标准编号:	GB 5009.247-2016
中文名称:	食品安全国家标准 食品中纽甜的测定
英文名称:	National Food Safety Standard - Determination of neotame in food
行业分类:	GB    国家标准
发布机构:	中华人民共和国国家卫生和计划生育委员会
发布日期:	2016-08-31
实施日期:	2017-3-1
标准状态:	valid
替代旧标准:	GB/T 23378-2009 食品中纽甜的测定方法 高效液相色谱法
翻译语言:	英文
文件格式:	PDF
中文字符数:	4000 字
翻译价格(元):	240.0
交付时间:	付款后 1 个工作日

This standard supersedes Method for the Determination of Neotame in Foods - High Performance Liquid Chromatography (GB/T 23378-2009). Compared with GB/T 23378-2009, this standard has the following main changes: - the standard name was revised as "National Food Safety Standard - Determination of Neotame in Foods". - the application scope was changed to the determination of neotame in beverage, preserved fruit, pastry, roasted seeds and nuts, pickled vegetable, candy, jam, jelly and compound flavouring. National Food Safety Standard Determination of Neotame in Foods 食品安全国家标准 食品中纽甜的测定 1 Scope This standard specifies the high performance liquid chromatography for the determination of neotame content in foods. This standard is applicable to the determination of neotame in foods such as beverage, preserved fruit, pastry, roasted seeds and nuts, pickled vegetable, candy, jam, jelly and compound flavouring. 2 Principle Specimen is determined with high performance liquid chromatograph after it is extracted with mixed extracting solution and purified with solid-phase extraction column. Qualitative analysis is conducted by retention time and quantitative analysis is conducted by external standard method of peak area. 3 Reagents and Materials Unless otherwise specified, the reagents adopted in this method are all analytically pure, and the water is Grade 1 water specified in GB/T 6682. 3.1 Reagents 3.1.1 Acetonitrile (CH3CN): chromatographically pure. 3.1.2 Sodium 1-octanesulfonate (C8H17NaO3S): chromatographically pure. 3.1.3 Phosphoric acid (H3PO4). 3.1.4 Formic acid (CH3COOH): chromatographically pure. 3.1.5 Methanol (CH3OH): chromatographically pure. 3.1.6 Triethylamine (C6H15N): chromatographically pure. 3.2 Reagent preparation 3.2.1 Mixed extracting solution: pipet 0.8mL of formic acid and 2.5mL of triethylamine respectively and add water to bring the volume to 1 000mL to make the pH of about 4.5. 3.2.2 Ion pair reagent buffer solution: weigh 2.00g of sodium 1-octanesulfonate and dissolve it with 500mL of water, then add 1.0mL of phosphoric acid and add water to bring the volume to 1 000mL. 3.3 Standard substance Neotame (C20H30N2O5, CAS No.: 165450-17-9), with purity greater than or equal to 99.0%. 3.4 Preparation of standard solution 3.4.1 Standard stock solution: accurately weigh 0.100 0g of neotame standard substance, dissolve in mixed extracting solution and bring the volume to 100ml; the neotame content in this solution is 1.00mg/mL. 3.4.2 Standard working solution: respectively pipet appropriate amount of neotame standard stock solution to prepare series of standard working solutions in concentrations of 0.2μg/mL, 1.0μg/mL, 5.0μg/mL, 10.0μg/mL ,50.0μg/mL and 100.0μg/mL with mixed extracting solution. 3.5 C18 solid-phase extraction column: 6mL, 500mg or equivalent; activate with 5mL of methanol and 10mL of water successively prior to use. 3.6 0.45μm filter membrane, organic system. 4 Instruments and Apparatus 4.1 Liquid chromatograph: equipped with ultraviolet detector or diode array detector. 4.2 Ultrasonic cleaner. 4.3 Analytical balance: with sensibility of 0.000 1g and 0.01g. 4.4 Tissue blender. 4.5 Swirl oscillator. 4.6 Pressured gas blowing concentrator. 4.7 Solid-phase extraction device. 4.8 Centrifuge: with rotation speed greater than or equal to 4 000r/m. 5 Analytical Procedures 5.1 Specimen preparation 5.1.1 Solid sample Weigh 10g (to the nearest of 0.01g) evenly pulverized specimen and put it into a 50mL plastic centrifuge tube with stopper, add 30mL of mixed extracting solution, oscillate for 10min in swirling way, treat is for 30min with ultrasound, and then bring the volume to the scale with mixed extracting solution; if the solution is turbid, centrifuge for 10min at the speed of no less than 4 000r/min and filter for use. 5.1.2 Liquid sample Accurately measure 10.0mL of specimen and put it into a 50mL plastic centrifuge tube with stopper, add 30mL of mixed extracting solution, oscillate and mix it uniformly. After 15min ultrasonic treatment, bring the volume to the scale with mixed extracting solution; if the solution is turbid, centrifuge for 10min at the speed of no less than 4 000r/min and filter for use. Note: For the sample containing gas, such as carbonated beverage and steam-water, subject it to tepidity and stirring to remove the carbon dioxide or carry out ultrasonic degasification firstly, then accurately weigh specimen. 5.2 Specimen purification Pipet 10.0mL of filtered solution and make it pass through solid-phase extraction column at the flow speed of 1mL/min~2mL/min, after the filtrate flows out entirely, rinse the extraction column with 5mL of mixed extracting solution at the flow speed of 1mL/min~2mL/min, discard all effluent, elute with 5mL of methanol at the flow speed of 1mL/min, concentrate the eluent with pressured gas blowing concentrator at 40℃ water bath, bring the volume to 2.0mL with mixed extracting solution, filter via 0.45μm filter membrane to serve as to-be-determined solution for the analysis with liquid chromatograph.

Foreword i 1 Scope 2 Principle 3 Reagents and Materials 4 Instruments and Apparatus 5 Analytical Procedures 6 Expression of Analysis Results 7 Precision 8 Others Appendix A Chromatogram

中华人民共和国国家标准 GB 5009.247—2016 食品安全国家标准 食品中纽甜的测定 2016—08—31发布 2017—03—01实施 中华人民共和国 国家卫生和计划生育委员会 发布 前言 本标准代替GB/T 23378—2009《食品中纽甜的测定方法 高效液相色谱法》。 本标准与GB/T 23378—2009相比，主要变化如下： ——标准名称修改为“食品安全国家标准 食品中纽甜的测定”。 ——适用范围更改为饮料、蜜饯、糕点、炒货、酱腌菜、糖果、果酱、果冻、复合调味料中纽甜的测定。 食品安全国家标准 食品中纽甜的测定 1 范围 本标准规定了食品中纽甜含量的高效液相色谱测定方法。 本标准适用于饮料、蜜饯、糕点、炒货、酱腌菜、糖果、果酱、果冻、复合调味料食品中纽甜的测定。 2 原理 试样经混合提取液提取，固相萃取柱净化后，采用高效液相色谱仪测定，保留时间定性，峰面积外标法定量。 3试剂和材料 除非另有说明，本方法所用试剂均为分析纯，水为GB/T 6682规定的一级水。 3.1 试剂 3.1.1 乙腈(CH3CN)：色谱纯。 3.1.2辛烷磺酸钠(C8H17NaO3S)：色谱纯。 3.1.3磷酸(H3PO4)。 3.1.4甲酸(CH3COOH)：色谱纯。 3.1.5甲醇(CH3OH)：色谱纯。 3.1.6三乙胺(C6H15N)：色谱纯。 3.2试剂配制 3.2.1 混合提取液：分别吸取0.8 mL甲酸和2.5 mL三乙胺，加水定容至1 000 mL，pH约4.5。 3.2.2离子对试剂缓冲液：称取2.00 g辛烷磺酸钠，用500 mL水溶解，加入1.0 mL磷酸，加水定容至1 000 mL。 3.3 标准品 纽甜(C20H30N2O5，CAS号：165450-17-9)，纯度≥99.0％。 3.4标准溶液配制 3.4.1 标准储备液：准确称取0.100 0 g纽甜标准品，加混合提取液溶解定容至100 mL，此溶液纽甜含量为1.00 mg/mL。 3.4.2 标准工作液：分别吸取适量纽甜标准储备液，用混合提取液配制成浓度分别为0.2 μg/mL、1.0 μg/mL、5.0 μg/mL、10.0 μg/mL、50.0 μg/mL、100.0 μg/mL的系列标准工作液。 3.5 C18固相萃取柱：6 mL、500 mg，或相当者，使用前依次用5 mL甲醇、10 mL水活化。 3.6 0.45 μm滤膜，有机系。 4仪器和设备 4.1 液相色谱仪，配有紫外检测器或二极管阵列检测器。 4.2超声波清洗仪。 4.3分析天平：感量0.000 1 g和0.01 g。 4.4组织捣碎机。 4.5旋涡振荡器。 4.6 氮吹仪。 4.7固相萃取装置。 4.8离心机：转速≥4 000 r/min。 5 分析步骤 5.1 试样制备 5.1.1 固态样品 称取10 g(精确到0.01 g)粉碎均匀试样于50 mL具塞塑料离心管中，加入30 mL混合提取液，旋涡振荡10 min，超声30 min后，用混合提取液定容至刻度，若溶液混浊，以不低于4 000 r/min离心10 min后，过滤待用。 5.1.2液态样品 准确量取10.0 mL试样于50 mL具塞塑料离心管中，加入30 mL混合提取液，振荡混匀。超声15 min后，用混合提取液定容至刻度，若溶液混浊，以不低于4 000 r/min离心10 min后，过滤待用。 注：含气样品如碳酸饮料、汽水等先微温，搅拌去除试样中的二氧化碳或超声脱气后，再准确量取试样。 5.2 试样净化 吸取10.0 mL的过滤液以1 mL/min～2 mL/min的流速通过固相萃取柱，待滤液完全流出后，用5 mL的混合提取液以1 mL/min～2 mL/min的流速淋洗萃取柱，弃去全部流出液，用5 mL甲醇以1 mL/min的流速洗脱，洗脱液在40℃水浴中用氮吹仪浓缩，用混合提取液定容至2.0 mL，经0.45 μm滤膜过滤后作为待测液供液相色谱仪分析。 5.3仪器参考条件 5.3.1 色谱柱：C18色谱柱，5μm，250 mm×4.6 mm(内径)或相当者。 5.3.2 柱温：30℃。 5.3.3流动相：A相：乙腈；B相：离子对试剂缓冲液。梯度洗脱程序见表1。 5.3.4流速：1.0 mL/min。 5.3.5检测波长：218 nm。 5.3.6进样量：50 μL。 表1 流动相梯度洗脱程序 时间/min 流动相A/％ 流动相B/％ 0.00 25.0 75.0 10.00 65.0 35.0 20.00 100.0 0.0 24.00 100.0 0.0 24.10 25.0 75.0 30.00 25.0 75.0 5.4标准曲线的制作 将标准系列工作液分别注入液相色谱仪中，测定相应的峰面积，以标准工作液的浓度为横坐标，以峰面积为纵坐标，绘制标准曲线(纽甜标准色谱图见图A.1)。 5.5试样溶液的测定 将试样溶液注入液相色谱仪中，以保留时间定性，同时记录峰面积，根据标准曲线得到待测液中纽甜的浓度，试样溶液中纽甜含量的响应值均应在仪器的检测线性范围内。 6分析结果的表述 试样中纽甜的含量按式(1)计算： …………………………(1) 式中： X——试样中纽甜含量，单位为毫克每千克(mg/kg)或毫克每升(mg/L)； c——试样溶液中纽甜的浓度，单位为微克每毫升(μg/mL)； V——样品洗脱液浓缩定容体积，单位为毫升(mL)； V1——试样提取液定容体积，单位为毫升(mL)； m——样品质量，单位为克(g)； V2——吸取过滤液体积，单位为毫升(mL)。 计算结果保留两位有效数字。 7 精密度 在重复性条件下获得的两次独立测定结果的绝对差值不得超过算术平均值的10％。 8 其他 本标准方法的定量限为0.2 mg/kg。 附 录 A 色 谱 图 纽甜标样色谱图见图A.1。 纽甜 图A.1 纽甜标样液相色谱图