5 3 Vol. 5 No. 3 2014 3 Journal of Food Safety and Quality Mar., 2014 * 陈 燕, 李 晓 雯, 刘 畅 (, 201203) 摘 要 : 目 的 - (HPLC-MS/MS) 方 法 0.25 mol/l (ph 4.8), - 结 果 10~300 μg/kg, r>0.999 10 μg/kg, 97.0%~109.4%, ( ) 0.7%~2.8% (n=6) 结 论, 关 键 词 : ; - ; ; Study on detection methods of ribavirin in tissues of livestock and poultry CHEN Yan, LI Xiao-Wen, LIU Chang * (Shanghai Institute of Food and Drug Control, Shanghai 201203, China) ABSTRACT: Objectives To establish a method for the determination of ribavirin in tissues of livestock and poultry by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methods The residues of ribavirin in the samples were extracted with buffer (ammonium acetate ph 4.8). After being cleaned up with SPE column, concentrated and reconstituted, the residues were detected by HPLC-MS/MS under multiple reaction monitoring (MRM) via positive ionisation mode. Results The limit of quantification was 10 μg/kg. The method showed a good linearity over the range of 10~300 μg/kg for ribavirin with r>0.999. The average recoveries at 3 spiked concentration levels were between 97.0%~109.4%, while the relative standard deviations (RSD) were between 0.7%~2.8% (n=6). Conclusion The established method is highly sensitive, accurate and reproducible. It is suitable for the detection of ribavirin in tissues of livestock and poultry. KEY WORDS: ribavirin; liquid chromatography-tandem mass spectrometry; solid phase extraction; tissues of livestock and poultry 1 引 言, : 1-β-D- -1H-1,2,4,- -3-, CAS : 36791-04-5, : C 8 H 12 N 4 O 5, 1,,,,, [1],,, * 通 讯 作 者 :,,, E-mail: ccchangchang@hotmail.com *Corresponding author: 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
906 5 [2] 5500QTRAP ( AB Sciex ); MS3 ( IKA ); R40 ( Thermo ); ( Organomation ); Milli-Q ( Milli-pore ); PBA (100 mg, 3 ml)( Agilent ) 2.2 材 料 与 试 剂 1 Fig. 1 The chemical structure of ribavirin 2005 560 [2005]33, ( ) ( ),,,,, [3] [4] [5] [6] [7][8] [9] [10], [11 ] [12],, -, 2 材 料 与 方 法 2.1 仪 器 与 设 备 Agilent 1290 ( Agilent ); ( 97%) - 13 C 5 TRC (HPLC ) (HPLC ) β- - Merck (HPLC ) Sigma ( ) Milli-Q ( Millipore ) (100 μg/ml) : 10 mg, 100 ml,,, (1 μg/ml) : (100 μg/ml) 1.0 ml 100 ml,, (1 μg/ml) : 1-13 C 5 ( 1 mg) - (1:1, v/v) 100 ml,, 5.0 ml 50 ml,, 2.3 方 法 2.3.1 样 品 处 理 2.3.1.1 2.0 g( 0.1 g) 50 ml, 0.25 mol/l (ph 4.8) 5 ml, (1 μg/ml) 50 μl, β- - 20 μl, 1 min, 37 16 h, 4600 r/min 10 min, 50 ml ph 8.5±0.1, 5 ml, 30 /min 1 min, 4600 r/min 5 min, 2.3.1.2 PBA (100 mg, 3 ml), 3 ml 3% - 3 ml 0.25 mol/l (ph 8.5)
3, : 907,,, 3 ml 0.25 mol/l (ph 8.5), 10 min, 1 ml 0.1 mol/l,, HPLC-MS/MS 2.3.2 液 相 色 谱 条 件 : Agilent Eclipse XDB-C 18 (250 mm 4.6 mm, 5.0 μm); : 0.1 % 5 mmol/l (A), (B); : 0~3.0 min, (B) 5% 60%; 3.0~5.5 min, (B) 60%; 5.5~7.0 min, (B) 60% 5%; 7.0~10.0 min, (B) 5%; : 600 μl/min; : 10 μl; : 30 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 供 试 品 溶 液 制 备 的 研 究 3.1.1 酶 解 的 选 择,, [13 ] [14 ], 37, 1 h 16 h, 16 h 1 h 10%, 1h 3% 37 16 h 3.1.2 固 相 萃 取 柱 选 择, HLB MCX PBA, HLB, MCX ( 5%), PBA( ), 3.1.3 定 量 方 法 的 选 择,,, 30% - 13 C 5,, 90%, 3.2 液 相 色 谱 - 串 联 质 谱 条 件 的 确 定 3.2.1 色 谱 柱 的 选 择 HILIC (100 mm 2.1 mm, 3.5 μm) AMIDE(100 mm 2.1 mm, 1.8 μm) Agilent Eclipse XDB-C 18 (250 mm 4.6 mm, 5 μm), XDB-C 18, 3.2.2 流 动 相 的 选 择,,,,, - 0.1% 5 mmol/l 3.2.3 质 谱 条 件 的 选 择 1 µg/ml,, m/z 113.0 m/z 95.9 m/z 133.1,, 1 m/z 133.1,, ; m/z 113.0,, 3.3 方 法 学 验 证 3.3.1 选 择 性 20,
908 5,,, 2 3 3.3.2 最 低 检 测 限,,, ( >3) 10 μg/kg, 3 表 1 利 巴 韦 林 利 巴 韦 林 内 标 质 谱 检 测 参 数 Table 1 MS parameters for the determination of ribavirin and ribavirin- 13 C 5 (m/z) (m/z) DP(v) EP(V) CE(eV) CXP(V) 113 21 19 245.1 95.9 70 9 40 45 133.1 12 33-13 C 5 249.9 95.9 70 9 49 12 2 Fig. 2 The total ion chromatogram of ribavirin 3 Fig. 3 The total ion chromatogram of ribavirin spiked in blank samples
3, : 909 3.3.3 线 性 范 围 10 20 50 100 200 300 μg/kg,,, 10 300 μg/kg, 0.9999 3.3.4 回 收 率 2.0 g, (1 μg/ml) 20 200 600 μl, 10 100 300 μg/kg 3, 2, 90%, 3.3.5 精 密 度 2.0 g,, 10 100 300 μg/kg 3, 6,,, 3 3, 3%, 3.3.6 方 法 应 用 90 ( ) 300, 4, 17~1037 μg/kg, 4, 4 结 果 -, β- -, (ph 4.8), PBA, 4 Fig. 4 The chromatogram of LOD of ribavirin 表 2 利 巴 韦 林 回 收 率 测 试 结 果 (n=5) Table 2 The recovery results of ribavirin (n=5) (μg/kg) % RSD% % RSD% 10 97.0 2.9 97.8 2.1 100 109.4 3.7 105.3 2.5 300 102.0 2.3 102.9 1.8
910 5 表 3 猪 肉 中 利 巴 韦 林 的 精 密 度 (n=6) Table 3 The precision of ribavirin in porks (n=6) % μg/kg 10 2.7 2.5 100 1.0 0.7 300 2.8 0.8 表 4 利 巴 韦 林 阳 性 样 品 的 检 出 结 果 Table 4 Positive results for the real case (μg/kg) 1 133 2 3 17 3 477 4 1037 10 μg/kg, 97.0%~109.4%, 0.7%~2.8% (n=6), Agilent Eclipse XDB-C 18,,, 参 考 文 献 [1],,. ( 17 )[M]. :, 2011. Chen XQ, Jin YY, Tang G. New Pharmacology (17th Edition) [M]. Beijing: People's Medical Publishing Press, 2011. [2],,. [J]., 2010, (6): 17 19. Cheng ZG, Fu WL, Liu SZ. Research progress of application of animal antiviral drugs [J]. J Tradit Chin Vet Med, 2010, (6): 17 19. [3] Austin RK, Hintz M. Sensitive radio-immunossay for the broad-spectrum antiviral agent ribavirin [J]. Anntimicrobiol Agents Chemoth, 1983, 24(5): 696 701. [4] Hamma M, Jayewardene AL, Gambertoglio J, et al. High-performance liquid chromatographic determination of ribavirin in whole blood to assess disposition in erythrocytes [J]. Antimicrob Agents Chemoth, 1999, 43(11): 2716 2719. [5] Roboz J, Suzuki R. Determination of 1-β-D-ribofuranosyl-1,2, 4-trizole-3-carboxamide (Virazole) in blood and urine by chemical ionization-mass fragmentography [J]. J Chomatogr, 1978, 160: 169 179. [6] Yeh LT, Nguyen M, Dadgostari S, et al. LC-MS/MS method for simultaneous determination of viramidine and ribavirin levels in monkey red blood cells [J]. J Pharm Biomed Anal, 2007, 43(3): 1057 1064. [7] Henk VDL, Jan WCA, Wim TK, et al. Determination of ribavirin in human serum using liquid chromatography tandem mass spectrometry [J]. Talanta, 2012, 88: 385 390. [8] Liu YF, Xu C, Yan RZ, et al. Sensitive and specific LC-MS/MS method for the simultaneous measurements of viramidine and ribavirin in human plasma [J]. J Chromatogr B, 2006, (832): 17 23. [9],. LC-MS [J]. ( ), 2011, (8): 28 29. Wu F, Ding L. Study on the bioequivalence of ribavirin granules in healthy volunteers by LC-MS [J]. Med Inform, 2011, (8): 28 29. [10],,,. [J]., 2011, (09): 38 41. Cui CF, Chen CH, Lin HD, et al. Study on the Determination method of ribavirin illegally added in the veterinary traditional Chinese herb medicine powders [J]. Chin J Vet Drug, 2011, (09): 38 41. [11]. LC-MS/MS [J]., 2013, 34(7): 8 9. Wang WX. Analysis of ribavirin residues in chicken by LC-MS/MS [J]. Shandong J Anim Sci Vet Med, 2013, 34(7): 8 9. [12],,,. - [J]., 2008, 42(7): 22 25. Zhu YL, Shao DJ, Jiang TM, et al. Determination of the total amount of ribavirin and its metabolite residues in chicken liver by HPLC-MS/MS [J]. Chin J Vet Drug, 2008, 42(7): 22 25. [13] Yeh LT, Nguyen M, Dadgosteri S, et al. LC-MS/MS method for simultaneous determination of viramidine and ribavirin levels in monkey red blood cells [J]. J Pharm Biomed Anal, 2007, 43(3): 1057 1064. [14] Lin CC, Yeh LT, Luu T, et al. Pharmacokinets and metabolism of [ 14 C]ribavirin in rats and cynomolgus monkeys [J]. Antimicrobiol Agents Chemoth, 2003, 47(4): 1395 1398. ( 责 任 编 辑 : 张 宏 梁 )
3, : 911 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 刘 畅, 博 士, 副 主 任 药 师, 主 要 研 究 方 向 为 食 品 与 药 品 分 析 E-mail: ccchangchang@hotmail.com