超高效液相色谱-四极杆/静电场轨道阱高分辨质谱快速筛查鱼和虾样品中200种药物残留
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摘要
建立了超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法,快速筛查鱼和虾样品中200种药物残留。取均质后的鱼或虾样品,加入适量0.1 mol/L EDTA-Na_2溶液,分别用乙腈和乙酸乙酯超声提取,经Oasis PRiME HLB通过式固相萃取柱净化,以0.1%甲酸水溶液和0.1%甲酸-乙腈溶液为流动相,Thermo AccucoreaQ色谱柱(100 mm×2.1 mm×2.6μm)分离;采用Full MS/dd-MS~2模式采集数据,利用自建的包含化合物保留时间、母离子和子离子质荷比等信息的数据库定性,一级质谱全扫描母离子定量。结果表明,本方法的检出限为1～50μg/kg,在不同的加标水平下,200种药物的加标回收率为30%～120%,精密度为5%～30%,其中,精密度在5%～15%、15%～20%和20%～30%的比例分别为70%、10%和20%,且精密度20%～30%主要分布于低加标水平样品。利用本方法对48个鱼和虾实际样品进行筛查,共定性确定9种药物,分别是乙氧喹啉、恩诺沙星、环丙沙星、氧氟沙星、诺氟沙星、甲氧苄啶、磺胺二甲基嘧啶、敌百虫和依维菌素。该方法前处理简单、可同时测定多种药物、检测效率高、筛查定性准确度高。
To monitor the safety of foods in terms of the restricted or banned compounds, it is especially preferable to develop generic methods which are capable of detecting as many different classes of concerned compounds as possible, with reliable confirm ability, satisfied sensitivity, good time-efficiency and high throughput. In this work, a method of ultra-high performance liquid chromatography(UPLC)-quadrupole/exactive orbitrap mass spectrometry was developed for the rapid screening and semi-quantification of 200 drug residues in fish and shrimp. The sample was firstly added with 0.1 mol/L EDTA-Na_2, extracted with acetonitrile and ethyl acetate, cleaned-up by solid-phase extraction using Oasis PRiME HLB column. Then, the target compounds were separated on Thermo AccucoreaQ column(100 mm×2.1 mm×2.6 μm) with a mobile phase consisting of 0.1% formic acid and 0.1% formic acid-acetonitrile. The mass spectrometry data were acquired under Full MS/dd-MS~2 mode. Self-built database screening containing retention time, precursor ion and product ions were performed for qualification. The m/z of precursor ion, retention time(t_R) and fragment ions(FI) were acquired through analysis, while the isotope pattern for precursor was automatically calculated by TraceFinder software. And then, a database containing m/z of precursor ion, t_R, m/z of FI and isotope pattern were built in TraceFinder. Screening of all the analytes were performed by TraceFinder with the self-built database. The rules of screening were established as follow: allowed m/z deviation of precursor ion was 3×10~(-6), allowed t_R deviation was ±15 s, at least one fragment ion match with allowed m/z deviation at 2×10~(-5), and the fit threshold for precursor isotope pattern was 75% with allowed mass deviation at 10~(-5), and allowed intensity deviation less than 25%. The full scan MS data was used for quantification, and the limits of detection was 1-50 μg/kg. The average spiked recoveries of 200 target compounds were 30%-120% with the relative standard derivations of 5%-30%, 70% of which were 5%-15%, and the higher relative standard deviations of 20%-30% were from samples with low spiked concentration. The developed method was successfully applied to the simultaneous determination of 200 drugs residues in fish and shrimp. The practical application showed that 9 compounds including ethoxyquin, enrofloxacin, ciprofloxacin, ofloxacin, norfloxacin, trimethoprim, sulfamethazine, dipterex, ivermectin were identified in 48 actual fish and shrimp samples. As a qualitative and semi-quantitative screening method, the features of simple pretreatment, simultaneous multiresidue detection, high efficiency and accuracy are demonstrated.
To monitor the safety of foods in terms of the restricted or banned compounds, it is especially preferable to develop generic methods which are capable of detecting as many different classes of concerned compounds as possible, with reliable confirm ability, satisfied sensitivity, good time-efficiency and high throughput. In this work, a method of ultra-high performance liquid chromatography(UPLC)-quadrupole/exactive orbitrap mass spectrometry was developed for the rapid screening and semi-quantification of 200 drug residues in fish and shrimp. The sample was firstly added with 0.1 mol/L EDTA-Na_2, extracted with acetonitrile and ethyl acetate, cleaned-up by solid-phase extraction using Oasis PRiME HLB column. Then, the target compounds were separated on Thermo AccucoreaQ column(100 mm×2.1 mm×2.6 μm) with a mobile phase consisting of 0.1% formic acid and 0.1% formic acid-acetonitrile. The mass spectrometry data were acquired under Full MS/dd-MS~2 mode. Self-built database screening containing retention time, precursor ion and product ions were performed for qualification. The m/z of precursor ion, retention time(t_R) and fragment ions(FI) were acquired through analysis, while the isotope pattern for precursor was automatically calculated by TraceFinder software. And then, a database containing m/z of precursor ion, t_R, m/z of FI and isotope pattern were built in TraceFinder. Screening of all the analytes were performed by TraceFinder with the self-built database. The rules of screening were established as follow: allowed m/z deviation of precursor ion was 3×10~(-6), allowed t_R deviation was ±15 s, at least one fragment ion match with allowed m/z deviation at 2×10~(-5), and the fit threshold for precursor isotope pattern was 75% with allowed mass deviation at 10~(-5), and allowed intensity deviation less than 25%. The full scan MS data was used for quantification, and the limits of detection was 1-50 μg/kg. The average spiked recoveries of 200 target compounds were 30%-120% with the relative standard derivations of 5%-30%, 70% of which were 5%-15%, and the higher relative standard deviations of 20%-30% were from samples with low spiked concentration. The developed method was successfully applied to the simultaneous determination of 200 drugs residues in fish and shrimp. The practical application showed that 9 compounds including ethoxyquin, enrofloxacin, ciprofloxacin, ofloxacin, norfloxacin, trimethoprim, sulfamethazine, dipterex, ivermectin were identified in 48 actual fish and shrimp samples. As a qualitative and semi-quantitative screening method, the features of simple pretreatment, simultaneous multiresidue detection, high efficiency and accuracy are demonstrated.
引文
[1] 孔聪,周哲,汪洋,等. 超高压液相色谱串联高分辨质谱筛查渔用投入品中禁用药物[J]. 分析化学,2017,45(2):245-252. KONG Cong, ZHOU Zhe, WANG Yang, et al. Screening of chemical drugsin fishery inputs by ultrahigh performance liquid chromatography-orbitrap high resolution mass spectroscopy[J]. Chinese J Anal Chem, 2017, 45(2): 245-252 (in Chinese).
[2] 孙晓杰,王苏玥,卢立娜,等. 水产养殖投入品中药物残留检测方法研究及现状分析[J]. 食品安全质量检测学报,2016,7(5):1 984-1 990. SUN Xiaojie, WANG Suyue, LU Li'na, et al. Detection methods for drug residues in aquacultureinputs and its status analysis[J]. Journal of Food Safety and Quality, 2016, 7(5): 1 984-1 990(in Chinese).
[3] VALESE A C, MOLOGNONI L, DE SOUZA N C, et al. Development, validation and different approaches for the measurement uncertainty of a multi-class veterin ary drugs residues LC-MS method for feeds[J]. J Chromatogr B, 2017, 1 053: 48-59.
[4] 周贻兵,吴坤,刘利亚,等. 超高效液相色谱-串联质谱法测定鱼中孔雀石绿、结晶紫及其代谢物[J]. 质谱学报,2013,34(5):294-299. ZHOU Yibing, WU Kun, LIU Liya, et al. Determination of malachite gree, crystal violet and their metabolites contents in fishes by UPLC-MS/MS[J]. Journal of Chinese Mass Spectrometry Society, 2013, 34(5): 294-299(in Chinese).
[5] CHEN D, YU J, TAO Y, et al. Qualitative screening of veterinary anti-microbial agents in tissues, milk, and eggs of food-producing animals using liquid chromatography coupled with tandem mass spectrometry[J]. J Chromatogr B, 2016, 1 017-1 018: 82-88.
[6] 于慧娟,张姗姗,张晓玲,等. 同位素稀释-液相色谱-串联质谱法同时测定水产品中雌激素、孕激素和雄激素[J]. 质谱学报,2014,25(2):129-137. YU Huijuan, ZHANG Shanshan, ZHANG xiaoling, et al. Simultaneous determination of the estrogen, progestogen and androgen in aquatic products by isotope dilution-LC-MS/MS[J]. Journal of Chinese Mass Spectrometry Society, 2014, 25(2): 129-137(in Chinese).
[7] 陈丽娜,宋风瑞,郑重,等. 人参中农药多残留的超高效液相色谱-串联质谱分析方法研究[J]. 化学学报,2012,70(7):843-851. CHEN Lina, SONG Fengrui, ZHENG Zhong, et al. Studies on the determination method of pesticide multi-residues in ginseng by ultra performance liquid chromatography tandem mass spectrometry[J]. Acta Chimica Sinica, 2012, 70(7): 843-851(in Chinese).
[8] 熊春兰,郭平,占春瑞,等. 高效液相色谱-串联质谱法同时测定水产品中8类38种兽药残留[J]. 分析测试学报,2013,32(2):193-198. XIONG Chunlan, GUO Ping, ZHAN Chunrui, et al. Simultaneous determination of 8 kinds of 38 veterinary drug residues in aquatic products by high performance liquid chromatography coupled with tandem mass spectrometry[J]. Journal of Instrumental Analysis, 2013, 32(2): 193-198(in Chinese).
[9] 于慧娟,蔡友琼,黄宣运,等. 10种麻痹性贝类毒素的固相萃取及液相色谱-串联质谱测定法[J]. 海洋渔业,2015,37(4):364-370. YU Huijuan, CAI Youqiong, HUANG Xuan-yun, et al. Determination of 10 poisoning toxins by SPE and liquid chromatography-tandem mass spectrometry[J]. Marine Fisheries, 2015, 37(4): 364-370(in Chinese).
[10] LOPES R P, REYES R C, ROMERO-GONZáLEZ R, et al. Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra high performance liquid chromatography coupled to tandem mass spectrometry[J]. Journal of Chromatography B, 2012, 895: 39-47.
[11] 卜明楠,石志红,康健,等. QuEChERS结合LC-MS/MS同时测定虾肉中72种兽药残留[J]. 分析测试学报,2012,31(5):552-558. BU Mingnan, SHI Zhihong, KANG Jian, et al. Simultaneous determination of 72 veterinary drugs in shrimp by modified QuEChERS and high performance liquid chromatography-tandem mass spectrometry[J]. Journal of Instrumental Analysis, 2012, 31(5): 552-558(in Chinese).
[12] MUNARETTO J S, MAY M M, SAIBT N, et al. Liquid chromatography with high resolution mass spectrometry for identification of organic contaminants in fish fillet:screening and quantification assessment using two scan modes for data acquisition[J]. J Chromatogr A, 2016, 1 456: 205-216.
[13] ZHANG Y, LI X, LIU X, et al. Multi-class, multi-residue analysis of trace veterinary drugs in milk by rapid screening and quantification using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry[J]. J Dairy Sci, 2015, 98: 8 433-8 444.
[14] 陈建波,马琳,黄兰淇. 超高效液相色谱-飞行时间质谱快速筛查农业投入品中违规添加的氨基甲酸酯类农药[J]. 现代农药,2016,15(3):25-28. CHEN Jianbo, MA Lin, HUANG Lanqi. UPLC-TOF MS method for rapid screening of carbamate insecticides in agricultural inputs[J]. Modem Agrochemicals, 2016, 15(3): 25-28(in Chinese).
[15] RAJSKI ?, GóMEZ-RAMOS M D M, FERNáNDEZ-ALBA A R. Large pticide multiresidue screening method by liquid chromatography-orbitrap massspectrometry in full scan mode applied to fruit and vegetables[J]. J Chromatogr A, 2014, 1 360: 119-127.
[16] LEóN N, PASTOR A, YUSà V. Target analysis and retrospective screening of veternary drugs, ergot alkaloids, plant toxins and other undesirable subatances in feed using liquid chromatography-high resolution massspectrometry[J]. Talanta, 2016, 149: 43-52.
[17] 郭萌萌,国佼,吴海燕,等. 通过式固相萃取-液相色谱-四极杆/静电场轨道阱高分辨质谱快速筛查鱼肉中全氟化合物及其前提物质[J]. 分析化学,2016,44(10):1 504-1 513. GUO Mengmeng, GUO Jiao, WU Haiyan, et al. Rapid screening and confirmation of perfluorinated compounds and their precursors in fish muscle by solid phase extraction purification and liquid chromatography coupled with quadrupole/exactive orbitrap mass spectrometry[J]. Chinese J Anal Chem, 2016, 44(10): 1 504-1 513(in Chinese).
[18] JIA W, CHU X, CHANG J, et al. High-throughput untargeted screening of veterinary drug residues and metabolites in tilapia using high resolution orbitrap mass spectrometry[J]. Anal Chim Acta, 2017, 957: 29-39.
[19] 牛增元,罗忻,王风美,等. UPLC-LTO/Orbitrap MS 快速筛查确证化妆品中89种禁用物质[J]. 质谱学报,2016,37(3):201-212. NIU Zengyuan, LUO Xin, WANG Fengmei, et al. Fast screening and confirmation of 89 prohibited compounds in cosmetics using UPLC-LTQ/Orbitrap MS[J]. Journal of Chinese Mass Spectrometry Society, 2016, 37(3): 201-212(in Chinese).
[20] 杨璐齐,李蓉,高永清,等. UPLC-Q-Orbitrap HRMS 同时检测水产品中磺胺和喹诺酮类药物残留[J]. 食品与机械,2016,33(8):38-43. YANG Luqi, LI Rong, GAO Yongqing, et al. Simultaneous determination of sulfonamides and quinolones in fish by UPLC-Q/Orbitrap HRMS[J]. Food & Machinery, 2016, 33(8): 38-43(in Chinese).
[21] TURNIPSEED S B, STOREY J M, LOHNE J J, et al. Wide-scope screening method for multiclass veterinary drug residues in fish, shrimp, and eel using liquid chromatography-quadrupole high-resolution mass spectrometry[J]. Journal of Agricultural and Food Chemistry, 2017, 65(34): 7 252-7 267.
[2] 孙晓杰,王苏玥,卢立娜,等. 水产养殖投入品中药物残留检测方法研究及现状分析[J]. 食品安全质量检测学报,2016,7(5):1 984-1 990. SUN Xiaojie, WANG Suyue, LU Li'na, et al. Detection methods for drug residues in aquacultureinputs and its status analysis[J]. Journal of Food Safety and Quality, 2016, 7(5): 1 984-1 990(in Chinese).
[3] VALESE A C, MOLOGNONI L, DE SOUZA N C, et al. Development, validation and different approaches for the measurement uncertainty of a multi-class veterin ary drugs residues LC-MS method for feeds[J]. J Chromatogr B, 2017, 1 053: 48-59.
[4] 周贻兵,吴坤,刘利亚,等. 超高效液相色谱-串联质谱法测定鱼中孔雀石绿、结晶紫及其代谢物[J]. 质谱学报,2013,34(5):294-299. ZHOU Yibing, WU Kun, LIU Liya, et al. Determination of malachite gree, crystal violet and their metabolites contents in fishes by UPLC-MS/MS[J]. Journal of Chinese Mass Spectrometry Society, 2013, 34(5): 294-299(in Chinese).
[5] CHEN D, YU J, TAO Y, et al. Qualitative screening of veterinary anti-microbial agents in tissues, milk, and eggs of food-producing animals using liquid chromatography coupled with tandem mass spectrometry[J]. J Chromatogr B, 2016, 1 017-1 018: 82-88.
[6] 于慧娟,张姗姗,张晓玲,等. 同位素稀释-液相色谱-串联质谱法同时测定水产品中雌激素、孕激素和雄激素[J]. 质谱学报,2014,25(2):129-137. YU Huijuan, ZHANG Shanshan, ZHANG xiaoling, et al. Simultaneous determination of the estrogen, progestogen and androgen in aquatic products by isotope dilution-LC-MS/MS[J]. Journal of Chinese Mass Spectrometry Society, 2014, 25(2): 129-137(in Chinese).
[7] 陈丽娜,宋风瑞,郑重,等. 人参中农药多残留的超高效液相色谱-串联质谱分析方法研究[J]. 化学学报,2012,70(7):843-851. CHEN Lina, SONG Fengrui, ZHENG Zhong, et al. Studies on the determination method of pesticide multi-residues in ginseng by ultra performance liquid chromatography tandem mass spectrometry[J]. Acta Chimica Sinica, 2012, 70(7): 843-851(in Chinese).
[8] 熊春兰,郭平,占春瑞,等. 高效液相色谱-串联质谱法同时测定水产品中8类38种兽药残留[J]. 分析测试学报,2013,32(2):193-198. XIONG Chunlan, GUO Ping, ZHAN Chunrui, et al. Simultaneous determination of 8 kinds of 38 veterinary drug residues in aquatic products by high performance liquid chromatography coupled with tandem mass spectrometry[J]. Journal of Instrumental Analysis, 2013, 32(2): 193-198(in Chinese).
[9] 于慧娟,蔡友琼,黄宣运,等. 10种麻痹性贝类毒素的固相萃取及液相色谱-串联质谱测定法[J]. 海洋渔业,2015,37(4):364-370. YU Huijuan, CAI Youqiong, HUANG Xuan-yun, et al. Determination of 10 poisoning toxins by SPE and liquid chromatography-tandem mass spectrometry[J]. Marine Fisheries, 2015, 37(4): 364-370(in Chinese).
[10] LOPES R P, REYES R C, ROMERO-GONZáLEZ R, et al. Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra high performance liquid chromatography coupled to tandem mass spectrometry[J]. Journal of Chromatography B, 2012, 895: 39-47.
[11] 卜明楠,石志红,康健,等. QuEChERS结合LC-MS/MS同时测定虾肉中72种兽药残留[J]. 分析测试学报,2012,31(5):552-558. BU Mingnan, SHI Zhihong, KANG Jian, et al. Simultaneous determination of 72 veterinary drugs in shrimp by modified QuEChERS and high performance liquid chromatography-tandem mass spectrometry[J]. Journal of Instrumental Analysis, 2012, 31(5): 552-558(in Chinese).
[12] MUNARETTO J S, MAY M M, SAIBT N, et al. Liquid chromatography with high resolution mass spectrometry for identification of organic contaminants in fish fillet:screening and quantification assessment using two scan modes for data acquisition[J]. J Chromatogr A, 2016, 1 456: 205-216.
[13] ZHANG Y, LI X, LIU X, et al. Multi-class, multi-residue analysis of trace veterinary drugs in milk by rapid screening and quantification using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry[J]. J Dairy Sci, 2015, 98: 8 433-8 444.
[14] 陈建波,马琳,黄兰淇. 超高效液相色谱-飞行时间质谱快速筛查农业投入品中违规添加的氨基甲酸酯类农药[J]. 现代农药,2016,15(3):25-28. CHEN Jianbo, MA Lin, HUANG Lanqi. UPLC-TOF MS method for rapid screening of carbamate insecticides in agricultural inputs[J]. Modem Agrochemicals, 2016, 15(3): 25-28(in Chinese).
[15] RAJSKI ?, GóMEZ-RAMOS M D M, FERNáNDEZ-ALBA A R. Large pticide multiresidue screening method by liquid chromatography-orbitrap massspectrometry in full scan mode applied to fruit and vegetables[J]. J Chromatogr A, 2014, 1 360: 119-127.
[16] LEóN N, PASTOR A, YUSà V. Target analysis and retrospective screening of veternary drugs, ergot alkaloids, plant toxins and other undesirable subatances in feed using liquid chromatography-high resolution massspectrometry[J]. Talanta, 2016, 149: 43-52.
[17] 郭萌萌,国佼,吴海燕,等. 通过式固相萃取-液相色谱-四极杆/静电场轨道阱高分辨质谱快速筛查鱼肉中全氟化合物及其前提物质[J]. 分析化学,2016,44(10):1 504-1 513. GUO Mengmeng, GUO Jiao, WU Haiyan, et al. Rapid screening and confirmation of perfluorinated compounds and their precursors in fish muscle by solid phase extraction purification and liquid chromatography coupled with quadrupole/exactive orbitrap mass spectrometry[J]. Chinese J Anal Chem, 2016, 44(10): 1 504-1 513(in Chinese).
[18] JIA W, CHU X, CHANG J, et al. High-throughput untargeted screening of veterinary drug residues and metabolites in tilapia using high resolution orbitrap mass spectrometry[J]. Anal Chim Acta, 2017, 957: 29-39.
[19] 牛增元,罗忻,王风美,等. UPLC-LTO/Orbitrap MS 快速筛查确证化妆品中89种禁用物质[J]. 质谱学报,2016,37(3):201-212. NIU Zengyuan, LUO Xin, WANG Fengmei, et al. Fast screening and confirmation of 89 prohibited compounds in cosmetics using UPLC-LTQ/Orbitrap MS[J]. Journal of Chinese Mass Spectrometry Society, 2016, 37(3): 201-212(in Chinese).
[20] 杨璐齐,李蓉,高永清,等. UPLC-Q-Orbitrap HRMS 同时检测水产品中磺胺和喹诺酮类药物残留[J]. 食品与机械,2016,33(8):38-43. YANG Luqi, LI Rong, GAO Yongqing, et al. Simultaneous determination of sulfonamides and quinolones in fish by UPLC-Q/Orbitrap HRMS[J]. Food & Machinery, 2016, 33(8): 38-43(in Chinese).
[21] TURNIPSEED S B, STOREY J M, LOHNE J J, et al. Wide-scope screening method for multiclass veterinary drug residues in fish, shrimp, and eel using liquid chromatography-quadrupole high-resolution mass spectrometry[J]. Journal of Agricultural and Food Chemistry, 2017, 65(34): 7 252-7 267.