1、中华人民共和国出入境检验检疫行业标准SN/T 1965-2007 鲤鱼及其制品中磺肢类药物残留量测定方法高效液相色谱法Determination of sulfonamides residues in eels and eel products High performance Iiquid chromatographic method 2007-08-06发布2008-03-01实施二弘32旷嚣iw中华人民共和国发布国家质量监督检验检菇总局r ?, 内Uottom of the container. J沪5.9 Acetic acid. 1% solution in water: Add 1
2、0 mL of acetic acid to a 1 L volumetric flask.dlute to volume with water and mix. to homogeneity. Filter the solution for use by O. 45m aqueous filter. 5.10 Acetic acid.5% solution in water: Add 5.0 mL of acetic acid to a 100 mL volumetric flask.di lute to volume wth water and mix to homogeneity. 5.
3、11 Acetonitrile.50% solution in water: Add 50 mL acetonitrile to a 100 mL volumetric flask.d帽lute to volume with water and mix to homogeneity. 5.12 Hydrochlorc acid.O. 1 mol/L solution in water: Measure 9 mL HCL.add an adequate amount of 10 SN/T 1965-2007 water, disslove and make up to 1 L 5.13 Ammo
4、nium acetate , 5 mol/L solution in water, Weigh 38.5 9 of ammonium acetate in a 100 mL cylinder,add water to dissolve and make up to the mark. 5.14 Eluent, methanol-acetonitrile-5 mol/L ammonium acetate (47.5+47.5+5, V/V/V) , add 47.5 mL of methanol ,47.5 mL of acetonitrile and 5 mL of 5 mol/L ammon
5、ium acetate solution into a 100 mL volumetric flask,mix before use 5.15 Oasis( MCX SPE or equivalent, 150 mg/6 m L. Condition the SPE column with 5 mL of metha nol follow回by5 mL of O. 1 molll:.-hor山ut;rnur11,; tt山5.16 Anhydrous sodium sulfate! Ign阳at650(; for 4 h and store ln a desiccator. / 5.17 Me
6、mbrane filter,aqueous and organic t)1tr ，0.45m./ / y / / 5.18 6 Apparatus and equipment 6. 1 High performance liquid chromatography with UV detector. 6.2日alance.6.3 High speed blender. 6.4 Homogenizer. 11 SN/T 1965-2007 6.5 Centrifuge.3500 r/min. 6.6 Rotary vacuum evaporator. 6.7 Ultrasonic bath. 6.
7、8 Vortex mixer. 6.9 Vacuum pump. 6.10 Solid phase extraction vacuum manifold. 6.11 Brown pear-shaped flask.100 mL. 6.12 Brown heart-shaped flask.25 mL. 6.13 Centrifuge tube.glass.50 mL. 7 Procedure 7. 1 Extraction Weigh 5日ofthe test sample (accurate to O. 01 g) and 5 9 of anhydrous sodium sulfate (5
8、. 16) into a 50 mL glass centrifuge tube.add 20 mL of dichloromethane. Homogenize the sample for 30 s. After centrifugation for 5 min at 3 500 r/min.the supernatant layer was passed through a funnel.which put degrease cotton underlay 1 9 of anhydrous sodium sulfate. to a 100 mL brown pear-shaped fla
9、sk. Rinse the probe by another 50 mL glass centrifuge tube with 20 mL of dichloromethane for 10 s. transfer the rinse solution to the first centrifuge tube. Stir the residues with a glass stick and soni cate for 5 min. Centrifuge for 5 min at 3 500 r/min and pass through the same funnel to combined
10、the supernatants to the same 100 mL brown pear-shaped flask. Evaporate the organic solvent to dryness with rotary evaporator at 38C. 7.2 Clean up 7.2.1 Eels Add 750L of acetonitrile to the residue of the brown pear-shaped flask and vortex to m山.Sonicate for 10 s. Add 750L of 5 % aquecus acetic acid.
11、 vortex to mix. Transfer the solution into a glass tube.add 3 mL of n-hexane saturated with acetonitrile (5.酌.vortex to mix for 30 s and centrifuge for 5 min at 3 500 r/min.discard supematant layer. Add 3 mL of n-hexane saturated with acetonitrile 12 SN/T 1965-2007 to the glass tube. repeat the proc
12、edure. discard supernatant layer. The aqueous layer is filtered through the O. 45rn organic filter for HPLC analysis. 7.2.2 Roasted eels Add 1.50 mL of acetonitrile to the residue of the brown pear-shaped flask and vortex to mix. 50阳，cate for 10 s. Add 1.50 mL of 5 % aqueous acetic acid.vortex to mi
13、x. Transfer the solution into a glass tube.add 3 mL of n-hexane saturated with acetonitrile.vortex to mix for 30 s and centrifuge for 5 min at 3 500 r/min.discard supernatant layer. Add 3 mL of n-hexane saturated with acetonitrile to the glass tube. repeat the procedure. Measure 1.50 mL aqueous laye
14、r into a glass tube.add 5 mL 0.1 mol/L hydrochloric acid. Tranfer the solution from glass tube to the 5PE column (5.15) at a rate less than 0.4 mL/min. Wash the 5PE with 2 mL of O. 1 mol/L hydrochloric acid. followed by 2 mL of methanol. Elute the 5PE with 12 mL eluent(5. 14) at a rate less than O.
15、6 mL/min. Transfer the eluate to a 25 mL brown heart-shaped flask. The eluate is evaporated to nearly dryness with rotary evapora tor at 38 C .supplement to 1.00 mL with acetonitrile-water (5.1 1) and filtered through the 0.45m organic filter for HPLC analysis. 7.3 Determination 7.3.1 HPLC operating
16、 conditions 7.3.1.1 Column: C18.5m particle 5ize.250 mm x 4.6 mm( i. d. ). 7.3.1.2 Mobile phase: 7.3.1.2.1 Mobile phase for determination of eels A: methanol. B: aqueou5 solution of 1 % acetic acid. The gradient program is 5hown in table 1. Table 1一Thegradient program of mobile phase(methanol-aqueou
17、s solution of 1 % acetic acid as mobile phase) Time/min A/% B/% 。20 80 26 50 50 27 20 80 40 20 80 7.3. 1. 2. 2 Mobile phase for determination of roasted eels A: acetonitrile. B: aqueous solution of 1 % acetic acid. The gradient program is shown in table 2. 13 SN/T 1965-27 Table 2-The gradient progra
18、m of mobile phases (acetonitrile aqueous solution of 1 % acetic acid as mobile phase) Time/min 。30 31 40 7.3. 1.3 Flow rate: 1. 0 mL/min. 7. 3. 1. 4 Detect ion wavelength: 265 nm. 7.3.1.5 Column temperature:5飞7.3.1.6 Injection volume: 30L. 7. 3. 2 HPLC determination A/% 15 30 15 15 / / / f 8/% 85 70
19、 85 85 _.,.,/ According to the approximate c伊lcentrationof胃咽可ErIn-5出Iution.selectthe standard work. ing solution with similar responesto that of sample solutioTh responses of the analyte in the standard working solution and t叶和samplesolution should be withinlthe linear range of the instrument detect
20、ion. The standard workingsotmron-丽thsFfT吉飞6Tiltin飞houldbe injected with equal vol-14 Analyte sulfadiazine sulfadimidine sulfameter sulfaquinoxaline 12.7 27.6 sulfadimethoxne Retention time/ 町、In10.2 18.0 25.9 Table 4一Referenceretention times of seven sulfonamides (acetonitrile-aqueous solution of 1
21、% acetic acid as mobile phase) Analyte Retention time/ min Analyte Retention time/ min sulfadiazine 8.1 sulfamerazine 10.7 sulfadimidine 14.4 sulfamonomethoxine 18.1 sulfameter 13.3 sulfadimethoxne 31.9 sulfaquinoxaline 32.6 SN/T 1965-2007 7.3.3 Blank test The operation of the blank test is the same
22、 as the procedures described in the method of determina. tion. but with the omission of sample addition. 8 Calculation and expression of result Calculation the c咱ntentof sulfonamides residues in the test sample by HPLC data processor or ac. cording to the formula (1). TtnMl 由民Italtisnoilld 6e subtra
23、tea from the above result of calcula. ton. l 9 Limit of quantificatio 9. 1 Limit of quantification _.A x C x V / . ( 1 ) ff As X作J/。lution.g.quantification of the method for sulfadiazine. sulfamerazine. sulfadimidine.sulfameter. sulfamonome. thoxine.sulfadimethoxine and sulfaquinoxaline are O. 020 m
24、g/kg in roasted eels 9. 2 Recovery According to the experimental data. the corresponding recoveries of fortified concentrations of eels are shown in tabe 5. The corresponding recoveries of fortified concentrations of roasted eels are shown in table 6. 15 SN/T 1965一2007Table 5-The recoveries of seven
25、 sulf。阳midesin eels Analyte Fortifi由dconcentration/(mg/kgl Recoverv/% 0.0100 86.5-94.4 0.0500 88.4-97.8 sulfadiaZfne 。10085.6-102 。15090.7-1 0.0100 82.0-101 0.0500 87.6-97.6 sulfamerazine 0.100 88.6-106 O. 150 96.0-105 0.0100 84.9-103 0.0500 93.8-116 sulfadimidne 0.100 82.5-107 。15088.0-103 0.0100 8
26、2.2-105 0.0500 85.0-98.0 sulfameter O. 100 90.4-103 0.15日96.7-105 0.0100 91.7-104 0.0500 86.8-98.8 sulfamonomethoxine 口10085.1-102 O. 150 88.7-9日70.0100 88.0-102 0.0500 92.8-102 sulfadimethoxine O. 100 85.0-104 。15093.3-103 0.010 0 88.4-103 0.0500 84.2-99.2 sulfaquinoxaline 。10080.2-93.2 0.150 89.3-
27、97.3 16 SN/T 1965-2007 Table 6一Therecoveries of seven sulfonamides in roasted eels Analyte Fortified concentration/(mg/kg) Reve叩/%o.口20088.1-97.5 sulfadiazine 。.050091.2-97.8 0.100 94.4-98.7 0.0200 82.5-95.5 sulfamerazine 0.0500 83.0-98.8 。10091.5-99.0 口020084.5-95.5 sulfadimidine 0.050 0 80.3-98.8
28、0.100 86.9-99.1 0.0200 85.5-96.5 sulfameter 0.0500 8日4-98.4O. 100 95.9-98.9 0.0200 82.5-95.0 sulfamonomethoxine 0.0500 81.6-99.8 O. 100 81.3-99.0 0.0200 80.5-97.0 sulfadimethoxine 。.050080.6-98.0 。10086.6-96.5 0.0200 83.0-96.5 sulfaquinoxaline 0.0500 81.8-94.0 0.100 82.5-93.4 17 SN/T 1965-2007 0.015
29、 0.010 D 。.0050.000 0.008 0.006 王0.0040.002 0.000 0.00 Annex A (informative annex) Liquid chromatogram of standards XD EC 30.00 35.00 sulfadiazien(SD) ; sulfamerazine(SM1) ; sulfadimidine(SM2) ; sulfamonomethoxine(SMM) ; sulfadimethoxine(SOM) ; sulfaquinoxalineSQXHacetonitrile-aqueous solution of 1% acetic acid as mobile phase). Figure A.2一Liquidchromatogram of 18 中华人民共和国出入境检验检疫行业标准鲤鱼及其制品中磺胶类药物残留量测定方法离效液相色谱法S:-.1/T 1965一-2007 中国标准出版社出版北京复兴门外三里河北街16号邮政编码，100045网址电话，6852394668517548 中国标准出版社秦皇岛印刷厂印刷 开本880X12301/16 印张1.5字数38千字2007年11月第一版2007年11月第一次印刷印数l一2000 书号，155066.2-18246 定价13.00元
