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食品安全国家标准 食品中有机酸的测定
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GB 5009.157-2016
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标准编号: GB 5009.157-2016 (GB5009.157-2016)
中文名称: 食品安全国家标准 食品中有机酸的测定
英文名称: Determination of organic acid in foods
行业: 国家标准
中标分类: C53
字数估计: 8,858
发布日期: 2016-08-31
实施日期: 2017-03-01
旧标准 (被替代): GB/T 5009.157-2003
标准依据: 国家卫生和计划生育委员会公告2016年第11号
GB 5009.157-2016: 食品安全国家标准 食品中有机酸的测定
GB 5009.157-2016 英文名称: Determination of organic acid in foods
1 范围
本标准规定了食品中酒石酸、乳酸、苹果酸、柠檬酸、丁二酸、富马酸和己二酸的测定方法。
本标准适用于果汁及果汁饮料、碳酸饮料、固体饮料、胶基糖果、饼干、糕点、果冻、水果罐头、生湿面制品和烘焙食品馅料中7种有机酸的测定。
2 原理
试样直接用水稀释或用水提取后,经强阴离子交换固相萃取柱净化,经反相色谱柱分离,以保留时
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规定的一级水。
3.1 试剂
3.1.1 甲醇(CH3OH):色谱纯。
3.1.2 无水乙醇(CH3CH2OH):色谱纯。
3.1.3 磷酸(H3PO4)。
3.2 试剂配制
3.2.1 磷酸溶液(0.1%):量取磷酸0.1mL,加水至100mL,混匀。
3.2.2 磷酸-甲醇溶液(2%):量取磷酸2mL,加甲醇至100mL,混匀。
3.3 标准品
3.3.1 乳酸标准品(C3H6O3),纯度≥99%。
3.3.2 酒石酸标准品(C4H6O6),纯度≥99%。
3.3.3 苹果酸标准品(C4H6O5),纯度≥99%。
3.3.4 柠檬酸标准品(C6H8N7),纯度≥98%。
3.3.5 丁二酸标准品(C4H6N4),纯度≥99%。
3.3.6 富马酸标准品(C4H4O4),纯度≥99%。
3.3.7 己二酸标准品(C6H10N4),纯度≥99%。
3.4 标准溶液配制
3.4.1 酒石酸、苹果酸、乳酸、柠檬酸、丁二酸和富马酸混合标准储备溶液:分别称取酒石酸1.25g、苹果酸2.5g、乳酸2.5g、柠檬酸2.5g、丁二酸6.25g(精确至0.01g)和富马酸2.5mg(精确至0.01mg)于
50mL小烧杯中,加水溶解,用水转移到50mL容量瓶中,定容,混匀,于4℃保存,其中酒石酸质量浓度
为2500μg/mL、苹果酸5000μg/mL、乳酸5000μg/mL、柠檬酸5000μg/mL、丁二酸12500μg/mL
和富马酸12.5μg/mL。
3.4.2 酒石酸、苹果酸、乳酸、柠檬酸、丁二酸、富马酸混合标准曲线工作液:分别吸取混合标准储备溶液(3.4.1)0.50mL、1.00mL、2.00mL、5.00mL、10.00mL于25mL容量瓶中,用磷酸溶液(3.2.1)定容
至刻度,混匀,于4℃保存。
3.4.3 己二酸标准储备溶液(500μg/mL):准确称取按其纯度折算为100%质量的己二酸12.5mg,置
25mL容量瓶中,加水到刻度,混匀,于4℃保存。
3.4.4 己二酸标准曲线工作液:分别吸取标准储备溶液(3.4.3)0.50mL、1.00mL、2.00mL、5.00mL、
10.00mL于25mL容量瓶中,用磷酸溶液(3.2.1)定容至刻度,混匀,于4℃保存。
3.5 材料
强阴离子固相萃取柱(SAX):1000mg,6mL。使用前依次用5mL甲醇、5mL水活化。
4 仪器和设备
4.1 高效液相色谱仪,带二极管阵列检测器或紫外检测器。
4.2 天平:感量为0.01mg和0.01g。
4.3 高速均质器。
4.4 高速粉碎机。
4.5 固相萃取装置。
4.6 水相型微孔滤膜:孔径0.45μm。
5 分析步骤
警告:实验人员在使用液氮时,应佩戴手套等防护工具,防止意外洒溅,造成冻伤。
5.1 试样制备及保存
5.1.1 液体样品
将果汁及果汁饮料、果味碳酸饮料等样品摇匀分装,密闭常温或冷藏保存。
5.1.2 半固态样品
对果冻、水果罐头等样品取可食部分匀浆后,搅拌均匀,分装,密闭冷藏或冷冻保存。
5.1.3 固体样品
饼干、糕点和生湿面制品等低含水量样品,经高速粉碎机粉碎、分装,于室温下避光密闭保存;对于
固体饮料等呈均匀状的粉状样品,可直接分装,于室温下避光密闭保存。
5.1.4 特殊样品
对于胶基糖果类黏度较大的特殊样品,现将样品用剪刀铰成约2mm×2mm大小的碎块放入陶瓷
研钵中,再缓慢倒入液氮,样品迅速冷冻后采用研磨的方式获取均匀的样品,分装后密闭冷冻保存。
5.2 试样处理
5.2.1 果汁饮料及果汁、果味碳酸饮料
称取5g(精确至0.01g)均匀试样(若试样中含二氧化碳应先加热除去),放入25mL容量瓶中,加
水至刻度,经0.45μm水相滤膜过滤,注入高效液相色谱仪分析。
5.2.2 果冻、水果罐头
称取10g(精确至0.01g)均匀试样,放入50mL塑料离心管中,向其中加入20mL水后在
15000r/min的转速下均质提取2min,4000r/min离心5min,取上层提取液至50mL容量瓶中,残
留物再用20mL水重复提取一次,合并提取液于同一容量瓶中,并用水定容至刻度,经0.45μm水相滤
膜过滤,注入高效液相色谱仪分析。
5.2.3 胶基糖果
称取1g(精确至0.01g)均匀试样,放入50mL具塞塑料离心管中,加入20mL水后在旋混仪上振
荡提取5min,在4000r/min下离心3min后,将上清液转移至100mL容量瓶中,向残渣加入20mL
水重复提取1次,合并提取液于同一容量瓶中,用无水乙醇(3.1.2)定容,摇匀。
准确移取上清液10mL于100mL鸡心瓶中,向鸡心瓶中加入10mL无水乙醇(3.1.2),在80℃±
2℃下旋转浓缩至近干时,再加入5mL无水乙醇(3.1.2)继续浓缩至彻底干燥后,用1mL×1mL水洗
涤鸡心瓶2次。将待净化液全部转移至经过预活化的SAX固相萃取柱中,控制流速在1mL/min~
2mL/min,弃去流出液。用5mL水淋洗净化柱,再用5mL磷酸-甲醇溶液(3.2.2)洗脱,控制流速在
1mL/min~2mL/min,收集洗脱液于50mL鸡心瓶中,洗脱液在45℃下旋转蒸发近干后,再加入
5mL无水乙醇(3.1.2)继续浓缩至彻底干燥后,用1.0mL磷酸溶液(3.2.1)振荡溶解残渣后过0.45μm
滤膜后,注入高效液相色谱仪分析。
5.2.4 固体饮料
称取5g(精确至0.01g)均匀试样,放入50mL烧杯中,加入40mL水溶解并转移至100mL容量
准确移取上清液20mL于100mL鸡心瓶中,向鸡心瓶中加入10mL无水乙醇(3.1.2),在80℃±
2℃下旋转浓缩至近干时,再加入5mL无水乙醇(3.1.2)继续浓缩至彻底干燥后,用1mL×1mL水洗
涤鸡心瓶2次。将待净化液全部转移至经过预活化的SAX固相萃取柱中,控制流速在1mL/min~
2mL/min,弃去流出液。用5mL水淋洗净化柱,再用5mL磷酸-甲醇溶液(3.2.2)洗脱,控制流速在
1mL/min~2mL/min,收集洗脱液于50mL鸡心瓶中,洗脱液在45℃下旋转蒸发近干后,再加入
5mL无水乙醇(3.1.2)继续浓缩至彻底干燥后,用1.0mL磷酸溶液(3.2.1)振荡溶解残渣后过0.45μm
滤膜后,注入高效液相色谱仪分析。
5.2.5 面包、饼干、糕点、烘焙食品馅料和生湿面制品
称取5g(精确至0.01g)均匀试样,放入50mL塑料离心管中,向其中加入20mL水后在
残渣加入20mL水重复提取1次,合并提取液于同一容量瓶中,用无水乙醇(3.1.2)定容,摇匀。
准确移取上清液10mL于100mL鸡心瓶中,向鸡心瓶中加入10mL无水乙醇(3.1.2),在80℃±
2℃下旋转浓缩至近干时,再加入5mL无水乙醇(3.1.2)继续浓缩至彻底干燥后,用1mL×1mL水洗
涤鸡心瓶2次。将待净化液全部转移至经过预活化的SAX固相萃取柱中,控制流速在1mL/min~
2mL/min,弃去流出液。用5mL水淋洗净化柱,再用5mL磷酸-甲醇溶液(3.2.2)洗脱,控制流速在
1mL/min~2mL/min,收集洗脱液于50mL鸡心瓶中,洗脱液在45℃下旋转蒸发近干后,用5.0mL
磷酸溶液(3.2.1)振荡溶解残渣后过0.45μm滤膜后,注入高效液相色谱仪分析。
5.3 仪器参考条件
5.3.1 酒石酸、苹果酸、乳酸、柠檬酸、丁二酸和富马酸的测定
5.3.1.2 流动相:用0.1%磷酸溶液-甲醇=97.5+2.5(体积比)比例的流动相等度洗脱10min,然后用较
短的时间梯度让甲醇相达到100%并平衡5min,再将流动相调整为0.1%磷酸溶液-甲醇=97.5+2.5
(体积比)的比例,平衡5min。
5.3.1.3 柱温:40℃。
5.3.1.4 进样量:20μL。
5.3.1.5 检测波长:210nm。
5.3.2 己二酸的测定
5.3.2.1 色谱柱:CAPECELLPAKMGS5C18柱,4.6mm×250mm,5μm,或同等性能的色谱柱。
5.3.2.2 流动相:0.1%磷酸溶液-甲醇=75+25(体积比)等度洗脱10min。
5.3.2.4 进样量:20μL。
5.3.2.5 检测波长:210nm。
5.4 标准曲线的制作
将标准系列工作液分别注入高效液相色谱仪中,测定相应的峰高或峰面积。以标准工作液的浓度
为横坐标,以色谱峰高或峰面积为纵坐标,绘制标准曲线。(标准图谱见附录A)
5.5 试样溶液的测定
将试样溶液注入高效液相色谱仪中,得到峰高或峰面积,根据标准曲线得到待测液中有机酸的
浓度。
GB 5009.157-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Organic 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 ... 6
5 Analytical Procedures ... 6
6 Expression of Analysis Results ... 9
7 Precision ... 10
8 Others ... 10
Appendix A Standard Chromatogram of Acidity Regulator ... 11
Foreword
This Standard is drafted as a replacement of GB/T 5009.157-2003 “Determination of
Organic Acid in Foods”.
In comparison with GB/T 5009.157-2003 “Determination of Organic Acid in Foods”,
there are several main modifications as follows.
-- The name of this Standard is modified into “National Food Safety Standard -
Determination of Organic Acid in Foods”;
-- The scope of application is extended to jelly, solid beverage and canned fruit,
etc.;
-- Certain tested objects are added to this Standard, such as lactic acid, fumaric
acid and adipic acid, etc.
National Food Safety Standard -
Determination of Organic Acid in Foods
1 Scope
This Standard specifies the method of determining tartaric acid, lactic acid, malic acid,
citric acid, succinic acid, fumaric acid and adipic acid in foods.
This Standard is applicable to the determination of seven types of organic acid in fruit
juice, fruit juice beverage, carbonated beverage, solid beverage, gum-based candy,
cookies, pastry, jelly, canned fruit, fresh dough products and fillings in baked goods.
2 Principle
Directly add water to dilute the sample or adopt water to extract the sample; adopt
strong anion exchange solid phase extraction column for purification; adopt reversed
phase column for separation. Determine the nature through the retention time;
quantify with the external standard method.
3 Reagents and Materials
Unless otherwise indicated, the reagents adopted under this method are of analytical
purity. The water is first-grade water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH). chromatographic purity.
3.1.2 Anhydrous ethanol (CH3CH2OH). chromatographic purity.
3.1.3 Phosphoric acid (H3PO4).
3.2 Preparation of Reagents
3.2.1 Phosphoric acid solution (0.1%). weigh-take 0.1 mL of phosphoric acid; add
water to dilute to 100 mL; mix it up.
3.2.2 Phosphoric acid - methanol solution (2 %). weigh-take 2 mL of phosphoric acid;
add methanol to dilute to 100 mL; mix it up.
3.3 Standards
5.2 Sample Processing
5.2.1 Fruit juice beverage, fruit juice and fruity carbonated beverage
Weigh-take 5 g (accurate to 0.01 g) of homogenized sample (heat up the sample to
remove carbon dioxide if the sample contains carbon dioxide), place it in 25 mL
volumetric flask; add water to the constant volume; adopt 0.45 μm aqueous
membrane to filter the sample; inject it into high-performance liquid chromatograph for
analysis.
5.2.2 Jelly and canned fruit
Weigh-take 10 g (accurate to 0.01 g) of homogenized sample, place it in 50 mL plastic
centrifuge tube, then, add 20 mL of water; homogenize and extract at 15,000 r/min for
2 min, start centrifugation at 4,000 r/min for 5 min; take the upper extract and place it
in 50 mL volumetric flask. Use 20 mL of water to re-extract the remains; combine the
0.45 μm aqueous membrane to filter the sample; inject it into high-performance liquid
chromatograph for analysis.
5.2.3 Gum-based candy
Weigh-take 1 g (accurate to 0.01 g) of homogenized sample, place it in 50 mL plastic
centrifuge tube which is equipped with a plug; add 20 mL of water, then, start
oscillation extraction on a swirling mixer for 5 min; start centrifugation for 3 min at
4,000 r/min, then, transfer the supernatant into 100 mL volumetric flask; add 20 mL of
water to re-extract the remains; combine the extract in the sample volumetric flask.
Use anhydrous ethanol (3.1.2) to dilute to the constant volume, shake it well.
bottle; add 10 mL of anhydrous ethanol (3.1.2) to the bottle, then, rotate and
concentrate it under 80°C±2°C till it approaches dryness; add 5 mL of anhydrous
ethanol (3.1.2) to continue the concentration, till it reaches absolute dryness. Use 1
mL x 1 mL of water to rinse the heart-shaped bottle twice. Transfer all the liquid to be
purified to solid phase extraction column (SAX) that’s pre-activated, control the flow
rate at 1 mL/min~2 mL/min, then, remove the effluent. Use 5 mL of water to rinse the
purification column; use 5 mL of phosphoric acid - methanol solution (3.2.2) to elute it,
control the flow rate at 1 mL/min~2 mL/min; gather the eluent and place it into 50 mL
heart-shaped bottle; rotate the eluent under 45 °C and evaporate till it approaches
reaches absolute dryness. Use 1.0 mL of phosphoric acid solution (3.2.1) to oscillate
and dissolve the remains; adopt 0.45 μm membrane to filter it; inject it into
high-performance liquid chromatograph for analysis.
5.2.4 Solid beverage
Weigh-take 5 g (accurate to 0.01 g) of homogenized sample, place it in 50 mL beaker.
Add 40 mL of water to dissolve it, and transfer it into 100 mL volumetric flask. Adopt
anhydrous ethanol (3.1.2) to dilute to the constant volume; shake it well, place it
evenly for 10 min.
Accurately remove-take 20 mL of supernatant and place it in 100 mL heart-shaped
5.3.1.3 Column temperature. 40 °C.
5.3.1.4 Inlet volume. 20 μL.
5.3.1.5 Detection wavelength. 210 nm.
5.3.2 Determination of adipic acid
5.3.2.1 Chromatographic column. CAPECELL PAK MG S5 C18 column, 4.6 mm x 250
mm, 5 μm, or other chromatographic columns with equivalent performance.
5.3.2.2 Mobile phase. use 0.1% phosphoric acid solution - methanol=75+25 (volume
ratio) equivalent flow to elute for 10 min.
5.3.2.3 Column temperature. 40 °C.
5.3.2.5 Detection wavelength. 210 nm.
5.4 Draw a Standard Curve Line
Respectively inject standard series of working fluid into high-performance liquid
chromatograph; determine the corresponding peak height or peak area. Take the
concentration of standard working fluid as x-coordinate, take the peak height or peak
area as y-coordinate, and draw a standard curve line (please refer to Appendix A for
the standard curve line).
5.5 Determination of Sample Solution
Inject the sample solution into high-performance liquid chromatograph, obtain the
in accordance with the standard curve line.
6 Expression of Analysis Results
The content of organic acid in the sample shall be calculated in accordance with
Formula (1).
Where.
X - The content of organic acid in the sample, expressed in (g/kg);
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