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食品安全国家标准 食品中脱氢乙酸的测定
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GB 5009.121-2016
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标准编号: GB 5009.121-2016 (GB5009.121-2016)
中文名称: 食品安全国家标准 食品中脱氢乙酸的测定
英文名称: Determination of dehydroacetic acid in foods
行业: 国家标准
中标分类: X09
字数估计: 10,198
发布日期: 2016-08-31
实施日期: 2017-03-01
旧标准 (被替代): GB/T 5009.121-2003
标准依据: 国家卫生和计划生育委员会公告2016年第11号
发布机构: 中华人民共和国国家卫生和计划生育委员会
GB 5009.121-2016: 食品安全国家标准 食品中脱氢乙酸的测定
GB 5009.121-2016 英文名称: Determination of dehydroacetic acid in foods
1 范围
本标准规定了果蔬汁、果蔬浆、酱菜、发酵豆制品、黄油、面包、糕点、烘烤食品馅料、复合调味料、预制肉制品及熟肉制品中脱氢乙酸含量的测定方法。
本标准适用于果蔬汁、果蔬浆、酱菜、发酵豆制品、黄油、面包、糕点、烘烤食品馅料、复合调味料、预制肉制品及熟肉制品中脱氢乙酸含量的测定,其他食品可参考执行。
第一法 气相色谱法
2 原理
固体(半固体)样品,沉降蛋白、经脱脂酸化后,用乙酸乙酯提取;果蔬汁、果蔬浆样品经酸化后,用乙
酸乙酯提取;用配氢火焰离子化检测器的气相色谱仪分离测定,以色谱峰的保留时间定性,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规定的二级水。
3.1 试剂
3.1.1 乙酸乙酯(C4H8O2):色谱纯。
3.1.2 正己烷(C6H14):色谱纯。
3.1.3 盐酸(HCl)。
3.1.4 硫酸锌(ZnSO4·7H2O)。
3.1.5 氢氧化钠(NaOH)。
3.2 试剂配制
3.2.1 盐酸溶液(1+1,体积比):量取50mL盐酸加入到50mL水中。
3.2.2 硫酸锌溶液(120g/L):称取12g硫酸锌,溶于水并稀释至100mL。
3.2.3 氢氧化钠溶液(20g/L):称取2g氢氧化钠,溶于水并稀释至100mL。
3.3 标准品
脱氢乙酸标准品:纯度≥99.5%。
3.4 标准溶液的制备
3.4.1 脱氢乙酸标准贮备液(1.0mg/mL):准确称取脱氢乙酸标准品0.1000g(精确至0.0001g)于
100mL容量瓶中,用乙酸乙酯溶解并定容。4℃保存,有效期为3个月。
3.4.2 脱氢乙酸标准工作液:分别精确吸取脱氢乙酸标准贮备液0.01mL、0.1mL、0.5mL、1.0mL、
2.0mL于10mL容量瓶中,用乙酸乙酯稀释并定容,配制成浓度为1.00μg/mL、10.0μg/mL、50.0μg/mL、
100μg/mL、200μg/mL标准工作液。4℃保存,有效期为1个月。
4 仪器和设备
4.1 气相色谱仪,配氢火焰离子化检测器。
4.2 天平:感量为0.1mg和1mg。
4.3 离心机:转速≥4000r/min。
4.4 超声波清洗器:功率35kW。
4.5 粉碎机。
4.6 不锈钢高速均质器。
4.7 pH计。
5 分析步骤
5.1 试样制备
5.1.1 果蔬汁、果蔬浆:称取样品2g~5g(精确至0.001g),置于50mL离心管中,加10mL水振摇
1min,加1mL盐酸溶液(3.2.1)酸化后,准确加入5.0mL乙酸乙酯,振摇提取2min,静置分层,取上清
液供气相色谱测定。
5.1.2 酱菜、发酵豆制品:样品用不锈钢高速均质器均质。称取样品2g~5g(精确至0.001g),置于
50mL离心管中,加入约15mL水、2.5mL硫酸锌溶液(3.2.2),用氢氧化钠溶液(3.2.3)调pH至7.5,
超声提取15min,转移至25mL容量瓶中,加水定容。样液移入离心管中,4000r/min离心10min。
取10mL上清液,加1mL盐酸溶液(3.2.1)酸化后,准确加入5.0mL乙酸乙酯,振摇2min,静置分层,
取上清液供气相色谱测定。
5.1.3 面包、糕点、烘烤食品馅料、复合调味料、预制肉制品及熟肉制品:样品用粉碎机粉碎或不锈钢高速均质器均质。称取样品2g~5g(精确至0.001g),置于50mL离心管中,加入约15mL水、2.5mL
硫酸锌溶液(3.2.2),用氢氧化钠溶液(3.2.3)调pH至7.5,超声提取15min,转移至25mL容量瓶中,加
水定容。样液移入分液漏斗中,加入5mL正己烷,振摇1min,静置分层,取下层水相置于离心管中,
4000r/min离心10min。取10mL上清液,加1mL盐酸溶液(3.2.1)酸化后,准确加入5.0mL乙酸
乙酯,振摇2min,静置分层,取上清液供气相色谱测定。
5.1.4 黄油:称取样品2g~5g(精确至0.001g),置于50mL离心管中,加入约15mL水、2.5mL硫酸
锌溶液(3.2.2),用氢氧化钠溶液(3.2.3)调pH至7.5,超声提取15min,转移至25mL容量瓶中,加水定
容。样液移入分液漏斗中,加入5mL正己烷,振摇1min,静置分层,取下层水相置于离心管中,
4000r/min离心10min。取10mL上清液,加1mL盐酸溶液(3.2.1)酸化后,准确加入5.0mL乙酸
乙酯,振摇2min,静置分层,取上清液供气相色谱测定。
5.2 仪器参考条件
5.2.1 毛细管柱:极性毛细柱(化学键和聚乙二醇固定相,30m×0.32mm×0.25μm),或相当者。
5.2.2 柱温升温程序:初温150℃,以10℃/min速率升至210℃,20℃/min速率升至240℃,保持
2min。
5.2.3 进样口温度:240℃。
5.2.4 检测器温度:300℃。
5.2.5 载气(N2)流量:1.0mL/min。
5.2.6 分流进样,分流比为5∶1,进样体积1.0μL。
5.3 标准曲线的制作
将脱氢乙酸标准工作液(3.4.2)分别注入气相色谱仪中,测定相应峰面积,以标准工作液的浓度为
横坐标,峰面积为纵坐标,绘制标准曲线。(脱氢乙酸标准色谱图见附录A)。
5.4 测定
将测定溶液注入气相色谱仪中,以保留时间定性,同时记录峰面积,根据标准曲线得到测定溶液中
的脱氢乙酸浓度。
5.5 空白试验
除不加试样外,空白试验应与样品测定平行进行,并采用相同的分析步骤分析。
6 分析结果的表述
果蔬汁、果蔬浆试样中脱氢乙酸含量按式(1)计算。
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
果蔬汁、果蔬浆取样量5g,确定检出限为0.0003g/kg,定量限为0.001g/kg;其他试样取样量5g,
样液定容体积25mL,取10mL样液提取,确定检出限为0.001g/kg,定量限为0.003g/kg。
用氢氧化钠溶液提取试样中的脱氢乙酸,经脱脂、去蛋白处理,过膜,用配紫外或二极管阵列检测器
的高效液相色谱仪测定,以色谱峰的保留时间定性,外标法定量。
10 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。
10.1 试剂
10.1.1 甲醇(CH4O):色谱纯。
10.1.2 乙酸铵(C2H6O2N):优级纯。
10.1.3 氢氧化钠(NaOH)。
10.1.4 正己烷(C6H14)。
10.1.6 硫酸锌(ZnSO4·7H2O)。
10.2 试剂配制
10.2.1 乙酸铵溶液(0.02mol/L):称取1.54g乙酸铵,溶于水并稀释至1L。
10.2.2 氢氧化钠溶液(20g/L):称取20g氢氧化钠,溶于水并稀释至1L。
10.2.3 甲酸溶液(10%):量取10mL甲酸,加水90mL,混匀。
10.2.4 硫酸锌溶液(120g/L):称取120g硫酸锌,溶于水并稀释至1L。
10.2.5 甲醇溶液(70%):量取70mL甲醇,加水30mL,混匀。
10.3 标准品
脱氢乙酸标准品:纯度≥99.5%。
10.4.1 脱氢乙酸标准贮备液(1.0mg/mL):准确称取脱氢乙酸标准品0.1000g(精确至0.0001g)于
100mL容量瓶中,用10mL氢氧化钠溶液(10.2.2)溶解,用水定容。4℃保存,有效期为3个月。
10.4.2 脱氢乙酸标准工作液:分别吸取脱氢乙酸贮备液0.1mL、1.0mL、5.0mL、10mL、20mL于100mL容量瓶中,用水定容。配制成浓度为1.00μg/mL、10.0μg/mL、50.0μg/mL、100μg/mL、200μg/mL标准工作液。4℃保存,有效期为1个月。
11 仪器和设备
11.1 高效液相色谱仪:配有紫外检测器或二极管阵列检测器。
11.2 分析天平:感量为0.1mg和1mg。
11.3 粉碎机。
11.4 不锈钢高速均质器。
11.5 超声波清洗器:功率35kW。
11.7 离心机:转速≥4000r/min。
11.8 pH计。
11.9 C18固相萃取柱:500mg,6mL(使用前用5mL甲醇、10mL水活化,使柱子保持湿润状态)。
12 分析步骤
12.1 试样制备与提取
12.1.1 果蔬汁、果蔬浆:称取样品2g~5g(精确至0.001g),置于50mL离心管中,加入约10mL水,
用氢氧化钠溶液(10.2.2)调pH至7.5,转移至50mL容量瓶中,加水稀释至刻度,摇匀。置于离心管
中,4000r/min离心10min。取20mL上清液用10%的甲酸溶液(10.2.3)调pH至5,定容到25mL。
取5mL过已活化固相萃取柱,用5mL水淋洗,2mL70%的甲醇溶液(10.2.5)洗脱,收集洗脱液2mL,
12.1.2 酱菜、发酵豆制品:样品用不锈钢高速均质器均质。称取样品2g~5g(精确至0.001g),置于
25mL离心管中,加入约10mL水、5mL硫酸锌溶液(10.2.4),用氢氧化钠溶液(10.2.2)调pH至7.5,
转移至25mL容量瓶中,加水稀释至刻度,摇匀。置于25mL离心管中,超声提取10min,4000r/min
离心10min,取上清液过0.45μm有机滤膜,供高效液相色谱测定。
12.1.3 面包、糕点、焙烤食品馅料、复合调味料:样品用粉碎机粉碎或不锈钢高速均质器均质。称取样品2g~5g(精确至0.001g),置于25mL离心管(如需过固相萃取柱则用50mL离心管)中,加入约
10mL水、5mL硫酸锌溶液(10.2.4),用氢氧化钠溶液(10.2.2)调pH至7.5,转移至25mL容量瓶(如
需过固相萃取柱则用50mL容量瓶)中,加水稀释至刻度,摇匀。置于离心管中,超声提取10min,转移
到分液漏斗中,加入10mL正己烷,振摇1min,静置分层,弃去正己烷层,加入10mL正己烷重复进行
一次,取下层水相置于离心管中,4000r/min离心10min。取上清液过0.45μm有机滤膜,供高效液相
容到25mL,取5mL过已活化的固相萃取柱,用5mL水淋洗,2mL70%的甲醇溶液(10.2.5)洗脱,收
集洗脱液2mL,涡旋混合,过0.45μm有机滤膜,供高效液相色谱测定。
12.1.4 黄油:称取样品2g~5g(精确至0.001g),置于25mL离心管(如需过固相萃取柱则用50mL
离心管)中,加入约10mL水、5mL硫酸锌溶液(10.2.4),用氢氧化钠溶液(10.2.2)调pH至7.5,转移至
25mL容量瓶(如需过固相萃取柱则用50mL容量瓶)中,加水稀释至刻度,摇匀。置于离心管中,超声
提取10min,转移到分液漏斗中,加入10mL正己烷,振摇1min,静置分层,弃去正己烷层,加入10mL
正己烷重复进行一次,取下层水相置于离心管中,4000r/min离心10min。取上清液过0.45μm有机
滤膜,供高效液相色谱测定。若高效液相色谱分离效果不理想,取20mL上清液,用10%的甲酸
(10.2.3)调pH至5,定容到25mL,取5mL过已活化的固相萃取柱,用5mL水淋洗,2mL70%的甲
GB 5009.121-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
Food safety national standard –
Determination of dehydroacetic acid in foods
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 1, 2017
Issued by. National Health and Family Planning Commission of the
People's Republic of China
Table of Contents
Foreword . 3
1 Scope .. 4
2 Principle.. 4
3 Reagents and materials .. 4
4 Instruments and equipment . 5
5 Analysis steps . 6
6 Expression of analytical results . 7
7 Precision. 8
8 Other . 8
9 Principle.. 9
10 Reagents and materials. 9
11 Instruments and equipment .. 10
12 Analysis steps .. 10
13 Expression of analytical results .. 13
14 Precision .. 13
15 Other .. 13
Annex A Dehydroacetic acid gas chromatograph . 14
Annex B Dehydroacetic acid liquid chromatography . 15
Foreword
This Standard replaces GB/T 5009.121-2003 "Determination of dehydroacetic
acid in foods".
Compared with GB/T 5009.121-2003, the main changes in this Standard are as
follows.
- modified the standard name as "Food safety national standard -
Determination of dehydroacetic acid in foods";
- added liquid chromatography;
- the liquid chromatography used the method of GB/T 23377-2009;
- added specimen classification;
- improved the preparation and extraction method of sample in gas
chromatography;
- modified the chromatographic conditions of gas chromatography.
Food safety national standard –
Determination of dehydroacetic acid in foods
1 Scope
This Standard specifies the determination method of dehydroacetic acid in fruit
and vegetable juice, fruit and vegetable pulp, pickle, fermented soy product,
butter, bread, cake, baked food stuffing, compound seasoning, pre-made meat
product, and cooked meat product.
This Standard is applicable to the determination of dehydroacetic acid in fruit
and vegetable juice, fruit and vegetable pulp, pickle, fermented soy product,
butter, bread, cake, baked food stuffing, compound seasoning, pre-made meat
product, and cooked meat product. Other foods shall refer for implementation.
Method One -- Gas chromatography
2 Principle
Solid (semi-solid) sample, after protein settlement and defatting, is extracted
with ethyl acetate. After acidified, the samples of fruit and vegetable juice and
fruit and vegetable pulp are extracted with ethyl acetate. Use a gas
chromatograph with hydrogen flame ionization detector to separate and
determine. Determine the nature with the retention time of chromatographic
peak. Quantify by external standard method.
3 Reagents and materials
Unless otherwise indicated, the reagents used in this method are all analytically
pure, and water is Grade Two water specified in GB/T 6682.
3.1 Reagents
3.1.1 Ethyl acetate (C4H8O2). chromatographically pure.
3.1.2 Hexane (C6H14). chromatographically pure.
3.1.3 Hydrochloric acid (HCl).
3.1.4 Zinc sulfate (ZnSO4·7H2O).
5 Analysis steps
5.1.1 Fruit and vegetable juice, fruit and vegetable pulp. weigh 2g ~ 5g of
sample (to the nearest of 0.001 g) and place in a 50mL centrifuge tube. Add 10
mL of water and shake 1 min. After adding 1 mL of hydrochloric acid solution
(3.2.1) for acidification, accurately add 5.0 mL of ethyl acetate. Shake and
extract 2 min. Place still and perform stratification. Take supernatant for the
determination of gas chromatography.
5.1.2 Pickle, fermented soy product. use stainless steel homogenizer for
sample homogenization. Weigh 2g ~ 5g of sample (to the nearest of 0.001 g)
and place in a 50mL centrifuge tube. Add about 15 mL of water, 2.5 mL of zinc
Perform ultrasonic extraction 15 min. Transfer to a 25mL volumetric flask. Add
water to set volume. Transfer the sample solution into a centrifuge tube and
centrifuge at 4000 r/min for 10 min. Take 10 mL of supernatant. After adding 1
mL of hydrochloric acid solution (3.2.1) for acidification, accurately add 5.0 mL
of ethyl acetate. Shake 2 min. Place still and perform stratification. Take
supernatant for the determination of gas chromatography.
5.1.3 Bread, pastries, baked goods filling, compound seasoning, pre-made
meat product and cooked meat product. use a grinder to grind the sample or
homogenize the sample with a stainless steel high-speed homogenizer. Weigh
tube. Add into 15 mL of water, 2.5 mL of zinc sulfate solution (3.2.2). Adjust pH
to 7.5 with sodium hydroxide solution (3.2.3). Perform ultrasonic extraction 15
min. Transfer to a 25mL volumetric flask. Add water to set volume. Transfer the
sample into a separatory funnel. Add 5 mL of n-hexane. Shake 1 min. Place still
and perform stratification. Take the lower aqueous phase and place it in a
centrifuge tube. Centrifuge at 4000 r/min for 10 min. Take 10 mL of supernatant.
After adding 1 mL of hydrochloric acid solution (3.2.1) for acidification,
accurately add 5.0 mL of ethyl acetate. Shake 2 min. Place still and perform
stratification. Take supernatant for the determination of gas chromatography.
a 50mL centrifuge tube. Add into 15 mL of water, 2.5 mL of zinc sulfate solution
(3.2.2). Adjust pH to 7.5 with sodium hydroxide solution (3.2.3). Perform
ultrasonic extraction 15 min. Transfer to a 25mL volumetric flask. Add water to
set volume. Transfer the sample into a separatory funnel. Add 5 mL of n-hexane.
Shake 1 min. Place still and perform stratification. Take the lower aqueous
phase and place it in a centrifuge tube. Centrifuge at 4000 r/min for 10 min.
Take 10 mL of supernatant. After adding 1 mL of hydrochloric acid solution
(3.2.1) for acidification, accurately add 5.0 mL of ethyl acetate. Shake 2 min.
9 Principle
solution, delipidated, deproteinized, and membraned. Determine it with high
performance liquid chromatography that is with UV or diode array detector.
Determine the nature with the retention time of chromatographic peak. Quantify
by external standard method.
10 Reagents and materials
Unless otherwise indicated, the reagents used in this method are all analytically
pure, and water is Grade One water specified in GB/T 6682.
10.1 Reagents
10.1.1 Methanol (CH4O). chromatographically pure.
10.1.3 Sodium hydroxide (NaOH).
10.1.4 Hexane (C6H14).
10.1.5 Formic acid (CH2O2).
10.1.6 Zinc sulfate (ZnSO4·7H2O).
10.2 Reagent preparation
10.2.1 Ammonium acetate solution (0.02 mol/L). weigh 1.54 g of ammonium
acetate, dissolve in water and dilute to 1 L.
10.2.2 Sodium hydroxide solution (20 g/L). weigh 20 g of sodium hydroxide,
dissolve in water and dilute to 1 L.
of water, mix well.
10.2.4 Zinc sulfate solution (120 g/L). weigh 120 g of zinc sulfate, dissolve in
water and dilute to 1 L.
10.2.5 Methanol solution (70%). measure 70 mL of methanol, add 30 mL of
water, mix well.
10.3 Standard product
Dehydroacetic acid (C8H8O4, CAS. 520-45-6) standard product. purity ≥99.5%.
with 5 mL of water and elute with 2 mL of 70% methanol solution (10.2.5).
Collect 2 mL of eluent. Perform vortex mixing. Filter with 0.45μm organic
12.1.2 Pickle, fermented soy product. use stainless steel homogenizer for
sample homogenization. Weigh 2g ~ 5g of sample (to the nearest of 0.001 g)
and place in a 25mL centrifuge tube. Add about 10 mL of water, 5 mL of zinc
sulfate solution (10.2.4). Adjust pH to 7.5 with sodium hydroxide solution
(10.2.2). Transfer to a 25mL volumetric flask. Add water to dilute to the scale.
Shake well. Place in a 25mL centrifuge tube. Perform ultrasonic extraction for
10 min. Centrifuge at 4000 r/min for 10 min. Take supernatant and make it
through 0.45μm organic membrane for the determination of high performance
liquid chromatography.
grinder to grind the sample or homogenize the sample with a stainless steel
high-speed homogenizer. Weigh 2g ~ 5g of sample (to the nearest of 0.001 g)
and place in a 25mL centrifuge tube (if it needs filtering through solid phase
extraction column, then use a 50mL centrifuge tube). Add about 10 mL of water,
5 mL of zinc sulfate solution (10.2.4). Adjust pH to 7.5 with sodium hydroxide
solution (10.2.2). Transfer to a 25mL volumetric flask (if it needs filtering through
solid phase extraction column, then use a 50mL volumetric flask). Add water to
dilute to the scale. Shake well. Place it in a centrifuge tube. Perform ultrasonic
extraction for 10 min. Transfer to the separatory funnel. Add 10 mL of n-hexane.
Add 10 mL of n-hexane to repeat once. Take the lower aqueous phase in the
centrifuge tube. Centrifuge at 4000 r/min for 10 min. Take supernatant and filter
it through 0.45μm organic membrane for the determination of high performance
liquid chromatography. If the separation of high performance liquid
chromatographic is not ideal, take 20 mL of supernatant. Adjust pH to 5 with 10%
formic acid (10.2.3). Set volume to 25 mL. Take 5mL of activated solid phase
extraction column. Rinse with 5 mL of water and elute with 2 mL of 70%
methanol solution (10.2.5). Collect 2 mL of eluent. Perform vortex mixing. Filter
with 0.45μm organic membrane for the determination of high performance liquid
12.1.4 Butter. weigh 2g ~ 5...
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