4 3 Vol. 4 No. 3 2013 6 Journal of Food Safety and Quality Jun., 2013 朱 * 舟, 刘 桂 华 (, 518055) 摘 要 : 目 的, 方 法,, ph 5.2 SCX, (BSTFA), (SIM) (Scan) NIST, 结 果, 1331 463 11200 4.3 μg/kg 结 论,, 关 键 词 : ; ; ; ; Emergency determination of clenbuterol hydrochloride residue first found in food poisoning accident induced by intake of snake ZHU Zhou, LIU Gui-Hua *. (Shenzhen Center for Disease Control & Prevention, Shenzhen 518055, China) ABSTRACT: Objective Samples concerned two consecutive food poisoning accidents occurred in the same restaurant located at Shenzhen, Guangdong province, were taken and sent to laboratory for emergency determination to find the cause of poisoning. Methods Samples were alkalified and extracted using ethyl acetate, then re-extracted by hydrochloric acid. After adjusted to ph 5.2, the acidic extraction was purified by solid-phase extraction using SCX cartridges. After isolated with BSTFA, clentuberol hydrochloride in the solution was detected by gas chromatography-mass spectrometry. Identification was achieved by electron impact ionization (EI) using selected ion monitoring (SIM) combined with full scan of mass spectrum. Quantification was performed by internal standard calibration using metoprolol. Results The muscle, blood and gall of snake (Vipera ruselli siamensis) were collected from the restaurant where the food poisoning accidents happened, as well as the urine of a patient were determined and the content of clenbuterol hydrochloride were 1331, 463, 11200 and 4.3 μg/kg, respectively. However, there was no positive result of clenbuterol hydrochloride in other food samples including subsequent snake samples collected for traceability. Conclusion The reason of two consecutive food poisoning accidents was finally determined to be the intake of snake (Vipera ruselli siamensis) contaminated by clenbuterol hydrochloride, which was first remarkably reported at home and abroad. * 通 讯 作 者 : E-mail: gliu_686@hotmail.com *Corresponding author: LIU Gui-Hua, Professor, Shenzhen Center for Disease Control & Prevention, No.8, Longyuan Road, Nanshan District, Shenzhen 518055, China. E-mail: gliu_686@hotmail.com
666 4 KEY WORDS: snake; clenbuterol hydrochloride; residue; food poisoning; emergency determination (clenbuterol hydrochloride), 2-[( ) ]-4- -3,5-,,, 2.5 μg [1],,, [2-4] [5] - (GC/MS) 2010 9 1 2,,,,,,,,, 1 材 料 与 方 法 1.1 试 剂 与 仪 器 ( ), 99%( Sigma ); 1000 mg/l, 20 ; 10 mg/l ; 2.5 mg/l ;, Merck ;, ; Supelco (SCX) (3 ml/ 500 mg, Supelco ); (BSTFA, Supelco ) 99.999%( ) (Shimadzu GC 17A/ MS QP5050A, ), (EI) GC solution, ; : DB-5MS 30 m 0.25 mm, 0.25 μm( Agilent ); 0.22 μm ( ); ( IKA ); ( Supelco ); ( IKA ); ( Beckman ); ( ±2, Binder ) 1.2 实 验 方 法 NY/T 468-2006 /,,,, ph 5.2 SCX, (BSTFA) GC/MS, (EI), (SIM) Scan, 1.3 GC/MS 分 析 条 件 : ; 1 μl, ; 250 ; 70 0.6 min, 25 /min 200 6 min, 25 /min 280 5 min, 14 min; 0.9 ml/min : I/F 280 ; 7 min; (EM) 200 V; (EI) 230 ; 150 ; (SIM) m/z 86 262 243 187, m/z 72 223, m/z 86 72; (full scan) m/z 50~350 SIM 1 1.4 样 品 前 处 理 1.4.1 提 取 (5±0.05) g
3, : 667 1 SIM ( 2.5 mg/l; 0.5 mg/l) Fig. 1 SIM chromatogram of clenbuterol (2.5 mg/l) and metoprolol (internal standard, 0.5 mg/l), 15 ml, 5 ml 10%, 10000 r/min 1 min, 6000 r/min 5 min,, 10 ml, 1 min, 6000 r/min 5 min, 5 ml 0.10 mol/l, 1 min, 6000 r/min 5 min,,,, 2.5 mol/l ph 5.2 1.4.2 净 化 SCX 5 ml 5 ml 5 ml 30 mmol/l,, 5 ml 5 ml,, SCX, 5 ml 4%, 1.4.3 氮 气 浓 缩 及 衍 生 化 2.5 mg/l ( ) 100 μl, 40, 50 μl 50 μl BSTFA, 1 min, 80 1 h( ); 0 25 50 100 250 μl 10 mg/l 4.5 ml 4%, 2.5 mg/l 100 μl, 400 μl, 0.22 μm, GC/MS 1.5 定 性 定 量 方 法 2 s,, 3 ( ) ±20% 3 ( ), GC/MS, EI NIST, 0 0.5 1.0 2.0 5.0 mg/l, 0.5 mg/l, (m/z 86) (m/z 72)
668 4 2 结 果 与 分 析 2.1 食 物 中 毒 事 件 回 顾 2010 9 1 2 2 : (1)9 1 7, 8, 7,,, ; (2)9 2 8, 6,,,, [6], 2.2 检 测 结 果 9 3 7 1, ;, ;, 9 10 1, 9 11 1, 21, 29 (0903-1~0903-8, 0910-1~0910-9, 0911-1~ 0911-12), >0.99, 1 29, 0903-5~0903-8, SIM( ), EI NIST, 90% ; ( ), 1331 463 11200 μg/kg; 4.3 μg/kg; 0903-1~0903-4 4, (<2.0 μg/kg), ( ), ( 600 μg/kg),, >80%,, 0903-5~0903-8, 0910-1~0910-9 0911-1~0911-12 21 (<2.0 μg/kg) SIM, NIST 2~5 表 1 29 份 样 品 中 克 伦 特 罗 的 GC/MS 测 定 结 果 Table 1 GC/MS analytical results of clenbuterol in 29 collected samples X (μg/kg) X (μg/kg) 0903-1 <2.0 0910-8 <2.0 0903-2 <2.0 0910-9 <2.0 0903-3 <2.0 0911-1 <2.0 0903-4 <2.0 0911-2 <2.0 0903-5 1331 0911-3 <2.0 0903-6 463 0911-4 <2.0 0903-7 11200 0911-5 <2.0 0903-8 4.3 0911-6 <2.0 0910-1 <2.0 0911-7 <2.0 0910-2 <2.0 0911-8 <2.0 0910-3 <2.0 0911-9 <2.0 0910-4 <2.0 0911-10 <2.0 0910-5 <2.0 0911-11 <2.0 0910-6 <2.0 0911-12 <2.0 0910-7 <2.0 : 2.0 μg/kg
第3期 朱 图2 Fig. 2 空白猪肉及克伦特罗加标猪肉(加标 600 μg/kg)的 SIM 模式色谱图 SIM chromatogram of clenbuterol in blank samples and spiked pork (600 μg/kg) 图3 Fig. 3 舟, 等: 首例蛇中盐酸克伦特罗残留引起的食物中毒应急检测 阴性八步蛇肉(0911-6)及阳性八步蛇肉(0903-5)的 SIM 模式色谱图 SIM chromatogram of clenbuterol in negative (0911-6) and positive (0903-5) snake muscle 669
670 4 Fig. 4 4 Scan EI NIST Full scan spectrum of clenbuterol in standard solution and corresponding similarity report of NIST library (EI source) search Fig. 5 5 0903-5 Scan EI NIST Full scan spectrum of clenbuterol in positive snake (Vipera ruselli siamensis) muscle (0903-5) and corresponding similarity report of NIST library (EI source) search 3 讨 论,, ;, ( ),
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