5 2 Vol. 5 No. 2 2014 2 Journal of Food Safety and Quality Feb., 2014 - 陈燕, 李晓雯, 王柯 * *, 刘畅 (, 201203) 摘要 : 目的 - 方法 -1% (1:1, v/v),, - 结果 5.0 μg/kg 1~100 μg/kg, r>0.999, 89.0%~98.7%, 0.3%~2.5%(n=5), 1.9%~5.5%(n=5) 结论 关键词 : ; - ; ; Determination of amantadine in chicken by liquid chromatography-tandem mass spectrometry CHEN Yan, LI Xiao-Wen, WANG Ke *, LIU Chang * (Shanghai Institute of Food and Drug Control, Shanghai 201203, China) ABSTRACT: Objective To establish a method for the determination of amantadine in chicken by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods The residues of amantadine in the test samples were extracted with methanol-1% trichloroacetic acid (1:1, v/v). After being cleaned up with SPE column, the analytes were detected by LC-MS/MS under multiple reaction monitoring (MRM) via positive ionisation mode. Results The limit of quantification was 5.0 μg/kg. The method showed a good linearity over the range of 1~100 μg/kg for amantadine with r>0.999. The average recoveries at 3 spiked concentration levels were between 89.0%~98.7%, while the relative standard deviations (RSD) were between 0.3%~2.5% (n=5) and bias were between 1.9%~5.5% (n=5). Conclusion The established method is highly sensitive, accurate and reproducible. It is suitable for the detection of amantadine in chicken. KEY WORDS: amantadine; liquid chromatography-tandem mass spectrometry; solid phase extraction; chicken 1 引言, [1], [2] (amantadine), [3.3.1.13,7] * 通讯作者 :,, E-mail: wksifdc@163.com,,, E-mail: ccchangchang@hotmail.com *Corresponding author: WANG Ke, Chief Pharmacist, Shanghai Institute for Food and Drug Control, No.1500, Zhangheng Road, Shinan District, Shanghai 201230, China. E-mail: wksifdc@163.com LIU Chang, Ph.D, Associate Chief Pharmacist, Shanghai Institute for Food and Drug Control, No.1500, Zhangheng Road, Shinan District, Shanghai 201230, China. E-mail: ccchangchang@hotmail.com
2, : - 409-1-,, 1, CAS : 665-66-7, : C 10 H 18 NCl, : 206~208, : 1.066 g/cm 3,,, [3],,, 2005 2005 33, 1 Fig. 1 The chemical structure of amantadine,,,, (gas chromatography, GC) [4] (capillary gas chromatography, CE) [5] - [6] [7] [8,9], LC-MS/MS [10, 11],,,,, 2 材料与方法 2.1 仪器与设备 Agilent 1290 ( Agilent ); 5500QTRAP ( AB Sciex ); MS3 ( IKA ); R40 ( Thermo ); ( Organomation ); Milli-Q ( Milli-pore ); Oasis MCX (150 mg, 6 ml)( Waters ) 2.2 材料与试剂 ( 97%) Aldrich -D 15 TRC (HPLC ) (HPLC ) Merck (HPLC ) Sigma Milli-Q ( Millipore ) :, 100.0 μg/ml :, 1.0 μg/ml : -D 15, 100.0 μg/ml, 1.0 μg/ml 2.3 方法 2.3.1 样品处理 2.3.1.1 提取 2.0 g( 0.01 g), 50 ml, 100 μl, 10 ml -1% (1:1, v/v), 1 min, 30 min 4600 r/min 15 min 50 ml, 5 ml 3 min, 4600 r/min 5 min, 5 min 2.3.1.2 净化 MCX 3 ml 3 ml
410 5, 0.5 ml/min, 3 ml 2% 3 ml, 5 min, 5 ml - - (5:80:15, v/v/v),, 45 1.0 ml,,, 13000 r/min 10 min, - 2.3.2 液相色谱条件 : Agilent Eclipse XDB-C 18 (250 mm 4.6 mm, 5.0 μm) : (A), 0.1% (B); : 0~3.0 min, (A) 20% 95%; 3.0~5.5 min, (A) 95%; 5.5~7.0 min, (A) 95% 20%; 7.0~10.0 min, (A) 20% : 500 μl/min; : 10 μl; : 40 2.3.3 质谱条件 : (electrospray ionization, ESI); : ; : 5.5 kv; : 500 ; (GS1): 50 psi; (GS2): 50 psi; (CUR): 35 psi; (CAD): 5 psi; : (MRM) 3 结果与分析 3.1 前处理条件的优化,,, ;,,, 0.1% 0.1%,, SPE, -0.1% (1:1, v/v),,, ;,,,,, HLB (500 mg, 6 ml) MCX (150 mg, 6 ml), MCX (150 mg, 6 ml) HLB (500 mg, 6 ml), MCX (150 mg, 6 ml), -,, 3.2 HPLC-MS/MS 3.2.1 色谱条件的选择, Agilent Eclipse XDB-C 18 (4.6 mm 250 mm, 5 μm),,,,,, - 0.1% 5 mmol/l 3.2.2 质谱条件的选择, 1 µg/ml,, m/z 135.2 m/z 93.1, 2 m/z 93.1,, 93.1 (MRM) : (CUR): 35 psi; (IS): 5500 V; (TEM): 500 ; : 50 psi; : 50 psi MRM 1 3, ( ) 4 3.3 方法学验证 3.3.1 待测试样的选择,
2, : - 411 Table 1 表 1 金刚烷胺质谱检测参数 MS parameters for the determination of amantadine (m/z) (m/z) DP(V) EP(V) CE(eV) CXP(V) 152 * 135.2 24 90 12 93.1* 38 20 14 2 Fig. 2 The proposed product ions used in MRM of amantadine 3.3.2 特异性 ( Sigma), 5 10 50 µg/kg,,, 5 25,, 3.3.3 检测限, 1 μg/ml 10 µl,, 5 μg/kg,, 88.5%~98.6%, 18.6~28.4 6.8 pg( 10 μl), 5.0 μg/kg 3.3.4 线性范围 1~100 μg/kg,, ( 2), 1~100 μg/kg, 3 MRM Fig. 3 The MRM chromatogram of blank sample
412 食品安全质量检测学报 Fig. 4 图 4 金刚烷胺阳性样本 MRM 色谱图 The MRM chromatogram of positive sample of amantadine 图 5 金刚烷胺干扰性实验 Fig. 5 Interference test for amantadine 第5卷
2, : - 413 Table 2 表 2 金刚烷胺的线性方程 相关系数 Linear equation, correlation coefficient of amantadine (r) (μg/kg) Y= 0.00871X+0.05 0.9994 1~100 表 3 金刚烷胺回收率试验结果 Table 3 The result of recovery of amantadine (µg/kg) (%) (%) RSD%(n=5) 5 38.6 93.4 5.4 10 40.1 89.0 1.9 50 42.5 98.7 4.3 3.3.5 精密度 15, 2.0 g, 1 μg/ml 10 20 100 μl, 5,, 0.3%~2.5%, 1.9%~5.5%, 3.3.6 准确度 15, 2.0 g, 1 μg/ml 10 20 100 μl, 5( ) 10 50 μg/kg 5,, : 38.6%~42.5%, 93.4%~98.7%,, 3 4 结论 - -1% (1:1, v/v),, -, 5.0 μg/kg, 89.0%~98.7%, 0.3%~2.5% (n=5), 1.9%~5.5%(n=5) 参考文献 [1]. [J]., 2012, 10: 188. Hu YB. The application of antiviral drugs in the veterinary clinic[j]. Tehnical Advisor for Animal Husbandry, 2012, 10: 188. [2],,,. [J]., 2011, 5: 19 25. Li W, Lv L, Qu J, et al. Design and synthesis the amantadine derivatives, and study the Anti-avian influenza virus activity of it[j]. Chin J Med Chem, 2011, 5: 19 25. [3]. [J]., 2007, (3): 56 57. Wang DK. The clinical application and research of Veterinary antivirals[j]. Stockbreeding Market, 2007, (3): 56 57. [4] Sioufi A, Pommier F. Gas chromatographic determination of amantadine hydrochloride in human plasma and urine [J]. J Chromatogr B, 1980, 183(1): 33 39. [5] Rakestraw D. Determination of amantadine in human plasma by capillary gas chromatography using electron-capture detection following derivatization with pentafluorobenzoyl chloride [J]. J Pharm Biomed A, 1993, 11(8): 699 703. [6] Zhou FX, KruU IS, Feibush B. Direct determination of amantadine in plasma and urine with automated solid phase derivatization [J]. J Chromatogr B, 1993, 619(1): 93 101. [7] Nagara J, Rahavendran SV, Karnes HT, et al. Visible diode laser induced fluorescence detection for capillary electrophoresis analysis of amantadine in human plasma following precolumn derivatization with Cy5.29.OSu [J]. J Pharm Biomed Anal, 1998, l8(3): 411 420. [8] Xu M, Ju W, Xia X, et al. Determination of rimantadine in rat plasma by liquid chromatography/electrospray mass spectrometry and its application in a pharmacokinetic study [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2008, 864(1-2): 123 128.
414 5 [9] Suski M, Bujak-Gizycka B, Madej J, et al. Evaluation of morphine metabolism in presence of amantadine and dextromethorphan in human serum by high performance liquid chromatography coupled with mass spectrometry (LC/MS) method [J]. Folia Med Cracov, 2008, 49(3 4): 111 121. [10],,,. /LC-MS/MS [J]., 2009, (6): 42 45. Yun H, Zhang ZH, Luo SL, et al. Determination of amantadine in foodstuffs of animal origin by SPE/LC-MS/MS[J]. Mod Instrum, 2009, (6): 42 45. [11],,,. - 5 [J]., 2012, 30(12): 1253 1259. Liu ZC, Yang F, Yu KJ, et al. Multi-residue determination of five antiviral drugs in chichen tissues by liquid chromatographyelectrospray ionization tandem mass spectrometry[j]. Chin J Chromatogr, 2012, 30(12): 1253 1259. ( 责任编辑 : 赵静 ) 作者简介 陈燕, 主管药师, 主要研究方向为食品与药品分析 E-mail: rammychen@hotmail.com 王柯, 主任药师, 主要研究方向为食品 药品分析 E-mail: wksifdc@163.com 刘畅, 博士, 副主任药师, 主要研究方向为食品 药品分析 E-mail: ccchangchang@hotmail.com