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1 7 2 Vol. 7 No Journal of Food Safety and Quality Feb., 2016 马帅, 冯晓元, 韩平, 戴莹, 平华, * 李杨, 王纪华 (, ( ),, ) 摘要 : 目的 (UPLC-MS/MS) 方法 PSA+C 18, UPLC-MS/MS (MRM), / (50:50, V:V) 0.1% 结果 1~200 µg/kg, (r 2 ) , (S/N=3) µg/kg µg/kg ~103.6%, RSD 7.7 结论, 关键词 : ; ; ; ; ; Determination of estrone, 17β-estradiol and 17α-ethinylestradiol in soil by ultra-high performance liquid chromatography-tandem mass spectrometry with dispersive solid phase extraction MA Shuai, FENG Xiao-Yuan, HAN Ping, DAI Ying, PING Hua, LI Yang, WANG Ji-Hua * (Beijing Research Center for Agricultural Standards and Testing, Risk Assessment Lab for Agro-products (Beijing), Ministry of Agriculture, Beijing Municipal Key Laboratory of Agriculture Environment Monitoring, Beijing , China) ABSTRACT: Objective To establish a method for the determination of estrone, 17β-estradiol and 17α-ethinylestradiol in soil by ultra-high performance liquid chromatography-tandemmass spectrometry (UPLC-MS/MS). Method According to dispersive solid phase extraction method, the soil samples were extracted by acetonitrile, purified by PSA+C 18 and analyzed by UPLC-MS/MS under the multiple reaction monitoring (MRM) mode with methanol/acetonitrile(50:50, V:V) and 0.1% ammonia water as mobile phase. Results Good linear relationships were obtained in the concentration range of 1.0~200 µg/kg with high correlation coefficients over , and the limits of detection and quantification for 3 kinds of estrogens were 基金项目 : ( ) ( ) ( ) (ZBZXKFKT201503) Fund: Supported by the Special Fund for Agro-scientific Research in the Public Interest ( ) and the Open Project of the Risk Assessment Lab for Agro-products (Beijing), Ministry of Agriculture (ZBZXKFKT201503) * 通讯作者 :,, wangjh@nercita.org.cn *Corresponding author: WANG Ji-Hua, Professor, Beijing Research Center for Agricultural Standards and Testing, Beijing , China. wangjh@nercita.org.cn

2 , and µg/kg, respectively. The average recoveries ranged from 82.9% to 103.6% under the addition levels of 10, 50 and 500 µg/kg and the relative standard deviation was below 7.7%. Conclusion This method is simple, rapid, sensitive and solvent saving, which is suitable for the determination of estrogens in soil samples. KEY WORDS: soil; dispersive solid phase extraction; ultra-high performance liquid chromatography-tandem mass spectrometry; estrone; 17β-estradiol; 17α-ethinylestradiol 1 引言, (estrone, E1) 17β- (17β-estradiol, E2) (17α-ethinylestradiol, EE2) [1] 90% [2,3], E1 E2 EE2 - (LogKow) 2.45~4, [4],,, [5], (0.1 ng/l) [6,7],,, (matrix solid-phase dispersion extraction, MSPD) (pressurized solvent extraction, PLE) [8] 6 ; Albero [9] - (MSPD-GC-MS) ; [10-13] ; Salvia [14] QuEChERS, QuEChERS Anastassiadas 2003,, [15], QuEChERS,, QuEChERS (QuEChERS-UPLC-MS/MS), 2 材料与方法 2.1 仪器与试剂 ACQUITY -Xevo TQ ( Waters ); ( Sigma ); Milli-Q ( Mimpore ); ( ); MH-3 ( ) E1 E2 EE2 Dr. Ehrensorfer, 1 mg/ml, -20, ; (HPLC, Fisher ); (HPLC, Sigma ); (, ), h; C 18 (40 μm, YMC ); PSA (40 μm, ) 2.2 土壤的提取和净化 (0~20 cm),,, 60 5 g 50 ml, 10 ml 15 ml ; 1.5 g NaOAc 6 g MgSO 4, 30 s, 30 s, 200 r/min 20 min, 5000 r/min 5min 1.5 ml 2 ml, 150 mg MgSO 4 25 mg PSA+25 mg C 18, 1 min, r/min 5 min, 1 ml, 0.22 μm, 4

3 2, : UPLC-MS/MS 分析 : (ESI), 150, 2.2 KV, 400, 800 L/h, (MRM), E1 E2 EE2 1 : Waters ACQUITY UPLC BEH C 18 (2.1 mm 100 mm, 1.7 μm), 40, 4, 5 µl, 0.3 ml/min, 2 3 结果与分析 3.1 质谱条件的优化, (ESI), E1 E2 EE2, E1 E2 EE2 ESI -, [M-H] -,,,,, 液相色谱条件的选择, E1 E2 EE2,, / (50:50,V:V) 3 0.1% 5 mmol/l 3, 0.1% 0.1% - / (50:50,V:V), 土壤提取方法的优化 AOAC (1.5 g NaOAc 6 g MgSO4) [16] EN15562 (1 g NaOCitrate 4 g MgSO 4 1 g NaCl 0.5 g disodium citrate sesquihydrate) [17] 2 [18], (PSA:150 mg MgSO 4 50 mg PSA; C 18 :150 mg MgSO 4 50 mg C 18 ; PSA+ C 18 :150 mg MgSO 4, 25 mg PSA+25 mg C 18 ), PSA+C 18, 99.4%~103.6%, (RSD) 3.1%~6.0%( 2) Table 1 表 1 目标化合物的质谱多反应监测 (MRM) 优化参数 Optimized parameters of the target compounds using MS-MS with MRM mode /min (m/z) (m/z) /V /ev E *, , 31 EE *, , 33 E *, , 48 : * Table 2 表 2 流动相梯度条件 Gradient mobile phase for UPLC /min (ml/min) /% 0.1% /%

508 7 Fig. 1 1 MRM E1 E2 EE2 (20150820-40) (20150820-46) The total ion chromatograms in MRM mode for estrogens standard (20150820-40) and sample spiked with standards (20150820-46) 3 1~200 µg/kg, 0.

4 508 7 Fig. 1 1 MRM E1 E2 EE2 ( ) ( ) The total ion chromatograms in MRM mode for estrogens standard ( ) and sample spiked with standards ( ) 3 1~200 µg/kg, ~0.9986,, E1 E2 EE2 (LOD) µg/kg 3.5 方法的准确度和精密度 Fig. 2 2 (n=3) Effect of different adsorbents on the recoveries of estrogens (n=3) 3.4 方法的线性范围和检出限 1~200 µg/kg E1 E2 EE2, 5 g 50 ml, 0.5 ml µg/l,,, 6,, %~ 103.6%, (RSD) 1.7%~7.7%, ( 3) 3.6 实际样品测定, 3 4, 5, 1.15~10.81 µg/kg, EE2, 71.4%

5 2, : Table 3 表 3 目标化合物的线性方程相关系数检出限定量下限与加标回收率 (n=6) Linear equations, correlation coefficients (r), limits of detection (LOD), and spiked recoveries for target compounds (n=6) /(µg/kg) /% RSD% 10 µg/kg 50 µg/kg 500 µg/kg E2 Y= X EE2 Y= X E1 Y= X 表 4 实际土壤样品中 3 种雌激素的含量 (n=3) Table 4 Concentrations of 3 kinds of estrogen in soil (n=3) (µg/kg) E ND ND ND 2.54 EE ND ND 3.83 E ND ND 7.63 ND ND 4 结论 E1 E2 EE2 - PSA+C 18,,,,,, 参考文献 [1] Khetan SK, Collins TJ. Human pharmaceuticals in the aquatic environment: a challenge to green chemistry [J]. Chem Rev, 2007, 107(6): [2] Khanal SK, Xie B, Thompson ML, et al. Fate, transport and biodegradation of natural estrogens in the environment and engineered systems [J]. Environ Sci Technol, 2006, 40 (21): [3],,,. [J]., 2010, 30 (6): Han W, Li YX, Yang M, et al. Effects, sources and behaviors of environmental androgens [J]. Acta Ecol Sin, 2010, 30 (6): [4]. EE2 [D]. :, Wang C. Study on electrocatalysis of EE2 and estrogenic activity of oxidation products [D]. Harbin: Harbin Institute of Technology, [5] Gibson R, Smith MD, Spary CJ, et al. Mixtures of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents [J]. Environ Sci Technol, 2005, 39(8): [6] Shappell NW, Hyndman KM, Bartell SE, et al. Comparative biological effects and potency of 17α and 17β-estradiol in fathead minnows [J]. Aquatic Toxicol, 2010, 100 (1): 1 8. [7] Froehner S, Machado KS, Stefan E, et al. Occurrence of selected estrogens in mangrove sediments [J]. Mar Pollut Bull, 2012, 64(1): [8],,,. SPE-HPLC-MS/MS [J]., 2011, 30 (3): Na GS, Chen T, Zhang YM, et al. Determination of six estrogens in water and sediment by SPE-HPLC-MS/MS [J]. Mar Environ Sci, 2011, 30 (3): [9] Albero B, Sánchez-Brunete C, Miguel E, et al. Analysis of natural-occurring and synthetic sexual hormones in sludge - amended soils by matrix solid-phase dispersion and isotope dilution gas chromatography tandem mass spectrometry [J]. J Chromatogr A, 2013 (1283): [10] Hansen M, Krogh KA, Halling-Sorensen B, et al. Determination of ten steroid hormones in animal waste manure and agricultural

510 7 soil using inverse and integrated clean-up pressurized liquid extraction and gas chromatography-tandem mass spectrometry [J]. Anal Methods, 2011, 3:1087 1095. [11],,,. [J]., 2013, 29(3): 380 385.

[12] Wang YH, Wang QY, Hu LF, et al. Occurrence of estrogens in water, sediment and biota and their ecological risk in Northern Tai hu Lake in China [J]. Environ Geochem Health, 2015, (37):147 156.

6 510 7 soil using inverse and integrated clean-up pressurized liquid extraction and gas chromatography-tandem mass spectrometry [J]. Anal Methods, 2011, 3: [11],,,. [J]., 2013, 29(3): Chen CB, Wang N, Kong DY, et al. Method for simultaneous determination of sulfonamides and hormone-type drugs in water and soil [J]. J Ecol Rural Environ, 2013, 29(3): [12] Wang YH, Wang QY, Hu LF, et al. Occurrence of estrogens in water, sediment and biota and their ecological risk in Northern Tai hu Lake in China [J]. Environ Geochem Health, 2015, (37): [13] Tso J, Dutta S, Inamdar S, et al. Simultaneous analysis of free and conjugated estrogens, sulfonamides, and tetracyclines in runoff water and soils using solid-phase extraction and liquid chromatography-tandem mass spectrometry [J]. Agric Food Chem, 2011, 59, [14] Salvia MV, Vulliet E, Wiest L, et al. Development of a multi-residue method using acetonitrile-based extraction followed by liquid chromatography tandem mass spectrometry for the analysis of steroids and veterinary and human drugs at trace levels in soil [J]. J Chromatogr A, 2012, 1245: [15] Anastassiades M, Lehotay SJ, Stajnbaher D, et al. Fast and easy multiresidue method employing acetonitrile extraction/ partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce [J]. J AOAC Int, 2003, 86(2): [16] AOAC Official Method Pesticides residues in food by acetonitrile extraction and partitioning with magnesium sulphate[z]. AOAC International, 2007: 9. [17] AFNOR (NF EN 15662), Méthode polyvalente de détermination des résidus des pesticides par GC-MS et SL/SM/SM avec extraction/partition avec de l acétonitrile et nettoyage par SPE disperses (Méthode QuEChERS) [Z]. France, 2009: 11. [18] Padilla Sáncheza JA, Plaza Bolañosab P, Romero Gonzáleza R, et al. Application of a quick, easy, cheap, effective, rugged and safe based method for the simultaneous extraction of chlorophenols, alkylphenols, nitrophenols and cresols in agricultural soils, analyzed by using gas chromatography triple quadrupole-mass spectrometry/mass spectrometry [J]. J Chromatogr A, 2010, 1217: 作者简介 ( 责任编辑 : 金延秋 ) 马帅, 硕士, 主要研究方向为农田环境与农产品质量安全 mashuai @126.com 王纪华, 博士, 研究员, 主要研究方向为农产品质量检测技术与信息技术的交叉与集成 wangjh@nercita.org.cn

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0.

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0. 118 217 5 38 1 DOI 1.3969/j.issn.5-6521.217.1.28 Food Research And Development 1 2 1 2 1 2 1 2 1 2 1 2 1. 731 2. 731 - UPLC-MS/MS 5. 1 mol/l ProElut PXC 5 % - Thermo Hypersil Gold mm 2.1 mm 1.9 μm ESI