全自动固相萃取结合UPLC-MS/MS测定茶叶中的草甘膦及其代谢物

doi:10.13305/j.cnki.jts.2023.06.010

摘要/Abstract

摘要： 建立超高效液相色谱-质谱/质谱法（UPLC-MS/MS）测定茶叶中草甘膦及其代谢物氨甲基膦酸的分析方法。用水-二氯甲烷体系对样品进行提取和初步除杂,在全自动固相萃取仪中通过聚苯乙烯键合苯磺酸混合型阳离子交换柱进一步净化样品溶液基质,利用正交试验和方差分析得出最优的衍生条件为5%硼酸钠缓冲溶液和10 mg·mL^-1的9-芴基甲基三氯甲烷（FMOC-Cl）衍生剂在室温下衍生2 h,衍生后的样品溶液在HSS T₃色谱柱和5 mmol·L^-1乙酸铵溶液（含0.1%甲酸）-乙腈流动相体系下进行梯度洗脱分离,经UPLC-MS/MS进行内标法定量分析。草甘膦和氨甲基膦酸在0.2~50 ng·mL^-1质量浓度范围内线性关系良好,相关系数（R²）均大于0.999,该方法的检出限为2 μg·kg^-1,定量限为5 μg·kg^-1。低、中、高3个加标水平（5、50 μg·kg^-1和250 μg·kg^-1）的回收率为94.2%~106.7%,相对标准偏差为3.8%~4.9%。该方法具有高效率、高准确度、高通量、基质干扰小和自动化程度高等优点,适用于实验室日常大批量茶叶样品筛查草甘膦及氨甲基膦酸残留的工作需求。

关键词: 草甘膦, 氨甲基膦酸, 全自动固相萃取仪, 超高效液相色谱-质谱联用仪

Abstract: An analytical method for the determination of glyphosate and its metabolite aminomethylphosphonic acid in tea was established by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted and preliminarily purified using a water dichloromethane system and the sample matrix was further purified by mixed-mode cation exchanger column filled with polystyrene bonded benzenesulfonic acid in an automatic solid phase extractor. The optimal derivation conditions obtained by orthogonal test and variance analysis were 5% sodium borate buffer solution and 10 mg·mL^-1 FMOC-Cl derivatizing agent for derivatization at room temperature for 2 h. The derived sample solution was subjected to gradient elution separation on HSS T₃ column and 5 mmol·L^-1 ammonium acetate solution (containing 0.1% formic acid)-acetonitrile mobile phase system. The targets were analyzed by UPLC-MS/MS and quantified by internal standard method. The linearity of glyphosate and aminomethylphosphonic acid was good in the concentration range of 0.2-50 ng·mL^-1 and the correlation coefficient (R²) was greater than 0.999. The detection limit of the method was 2 μg·kg^-1, and the quantitation limit was 5 μg·kg^-1. The recoveries of 5, 50 and 250 μg·kg^-1 were in range of 94.2%-106.7% with relative standard deviations of 3.8%-4.9%, at low, medium and high spiked levels. The method is suitable for the determination of glyphosate and aminomethylphosphonic acid residues in daily large quantities of tea samples in the laboratory with the advantages of high efficiency, high accuracy, high throughput, low matrix interference and high degree of automation.

Key words: glyphosate, aminomethylphosphonic acid, automatic solid phase extractor, UPLC-MS/MS

中图分类号:

S571.1
S482

易华娟, 朱洁灵, 周瑞铮, 郑耀林, 张树权, 杨乐, 苏雍伦. 全自动固相萃取结合UPLC-MS/MS测定茶叶中的草甘膦及其代谢物[J]. 茶叶科学, 2023, 43(6): 857-869. doi: 10.13305/j.cnki.jts.2023.06.010.

YI Huajuan, ZHU Jieling, ZHOU Ruizheng, ZHENG Yaolin, ZHANG Shuquan, YANG Le, SU Yonglun. Determination of Glyphosate and Its Metabolites in Tea by Automatic Solid Phase Extraction Combined with UPLC-MS/MS[J]. Journal of Tea Science, 2023, 43(6): 857-869. doi: 10.13305/j.cnki.jts.2023.06.010.

参考文献 39

[1]	Gandhi K, Khan S, Patrikar M, et al.Exposure risk and environmental impacts of glyphosate: highlights on the toxicity of herbicide co-formulants[J]. Environmental Challenges, 2021, 4: 100149. doi: 10.1016/j.envc.2021.100149.
[2]	Williams G M, Kroes R, Munro I C.Safety evaluation and risk assessment of the herbicide roundup and its active ingredient, glyphosate, for humans[J]. Regulatory Toxicology and Pharmacology, 2000, 31(2): 117-165.
[3]	林静雯, 李莹, 罗洁文, 等. 林用除草剂草甘膦的环境行为及生态风险研究进展[J]. 世界林业研究, 2015, 28(4): 58-63.Lin J W, Li Y, Luo J W, et al.Research progress on environmental behavior and ecological risk of glyphosate in forestry[J]. World Forestry Research, 2015, 28(4): 58-63.
[4]	高瑾, 熊六凤, 阮记明, 等. 草甘膦的水环境行为及其对水生生物毒性的研究进展[J]. 生态毒理学报, 2022, 17(3): 422-433.Gao J, Xiong L F, Ruan J M, et al.Advances of aquatic environmental behaviors and toxicity of glyphosate to aquatic organisms[J]. Asian Journal of Ecotoxicology, 2022, 17(3): 422-433.
[5]	Schuster I.Cytochromes P450 are essential players in the vitamin D signaling system[J]. Biochim Biophys Acta (BBA)-Proteins and Proteomics, 2011, 1814(1): 186-199.
[6]	南旭阳. 除草剂“草甘膦”对鲫鱼外周血红细胞微核及核异常的影响[J]. 安徽师范大学学报(自然科学版), 2001, 24(4): 329-331.Nan X Y.Effect of herbicide (glyphosate) on micronuclei and nuclear anomalies in erythrocyte in Carassius auratus[J]. Journal of Anhui Normal University (Natural Science), 2001, 24(4): 329-331.
[7]	康菊芳, 曾明, 关岚, 等. 草甘膦对小鼠的致突变作用研究[J]. 癌变·畸变·突变, 2008, 20(3): 227-230.Kang J F, Zeng M, Guan L, et al.Study on mutagenesis induced by glyphosate in mice[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2008, 20(3): 227-230.
[8]	Van Bruggen A H C, He M M, Shin K, et al. Environmental and health effects of the herbicide glyphosate [J]. Science of the Total Environment, 2018, 616/617: 255-268.
[9]	Franke A A, Li X N, Shvetsov Y B, et al.Pilot study on the urinary excretion of the glyphosate metabolite aminomethylphosphonic acid and breast cancer risk: the Multiethnic Cohort study[J]. Environmental Pollution, 2021, 277: 116848. doi: 10.1016/j.envpol.2021.116848.
[10]	Woźniak E, Reszka E, Jabłońska E, et al.Glyphosate and AMPA induce alterations in expression of genes involved in chromatin architecture in human peripheral blood mononuclear cells (in vitro)[J]. International Journal of Molecular Sciences, 2021, 22(6): 2966. doi: 10.3390/ijms22062966.
[11]	赵丽, 陈瑞, 岑铭松, 等. 我国出口欧盟茶叶农药残留状况与控制对策[J]. 中国植保导刊, 2021, 41(5): 69-74.Zhao L, Chen R, Ceng M S, et al.Pesticide residues in tea exported from China to EU and control countermeasures[J]. China Plant Protection, 2021, 41(5): 69-74.
[12]	王如坤, 方爱琴, 赵海英, 等. 超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法快速检测茶叶中草甘膦、草铵膦及其代谢物残留[J]. 食品安全质量检测学报, 2021, 12(10): 3954-3959.Wang R K, Fang A Q, Zhao H Y, et al.Rapid determination of glyphosate, glufosinate-ammonium and their metabolite residues in tea by ultra performance liquid chromatography-quadrupole electrostatic field orbital trap high resolution mass spectrometry[J]. Journal of Food Safety and Quality, 2021, 12(10): 3954-3959.
[13]	李燕虹, 王耀, 裴亚峰, 等. 免疫分析技术在草甘膦残留检测中的应用[J]. 中国免疫学杂志, 2018, 34(2): 291-295.Li Y H, Wang Y, Pei Y F, et al.Application of immunoassay techniques in glyphosate residue detection[J]. Chinese Journal of Immunology, 2018, 34(2): 291-295.
[14]	胡继业, 赵殿英, 宁君, 等. 气相色谱-氮磷检测器测定草甘膦在土壤和苹果中的残留量[J]. 农药学学报, 2007, 9(3): 285-290.Hu J Y, Zhao D Y, Ning J, et al.Determination of glyphosate residues in soil and apple by capillary gas chromatography with nitrogen-phosphorus detection[J]. Chinese Journal of Pesticide Science, 2007, 9(3): 285-290.
[15]	方芳, 徐会, 魏荣卿, 等. 草甘膦的邻硝基苯磺酰氯柱前衍生高效液相色谱分析[J]. 分析测试学报, 2011, 30(6): 683-686.Fang F, Xu H, Wei R Q, et al.Determination of glyphosate by high performance liquid chromatography with o-nitrobenzenesulfonyl chloride as derivatization reagent[J]. Journal of Instrumental Analysis, 2011, 30(6): 683-686.
[16]	曹丽伟, 梁丝柳, 谭小芳, 等. 毛细管电泳-激光诱导荧光法测定草甘膦、草胺膦及氨甲基膦酸[J]. 色谱, 2012, 30(12): 1295-1300.Cao L W, Liang S L, Tang X F, et al.Capillary electrophoresis analysis for glyphosate, glufosinate and aminomethylphosphonic acid with laser-induced fluorescence detection[J]. Chinese Journal of Chromatography, 2012, 30(12): 1295-1300.
[17]	罗彤, 付文雯, 郭卢云, 等. 离子色谱法测定不同谷物中草甘膦残留[J]. 食品安全质量检测学报, 2018, 9(1): 161-166.Luo T, Fu W W, Guo L Y, et al.Determination of glyphosate residues in different kinds of grains by ion chromatography[J]. Journal of Food Safety and Quality, 2018, 9(1): 161-166.
[18]	张燕, 舒平, 陆敏, 等. 分散固相萃取/衍生化-高效液相色谱-串联质谱法同时测定水和食品中草甘膦、草铵膦和氨甲基膦酸残留[J]. 食品安全质量检测学报, 2017, 8(4): 1422-1428.Zhang Y, Shu P, Lu M, et al.Simultaneous determination of glyphosate, glufosinate and aminomethyl residues in water and foods by dispersive solid phase extraction/derivatization-high performance liquid chromatography-tandem mass spectrometry[J]. Journal of Food Safety and Quality, 2017, 8(4): 1422-1428.
[19]	Sorokin A V, Ovcharenko V V.LC-MS/MS analysis of glyphosate, aminomethylphosphonic acid and glufosinate in honey[J]. Asian Journal of Chemistry, 2022, 34(8): 2128-2132.
[20]	Jansons M, Pugajeva I, Bartkevics V, et al.LC-MS/MS characterisation and determination of dansyl chloride derivatised glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in foods of plant and animal origin[J]. Journal of Chromatography B, 2021, 1177: 122779. doi: 10.1016/j.jchromb.2021.122779.
[21]	孙能惠. 固相萃取及液质联用在茶叶农药残留检测中的应用[J]. 现代食品, 2016(1): 57-58.Sun N H.Application of solid phase extraction and liquid chromatography in detection of pesticide residues in tea[J]. Modern Food, 2016(1): 57-58.
[22]	罗本越. QuEchERS-LC/MS/MS快速测定不同茶叶中草甘膦、氨甲基膦酸及草铵膦的残留[J]. 福建分析测试, 2022, 31(3): 14-20.Luo B Y.Fast determination of glyphosate, aminomethyl phosphonic acid and glufosinate in different teas by QuEchERS and UPLC/MS/MS[J]. Fujian Analysis and Testing, 2022, 31(3): 14-20.
[23]	李自强, 杨梅, 张新忠, 等. 改良QuEChERS方法与UPLC-MS/MS联用测定茶叶中草甘膦、草铵膦及氨甲基膦酸[J]. 茶叶科学, 2023, 43(2): 263-274.Li Z Q, Yang M, Zhang X Z, et al.Simultaneous determination of glyphosate, glufosinate and aminomethyl phosphonic acid residues in tea by modified QuEChERS method coupled with UPLC-MS/MS[J]. Journal of Tea Science, 2023, 43(2): 263-274.
[24]	邱世婷, 侯雪, 韩梅, 等. 基于亲水作用色谱-串联质谱的非衍生化法测定绿茶中草甘膦、草铵膦及其代谢物残留[J]. 农产品质量与安全, 2021(4): 30-35.Qiu S T, Hou X, Hang M, et al.Determination of glyphosate, glufosinate and its metabolites in tea by hydrophilic interaction chromatography-tandem mass spectrometry without derivatization[J]. Quality and Safety of Agro-Products, 2021(4): 30-35.
[25]	周茜, 黄倩, 尧聪, 等. UPLC-MS/MS非衍生化法测定茶叶中草甘膦及其代谢产物[J]. 食品工业, 2022, 43(9): 284-287.Zhou Q, Huang Q, Yao C, et al.Determination of glyphosate and its metabolite aminomethyl phosphonic acid in tea by non-derivatization ultra performance liquid chromatography tandem mass spectrometry[J]. The Food Industry, 2022, 43(9): 284-287.
[26]	许有诚, 赵庄, 李丹凤. 柱前在线衍生-高效液相色谱法同时测定复方丹参胶囊中19种氨基酸[J]. 化学分析计量, 2021, 30(10): 1-6.Xu Y C, Zhao Z, Li D F.Simultaneous determination of 19 amino acids in composite salvia miltiorrhiza capsulesby high performance liquid chromatography with online pre-column derivatization[J]. Chemical Analysis and Meterage, 2021, 30(10): 1-6.
[27]	倪先哲, 王刚, 顾菊菊. OPA/FMOC柱前衍生高效液相色谱法检测东太湖原水中游离氨基酸含量[J]. 给水排水, 2021, 57(s2): 32-36.Ni X Z, Wang G, Gu J J.Determination of amino acids in East Taihu Lake water by OPA/FMOC pre-column derivatization high performance liquid chromatography[J]. Water and Wastewater Engineering, 2021, 57(s2): 32-36.
[28]	殷娇阳, 阎姝, 田书霞. 柱前衍生高效液相色谱法测定灵芝孢子粉中氨基酸的含量[J]. 中国中西医结合外科杂志, 2021, 27(2): 182-188.Yin J Y, Yan S, Tian S X.Assay of amino acids in Ganoderma lucidum spore powder by pre-column derivatization HPLC[J]. Chinese Journal of Surgery of Integrated Traditional and Western Medicine, 2021, 27(2): 182-188.
[29]	杨宇锋, 陈玉浩, 蓝康华, 等. 超高效液相色谱串联质谱法测定茶叶中的草铵膦、草甘膦及其代谢物[J]. 广东化工, 2018, 45(10): 231-232, 242.Yang Y F, Chen Y H, Lan K H.Determination of glufosinate-ammonium, glyphosate and its metabolites in tea by UPLC-MS/MS method[J]. Guangdong Chemical Industry, 2018, 45(10): 231-232, 242.
[30]	王军, 郑妙楷, 李晓婷, 等. 超高效液相色谱-高分辨质谱测定茶叶中的草甘膦和草铵膦[J]. 食品与发酵工业, 2022, 48(1): 253-260.Wang J, Zheng M K, Li X T, et al.Determination of glyphosate and glufosinate in tea by ultra-high performance liquid[J]. Food and Fermentation Industries, 2022, 48(1): 253-260.
[31]	叶美君, 陆小磊, 刘相真, 等. 柱前衍生-超高效液相色谱-串联质谱测定茶叶中草甘膦、草铵膦及主要代谢物氨甲基膦酸残留[J]. 色谱, 2018, 36(9): 873-879.Ye M J, Lu X L, Liu X Z, et al.Determination of glyphosate, glufosinate, and main metabolite aminomethylphosphonic acid residues in dry tea using ultra-high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2018, 36(9): 873-879.
[32]	肖泳, 唐吉旺, 袁列江, 等. 高效液相色谱-质谱/质谱法测定茶叶中的草甘膦和氨甲基膦酸残留[J]. 食品与发酵工业, 2016, 42(9): 217-222.Xiao Y, Tang J W, Yuan L J, et al.Determination of glyphosate and aminomethylphosphonic acid residues in tea using high performance liquid chromatography-mass spectrometry/mass spectrometry[J]. Food and Fermentation Industries, 2016, 42(9): 217-222.
[33]	杨梅, 孙思, 刘文锋, 等. 超高效液相色谱-串联质谱法测定茶叶中草甘膦和草铵膦的残留量[J]. 食品科学, 2019, 40(10): 337-343.Yang M, Sun S, Liu W F, et al.Determination of glyphosate and glufosinate ammonium residues in tea by UPLC-MS[J]. Food Science, 2019, 40(10): 337-343.
[34]	刘文静, 林晶, 傅建炜, 等. 超高效液相-串联质谱法测定茶叶中草甘膦、草铵膦、氨甲基膦酸残留[J]. 中国农学通报, 2020, 36(25): 129-136.Liu W J, Lin J, Fu J W, et al.Determination of glyphosate, ammonium phosphate and ammonia in tea by UHPLC-MS[J]. Chinese Agricultural Science Bulletin, 2020, 36(25): 129-136.
[35]	周茜, 黄倩, 尧聪, 等. UPLC-MS/MS非衍生化法测定茶叶中草甘膦及其代谢产物[J]. 食品工业, 2022, 43(9): 284-287.Zhou Q, Huang Q, Yao C, et al.Determination of glyphosate and its metabolite aminomethyl phosphonic acid in tea by non-derivatization ultra performance liquid chromatography tandem mass spectrometry[J]. The Food Industry, 2022, 43(9): 284-287.
[36]	钟茂生, 梁剑, 朱品玲, 等. HPLC-MS-MS法测定茶叶中的草甘膦和氨甲基膦酸[J]. 广州化工, 2014, 42(22): 128-130.Zhong M S, Liang J, Zhu P L, et al.Determination of glyphosate and aminomethylphosphonic acid residues in tea by HPLC-MS/MS[J]. Guangzhou Chemical Industry, 2014, 42(22): 128-130.
[37]	吴晓刚, 陈孝权, 肖海军, 等. 柱前衍生-超高效液相色谱-串联质谱法同时检测茶叶中草甘膦和草铵膦的残留量[J]. 色谱, 2015, 33(10): 1090-1096.Wu X G, Chen X Q, Xiao H J, et al.Simultaneous determination of glyphosate and glufosinateammonium residues in tea by ultra performance liquid chromatography-tandem mass spectrometry coupled with pre-column derivatization[J]. Chinese Journal of Chromatography, 2015, 33(10): 1090-1096.
[38]	熊潇垚, 任岗, 董照锋, 等. QuEChERS-气相色谱质谱法测定果蔬菌类中10种农药残留及基质效应研究[J]. 江西农业学报, 2021, 33(5): 90-95.Xiong X Y, Ren G, Dong Z F, et al.Determination of 10 pesticide residues in edible fungi of vegetables and fruits by QuEChERS-GC-MS and their matrix effects[J]. Acta Agriculturae Jiangxi, 2021, 33(5): 90-95.
[39]	代春梅, 李卿沛, 焦绍明, 等. 液质联用法测定鸡肉中9种激素类药物的基质效应研究[J]. 甘肃畜牧兽医, 2020, 50(10): 51-53, 59.Dai C M, Li Q P, Jiao S M, et al.Study on matrix effect of 9 hormone drugs in chicken by LC-MS[J]. Gansu Animal Husbandry and Veterinary, 2020, 50(10): 51-53, 59.