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食品安全国家标准 食品中胆固醇的测定
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GB 5009.128-2016
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标准编号: GB 5009.128-2016 (GB5009.128-2016)
中文名称: 食品安全国家标准 食品中胆固醇的测定
英文名称: National food safety standard -- Determination of cholesterin in foods
行业: 国家标准
中标分类: X09
字数估计: 11,116
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB/T 22220-2008; GB/T 5009.128-2003; GB/T 9695.24-2008
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 5009.128-2016: 食品安全国家标准 食品中胆固醇的测定
GB 5009.128-2016 英文名称: National food safety standard -- Determination of cholesterin in foods
1 范围
本标准规定了食品中胆固醇的测定方法。
本标准适用于食品中胆固醇的测定,第一法气相色谱法适用于肉及肉制品、蛋及蛋制品、乳及乳制
品等各类动物性食品以及植物油脂中胆固醇的测定;第二法高效液相色谱法适用于肉及肉制品、蛋及蛋制品、乳及乳制品等各类动物性食品中胆固醇的测定;第三法比色法适用于肉及肉制品、蛋及蛋制品等动物性食品中胆固醇的测定。
第一法 气相色谱法
2 原理
样品经无水乙醇-氢氧化钾溶液皂化,石油醚和无水乙醚混合提取,提取液浓缩至干,无水乙醇溶解
定容后,采用气相色谱法检测,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(C2H5OH)。
3.1.3 石油醚:沸程30℃~60℃。
3.1.4 无水乙醚(C4H10O)。
3.1.5 无水硫酸钠(Na2SO4)。
3.1.6 氢氧化钾(KOH)。
3.2 试剂配制
3.2.1 60%氢氧化钾溶液:称取60g氢氧化钾,缓慢加水溶解,并定容至100mL。
3.2.2 石油醚-无水乙醚混合液(1+1,体积比):将石油醚和无水乙醚等体积混合均匀。
3.3 标准品
胆固醇标准品(C27H46O,CAS号:57-88-5):纯度≥99%。
3.4 标准溶液配制
3.4.1 胆固醇标准储备液(1.0mg/mL)
称取胆固醇标准品0.05g(精确至0.1mg),用无水乙醇溶解并定容至50mL,放置0℃~4℃密封
可贮藏半年。
3.4.2 胆固醇标准系列工作液
分别吸取标准储备液(1.0mg/mL)25μL、50μL、100μL、500μL、2000μL,用无水乙醇定容至10mL,
该标准系列工作液的浓度分别为2.5μg/mL、5μg/mL、10μg/mL、50μg/mL、200μg/mL。现用现配。
4 仪器和设备
4.1 气相色谱仪:配有氢火焰离子化检测器(FID)。
4.2 电子天平:感量为1mg和0.1mg。
4.3 匀浆机。
4.4 皂化装置。
5 分析步骤
5.1 试样制备
5.1.1 肉及肉制品等各类固体试样
取样品的可食部分200g进行均质。将试样装入密封的容器里,防止变质和成分变化。试样应在
均质化24h内尽快分析。
5.1.2 植物油脂、乳品等液体试样
取混匀后的均匀液体试样装入密封容器里待测。
5.2 样品处理
5.2.1 皂化
称取制备后的样品0.25g~10g(准确至0.001g,胆固醇含量约为0.5mg~5mg),于250mL圆底
烧瓶中,加入30mL无水乙醇,10mL60%氢氧化钾溶液,混匀。将试样在100℃磁力搅拌加热电热套
皂化回流1h,不时振荡防止试样黏附在瓶壁上,皂化结束后,用5mL无水乙醇自冷凝管顶端冲洗其内
部,取下圆底烧瓶,用流水冷却至室温。
5.2.2 提取
定量转移全部皂化液于250mL分液漏斗中,用30mL水分2次~3次冲洗圆底烧瓶,洗液并入分
液漏斗,再用40mL石油醚-无水乙醚混合液(1+1,体积比)分2次~3次冲洗圆底烧瓶并入分液漏斗,
振摇2min,静置,分层。转移水相,合并三次有机相,用水每次100mL洗涤提取液至中性,初次水洗时
轻轻旋摇,防止乳化,提取液通过约10g无水硫酸钠脱水转移到150mL平底烧瓶中。
5.2.3 浓缩
将上述平底烧瓶中的提取液在真空条件下蒸发至近干,用无水乙醇溶解并定容至5mL,待气相色
谱仪测定。
不同试样的前处理需要同时做空白试验。
5.3 测定
5.3.1 仪器参考条件
a) 色谱柱:DB-5弹性石英毛细管柱,柱长30m,内径0.32mm,粒径0.25μm,或同等性能的色
谱柱;
b) 载气:高纯氮气,纯度≥99.999%;恒流2.4mL/min;
c) 柱温(程序升温):初始温度为200℃,保持1min,以30℃/min速率升至280℃,保持10min;
d) 进样口温度280℃;
e) 检测器温度:290℃;
f) 进样量:1μL;
g) 进样方式:不分流进样,进样1min后开阀;
h) 空气流量:350mL/min;
i) 氢气流量:30mL/min。
5.3.2 标准曲线的制作
分别取胆固醇标准系列工作液注入气相色谱仪,在上述色谱条件下测定标准溶液的响应值(峰面
积),以浓度为横坐标、峰面积为纵坐标,制作标准曲线。
5.3.3 测定
试样溶液注入气相色谱仪,测定峰面积,由标准曲线得到试样溶液中胆固醇的浓度。根据保留时间
定性,外标法定量。胆固醇标准溶液的色谱图见图A.1。
6 分析结果的表述
试样中胆固醇的含量按式(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%。
当称样量为0.5g,定容体积为5.0mL,方法的检出限为0.3mg/100g,定量限为1.0mg/100g。
第二法 高效液相色谱法
9 原理
样品经无水乙醇-氢氧化钾溶液皂化,石油醚和无水乙醚混合提取,提取液浓缩至干,无水乙醇溶解
定容后,采用高效液相色谱仪检测,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(CH3OH):色谱纯。
10.1.3 石油醚:沸程30℃~60℃。
10.1.4 无水乙醚(C4H10O)。
10.1.5 无水硫酸钠(Na2SO4)。
10.1.6 氢氧化钾(KOH)。
10.2 试剂配制
10.2.1 60%氢氧化钾溶液:称取60g氢氧化钾,缓慢加水溶解,并定容至100mL。
10.2.2 石油醚-无水乙醚混合液(1+1,体积比):将石油醚和无水乙醚等体积混合均匀。
10.3 标准品
胆固醇标准品(C27H46O,CAS号:57-88-5):纯度≥99%。
10.4.1 胆固醇标准储备液(1.0mg/mL):称取胆固醇标准品0.05g(精确至0.1mg),用无水乙醇溶解
并定容至50mL,放置0℃~4℃密封可贮藏半年。
10.4.2 胆固醇标准系列工作液:分别吸取标准储备液(1.0mg/mL)25μL、50μL、100μL、500μL、
2000μL,用无水乙醇定容至10mL,该标准系列工作液的浓度分别为2.5μg/mL、5μg/mL、10μg/mL、
50μg/mL、200μg/mL。现用现配。
11 仪器和设备
11.1 匀浆机。
11.2 高效液相色谱仪:配有紫外检测器或相当的检测器。
11.3 电子天平:感量为1mg和0.1mg。
12.1 试样制备
12.1.1 肉及肉制品等各类固体试样
样品取可食部分200g,使用绞肉机或匀浆机将试样均质。将试样装入密封的容器里,防止变质和
成分变化。试样应在均质化24h内尽快分析。
12.1.2 乳品等液体试样
取混匀后的均匀液体试样装入密封容器里待测。
12.2 样品处理
12.2.1 皂化
称取制备后的样品0.25g~10g(精确至0.001g,胆固醇含量约为0.5mg~5mg),于250mL圆底
皂化回流1h,不时振荡防止试样黏附在瓶壁上,皂化结束后,用5mL无水乙醇自冷凝管顶端冲洗其内
部,取下圆底烧瓶,用流水冷却至室温。
12.2.2 提取
定量转移全部皂化液于250mL分液漏斗中,用30mL水分2次~3次冲洗圆底烧瓶,洗液并入分
液漏斗,再用40mL石油醚-无水乙醚混合液(1+1,体积比)分2次~3次冲洗圆底烧瓶并入分液漏斗,
振摇2min,静置,分层。转移水相,合并三次有机相,用水每次100mL洗涤提取液至中性,初次水洗时
轻轻旋摇,防止乳化,提取液通过约10g无水硫酸钠脱水转移到150mL平底烧瓶中。
12.2.3 浓缩
将上述平底烧瓶中的提取液在真空条件下蒸发至近干,用无水乙醇溶解并定容至5mL,溶液通过
不同试样的前处理需要同时做空白试验。
12.3 测定
12.3.1 仪器参考条件
a) 色谱柱:C18反相色谱柱,柱长4.6mm,内径150mm,粒径5μm,或同等性能的色谱柱;
b) 柱温:38℃;
c) 流动相:甲醇;
d) 流速:1.0mL/min;
e) 测定波长:205nm;
f) 进样量:10μL。
分别取10μL胆固醇标准工作液注入高效液相色谱仪,在上述色谱条件下测定标准溶液的响应值
(峰面积),以浓度为横坐标、峰面积为纵坐标,制作标准曲线。
12.3.3 测定
将10μL试样溶液注入高效液相色谱仪,测定峰面积,由标准曲线得到试样溶液中胆固醇的浓度。
胆固醇标准溶液的色谱图见图A.2。
GB 5009.128-2016
GB
NATIONAL STANDARD OF
THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Determination of cholesterol in foods
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People's Republic of China;
China Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle... 4
3 Reagents and materials ... 4
4 Instruments and equipment ... 5
5 Analysis steps ... 5
6 Expression of analysis result ... 7
7 Precision... 8
8 Other ... 8
9 Principle... 8
10 Reagents and materials... 8
11 Instruments and equipment ... 9
12 Analysis steps ... 9
13 Expression of analysis result ... 11
14 Precision ... 12
15 Other ... 12
16 Principle... 12
17 Reagents and materials... 12
18 Instruments and equipment ... 14
19 Analysis steps ... 14
20 Expression of analysis result ... 15
21 Precision ... 15
22 Other ... 15
Annex A Cholesterol standard chromatogram ... 16
Foreword
This Standard replaces GB/T 5009.128-2003 Determination of cholesterol in
foods, GB/T 22220-2008 Determination of cholesterol in foods - High-
performance liquid chromatography and GB/T 9695.24-2008 Meat and meat
products - Determination of cholesterol.
Compared with GB/T 5009.128-2003, the main changes in this Standard are as
follows.
- modified the standard’s name to “National food safety standard -
Determination of cholesterol in foods”;
- added gas chromatography as method one and high-performance liquid
chromatography as method two; modified colorimetric method as method
three;
- modified extraction solvent, the amount of ethanol and volume constant
volume in pre-treatment method of gas chromatography in GB/T 9695.24-
2008.
National food safety standard -
Determination of cholesterol in foods
1 Scope
This Standard specifies the determination methods of cholesterol in foods.
This Standard applies to the determination of cholesterol in foods. Method One
Gas chromatography is applicable to the determination of cholesterol in meat
and meat products, eggs and egg products, milk and dairy products and other
animal foods and vegetable oils and fats. Method Two High performance liquid
chromatography is applicable to the determination of cholesterol in meat and
meat products, eggs and egg products, milk and dairy products and other
animal foods. Method Three Colorimetric method is applicable to the
determination of cholesterol in meat and meat products, eggs and egg products
and other animal foods.
Method One -- Gas chromatography
2 Principle
Saponify the sample by absolute ethanol - potassium hydroxide solution.
Extract by petroleum ether and anhydrous ether mixture. Concentrate the
extract to dry. After dissolving and setting volume of absolute ethanol, use gas
chromatography to detect, external standard method to quantify.
Unless otherwise indicated, the reagents used in this method are of analytical
grade, water is grade one water specified in GB/T 6682.
3.1 Reagents
3.1.1 methanol (CH3OH). chromatographic pure
3.1.2 anhydrous ethanol (C2H5OH)
3.1.3 petroleum ether. boiling range 30°C ~ 60°C
3.1.4 anhydrous ether (C4H10O).
Take 200 g of edible part of sample to homogenize. Place the sample in a
sealed container to prevent deterioration and composition changes. The
homogenization.
5.1.2 Vegetable oil, dairy products and other liquid samples
Take the uniform liquid sample after mixing into a sealed container to be tested.
5.2 Sample processing
5.2.1 Saponification
Weigh 0.25g ~ 10g of prepared sample (nearest to 0.001 g, cholesterol content
of about 0.5mg ~ 5mg) in a 250-mL round-bottomed flask. Add 30 mL of
absolute ethanol, 10 mL of 60% potassium hydroxide solution. Well mix. Heat
the sample at 100°C magnetic stirring heater for saponification reflux 1h. Shake
saponification ends, use 5 mL of absolute ethanol to rinse its interior from the
condenser top. Remove the round-bottomed flask. Cool to room temperature
with running water.
5.2.2 Extraction
Quantitatively transfer of all saponified liquid in a 250-mL separatory funnel.
Use 30 mL of water to rinse the round-bottomed flask in 2~3 times. Merge the
washing liquid into the separatory funnel. Then use 40 mL of petroleum ether -
anhydrous ether mixture (1+1, volume ratio) to rinse the round-bottomed flask
in 2~3 times. Then merge into the separatory funnel. Shake 2 min, place still
the extract to neutral with 100 mL of water each time. Swirl gently for the first
time to prevent emulsification. The extract shall be dehydrated with about 10 g
of anhydrous sodium sulfate to a 150-mL flask.
5.2.3 Concentration
Evaporate the extract in the flat-bottomed flask to near dryness under vacuum
condition. Use absolute ethanol to dissolve and set volume to 5 mL. Determine
with gas chromatograph.
The pretreatment of different sample requires blank test at the same time.
5.3 Determination
a) Chromatographic column. DB-5 flexible quartz capillary column, column
length of 30 m, inner diameter of 0.32 mm, particle size of 0.25 μm, or
Take 200 g of edible part of sample. Use a meat grinder or homogenizer to
homogenize the sample. Place the sample in a sealed container to prevent
deterioration and composition changes. The sample shall be analyzed as soon
as possible within 24 hours of homogenization
12.1.2 Dairy and other liquid samples
Take well mixed uniform liquid sample into a sealed container for testing.
12.2 Sample processing
Weigh 0.25g ~ 10g of prepared sample (nearest to 0.001 g, cholesterol content
of about 0.5mg ~ 5mg) in a 250-mL round-bottomed flask. Add 30 mL of
absolute ethanol, 10 mL of 60% potassium hydroxide solution. Well mix. Heat
the sample at 100°C magnetic stirring heater for saponification reflux 1h. Shake
from time to time so as to prevent sample sticking to the bottle wall. After the
saponification ends, use 5 mL of absolute ethanol to rinse its interior from the
condenser top. Remove the round-bottomed flask. Cool to room temperature
with running water.
12.2.2 Extraction
Use 30 mL of water to rinse the round-bottomed flask in 2~3 times. Merge the
washing liquid into the separatory funnel. Then use 40 mL of petroleum ether -
anhydrous ether mixture (1+1, volume ratio) to rinse the round-bottomed flask
in 2~3 times. Then merge into the separatory funnel. Shake 2 min, place still
and stratify. Transfer the water phase. Combine the three organic phases. Wash
the extract to neutral with 100 mL of water each time. Swirl gently for the first
time of water washing to prevent emulsification. The extract shall be dehydrated
with about 10 g of anhydrous sodium sulfate to a 150-mL flask.
12.2.3 Concentration
condition. Use absolute ethanol to dissolve and set volume to 5 mL. The
solution passes through a 0.45 μm filter membrane. Collect the filtrate in the
sample bottle. Determine with high performance liquid chromatograph.
The pretreatment of different sample requires blank test at the same time.
12.3 Determination
12.3.1 Instrument reference conditions
a) Chromatographic column. C18 reversed-phase column, column length
17.4.2 Cholesterol standard working solution (100 μg/mL). pipette 10 mL of
cholesterol standard stock solution (1.0 mg/mL); use glacial ac...
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