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食品安全国家标准 食品中维生素K1的测定
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GB 5009.158-2016
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标准编号: GB 5009.158-2016 (GB5009.158-2016)
中文名称: 食品安全国家标准 食品中维生素K1的测定
英文名称: National Food Safety Standard -- Determination of Vitamin K1 in Foods
行业: 国家标准
中标分类: X09
字数估计: 15,160
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB 5413.10-2010; GB/T 5009.158-2003
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 5009.158-2016: 食品安全国家标准 食品中维生素K1的测定
GB 5009.158-2016 英文名称: National Food Safety Standard -- Determination of Vitamin K1 in Foods
1 范围
本标准规定了食品中维生素K1 的测定方法。
本标准第一法为高效液相色谱-荧光检测法,第二法为液相色谱-串联质谱法,均适用于各类配方食
品、植物油、水果和蔬菜中维生素K1 的测定。
第一法 高效液相色谱-荧光检测法
2 原理
婴幼儿食品和乳品、植物油等样品经脂肪酶和淀粉酶酶解,正己烷提取样品中的维生素K1 后,用
C18液相色谱柱将维生素K1 与其他杂质分离,锌柱柱后还原,荧光检测器检测,外标法定量。
水果、蔬菜等低脂性植物样品,用异丙醇和正己烷提取其中的维生素K1,经中性氧化铝柱净化,去
除叶绿素等干扰物质。用C18液相色谱柱将维生素K1 与其他杂质分离,锌柱柱后还原,荧光检测器检
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 无水乙醇(CH3CH2OH)。
3.1.2 碳酸钾(K2CO3)。
3.1.3 无水硫酸钠(Na2SO4)。
3.1.4 异丙醇(C3H8O)。
3.1.5 正己烷(C6H14)。
3.1.6 甲醇(CH3OH):色谱纯。
3.1.7 四氢呋喃(C4H8O):色谱纯。
3.1.8 乙酸乙酯(C4H8O2)。
3.1.9 冰乙酸(CH3COOH):色谱纯。
3.1.10 氯化锌(ZnCl2):色谱纯。
3.1.11 无水乙酸钠(CH3COONa)。
3.1.12 氢氧化钾(KOH)。
3.1.13 脂肪酶:酶活力≥700U/mg。
3.1.14 淀粉酶:酶活力≥1.5U/mg。
3.1.15 锌粉:粒径50μm~70μm。
3.2 试剂配制
3.2.1 40%氢氧化钾溶液:称取20g氢氧化钾于100mL烧杯中,用20mL水溶解,冷却后,加水至
50mL,储存于聚乙烯瓶中。
3.2.2 磷酸盐缓冲液(pH8.0):溶解54.0g磷酸二氢钾于300mL水中,用40%氢氧化钾溶液调节pH
至8.0,加水至500mL。
3.2.3 正己烷-乙酸乙酯混合液(90+10):量取90mL正己烷,加入10mL乙酸乙酯,混匀。
3.2.4 流动相:量取甲醇900mL,四氢呋喃100mL,冰乙酸0.3mL,混匀后,加入氯化锌1.5g,无水乙
酸钠0.5g,超声溶解后,用0.22μm有机系滤膜过滤。
3.3 标准品
维生素K1(C31H46O2,CAS号:84-80-0):纯度≥99%,或经国家认证并授予标准物质证书的标准
物质。
3.4 标准溶液配制
3.4.1 维生素K1 标准贮备溶液(1mg/mL):准确称取50mg(精确至0.1mg)维生素K1 标准品于50
mL容量瓶中,用甲醇溶解并定容至刻度。将溶液转移至棕色玻璃容器中,在-20℃下避光保存,保存
期2个月。标准储备液在使用前需要进行浓度校正,校正方法参照附录A。
3.4.2 维生素K1 标准中间液(100μg/mL):准确吸取标准贮备溶液10.00mL于100mL容量瓶中,加
甲醇至刻度,摇匀。将溶液转移至棕色玻璃容器中,在-20℃下避光保存,保存期2个月。
3.4.3 维生素K1 标准使用液(1.00μg/mL):准确吸取标准中间液1.00mL于100mL容量瓶中,加甲
醇至刻度,摇匀。
3.4.4 标准系列工作溶液:分别准确吸取维生素K1 标准使用液0.10mL、0.20mL、0.50mL、1.00mL、
2.00mL、4.00mL于10mL容量瓶中,加甲醇定容至刻度,维生素 K1 标准系列工作溶液浓度分别为
10ng/mL、20ng/mL、50ng/mL、100ng/mL、200ng/mL、400ng/mL。
3.5 材料
3.5.1 中性氧化铝:粒径50μm~150μm。
3.5.2 中性氧化铝柱:2g/6mL,填料中含10%水,可直接购买商品柱,也可自行装填。
注:中性氧化铝柱装填方法:
a) 填料处理:取200g中性氧化铝于500mL的广口瓶中,于150℃干燥箱中烘烤2h,加盖后于干燥器中冷
却至室温,缓慢加入20mL纯水,边加边摇,加完后加上瓶盖,放入80℃烘箱中3min~5min,取出后,剧
烈振摇,直至瓶内氧化铝自由流动,无结块,置于干燥器中冷却30min,备用。
b) 层析柱装填:取6mL的针筒式柱套,加入筛板,称取2.00g上述经脱活化处理的填料,再加入一块筛板,
用装填工具压紧。
3.5.3 锌柱:柱长50mm,内径4.6mm,锌柱可直接购买商品柱,也可自行装填。
注:
a) 锌柱填装方法:将锌粉密集装入不锈钢材质的柱套(柱长50mm,内径4.6mm)中。装柱时,应连续少量多次将锌粉装入柱中,边装边轻轻拍打,以使装入的锌粉紧密。
b) 锌柱接入仪器前,须将液相色谱仪所用管路中的水排干。
3.5.4 微孔滤头:带0.22μm有机系微孔滤膜。
4 仪器和设备
4.1 高效液相色谱仪:带荧光检测器。
4.2 匀浆机。
4.3 高速粉碎机。
4.4 组织捣碎机。
4.5 涡旋振荡器。
4.6 恒温水浴振荡器。
4.7 pH计:精度0.01。
4.8 天平:感量为1mg和0.1mg。
4.9 离心机:转速≥6000r/min。
4.10 旋转蒸发仪。
4.11 氮吹仪。
4.12 超声波振荡器。
5.1 试样制备
米粉、奶粉等粉状样品经混匀后,直接取样;片状、颗粒状样品,经样本粉碎机磨成粉,储存于样品袋
中备用;液态乳、植物油等液态样品摇匀后,直接取样;水果、蔬菜等取可食部分,水洗干净,用纱布擦去
表面水分,经匀浆器匀浆,储存于样品瓶中备用。制样后,需尽快测定。
5.2 试样处理
警示:处理过程应避免紫外光直接照射,尽可能避光操作。
5.2.1 婴幼儿食品和乳品、植物油
5.2.1.1 酶解
准确称取经均质的试样1g~5g(精确到0.01g,维生素K1 含量不低于0.05μg)于50mL离心管
5mL,混匀,加入0.2g脂肪酶和0.2g淀粉酶(不含淀粉的样品可以不加淀粉酶),加盖,涡旋2min~
3min,混匀后,置于37℃±2℃恒温水浴振荡器中振荡2h以上,使其充分酶解。
5.2.1.2 提取
取出酶解好的试样,分别加入10mL乙醇及1g碳酸钾,混匀后加入10mL正己烷和10mL水,涡
旋或振荡提取10min,6000r/min离心5min,或将酶解液转移至150mL的分液漏斗中萃取提取,静
置分层(如发生乳化现象,可适当增加正己烷或水的加入量,以排除乳化现象),转移上清液至100mL
旋蒸瓶中,向下层液再加入10mL正己烷,重复操作1次,合并上清液至上述旋蒸瓶中。
5.2.1.3 浓缩
将上述正己烷提取液旋蒸至干(如有残液,可用氮气轻吹至干),用甲醇转移并定容至5mL容量瓶
不加试样,按同一操作方法做空白试验。
5.2.2 水果、蔬菜样品
5.2.2.1 提取
准确称取1g~5g(精确到0.01g,维生素K1 含量不低于0.05μg)经均质匀浆的样品于50mL离
心管中,加入5mL异丙醇,涡旋1min,超声5min,再加入10mL正己烷,涡旋振荡提取3min,
6000r/min离心5min,移取上清液于25mL棕色容量瓶中,向下层溶液中加入10mL正己烷,重复提
取1次,合并上清液于上述容量瓶中,正己烷定容至刻度,用移液管准确分取上清液1mL~5mL(视样
品中维生素K1 含量而定)至10mL试管中,氮气轻吹至干,加入1mL正己烷溶解,待净化。
5.2.2.2 净化、浓缩
至近干时,5mL正己烷淋洗,6mL正己烷-乙酸乙酯混合液洗脱至10mL试管中,氮气吹干后,用甲醇
定容至5mL,过0.22μm滤膜,滤液供分析测定。
不加试样,按同一操作方法做空白试验。
5.3 色谱参考条件
a) 色谱柱:C18柱,柱长250mm,内径4.6mm,粒径5μm,或具同等性能的色谱柱;
b) 锌还原柱:柱长50mm,内径4.6mm;
c) 流动相:按3.2.4配制;
d) 流速:1mL/min;
e) 检测波长:激发波长为243nm,发射波长为430nm;
g) 标准溶液的色谱图见附录B。
5.4 标准曲线的制作
采用外标标准曲线法进行定量。将维生素K1 标准系列工作液分别注入高效液相色谱仪中,测定
相应的峰面积,以峰面积为纵坐标,以标准系列工作液浓度为横坐标绘制标准曲线,计算线性回归方程。
5.5 试样溶液的测定
在相同色谱条件下,将制备的空白溶液和试样溶液分别进样,进行高效液相色谱分析。以保留时间
定性,峰面积外标法定量,根据线性回归方程计算出试样溶液中维生素K1 的浓度。
6 分析结果的表述
试样中维生素K1 的含量按式(1)计算。
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
婴幼儿食品和乳品、植物油,当取样量为1g,定容5mL时,检出限为1.5μg/100g,定量限为
5μg/100g;果蔬样品当取样量为5g,提取液分取5mL,定容5mL时,检出限为1.5μg/100g,定量限
为5μg/100g。
第二法 液相色谱-串联质谱法
9 原理
婴幼儿食品和乳品、植物油等样品经脂肪酶和淀粉酶酶解,用正己烷提取样品中的维生素K1 后,
用C18液相色谱柱将维生素K1 与其他杂质分离,串联质谱检测,同位素内标法定量。
除叶绿素等干扰物质。用C18液相色谱柱将维生素K1 与其他杂质分离,串联质谱检测,同位素内标法
定量。
GB 5009.158-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Vitamin K1 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;
State Food and Drug Administration of the People’s Republic
of China.
Table of Contents
Foreword ... 3
1 Scope ... 4
Method I High-performance Liquid Phase Chromatography - Fluorescence
Detection ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Instruments and Equipment ... 7
5 Analytical Procedures ... 7
6 Expression of Analysis Results ... 10
7 Precision ... 10
8 Others ... 11
Method II Liquid Phase Chromatography - Tandem Mass Spectrometry ... 11
9 Principle ... 11
10 Reagents and Materials... 11
11 Instruments and Equipment ... 13
12 Analytical Procedures ... 14
13 Expression of Analysis Results ... 18
14 Precision ... 18
15 Others ... 18
Appendix A Methods of Correcting Vitamin K1 Standard Concentration ... 19
Appendix B High-performance Liquid Phase Chromatogram ... 20
Appendix C Mass Spectrometry Reference Figure ... 21
Foreword
This Standard is drafted as a replacement of GB/T 5009.158-2003 “Determination of
Vitamin K1 in Vegetables” and GB 5413.10-2010 “National Food Safety Standard -
Determination of Vitamin K1 in Foods for Infants and Young Children, Milk and Milk
Products”.
In comparison with GB/T 5009.158-2003, there are several main modifications as
follows.
-- The name of this Standard is modified into “National Food Safety Standard -
Determination of Vitamin K1 in Foods”;
-- The method of high-performance liquid phase chromatography - fluorescence
detection is added;
-- The method of liquid phase chromatography - tandem mass spectrometry is
added;
-- The method of high-performance liquid phase chromatography - UV detection is
deleted.
National Food Safety Standard -
Determination of Vitamin K1 in Foods
1 Scope
This Standard specifies the method of determining vitamin K1 in foods.
In this Standard, Method I is high-performance liquid phase chromatography -
fluorescence detection, Method II is liquid phase chromatography - tandem mass
spectrometry, which are both applicable to the determination of vitamin K1 in all kinds
of formula food, vegetable oil, fruit and vegetable.
Method I -- High-performance Liquid Phase
Chromatography - Fluorescence Detection
2 Principle
Through lipase and amylase enzymolysis of samples like foods for infants and young
children, dairy products and vegetable oil, adopt N-hexane to extract vitamin K1 from
the sample. Adopt C18 liquid phase chromatographic column to separate vitamin K1
from other impurities. Adopt zinc column for reduction, adopt fluorescence detector for
detection; quantify with the external standard method.
Adopt isopropanol and N-hexane to extract vitamin K1 from low-fat plant samples like
fruit and vegetable; adopt neutral alumina column for purification, remove interfering
substances like chlorophyll. Adopt C18 liquid phase chromatographic column to
separate vitamin K1 from other impurities. Adopt zinc column for reduction, adopt
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 Anhydrous ethanol (CH3CH2OH).
sample grinder to grind flaky and granular samples into powder, store it in a sample
bag for later usage; shake liquid samples like liquid milk and vegetable oil, directly
take the ample; take edible part from fruit and vegetable, use water to rinse it, then,
use gauze to remove water on the surface; homogenize with the homogenizer, store
sample is prepared.
5.2 Processing of Samples
Note. during the processing, avoid direct UV light irradiation, keep away from light.
5.2.1 Foods for infants and young children, dairy products and vegetable oil
5.2.1.1 Enzymolysis
Accurately weigh-take 1 g ~5 g (accurate to 0.01 g, the content of vitamin K1 shall be
≥0.05 μg) of homogenized sample, place it in 50 mL centrifuge tube. Add 5 mL of
warm water to dissolve it (directly absorb 5 mL of liquid sample; no water shall be
added to dilute vegetable oil), add 5 mL of phosphate buffer solution (pH8.0), mix it up.
doesn’t contain starch); put on the lid, start vortex for 2 min~3 min. Mix it up, place it in
37°C±2°C constant-temperature water bath oscillator and start oscillation for over 2 h,
so that it can go through thorough enzymolysis.
5.2.1.2 Extraction
After enzymolysis, take out the sample, respectively add 10 mL of ethanol and 1 g of
potassium carbonate, then, mix it up; add 10 mL of N-hexane and 10 mL of water,
start vortex or oscillation extraction for 10 min, start centrifugation at 6,000 r/min for 5
min; or transfer enzymolysis solution to 150 mL separating funnel for extraction, start
static stratification (if emulsification emerges, more N-hexane or water can be properly
evaporation bottle, add 10 mL of N-hexane to the underlayer fluid; repeat the above
operation once, then, combine the supernatant into the above-mentioned rotary
evaporation bottle.
5.2.1.3 Concentration
Start rotary evaporation of the above-mentioned N-hexane extract, till it reaches
dryness (if there’s any residual solution, slightly blow with nitrogen till it reaches
dryness); use methanol to transfer and dilute to the constant volume in 5 mL
volumetric flask, mix it up. Use 0.22 μm membrane to filter it, reserve the filtrate for
inlet.
5.2.2 Fruit and vegetable
8 Others
Foods for infants and young children, dairy products and vegetable oil. when the
amount of sampling is 1 g and the constant volume is 5 mL, the detection limit is 1.5
μg/100 g and the quantitation limit is 5 μg/100 g; fruit and vegetable. when the amount
of sampling is 5 g, the volume of dispensed extract is 5 mL and the constant volume is
5 mL, the detection limit is 1.5 μg/100 g and the quantitation limit is 5 μg/100 g.
Method II -- Liquid Phase Chromatography - Tandem
Mass Spectrometry
Through lipase and amylase enzymolysis of samples like foods for infants and young
children, dairy products and vegetable oil, adopt N-hexane to extract vitamin K1 from
the sample. Adopt C18 liquid phase chromatographic column to separate vitamin K1
from other impurities. Adopt tandem mass spectrometry for detection; quantify with
the isotope-labeled internal standard method.
Adopt isopropanol and N-hexane to extract vitamin K1 from low-fat plant samples like
fruit and vegetable; adopt neutral alumina column for purification, remove interfering
substances like chlorophyll. Adopt C18 liquid phase chromatographic column to
separate vitamin K1 from other impurities. Adopt tandem mass spectrometry for
10 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.
10.1 Reagents
10.1.1 Anhydrous ethanol (CH3CH2OH).
10.1.2 Potassium carbonate (K2CO3).
10.1.3 Potassium hydroxide (KOH).
10.1.4 Formic acid (HCOOH). chromatographic purity.
10.1.5 Ammonium formate (HCOONH4). chromatographic purity.
standard intermediate fluid of vitamin K1 and place it in 100 mL volumetric flask; add
methanol to the constant volume, mix it up.
10.4.4 Isotope-labeled internal standard stock solution of vitamin K1-D7 (100 μg/mL).
accurately weigh-take 1 mg (accurate to 0.01 mg) of isotope-labeled internal standard
of vitamin K1-D7; use methanol to dissolve and dilute to the constant volume of 10 mL.
10.4.5 Isotope-labeled internal standard working fluid of vitamin K1-D7 (1 μg/mL).
absorb 1.00 mL of isotope-labeled internal standard stock solution of vitamin K1-D7,
then, place it in 100 mL volumetric flask; add methanol to the constant volume, mix it
up.
0.20 mL, 0.50 mL, 1.00 mL, 2.00 mL and 4.00 mL of standard working fluid of vitamin
K1, place it in 10 mL volumetric flask; respectively add 0.50 mL of isotope-labeled
internal standard working fluid; add methanol to dilute to the constant volume. The
concentration of the standard series of working fluid of vitamin K1 is 10 ng/mL, 20
ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL and 400 ng/mL respectively.
10.5 Materials
10.5.1 Neutral alumina. particle size 50 μm~150μm.
10.5.2 Neutral alumina column. 2 g/6 mL, 10% of water is contained in the filling.
Commodity column can be purchased directly, or it can be replaced by filling.
a) Filling processing. take 200 g of neutral alumina and place it in 500 mL wide-mouth bottle;
bake for 2 h in a drying oven under 150 °C; put on the lid, then, cool it down in the drying
oven, till it reaches the room temperature. Slowly add 20 mL of pure water, shake it
while adding the water; put on the lid, place it in the oven for 3 min~5 min under 80 °C;
take it out, violently shake it, till alumina in the bottle can flow freely without showing any
agglomeration; place it in the drying oven and cool it down for 30 min, reserve for later
usage.
b) Filling of chromatographic column. take 6 mL syringe-shaped column sleeve, add sieve
plate; weigh-take 2.00 of the above-mentioned filling that’s deactivated; add another
10.5.3 Microporous filter head. equipped with 0.22 μm microporous membrane.
11 Instruments and Equipment
11.1 Liquid phase chromatography - tandem mass spectrometer. equipped with
electrospray ionization (ESI).
starch); put on the lid, start vortex for 2 min~3 min. Mix it up, place it in 37°C±2°C
constant-temperature water bath oscillator and start oscillation for over 2 h, so that it
can go through thorough enzymolysis.
12.2.1.2 Extraction
After enzymolysis, take out the sample, respectively add 10 mL of ethanol and 1 g of
for 10 min, start centrifugation at 6,000 r/min for 3 min. Transfer the supernatant to
another 50 mL centrifuge tube, add 10 mL of N-hexane to the underlayer fluid. Start
vortex for 5 min and centrifugation at 6,000 r/min for 3 min. Combine the supernatant,
add N-hexane solution to dilute to the constant volume of 25 mL, reserve for
purification.
12.2.1.3 Purification
Add 20 mL of water to the above-mentioned extract, shake for 0.5 min. After static
stratification, respectively take 5 mL of the supernatant and place it in 10 mL glass
tube. Use nitrogen to blow it, till it reaches dryness. Add 1 mL of methanol to dissolve
Add no sample, comply with the same method of operation and start a blank test.
12.2.2 Fruit and vegetable
12.2.2.1 Extraction
Accurately weigh-take 1 g~5 g (accurate to 0.01 g, the content of vitamin K1 shall be
≥0.02 μg) of homogenized sample, place it in 50 mL centrifuge tube. Add 0.25 mL of
isotope-labeled internal standard working fluid (1 μg/mL); add 5 mL of isopropanol.
Start vortex for 1 min and ultrasonic for 5 min. Add 10 mL of N-hexane, start vortex or
oscillation extraction for 3 min, start centrifugation at 6,000 r/min for 5 min.
Remove-take the supernatant in 25 mL brown volumetric flask. Add 10 mL of
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