniline in honey by solid-phase microextraction coupled with gas chromatography-tandem mass spectrometry[J]. Journal of Yanshan University, 2016, 40(3): 236-240(in Chinese).
[18] 薛晓锋,赵静,邱静,等. 气相色谱-质谱法同时测定蜂蜜中的双甲脒及其代谢物残留[J]. 现代科学仪器,2005,(1):65-67. XUE Xiaofeng, ZHAO Jing, QIU Jing, et al. Simultaneous determination of amitraz and its metabolite (2,4-xylidine) residues in honey by gas chromatography-mass spectrometry[J]. Modern Scientific Instruments, 2005, (1): 65-67(in Chinese).
[19] 周召千,丁慧瑛,吴娟,等. 动物源产品中双甲脒及其代谢产物残留量的气相色谱-质谱法测定[J]. 农药研究与应用,2009,13(4):23-26. ZHOU Zhaoqian, DING Huiying, WU Juan, et al. Determination of amitraz and its metabolites residues in foods of aminal origin by gas chromatography-mass spectrometry[J]. Agrochemicals Research & Application, 2009, 13(4): 23-26(in Chinese).
[20] YAMINI Y, FARAJI M, GHAMBARIAN M. Hollow-fiber liquid-phase microextraction followed by gas chromatography glame ionization detection for the determination of amitraz in honey and water samples[J]. Food Analytical Methods, 2015, 8(3): 758-766.
[21] BASHIRI JUYBARI M, MEHDINIA A, JABBARI A, et al. Determination of amitraz in the honey samples by dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection[J]. American Journal of Analytical Chemistry, 2011, 2(5): 632-637.
[22] GB 23200.103—2016 食品安全国家标准蜂王浆中双甲脒及其代谢产物残留量的测定气相色谱-质谱法[S]. 北京:中国标准出版社,2016.
[23] 赵增运,吴斌,沈崇钰,等. 高效液相色谱法测定蜂蜜中的双甲脒残留[J]. 中国养蜂,2005,56(5):4-6. ZHAO Zengyun, WU Bin, SHEN Chongyu, et al. Determination of amitraz residue in honey by HPLC with UV detector[J]. Apiculture of China, 2005, 56(5): 4-6(in Chinese).
[24] GB/T 21169—2007 蜂蜜中双甲脒及其代谢物残留量测定-液相色谱法[S]. 北京:中国标准出版社,2007.
[25] TOKMANA N, SOLERA C, FARRé M, et al. Determination of amitraz and its transformation products in pears by ethyl acetate extraction and liquid chromatography-tandem mass spectrometry[J]. Journal of Chromatography A, 2009, 1 216(15): 3 138-3 146.
[26] XU J Z, MIAO J J, LIN H, et al. Determination of amitraz and 2,4-dimethylaniline residues in honey by using LC with UV detection and MS/MS[J]. Journal of Separation Science, 2009, 32(23/24): 4 020-4 024.
[27] 郭浩,郭东东,李恒,等. 固相萃取/液相色谱-串联质谱法测定鱼塘水中双甲脒及其代谢产物[J]. 分析测试学报,2014,33(12):1 416-1 420. GUO Hao, GUO Dongdong, LI Heng, et al. Detection of amitraz and its metabolites in pond-water using SPE/LC-MS/MS[J]. Journal of Instrumental Analysis, 2014, 33(12): 1 416-1 420(in Chinese).
[28] GUO H, ZHANG P, WANG J W, et al. Determination of amitraz and its metabolites in whole blood using solid-phase extraction and liquid chromatography-tandem mass spectrometry[J]. Journal of Chromatography B, 2014, 951/952(1): 89-95.
[29] KORTA E, BAKKALI A, BERRUETA L A, et al. Study of acaricide stability in honey. Characterization of amitraz degradation products in honey and beeswax[J]. Journal of Agricultural and Food Chemistry, 2001, 49(12): 5 835-5 842.
[30] STAHNKE H, KITTLAUS S, KEMPE G N, et al. Reduction of matrix effects in liquid chromatography-electrospray ionization-mass spectrometry by dilution of the sample extracts: how much dilution is needed?[J]. Analytical Chemistry, 2012, 84(3): 1 474-1 482.
[31] FERRER C, LOZANO A, AGUERA A, et al. Overcoming matrix effects using the dilution approach in multiresidue methods for fruits and vegetables[J]. Journal of Chromatography A, 2011, 1 218(42): 7 634-7 639.