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1 5 3 Vol. 5 No Journal of Food Safety and Quality Mar., 2014 * 陈燕, 李晓雯, 刘畅 (, ) 摘要 : 目的 - (HPLC-MS/MS) 方法 0.25 mol/l (ph 4.8), - 结果 10~300 μg/kg, r> 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 , 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> 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 : , : C 8 H 12 N 4 O 5, 1,,,,, [1],,, * 通讯作者 :,,, 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 , China. ccchangchang@hotmail.com

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

2 906 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]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 方法样品处理 g( 0.1 g) 50 ml, 0.25 mol/l (ph 4.8) 5 ml, (1 μg/ml) 50 μl, β μl, 1 min, h, 4600 r/min 10 min, 50 ml ph 8.5±0.1, 5 ml, 30 /min 1 min, 4600 r/min 5 min, PBA (100 mg, 3 ml), 3 ml 3% - 3 ml 0.25 mol/l (ph 8.5)

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.

3 3, : 907,,, 3 ml 0.25 mol/l (ph 8.5), 10 min, 1 ml 0.1 mol/l,, HPLC-MS/MS 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; : 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酶解的选择,, [13 ] [14 ], 37, 1 h 16 h, 16 h 1 h 10%, 1h 3% h 固相萃取柱选择, HLB MCX PBA, HLB, MCX ( 5%), PBA( ), 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, 流动相的选择,,,,, - 0.1% 5 mmol/l 质谱条件的选择 1 µg/ml,, m/z m/z 95.9 m/z 133.1,, 1 m/z 133.1,, ; m/z 113.0,, 3.3 方法学验证选择性 20,

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.

4 908 5,,, 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) C Fig. 2 The total ion chromatogram of ribavirin 3 Fig. 3 The total ion chromatogram of ribavirin spiked in blank samples

the determination of ribavirin and ribavirin- 13 C 5 (m/z) (m/z) DP(v) EP(V) CE(eV) CXP(V) 113 21

5 3, : 线性范围 μg/kg,,, μg/kg, 回收率 2.0 g, (1 μg/ml) μl, μg/kg 3, 2, 90%, 精密度 2.0 g,, μg/kg 3, 6,,, 3 3, 3%, 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%

6 910 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 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) μg/kg, 97.0%~109.4%, 0.7%~2.8% (n=6), Agilent Eclipse XDB-C 18,,, 参考文献 [1],,. ( 17 )[M]. :, Chen XQ, Jin YY, Tang G. New Pharmacology (17th Edition) [M]. Beijing: People's Medical Publishing Press, [2],,. [J]., 2010, (6): Cheng ZG, Fu WL, Liu SZ. Research progress of application of animal antiviral drugs [J]. J Tradit Chin Vet Med, 2010, (6): [3] Austin RK, Hintz M. Sensitive radio-immunossay for the broad-spectrum antiviral agent ribavirin [J]. Anntimicrobiol Agents Chemoth, 1983, 24(5): [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): [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: [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): [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: [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): [9],. LC-MS [J]. ( ), 2011, (8): Wu F, Ding L. Study on the bioequivalence of ribavirin granules in healthy volunteers by LC-MS [J]. Med Inform, 2011, (8): [10],,,. [J]., 2011, (09): 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): [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): 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): [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): [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): ( 责任编辑 : 张宏梁 )

( 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.

7 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 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 ccchangchang@hotmail.com

Microsoft Word 陈燕-.doc

Microsoft Word 陈燕-.doc 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)