【商检行业标准(SN)】 进出口食醋中苯甲酸、山梨酸检测方法 液相色谱法

中华人民共和国出入境检验检疫行业标准SN/T 2012—2007
进出口食醋中苯甲酸、山梨酸的检测方法液相色谱法
Determination of benzoic acid and sorbic acid in vinegar for export andimportLiquid chromatography
2007-12-24发布
中华人民共和国
国家质量监督检验检疫总局
2008-07-01实施
本标准的附录A为资料性附录。
本标准由国家认证认可监督管理委员会提出并归口。本标准由中华人民共和国山西出人境检验检疫局负责起草本标准主要起草人：宋欢、连庚寅、薛平、杜利君、宋洁、苑莉。本标准系首次发布的检验检疫行业标准SN/T2012—2007
1范围
进出口食醋中苯甲酸、山梨酸的检测方法液相色谱法
本标准规定了进出口食醋中苯甲酸、山梨酸的液相色谱检测方法。本标准适用于食醋中苯甲酸、山梨酸的检测，2检测方法
2.1方法提要
试样在酸性条件下蒸馏，馏出液采用液相色谱紫外检测器直接测定，外标法定量。2.2试剂和材料
除有特殊说明以外，所用试剂均为分析纯，水为去离子水，2.2.1甲醇：高效液相色谱级。
2.2.2乙睛：高效液相色谱级。
2.2.3柠檬酸(C,H.O,·H,O)。
2.2.4柠檬酸三钠(NaC,H,O,·2H,O)。SN/T2012—2007
2.2.5柠檬酸缓冲溶液.5mmol/L：称取柠檬酸0.7g和柠檬酸三钠0.6g加水溶解定容至1000mL酒石酸。
酒石酸溶液，15%：取15g酒石酸用水定容到100mL。2.2.8
消泡剂：硅酮树脂。
碳酸氢钠。
20g/L碳酸氢钠溶液：称取2g碳酸氢钠，加水至100mL，振摇溶解。氯化钠。
苯甲酸标准品：纯度大于等于99%。山梨酸标准品：纯度大于等于99%2.2.14苯甲酸标准储备溶液：准确称取0.1000g苯甲酸，加入5mL碳酸氢钠溶液，加热溶解，用水定容至100mL，储备液浓度为1mg/mL。5山梨酸标准储备溶液：准确称取0.1000g山梨酸，加入5mL碳酸氢钠溶液，加热溶解，用水2.2.15
定容至100mL，储备液浓度为1mg/mL。2.2.16苯甲酸、山梨酸标准混合液：准确移取苯甲酸、山梨酸标准储备液各10.0mL，移入100mL容量瓶中，用水定容。此溶液苯甲酸、山梨酸的浓度均为0.1mg/mL。2.3仪器和设备
2.3.1高效液相色谱仪：配紫外检测器。2.3.2蒸馏装置。
2.4测定步骤
2.4.1试样的处理
准确量取50.0mL试样，加人40g氯化钠.5ml15%酒石酸溶液，1滴消泡剂，加水至约200mL，混匀后，进行蒸馏（蒸馏速度约为2.5mL/min），收集约90mL馏出液，用水定容至100mL。用0.45um的过滤膜过滤后，待液相色谱测定。1
SN/T2012—2007
2.4.2测定
液相色谱条件
色谱柱：Cis柱（150mm×4.6mm，5μm，或相当者）；流动相：甲醇-乙腈-5mmol/L柠檬酸缓冲溶液（10：20：70体积比）；流速：1.0mL/min：
检测波长：230nm；
柱温：30℃：
进样量：25μL。
液相色谱测定
2. 4. 2.2
根据样液中苯甲酸和山梨酸的含量，选定浓度相近的标准混合工作液，保证样品溶液中苯甲酸、山梨酸的响应值在线性范围内，标准工作液与样液等体积参差进样进行测定。在上述色谱条件下，苯甲酸和山梨酸的参考保留时间分别约为5.13min和6.41min。苯甲酸、山梨酸标准溶液色谱图参见附录A.1。
2.4.3空白试验
除不加试样外，均按上述步骤进行。2.5结果计算和表述
用色谱数据处理软件外标法或按式（1)计算，计算结果需扣除空白值：X=
式中：
试样中苯甲酸或山梨酸含量，单位为毫克每升（mg/L）；样液中苯甲酸或山梨酸的峰面积；标准工作液中苯甲酸或山梨酸的浓度，单位为微克每毫升（ug/mL）；Vi
-样液最终定容体积，单位为毫升（mL)；As
标准工作液中苯甲酸或山梨酸的峰面积；试样的体积，单位为毫升（mL）。3方法的测定低限和回收率
3.1测定低限
本方法苯甲酸和山梨酸的测定低限均为0.1mg/L。3.2回收率
3.2.1食醋中苯甲酸的添加浓度及其回收率：添加浓度在0.1mg/L，回收率为88%~104%；添加浓度在5mg/L，回收率为89.0%~99.4%；一添加浓度在50mg/L.回收率为91.2%~101.6%。3.2.2食醋中山梨酸的添加浓度及其回收率：添加浓度在0.1mg/L，回收率为83%~103%；一添加浓度在5mg/L，回收率为87.8%~97.0%；添加浓度在50mg/L回收率为89.5%~98.2%。2
.（1)
苯甲酸；
山梨酸。
附录A
（资料性附录）
标准品液相色谱图
图A.1苯甲酸、山梨酸标准品液相色谱图SN/T2012—2007
SN/T2012—2007
Foreword
AnnexAofthis standardisaninformativeannex.This standardwasproposed by and isunderthecharged of Certification and Accreditation AdministrationofthePeople'sRepublicofChina.This standard was drafted by Shanxi Entry-Exit Inspection and Quarantine Bureauof thePeople's Republic of China.
The standard was mainly drafted by Huan Snog,Geng-Yin Lian,Ping Xue,Li-Jun Du, Jie Song, LiYuan.
This standard is a professional standard for entry-exit inspection and quarantine promulgated forthefirst time.
SN/T2012—2007
Determinationofbenzoicacid and sorbicacid invinegarforexportandimport-
Liquid chromatography
This standard specifiies thedetermination of benzoicacid and sorbic acidbyhigh performance liquidchromatographyinvinegarforexportandimport.This standard is applicable to the determination of benzoic acid and sorbic acid in vinegar.2Methodof determination
Abstract of method
Benzoic acid and sorbic acid wereextacted by distilled in acid solution and determined byhigh per-formanceliquid chromatography,using external standardmethod.2.2
Reagentsandmaterials
Unless otherwise specifie,all the reagents used should be analytical grade and\water\is deionizedwater.
Methanol:HPLCgrade
Acetonitrile:HPLCgrade
Citric acid(C.HeO,H2O).
Sodiumcitrate(NasC.HsO,2H,O)Citric acid buffer.5mmol/L:Weigh0.7g citricacid and 0.6 g sodium citrate,dissolvewith2.2.50
waterin1o0omLvolumetricflask.2.2.6
Tartaric acid.
Tartaric acid solution,15% :Dissolve 15 g tartaric acid with water in 100 mL volumetric5
SN/T2012—2007
Antifoaming agent:silicone.
Sodiumhydrogencarbonate
Sodiumhydrogencarbonatesolution,20g/L:Dissolve2gsodiumhydrogencarbonatewithwaterin100mL volumetricflask2.2.11
Sodium chloride.
Benzoicacid:Purity≥99%
Sorbic acid :Purity≥99%.
Stockstandardsolutionofbenzoicacid:Accuratelyweigh0.1000gbenzoicacid,add5mL2.2.14
Sodium hydrogen carbonate solution and dissolve by heating,then dissolve with water and quantita-tivelyon1o0mLvolumetricflask,theconcentrationis1mg/mL.2.2.15
Stock standard solution of sorbic acid:Accuratelyweigh0.100 0 g sorbic acid,add 5 mLSodium hydrogen carbonate solution and dissolve by heating,then dissolve with water and quantitativelyon1oomLvolumetricflask，theconcentrationis1mg/mL.2.2.16
Mixed standard working solution of benzoic acid and sorbic acid:Transfer Accurately10.0mLstockstandardsolutionofbenzoicacidand sorbicacidin100mLvolumetricflask,dilutethecontent to volume with water individually.The concentration of solution are 0. 1 mg/mL.2.3
Apparatusandequipment
HighperformanceliquidchromatographywithUVdetection.2.3.1
Distilling apparatus.
Procedure
2.4.1Preparation of test sampleAccuratelytransfer50.0mLofthetestsampleindistillingflask，add40gNaCl.5mL15%tartaricacid solution,1 d antifoaming agent,then add water about to 200 mL and mix, finally distill(about2.5mL/min).Collectabout90mLdistillingsolution,transferin100mLvolumetricflask,andaddwa-tertomark.The solution was filtered though0.45μmmembraneand determined byhighperform-ance liquid chromatography.
2.4.2Determination
2.4.2.1HPLC operating conditionsa)
Chromatographiccolumn:Cia（150mmX4.6mm,5μm)ortheequivalent；SN/T2012—2007
Mobile phase:methanol-acetonitril-5mmol/L citric acid buffer(10:20:70,V/V/V)Flow:1.0mL/min;
Detectionwavelength:230nm;
Columntemperature:30℃;
Injectionvolume:25μL.
2.4.2.2HPLC determination
According to the HPLC operating condition,the standard working solution and the sample solutionwere determined. Quantitative analysis of the benzoic acid and sorbic acid in sample solution wasdone using standard curve method. The responses of benzoic acid and sorbic acid in sample solutionshould be within the linear range of the instrumental detection. Under the above chromatographiccondition,the retention time of benzoic acid and sorbic acid are 5.13 min and 6. 41 min. HPLC chro-matogram of thestandard seeFigureA.1inannexA.2.4.3Blank
The opration of blank test is the same as that described in the method of determination but withoutaddition of sample.
2.5Calculationandexpressionof resultCalculation the content of benzoic acid and sorbic acid in the test sample by HPLc data processor oraccording to the formula(1).The blank value should be subtracted from the above result of cacula-tion.
A×cxV,
X-the content of benzoic acid or sorbic acid in the test sample,mg/L;A-thepeakareaofbenzoicacidorsorbicacid insamplesolution;c-the concentrationof benzoic acid or sorbic acid in standard work solution,ug/mL;.（1)
SN/T2012—2007
V,the final volume of the sample solution,mLAs-thepeakareaofbenzoicacid orsorbicacid instandardworking solution;V-volumeofthetestsample,mL
Limitof quantificationand recovery3.1
Limit of quantification
The limit of determination of the method for benzoic acid or sorbic acid is O. 1 mg/LRecovery
According the experiment data,the fortified concentrations of benzoic acid in vinegar and its3.2.1
corresponding recoveries are:Spike 0.1mg/L,the recovery is 88%~104%;Spike5mg/L,therecoveryis89.0%~99.4%;-Spike50mg/L.therecoveryis91.2%101.6%3.2.2
According the exper iment data,the fortified concentrations of sorbic acid in vinegar and itscorresponding recoveries are:Spike 0.1mg/L,the recovery is 83%~103%;-Spike5mg/L,therecoveryis87.8%~97.0%；-Spike50mg/L，therecoveryis89.5%98.2%.oo
1-Benzoic acid;
2—Sorbic acid.
AnnexA
（informative）
Chromatogramsofbenzoicacidand sorbicacid standardsA
SN/T2012—2007
Figure A.1-Chromatograms of benzoic acid and sorbic acid standards9
SN/T 2012-2007
中华人民共和国出人境检验检疫行业标准
进出口食醋中苯甲酸、山梨酸的检测方法液相色谱法
SN/T2012—2007
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