本标准规定了进出口蜂王浆及蜂王浆冻干粉中链霉素残留量检测的制样和液相色谱测定方法。本标准适用于进出口蜂王浆及蜂王浆冻干粉中链霉素残留量的检测。 SN/T 1775-2006 进出出口蜂王浆及蜂王浆冻干粉中链霉素残留量检测方法 液相色谱法 SN/T1775-2006 标准下载解压密码：www.bzxz.net

中华人民共和国出入境检验检疫行业标准SN/T 1775--2006
进出口蜂王浆及蜂王浆冻干粉中链霉素残留量检测方法液相色谱法
Determination of streptomyein residues in royal jelly and royal jellpowder for import and export-Liquid chromatographic method2006-04-25发布
中华人民共和国
国家质量监督检验检疫总局
2006-11-15实施
本标准的附录A为资料性断录，
本标准由区家认证认可监督管理委员会提出并归口，本标滩起草单位：中华人民共和国渐江山入境检验检疫局。本标准主起草人：陈笑梅、泡沿超、陈晓霞、二栋、施旭霞刘游口、石黏。本标准系首次发布的出入境检验检疫行业标准。SN/T1775—2006
１范围
进出口蜂王浆及蜂王浆冻于粉中链霉暴残留量检测方法液相色谱法
SN/T 1775—2006
本标雅规定了进山口蜂下浆及炼下浆冻十粉中链每素残留量检测的制祥和液相色谱洲定方汰。本标准尼二进出口蜂正浆及蜂工浆冻干粉中链希素残留量的检测。2制样
2.1试样的制备
将排取的样品充分混勾，均分成两份，分别装人洁净容器内，一份作为留样保存，乃一份作为试样供检测用。在执样和制样的操作过程中，应陆止样品受到污染或发生含量的变化。2.2试样的保存
试栏宜及讨检测，在不能及时检测的情况下，应置于二8℃以下冷冻保存。3测定方法
3.1方法提要
试栏中的铠霉素用稀磷酸提，三氯乙酸沉淀样品中的聋白质，清液过阳离子交换柱和HLB固萃取小柱净化。液相色谱栏后衍生荧光检测器测定样品中的链需素，外标法定量。3.2试剂和材料
除刃有规定外.试剂均为分析纯,水为蒸水或去离子水，3.2.1中醇：液树色谱级试剂
3.2.2乙晴：澈相包谱缀试剂。
3.2.3磷酸：8%
3.2.4乙酸。
3.2.5磷酸氢一钾,
3.2.6磷酸二氢细。
3.2.7氢氧化钠。
3.2. 8 三氯乙酸：
3.2. 9庚烷磺酸钠(C,HNaU.S.H,0):液相色谱纯。3.2.131,2-萘醛-1-磺酸钠。
30为醇水溶液：醇+水（30+70,V/V)3.2. 11
稀磷骏溶浚;pI=2。1009 mL 水中滴加磷殷,在 pII 二调溶液的pI为2.3.2.12
磷酸盐缓冲溶液：0.2mol/I.,pH一8，称取38.46g磷酸氢二钾和1.05g磷酸二氢钾丁烧杯3.2. 13
,加水溶解后定容100L：用磷酸调节溶液H为8,3.2.14氢氧化钠济液:0.2 mol/1.,称取 8 %氢氢化钠溶丁适量水，冉定容至1 G00 ml3.2.15三氯乙酸溶液：5C乐水溶液。称取100&三氛乙酸溶二适量水.再定容至100mL。3.2.16饱和氢氧化钠溶液、
3.2.17 质烷破酸溶被;0.5 mol/1,称取1lg庚烷磺酸钠溶二109 ml.水中。庚烷磺酸钠酸溶液:0. U:r:o1/L.pH 3. 3。称取 2. 2 g庚烷潢酸钠溶于 900 tmL.水巾,滴乙3.2.18
SN/T 1775—2006
酸,在 pH计上调节溶液的 pH为 3. 3，血水容至 1 0 nL.。3.2.19阴离子交换杜：苯磺酸型固相萃敢杜，500 tng31rl.a使用前分别用5Tml.甲酵和10 t:l.水预洗并保持性体湿润，
3.2.23IILB固相萃取柱：500 rmg.3nL。使用前分别用5 mL甲婷和10 ImL水预洗并保诗栏体湿润。
链毒素索标雅品：95.8%。
链霉索标准储密液：精硝称取适量链霉索标准，用水溶解，配制成100eg/mL。4℃催存3.2.23链霉系工作溶液：根据需要.将储备液用0.011mol/L庚烷磺酸钠酸溶凌稀释至适当浓度的工作溶液。
3.3器和设备
3.3.1高效液相色谱仪：配有柱后衍生装置和荧光检测器。3.3.2离心机（400r/rin）。
3.3.3混勾器，
3.3.4真空固相幸取装置.
3. 3. 5p11 :硫牟 0, 02.
3.3.6 H盖塑料离心管:100 ml.。3.4测定步骤
3.4.1提取
称取蜂王浆_0 g（精确至0.01 ）置于 50 rrL容量板中,加 40 rrL稀磷酸溶液在滤器「:混勺,再加人5c%三氯乙酸2rrL,用稀磷酸定容.轻轻滤孕,将溶液转移到离心管中,离心(000 /rnin)5 iz过滤。：取滤液25m展饱和氢氣花钩溶浚调节3为2.0净化而称取蛇+浆冻下粉5g(精确至C.01 g)置二离心管中，加入58 ml.稀磷酸溶液在混匀器上混匀，币川人5c%一氯乙骏2mL,轻轻混匀，离心（40r/min)mir,过滤。段滤液0 ml.用饱和氢氧化钠溶液调节pH为2.0,待净化用。
3. 4.2阳离子交换柱净化
将1述滤液过顽光逊洗好的苯酸型固相萃取杜·分别用5L稀磷酸溶液和1CrrL水淤洗固相举取杆.充云全部渐出液。用30rL磷酸盐缓冲溶液洗脱链需素。在洗脱液中加人05r11/L爽烷酸溶液 3 ml,擢勾,再用磷羧调节洗脱液 pH 3. 3,3. 4. 3HIB 固相萃取柱净化
将上述溶液过琐洗好的 HLB质相萃取性柱,丧用稀磷酸溶液 5 nL 淋洗,在真空负压下,减压抽十5 zrir1弃去全部淋出液。用 5 mL30%中醇水溶液淋洗 IILB固相萃取柱,在真空负压下，减压排干5min。而2ml.下醇溶以1.5mL/min剂流迷洗脱链得素，洗脱液在小于45℃求裕上氮气流收至近下。用0.Clmol/I.质烯碳酸骏溶液溶解并定容至：,Cml,溶液供液柜色岩测定，3. 4.4测定
3.4.4.1色谱条件
谱柱:LiehrospherC5μi,250 innX4,n(内径）,或相当的色谱柱；流动相：称取1._0 庚烷磺酸钠和9.052 1,2-茶酶-4-磺酸钠，溶于 500 1mL乙利水的滤合h)
液（28一72)中，操匀，川乙骏调节溶至pI4.3，当天配制：流速：1.0ml./min
检测波长：激发波长263ml.发射波长435nmz进栏量：1031.
F柱温:50℃;
衍生管:1C =nXC,25 imin(内径）,不诱管或 peek 塑料管：g
h）衍牛剂:0.2 mnl/l.氢氧化钠溶液：i）街生剂流速：0.2ttl/ttti:r；j）衍牛管温漫：℃。
3.4.4.2色谱测定
SN/T 1775—2006
根据样液中链素残留量，选择浓度相近的标雄丁炸济液，标雅工作溶液和样液中链霉素的响应值均做在仪器检测的线性池围内。对标谁！作液和样液等件积参插班样测定。在上述色谱条件下，链霉索的保留冠间约为13.3min标准崩的负谱图参死附录A市图A.1.3.5空自试验
除不加运样外，与接二述少骤进行，3.6结果计算和表述
按式()计算试栏中链霉素含量,计算结果需扣除空白值。X=Axexv
式中：
试样链素的残留量，单位为毫克每「克(rig/kg)：样液穴链霉索的峰面积：
标准二作液链霉素淤度，总位为微克有毫升(μg/inL)：样液最终定容体积，单位为毫升（rmL）：As
标雅二作液牛链霉素的峰面积：最终样液新代哀的示样量，单位为克（）)4测定低限、回收率
4.1测定低限
本方法对峰+浆的测定低限为c.0：ng/kg；刘蜂+浆冻下粉的测定低限为3.02mg/kg。4.2回收率
4.2.1峰土浆样品的添加浓度及其向收率的实验数如下：在添加0,C1 rmg/kg浓度水平时,链霉素的回改率范围为83.7%~92.5%;在添加0,02 rng/kg浓度水平吋,链霉素的回改率范围为 90.1%～98.5%;在漆加0.C5 mg/kg浓度水平时,链宽素的回改率范周为86.1%~-93.8%。4.2.2蜂+浆冻「粉样品的添加滚度改其问收率的实验数据如下：在添加0.C2mg/kg浓度水平附,链霉素的回改率范围为82.0%-~92.0%;在添加0.05 mg/kg浓度水平时，链幕素的回改率范国为85.4%~96.2%；在添加0.10mg/kg浓度水平时，链筛素的回改率范围为85.6%~96.8%(
SV/T 1775—2006
1.[31 A, 1x-26], [:--435
附录A
(资料性附录）
标准品色谱图
strcjpomyci
链霉素标准液相色谱图
Annex Ais an informative annexForeword
SN/T 1775—2006
This standard was proposed by and is under the charge of Certification and Accreditation administra-tion of thePeople's Republic of China.This standard was drafted by Zhejiang Entry-Exit Inspection and Quarantine Bureau of the People'sRepurblic of China.
The main drafters of this standard are Chen Xiaotnei, Chi haachao, Chen Xiaaxia, Wang dong, Liuhaishan, Shi xuxia、and Shi lei.This standard is a professional standard for entry-exit inspection and quarantine promulgated for thefirst time.
SN/T 1775—2006
Determination of streptomycin residues in royal jelly and royal jellypowder for import and exportLiquid chromatographic method1Scope
This standard specifies the method of sampling . sarnple preparation and determination of streptomycin residues in royal jelly and royal jelly powder by high performance liquid chromatographyThis standard is applicable ta the inspectian of streptomycin residues in royal jelly and royal jellypowder.
2Sample preparation
2. 1 Preparation of test sarmpleMix the sample, divide into two equal portions, and then place in clean containers, One uses as apreserved sample and the other uses as a test sample. In the caurse of sampling and sample prepara-tion, precaution must be taken to avoid the contamnination or any factors which may cauise the chan-ges of residue content.
2. 2Storage of sample
The test samples should be detected in time, if they cant, they should be stared belaw - 1'c.3Method of deterrmination
3.1Principle
Streptomycin residue in test sample is extracted with phosphoric acid solution. Precipitate the pro.tein in sample by trichloroacetic acid and purify the filter by cation exchange column and HLB solidphase extraction cartridge, Determine the streptomycin by HPLC with post-column derivation and flu-orescencedetector，using external standard method.3.2 Reagents and materials
Unless otherwise specified, all reagents shauld be analytically pure, water used should be redistilledor deianized.
3.2. 1Methanol: HPLC grade.
3. 2.2Acetonitrile: HPLC grade.3.2.3Phosphoric acid: :85%.
3.2. 4Acetic acid.
Di-potassium hydrogen phosphate.3.2.5
3.2. 6 Potassium dihydrogen phasphate.3.2.7 Sodium hydroxide
3Trichloroacetic acid.
3. 2, 91-Heptane sulphanic acid sodiurn salt (C, Hi, NaO, S. H,O) : HPLC grade.3.2. 101,2-Naphthoquinone-4-sulfonic acid sodium salt.3.2. 11 30% methanol water solution:methanol-water(30+70, V/ V).SN/T 1775—2006
3. 2. 12 Phosphoric acid solution: pH= 2. Adjust pH= 2 by drop-wise addition of phosphoric acid3.2. 13 Phosphate buffer: 0. 2 mol/L. pH = 8. Dissolve 33. 46 g di-potassium hydrogen phosphateand 1. 05 g potassium dihydrogen phosphate in water. Adjust pH=8 with phosphoric acid and dilutedto 1 000 rmL.
3.2. 14 sodium hydroxide: 0. 2 mol/L. Dissolve 8 g sodium hydroxide in 1 000 mL water.3.2.15Trichloroacetic acid solution:50%water solution. Dissalve 100 g Trichlaraacetic acid in100 mL water.
Saturated NaoH solurtion.
1-Heptanesulphonic acid sodium solution: 0. 5 mal/L. Dissolve 11 g 1-Heptanesulphonic3.2. 17
acidsodiumsaltinwateranddiluteto1oomiLwithwater.3.2. 18 1-Heptanesulphonic acid sodium acidic solution: 0. 01 mol/L. pH = 3. 3. Dissolve 2. 2 g1-Heptanesulphonic acid sodium salt in 900 mL water, adjust pH = 3. 3 with acetic acid and dilute to1000 mL withwater.
SV/T 1775—2006
3.2.19 Cation exchange column: aromatic sulphonic solid phase extraction(PE) cartridg, 500 mg,3 mL. Condition aromatic sulphonic sPE cartridge With 5 mL methanol and 10 ml waterbefore using.
3.2.20 HLB Solid phase extraction(SPE)Cartridge: 500 mg,3 mL. Condition HLB SPE cartridgewith5mLmethanoland10mLwaterbeforeusing.Streptornycin standard: 95. 8%.3.2.21
3. 2.22 Streptomycin standard stock solution: Accurately weigh an appropriate amount of strepto-mycin standard and dissolve with water to prepare a standard stock solution of 100 μg/mL, Thisstandard stock solution should be stored at 4'c.3.2.23Streptomycin standard working solution:According to the requirement.pipette adequateamount of standard stock solution and dilute with 0. D1 mol/L 1-Heptanesulphonic acid sodiuim solution to prepare standard working solution of suitable concentrations.3.3Apparatus and equipment
3.3. 1 High-performance liquid chromatograph; equipped with fluorescence detector and post-column derivation apparatus.
3, 3.2 Centrifuge,wwW.bzxz.Net
3.3.3Vortex mixer.
3.3.4Solid phase extraction with vacuum pump.3. 3. 5 pH measurer; Capable of measuring ±0. 02 unit.3.3.6 Plastic centrifuge turbe with cap: 100 mL3. 4Determination Procedure
3.4.1 Extraction
Weigh 10 g royal jelly (accurate ta 0. o1g) in 50ml volumetric flask . Add 40 ml phosphoric acidsolution and agitate or a vortex rnixer unitil the sarnple was dissolved cornpletely. 2. 0 rnL of 50%Trichloroacetic acid solution was addecd to the sample solution and diluted to volume by phosphoricacid solution, Shake lightly and transfer the solution to centrifuge tube. Centrifuge the sample solu-tion for 5 mins(4 000 r/min). The supernatant was filtered , Piptte 25 mL filtrate into a clean contain-er and adjusted it to pH 2. 0 by saturated NaoH solution.8
SN/T 1775—2006
Weigh 5 g royal jelly powder (accurate to 0. 01g) in centrifuge tube. Add 58 mL phosphoric acid so-lution and agitate on a vortex mixer until the sample was dissolved completely. 2. 0 mL of 50% Tri-chloroacetic acid solution was aclded to the sarnple solution, Shake lightly. Centrifuge the sample so-lution for 5 mins (4 000 r/min). The supernatant was filtered , Piptte 30 mL filtrate into a clean con-tainer and adjusted it to pH 2. o by saturated NaoH solution.3. 4. 2Cleanup by cation exchange colurmnDraw the above solution through a per-canditioned aromatic sulphonic SPE cartridge. Wash the SPEcartridge with 5 mL phosphoric acid solution and 10 mL water. Discard all of the above effluents. E-lute streptomycin from the SPE cartridge with 30 mL phosphate buffer, Add 3 mL 0, 5 mol/L 1-Hep-tanesulphonic acid sodium solution to the eluate and shake well, adjust pH to 3. 3 by drop-wise addit-on of phosphoric acid.
3.4.3Cleanup byHLBPEcartridgeLoad the prepared solution into a pre-conditianed HLB SPE cartridge. Wash the HLB SPE cartridgewith 5 mL pH 2 phosphoric acid solution. Dry the HLB SPE cartridge for 5 min by vacuurn purnp.5 mL30% methanol water solution were loaded to the the HLB SPE cartridge and continue dry for5 min. Elute the streptomycin residue with 2 mL methanol into an evaporation flask. Blow it to nearlydry under a nitrogen flow below 45'c. The residue is dissolved in 1. 0 mL 0. a1 mol/L 1-Heptanesul-phonic acid sodiurm solution anid used for liquid chrornatographic determination.3.4.4
Determination
3.4, 4, 1HPLC conditions
Chromatographic column: Lichrospher Cis 5 pm, 250 × 4. 6 mm(id), or equivalent :b) Mobil phase: Dissolve 1. 10g 1-Heptanesulphonic acid sodiurn salt and 0. 052 g 1.2-Naphthoqui-none-4-sulfonic acid sodium salt in 500 mL acetonition -water(28 + 72) . Adjust pH 4. 3 with ace-tic acid. Prepared daily:
Mabil phase flow rate: 1. 0 mL/min:c)
Detected wavelength: Excitation 263 nm, Emitting 435 nm;e)Injection volumn:100 μL;
Column temperature:50'c
Derivation column: 10 m x 0. 25 mm(id), stainless steel or peek plastic tube;Derivation reagent: o. 2 mol/L sodiurn hydroxide solution;h)
Derivation reagentflow rate:0.2mL/min:j) Derivation temperature: 50℃.3, 4, 4, 2HPLC determinationAccording to the approximate concentration of streptomycin in the sample solution, select thestandard working solution with similar concentratian to that of sample solution. The respanses ofSN/T 1775—2006
streptomycin in the standard working salution and the sample solution should be in the linear range ofthe instrumental detection. The standard solution should be randomly injected between the injectionsof sample solution of equal volume, Under the above operating condition, the retention time ofstreptomycin is about 13, 3 min, For the chromatogram of the standard, reference fig A1 in annex A.3.5Blank test
The operation of the blank test is the sarme as that described in the method of determination, butwithout addition of sample.
3. 6 Calculation and expression of resultThe calculation of result is carried ourt according to the following formulaX
As× m
the residue content of streptomycin in the test sample, mg/kg:the peak area of streptomycin in sample solution;the concentration of streptomycin in the standard working solution,μg/mL:the final volume of sample solution, mL;the peak area of streptomycin in the standard working solution#the corresponding mass af the test sample in the final sample salutian, g.4Limitofdetermination and recovery4. 1 Limit of determination
The limit of determination of this method is 0. D10 mg/kg for royal jelly and 0. 020 mg/kg for royaljellypowder
4.2Recovery
4. 2. 1 According to the experiment data, the fortifying concentrations of streptomycin and its corresponding recoveries in royal jelly are:0.010 mg/kg. The recovery of streptomycin is between 83.7% ~94.5%;-0,020 mg/kg. The recovery of streptomycin is between 90,4%~98.5% :-0, 050 mg/kg. The recovery of streptomycin is between 86, 4 % ~93. 8%.4.2.2According to the experiment data, the fortifying concentrations af streptomycin and its cor-responding recoveries in royal jelly powder are:-0. 02 mg/kg. The recovery of streptomycin is between 82. 0 % -94. 0% ; -0. 05 mg/kg. The recovery of streptomycin is between 85, 4% ~96. 2% : 0. 10 mg/kg. The recovery of streptomycin is between 85. 6% ~96. 8%.10
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