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GB 5009.248-2016

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标准编号: GB 5009.248-2016 (GB5009.248-2016)
中文名称: 食品安全国家标准 食品中叶黄素的测定
英文名称: Determination of lutein in milk powder--HPLC-UV method
行业: 国家标准
中标分类: X09
字数估计: 8,826
发布日期: 2016-08-31
实施日期: 2017-03-01
旧标准 (被替代): GB/T 23209-2008
标准依据: 国家卫生和计划生育委员会公告2016年第11号

GB 5009.248-2016
(Food safety national standard - Determination of lutein in foods)
中华人民共和国国家标准
食品安全国家标准
食品中叶黄素的测定
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 23209-2008《奶粉中叶黄素的测定 液相色谱-紫外检测法》。
本标准与GB/T 23209-2008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叶黄素的测定”;
---扩大适用范围,增加在冷冻饮品、米面制品、焙烤食品、果酱、果冻和饮料中叶黄素的液相色谱
测定方法;
---对原标准的原理进行了修改,把原用丙酮为溶剂提取改为用乙醚-正己烷-环已烷溶剂体系进
行提取;
---增加操作过程的注意点;
---增加了对包括奶粉在内的脂肪含量高的样品的皂化步骤,并提供了不同前处理方法以适应各
种样品基质分析需要;
---增加了对叶黄素标准溶液浓度的校正要求;
---对于在试验操作过程中叶黄素可能产生异构化的现象,增加了对因异构化生成的顺式叶黄素
的定性与定量要求。
食品安全国家标准
食品中叶黄素的测定
1 范围
本标准规定了食品中叶黄素的液相色谱测定方法。
本标准适用于婴幼儿配方奶粉、乳品、冷冻饮品、米面制品、焙烤食品、果酱、果冻和饮料中叶黄素的
液相色谱测定。
2 原理
脂肪含量高(脂肪含量以干基计不低于3%)的食品经氢氧化钾溶液室温皂化使叶黄素游离后,再
以乙醚-正己烷-环己烷(40+40+20,体积比)提取,液相色谱法分离,紫外检测器或二极管阵列检测器
检测,外标法定量。
其他食品直接以乙醚-正己烷-环己烷(40+40+20,体积比)提取样品中叶黄素。提取液经中性氧
化铝固相萃取小柱净化后,液相色谱法分离,紫外检测器或二极管阵列检测器检测,外标法定量。
样品在提取与分析过程中,反式结构的叶黄素可能发生异构化,转化为顺式叶黄素。对于转化产生
的顺式叶黄素,可通过保留时间定性、峰面积加合定量。
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 环己烷(C6H12):色谱纯。
3.1.2 乙醚[(C2H5)2O]:色谱纯。
3.1.3 正己烷(C6H10):色谱纯。
3.1.4 无水乙醇(C2H5OH):色谱纯。
3.1.5 甲基叔丁基醚[CH3OCC(CH3)3,MTBE]:色谱纯。
3.1.6 二丁基羟基甲苯(C15H24O,BHT)。
3.1.7 氢氧化钾(KOH)。
3.1.8 碘(I2)。
3.2 试剂配制
3.2.1 10%氢氧化钾溶液:称取10g氢氧化钾(3.1.7),加水溶解稀释至100mL。
3.2.2 20%氢氧化钾溶液:称取20g氢氧化钾(3.1.7),加水溶解稀释至100mL。
3.2.3 萃取溶剂:称取1gBHT(3.1.6),以200mL环己烷(3.1.1)溶解,加入400mL乙醚(3.1.2)和
400mL正己烷(3.1.3),混匀。
3.2.4 0.1%BHT乙醇溶液:称取0.1gBHT(3.1.6),以100mL乙醇(3.1.4)溶解,混匀。
3.2.5 碘的乙醇溶液:称取1mg碘(3.1.8),加乙醇(3.1.4)溶解稀释至1L。
3.3 标准品
叶黄素(CAS号:127-40-2),纯度不低于98.0%。
3.4 标准溶液配制
3.4.1 标准储备液(50μg/mL):准确称取5mg(精确至0.01mg)叶黄素(3.1.3),以0.1%BHT乙醇溶
液(3.2.4)溶解并定容至100mL。该标准储备液充氮避光置于-20℃或以下的冰箱中可保存六个月。
注:叶黄素标准储备液使用前需校正,具体操作见附录A。
3.4.2 标准工作液:从叶黄素标准贮备液(3.4.1)中准确移取0.050mL、0.100mL、0.200mL、0.400mL、
1.00mL溶液入5个25mL棕色容量瓶中,用0.1% BHT乙醇溶液(3.2.4)定容至刻度,得到浓度为
0.100μg/mL、0.200μg/mL、0.400μg/mL、0.800μg/mL、2.00μg/mL的系列标准工作液。标准工作
液充氮避光置于-20℃或以下的冰箱中可保存一个月。
3.5 中性氧化铝固相萃取小柱,500mg/3mL,使用前以5mL萃取溶剂(3.2.3)淋洗,保持柱体湿润。
3.6 0.45μm滤膜,有机系。
4 仪器和设备
4.1 液相色谱仪,带二极管阵列检测器或紫外检测器。
4.2 紫外可见分光光度计。
4.3 分析天平:感量0.01mg和0.01g。
4.4 组织捣碎机。
4.5 旋涡振荡器。
4.6 振荡器。
4.7 减压浓缩装置。
4.8 固相萃取装置。
4.9 离心机:转速不低于4500r/min。
5 分析步骤
注:由于叶黄素对光敏感,除非另行说明,所有试验操作应在无500nm以下紫外光的黄色光源或红色光源环境中
进行。
将一定数量的样品按要求经过粉碎、均质、缩分后,储存于样品瓶中。制备好的试样应充氮密封后
置于-20℃或以下的冰箱中保存。
5.2 提取
5.2.1 脂肪含量高的食品(如婴幼儿配方奶粉、乳粉、冰淇淋、焙烤坚果类食品等)
准确称取2g(精确至0.01g)均匀试样于50mL聚丙烯离心管中,加入约0.2gBHT(3.1.6)和
10mL乙醇(3.1.4),混匀,加入10mL10%氢氧化钾溶液(3.2.1),涡旋振荡1min混匀,室温避光振荡
皂化30min,以10mL萃取溶剂(3.2.3)避光涡旋振荡提取3min,4500r/min离心3min,重复提取
2次,合并提取液,以10mL水洗涤,4500r/min离心3min分层,重复洗涤1次,合并有机相于室温减
压浓缩至近干,以0.1% BHT乙醇溶液(3.2.4)涡旋振荡溶解残渣并定容至5mL,过0.45μm 滤膜
液态奶:准确称取10g(精确至0.01g)样品于50mL聚丙烯离心管中,加入约0.2gBHT(3.1.6)和
10mL乙醇(3.1.4),混匀,加入2mL20%的氢氧化钾溶液(3.2.2),涡动1min混匀,室温避光振荡皂化
30min,以10mL萃取溶剂(3.2.3)避光涡旋振荡提取3min,4500r/min离心3min,重复提取2次,合
并提取液,以10mL水洗涤,4500r/min离心3min分层,重复洗涤1次,合并有机相于室温减压浓缩
至近干,以0.1%BHT乙醇溶液(3.2.4)涡旋振荡溶解残渣并定容至25mL,过0.45μm滤膜(3.6),供液
相色谱测定。
5.2.2 其他食品(如米、面制品、果酱等)
准确称取5g(精确至0.01g)均匀样品置于50mL聚丙烯离心管中,以10mL萃取溶剂(3.2.3)避
光涡旋振荡提取3min,4500r/min离心3min,重复提取2次,合并提取液,于室温减压浓缩至近干,
将上述溶液以约1mL/min的流速过已活化的中性氧化铝固相萃取小柱(3.5),用3mL萃取溶剂
(3.2.3)洗脱,合并流出液与洗脱液,于室温减压浓缩至近干,以0.1%BHT乙醇溶液(3.2.4)涡旋振荡溶
解残渣并定容至10mL,过0.45μm滤膜(3.6),供液相色谱测定。
5.3 仪器参考条件
5.3.1 色谱柱:C30色谱柱,5μm,250mm×4.6mm(内径)或相当者;
5.3.2 柱温:30℃;
5.3.3 流动相:甲醇/水(88+12,体积比,含0.1% BHT)-甲基叔丁基醚(含0.1% BHT),梯度洗脱,
0min~18min,甲醇/水由100%变换至10%;18.1min,甲醇/水由10%变换至100%,保留10min。
5.3.4 流速:1.0mL/min;
5.3.6 进样量:50μL。
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。
5.5 试样溶液的测定
待测样液中叶黄素的响应值应在仪器线性响应范围内,否则应适当稀释或浓缩。标准工作液与待
测样液等体积进样。根据标准溶液色谱峰的保留时间和峰面积,对试样溶液的色谱峰根据保留时间进
行定性(待测样品中化合物色谱峰的保留时间与标准溶液相比变化范围应在±2.5%之内),外标法定
量。平行测定次数不少于两次。叶黄素标准溶液液相色谱图参见附录B图B.1。
可按以下步骤获得顺式叶黄素:以乙醇为溶剂,配制800μg/L的叶黄素标准溶液50mL,加入2mL碘的乙醇
溶液(3.2.5),摇匀,混合液在日光或日光灯下放置30min。可获得顺式结构的叶黄素。由此制备的含顺式结构
的叶黄素在检测时可作为对照品。经光碘异构化的反式叶黄素标准溶液色谱图参见附录B图B.2。
6 分析结果的表述
试样中叶黄素含量按式(1)计算:
X=
c×V
m ×
F ×100
式中:
X ---试样中叶黄素的含量,单位为微克每百克(μg/100g);
c ---由标准曲线而得的样液中标准品的含量,单位为微克每毫升(μg/mL);
V ---样品最终定容体积,单位为毫升(mL);
m ---称样量,单位为克(g);
F ---校正系数。可通过以下方式获得:用液相色谱分析试样溶液,将顺式与反式叶黄素色谱峰
面积加合作为总峰面积,其中反式叶黄素峰面积除以总峰面积所得值为校正系数。
以重复性条件下获得的两次独立测定结果的算术平均值表示,计算结果保留三位有效数字。
7 精密度
8 其他
本方法的检出限:婴幼儿配方奶粉、乳粉、冰淇淋、焙烤坚果类等食品,当取样量为2g、定容体积为
5mL时,检出限为3μg/100g;液态奶等,当取样量为10g、定容体积为25mL时,检出限为3μg/100g;
米、面制品、果酱等食品,当取样量为5g、定容体积为10mL时,检出限为3μg/100g。
本方法的定量限:婴幼儿配方奶粉、乳粉、冰淇淋、焙烤坚果类等食品,当取样量为2g、定容体积为
5mL时,定量限为10μg/100g;液态奶等当取样量为10g、定容体积为25mL时,定量限为10μg/100g;
米、面制品、果酱等食品,当取样量为5g、定容体积为10mL时,定量限为10μg/100g。
附 录 A
标准溶液浓度校正方法
1cm的石英比色皿中,以乙醇为空白,以分光光度计在445nm波长下测定吸光值A。按式(A.1)计算
标准溶液浓度:
c=
E1%1cm
×2500×F (A.1)
式中:
c ---标准溶液浓度,μg/mL;
A ---标准溶液的吸光值;
2500---转换系数;
F ---校正系数,可按下述方式获得:用液相色谱分析校准后的标准溶液,将顺式与反式叶黄素
色谱峰面积加合作为总峰面积,其中反式叶黄素峰面积除以总峰面积所得值为校正
系数。
附 录 B
标准溶液液相色谱图
B.1 叶黄素(反式)标准溶液液相色谱图
叶黄素(反式)标准溶液液相色谱图见图B.1。
图B.1 叶黄素(反式)标准溶液液相色谱图
B.2 经光碘异构化的叶黄素(反式)标准溶液液相色谱图
说明:
1---顺式结构的叶黄素;
2---反式结构的叶黄素。
图B.2 经光碘异构化的叶黄素(反式)标准溶液液相色谱图

GB 5009.248-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of lutein in foods
食品安全国家标准
食品中叶黄素的测定
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 01, 2017
Issued by. National Health and Family Planning Commission of the PRC
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Principles ... 4 
3 Reagents and materials ... 4 
4 Instruments and equipment... 6 
5 Analytical procedures ... 6 
6 Analysis results expression ... 9 
7 Precision ... 9 
8 Other ... 9 
Appendix A Method for correction of standard solution concentration ... 11 
Appendix B Liquid chromatogram of standard solution ... 12 
Foreword
This Standard replaces GB/T 23209-2008 “Determination of lutein in milk
powder - HPLC-UV method”.
As compared with GB/T 23209-2008, the main changes of this Standard are as
follows.
- CHANGE the standard’s name to “National Food Safety Standard -
Determination of lutein in foods”;
- EXPAND the scope of application; ADD the liquid chromatography for the
determination of lutein in frozen drinks, rice and flour products, bakery
products, jams, jellies, and beverages;
- MODIFY the “Principles” of the former standard. The former “USE acetone
as a solvent to extract” is changed to “USE diethyl ether-n-hexane-
cyclohexane solvent system to extract”;
- ADD the points worthy of notice for the operation process;
- ADD a saponification step for samples with high fat content including milk
powder; and PROVIDE different pretreatment methods to accommodate
various sample matrix analysis needs;
- ADD the correction requirement for the concentration of lutein standard
solution;
- For the phenomenon that lutein may be isomerized during the test operation,
ADD the qualitative and quantitative requirements for cis-lutein produced
due to isomerization.
National Food Safety Standard -
Determination of lutein in foods
1 Scope
This Standard specifies the liquid chromatography for the determination of
lutein in foods.
This Standard applies to liquid chromatography determination of lutein in infant
formula, dairy products, frozen drinks, rice and flour products, bakery products,
jams, jellies, and beverages.
2 Principles
After foods with high fat content (fat content of not less than 3% on a dry basis)
are saponified with potassium hydroxide solution at room temperature to free
lutein, then USE diethyl ether-n-hexane-cyclohexane (40+40+20, volume ratio)
to extract; USE liquid chromatography to separate; USE UV-detector or diode
array detector to detect; and USE external standard method for quantification.
Lutein in the samples of the other foods is directly extracted by diethyl ether-n-
hexane-cyclohexane (40+40+20, volume ratio). After the extract is purified by a
neutral alumina solid phase extraction cartridge, USE liquid chromatography to
separate; USE UV-detector or diode array detector to detect; and USE external
standard method for quantification.
During the extraction and analysis of sample, the trans-structured lutein may
be isomerized and converted to cis-lutein. The cis-lutein produced by the
transformation can be qualitatively determined by retention time and quantified
by peak area adduction.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytically
3.1 Reagents
3.1.1 Cyclohexane (C6H12). chromatographically pure.
cream, bakery nuts, etc.)
Accurately WEIGH 2 g (accurate to 0.01 g) of uniform sample in a 50 mL
polypropylene centrifuge tube; ADD about 0.2 g of BHT (3.1.6) and 10 mL of
ethanol (3.1.4), and MIX well. ADD 10 mL of 10% potassium hydroxide solution
(3.2.1); vortex oscillate for 1 min, and MIX well. Oscillate and saponify at room
temperature for 30 min in the dark; USE 10 mL of extraction solvent (3.2.3) to
vortex oscillate and extract in the dark for 3 min; CENTRIFUGE at 4500 r/min
of water to wash; CENTRIFUGE at 4500 r/min for 3 min for delamination; wash
once again. The organic phases are combined and concentrated under reduced
pressure at room temperature until nearly dry. USE 0.1% BHT ethanol solution
(3.2.4) to vortex oscillate to dissolve the residue and dilute to 5 mL; pass
through a 0.45 μm filter membrane (3.6), for liquid chromatography
determination.
Liquid milk. Accurately WEIGH 10 g (accurate to 0.01 g) of the sample in a 50
mL polypropylene centrifuge tube; ADD about 0.2 g of BHT (3.1.6) and 10 mL
of ethanol (3.1.4), and MIX well. ADD 2 mL of 20% potassium hydroxide solution
temperature for 30 min in the dark; USE 10 mL of extraction solvent (3.2.3) to
vortex oscillate and extract in the dark for 3 min; CENTRIFUGE at 4500 r/min
for 3 min; repeatedly EXTRACT twice; and combine the extracts. USE 10 mL
of water to wash; CENTRIFUGE at 4500 r/min for 3 min for delamination; wash
once again. The organic phases are combined and concentrated under reduced
pressure at room temperature until nearly dry. USE 0.1% BHT ethanol solution
(3.2.4) to vortex oscillate to dissolve the residue and dilute to 25 mL; pass
through a 0.45 μm filter membrane (3.6), for liquid chromatography
determination.
Accurately WEIGH 5 g (accurate to 0.01 g) of uniform sample in a 50 mL
polypropylene centrifuge tube. USE 10 mL of extraction solvent (3.2.3) to vortex
oscillate and extract in the dark for 3 min; CENTRIFUGE at 4500 r/min for 3
min; repeatedly EXTRACT twice. The extracts are combined and concentrated
under reduced pressure at room temperature until nearly dry. USE 3 mL of
extraction solvent (3.2.3) to vortex oscillate and dissolve; REPEAT the
operation once; combine the extraction solvents, and mix well, for purification.
The above solution, at a flow rate of about 1 mL/min, is passed through an
activated neutral alumina solid phase extraction cartridge (3.5). USE 3 mL of
concentrated under reduced pressure at room temperature until nearly dry.
USE 0.1% BHT ethanol solution (3.2.4) to vortex oscillate to dissolve the
mL of ethanol solution of iodine (3.2.5), shake well; and PLACE the mixture under
sunlight or fluorescent lamp for 30 min. Cis-structured lutein can be obtained. The
thus prepared lutein containing cis-structure, when testing, can be used as a
reference substance. The chromatogram of standard solution of trans-lutein
isomerized by light iodine is shown in Appendix B, Figure B.2.
6 Analysis results expression
The content of lutein in the sample shall be calculated according to formula (1).
X - The content of lutein in the sample, in micrograms per hundred grams
(μg/100 g);
c - The content of the standard in the sample solution obtained from the
standard curve, in micrograms per milliliter (μg/mL);
V - The final constant volume of sample, in milliliters (mL);
m - Sample weighing amount, in grams (g);
F - Correction factor. It can be obtained by the following method. USE liquid
chromatography to analyze the sample solution. The adduct of the cis and trans
lutein chromatographic peak areas is taken as the total peak area, wherein the
factor.
The calculation result is expressed as the arithmetic mean of the two
independent determination results obtained under repeated conditions. The
result retains three significant figures.
7 Precision
The absolute difference between the two independent determination results,
obtained under repeated conditions, shall not exceed 15% of the arithmetic
mean.
8 Other
   
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