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1 5 11 Vol. 5 No Journal of Food Safety and Quality Nov., , 吴佳俊 2, 黄文雯 1, 肖陈贵 3, 岳振峰 3 1,, 林群声 2, 陈荔 (1.,, ; 2., ; 3., ) 1, 2* 摘要 : 目的 (TTX) 方法, 0.03 mol/l, 0.22 µm Waters ACQUITY UPLC BEH Amide(2.1 mm 100 mm, 1.7 µm), 0.1% MilliQ, 结果 5~1000 ng/ml, 83.3%~93.2%, 47.8%~48.9%, 5 µg/kg, 2 0µg/kg 结论 TTX LC-MS/MS,, TTX 关键词 : ; ; ; Determination of tetrodotoxin by high performance liquid chromatography-tandem mass spectrometry WU Jia-Jun 1, 2, HUANG Wen-Wen 1, XIAO Chen-Gui 3, YUE Zhen-Feng 3, LAM Kwan-Sing 1, 2 1, 2*, CHAN Lai (1. Shenzhen Key Laboratory for Sustainable Use of Marine Biodiversity, Research Centre for the Oceans and Human Health, City University of Hong Kong Shenzhen Research Institute, Shenzhen , China; 2. State Key Laboratory in Marine Pollution, City University of Hong Kong, Hong Kong , Hong Kong S.A.R.; 3. Key Laboratory of Detection Technology R & D on Food Safety, Food Inspection and Quarantine Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen , China) ABSTRACT: Objective To establish a new method for determination of tetrodotoxin (TTX) in puffer fish by high performance liquid chromatography - tandem mass spectrometry. Methods The sample was sonicated after homogenizing with 0.03 mol/l acetic acid solution. After a low-speed centrifugation, the supernatant was mixed with equivalent methanol. With a subsequent high-speed centrifugation, the supernatant was filtered through a 0.22 μm membrane filter before injection. LC-MS/MS analysis was conducted by Waters ACQUITY UPLC BEH Amide (2.1 mm 100 mm, 1.7 μm) column with the mobile phase of 0.1% formic acid in water and acetonitrile at positive mode. Results There was a good linearity of TTX in the range of 5~1000 ng/ml. The matrix recoveries was 85.8%~93.2% and the absolute recoveries was 47.8%~48.9%. The detection limit is 5 * 通讯作者 :,,, leochan@cityu.edu.hk *Corresponding author: CHAN Lai, Research Professor, Shenzhen Key Laboratory for Sustainable Use of Marine Biodiversity, Research Centre for the Oceans and Human Health, City University of Hong Kong Shenzhen Research Institute, Shenzhen , China. leochan@cityu.edu.hk

2 µg/kg and the limit of quantification is 2 0 µg/kg. Conclusion Compared with the previous methods, the new method is simple, rapid, accurate, sensitive and at low cost for the determination of TTX in aquatic products. KEY WORDS: high performance liquid chromatography-tandem mass spectrometry; tetrodotoxin; aquatic products; hydrophilic interaction chromatography column 1 引言 (tetrodotoxin, TTX) [1], [2],,,, 60% [3], 100 [4], 10~45 min, 4~8 h [5], TTX ( 800 ng/g) [6] TTX (LC-MS/MS) [7-9] (HPLC-FLD) [10-14] LC-MS/MS, TTX,,,,, HPLC-FLD,,, LC-MS/MS,, 2 材料与方法 2.1 仪器与试剂 AB Sciex 3200QTRAP (AB SCIEX, USA), Agilent 1290(Agilent Technologies, USA); ; Waters ACQUITY UPLC BEH Amide (2.1 mm 100 mm, 1.7 µm); IKA T18 (IKA, Germany); Beckman Coulter Allegra X-22R Microfuge 22R (Beckman Coulter, USA); CREST 950D (CREST, USA) (Sigma-Aldrich, St.Louis,MO, USA), ; RCI Labscan (Bangkok, Thailand),, TTX (1 mg/ ) Calbiochem, 99.8% : TTX, 0.03 mol/l 200 μg/ml 2.2 检测条件 : Waters ACQUITY UPLC BEH Amide (2.1 mm 100 mm, 1.7 µm), 25, 10 μl : A 0.1% MilliQ, B 0.1% ; 0~3 min, B 90% 60%; 4~9 min, B 60%; 9~12 min, B 60% 90%, B 90% 4 min, :,, 5500 V, 400,, 10 L/min TTX m/z 320.1>302.2, DP 51, CE 33; m/z 320.1>162.1, DP 51 CE 样品前处理 (Takifugu obscurus, ) 2 g( 0.01 g) 50 ml, TTX (10 µg/ml)4 µl 80 µl 160 µl, 20 ng/g 400 ng/g 800 ng/g 30 min, 10 ml 0.03 mol/l, 10 s, 3 10 min 4500 r/min, 4 10 min 0.5 ml, 0.5 ml, r/min, 4 20 min 0.22 µm, -

3 11, : 结果与分析 3.1 分析色谱柱的选择 TTX, [6, 16-17], 4 (HILIC ) TTX, Tosoh TSK-GEL amide 80 (2.0 mm 250 mm, 5 µl) ; Phenomenex Kinetex HILIC (3.0 mm 150 mm, 2.6 µl) ; Merck ZIC-HILIC (2.1 mm 150 mm, 3.5µL) Waters ACQUITY UPLC BEH Amide (2.0 mm 250 mm, 1.7 µl) Tosoh ( 1 a), TTX,,,, Tosoh 2200 Psi, UPLC-MS/MS Phenomenex Merck, TTX, phenomenex,, ( 1 b 1 c) Waters ( 1 d) TTX HILIC, TTX Tosoh Waters, Merck Phenomenex, TTX, = ( / ) 100% = ( / ) 100% 1,, 80%,, Tosoh Waters 50%, 5% Phenomenex Merck, TTX,,, Tosoh,, Tosoh Merck Phenomenex, TTX,,,, Waters, Waters 1 4 (a-tosoh, b-phenomenex, c-meck, d-waters, 400 ng/g) Fig. 1 Chromatograms of the spiked sample on the four kinds of columns (a- Tosoh column, b- Phenonomenex column, c-merck column, d- Waters column, spike concentration-400 ng/g)

5 11, : 表 1 四根色谱柱的空白鱼肉加标样品回收率 ( 加标量为 400 ng/g) Table 1 Spike recovery by the nontoxic puffer fish of the four columns (spike concentration is 400 ng/g) (%) (%) Tosoh 80±5 50±3 Phenomenex 85±5 6±2 Merck 80±10 5±4 Waters 85±3 45±1 3.2 流动相和进样溶剂的选择, 0.1% 1% 0.1% +5 mmol/l 0.1% +10 mmol/l TTX, TTX,,,, 0.1% (0.03 mol/l ), TTX, 2(a) Waters, TTX 50%, TTX,, 2(b) 50% TTX 3.3 样品前处理方法的选择 TTX,,, TTX [18-20], , ; SPE,, LC-MS/MS [21-23] (1) TTX, 0.03 mol/l, mol/l 50%, 0.03 mol/l 50%, 2, %~35.7%, 45%, 0.03mol/L (2), 0.03 mol/l, ( 2), (3) SPE, : Waters Oasis MAX, Waters Sep-Pak C 18, Phenomenex Strata NH 2, Supelco PSA and Supelco Envi-carb 5, SPE 33% 5.9% 14.8% 29.4% 12.7% MAX, 33%, 50%,, SPE (4) TTX,,,, (5), TTX, (6),,, 3.4 方法的线性定量限及检测限, 50% 5 ng/ml 20 ng/ml 200 ng/ml 600 ng/ml 1000 ng/ml, 8 ng/ml 80 ng/ml 300 ng/ml 500 ng/ml 1000 ng/ml, 2.2 TTX, 3,, 48% S/N 3 TTX 5 ng/g; S/N 10 TTX 20 ng/g, [6]

6 方法回收率, TTX ng/g, 3, 3, ,, 3 Fig. 2 2 TTX (a-, b-50% ) Effect of MeOH on the peak shape of TTX (a-without MeOH, b-50%meoh)

7 11, : 表 2 不同提取剂及热处理操作对提取效果的影响 (n=3) Table 2 The extraction efficiency of different extract agents and hot treatment (n=3) RSD RSD 0.03 mol/l 88.8% 1.7% 46.2% 1.7% mol/l 50% 85.9% 2.5% 33.5% 2.4% 0.03 mol/l 50% 81.7% 0.6% 35.7% 0.6% 0.03 mol/l 82.3% 1.0% 43.2% 3.0% 82.7% 2.6% 43.1% 2.6% Hexane 85.3% 0.3% 39.8% 0.3% CH 2 CL % 3.1% 30% 3.1% 表 3 TTX 加标样品回收率分析 Table 3 Spike recoveries of TTX ( -R, -A, -RSD)-% R- RSD A- RSD R A R A R A 20 ng/g_ ng/g_ ng/g_ ng/g_ ng/g_ ng/g_ ng/g_ ng/g_ ng/g_ 结论 3 Fig. 3 Calibration curves in solvent and in matrix, 20 ng/g 81.0%~90.1%, 400 ng/g 81.6%~89.6%, 800 ng/g 87.7%~96.4% 2.8%~5.6% TTX LC-MS/MS, SPE, 83.3%~93.2%, 47.8%~48.9%, 5 µg/kg, 20 µg/kg,,,, [17],,,,, TTX,

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材料与方法仪器 ACQUITY UPLC Xevo TQ-S 三重四级杆液质联用仪 ( 美国 Waters 公司 ); 电喷雾离子源 (ESI), Masslynx 4.1 数据处理系统 (Waters);Thermo 低温离心机 ;KQ

材料与方法仪器 ACQUITY UPLC Xevo TQ-S 三重四级杆液质联用仪 ( 美国 Waters 公司 ); 电喷雾离子源 (ESI), Masslynx 4.1 数据处理系统 (Waters);Thermo 低温离心机 ;KQ [ 应用纪要 ] Rapid identification of 5 illegal additives in slimming health food by Xevo TQ-S 郝刚苏州市食品药品检验所, 苏州 15104 关键词超高效液相色谱 - 质谱, 多反应监测, 减肥类保健食品非法添加, 快速定性定量