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食品安全国家标准 食品中维生素B6的测定
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GB 5009.154-2016
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标准编号: GB 5009.154-2016 (GB5009.154-2016)
中文名称: 食品安全国家标准 食品中维生素B6的测定
英文名称: National food safety standard -- Determination of vitamin B6 in foods
行业: 国家标准
中标分类: X09
字数估计: 12,17
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB 5413.13-2010; GB/T 5009.154-2003
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 5009.154-2016: 食品安全国家标准 食品中维生素B6的测定
GB 5009.154-2016 英文名称: National food safety standard -- Determination of vitamin B6 in foods
1 范围
本标准规定了食品中维生素B6 的测定方法。
本标准第一法为高效液相色谱法,适用于添加了维生素B6 的食品测定;第二法为微生物法,适用于
各类食品中维生素B6 的测定。
第一法 高效液相色谱法
2 原理
试样经提取等前处理后,经C18色谱柱分离,高效液相色谱-荧光检测器检测,外标法定量测定维生
素B6(吡哆醇、吡哆醛、吡哆胺)的含量。
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 辛烷磺酸钠(C8H17NaO3S)。
3.1.2 冰乙酸(C2H4O2)。
3.1.3 三乙胺(C6H15N):色谱纯。
3.1.4 甲醇(CH4O):色谱纯。
3.1.5 盐酸(HCl)。
3.1.6 氢氧化钠(NaOH)。
3.1.7 淀粉酶:酶活力≥1.5U/mg。
3.2 试剂配制
3.2.1 盐酸溶液(5.0mol/L):量取45mL盐酸,用水稀释并定容至100mL。
3.2.2 盐酸溶液(0.1mol/L):吸取9mL盐酸,用水稀释并定容至1000mL。
3.2.3 氢氧化钠溶液(5.0mol/L):称取20g氢氧化钠,加50mL水溶解,冷却后,用水定容至100mL。
3.2.4 氢氧化钠溶液(0.1mol/L):称取0.4g氢氧化钠,加50mL水溶解,冷却后,用水定容至
100mL。
3.3 标准品
3.3.1 盐酸吡哆醇(C8H12ClNO3,CAS号:58-56-0):纯度≥98%,或经国家认证并授予标准物质证书
的标准物质。
3.3.2 盐酸吡哆醛(C8H10ClNO3,CAS号:65-22-5):纯度≥99%,或经国家认证并授予标准物质证书
的标准物质。
3.3.3 双盐酸吡哆胺(C8H14Cl2N2O3,CAS号:524-36-7):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 吡哆醇标准储备液(1mg/mL):准确称取60.8mg盐酸吡哆醇标准品,用0.1mol/L盐酸溶液溶
解后定容到50mL,在-20℃下避光保存,有效期1个月。
3.4.2 吡哆醛标准储备液(1mg/mL):准确称取60.9mg盐酸吡哆醛标准品,用0.1mol/L盐酸溶液溶
解后定容到50mL,在-20℃下避光保存,有效期1个月。
3.4.3 吡哆胺标准储备液(1mg/mL):准确称取71.7mg双盐酸吡哆胺标准品,用0.1mol/L盐酸溶液
溶解后定容到50mL,在-20℃下避光保存,有效期1个月。
3.4.4 维生素B6 混合标准中间液(20μg/mL):分别准确吸取吡哆醇、吡哆醛、吡哆胺的标准储备液各
1.00mL,用0.1mol/L盐酸溶液稀释并定容至50mL。临用前配制。
3.4.5 维生素B6 混合标准系列工作液:分别准确吸取维生素B6 混合标准中间液0.5mL、1.0mL、
2.0mL、3.0mL、5.0mL,至100mL容量瓶中,用水定容。该标准系列浓度分别为0.10μg/mL、
0.20μg/mL、0.40μg/mL、0.60μg/mL、1.00μg/mL。临用前配制。
注: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。
4 仪器和设备
4.1 高效液相色谱仪:带荧光检测器。
4.2 天平:感量1mg和0.01mg。
4.3 pH计:精度0.01。
4.4 涡旋混合器。
4.5 超声波振荡器。
4.6 分光光度计。
4.7 恒温培养箱,或性能相当者。
5 分析步骤
5.1 试样制备
5.1.1 含淀粉的试样
a) 固体试样:称取混合均匀的固体试样约5g(精确至0.01g),于150mL锥形瓶中,加入约25
mL45℃~50℃的水,混匀。加入约0.5g淀粉酶,混匀后向锥形瓶中充氮,盖上瓶塞,置50
℃~60℃培养箱内约30min。取出冷却至室温。
b) 液体试样:称取混合均匀的液体试样约20g(精确至0.01g)于150mL锥形瓶中,混匀。加入
约0.5g淀粉酶,混匀后向锥形瓶中充氮,盖上瓶塞,置50℃~60℃培养箱内约30min。取出
冷却至室温。
5.1.2 不含淀粉的试样
a) 固体试样:称取混合均匀的固体试样约5g(精确至0.01g),于150mL锥形瓶中,加入约25
mL45℃~50℃的水,混匀。静置5min~10min,冷却至室温。
b) 液体试样:称取混合均匀的液体试样约20g(精确至0.01g)于150mL锥形瓶中。静置5min
~10min。
5.1.3 待测液的制备
用盐酸溶液,调节上述试样溶液的pH至1.7±0.1,放置约1min。再用氢氧化钠溶液调节试样溶
液的pH至4.5±0.1。把上述锥形瓶放入超声波振荡器中,超声振荡约10min。将试样溶液转移至
50mL容量瓶中,用水冲洗锥形瓶。洗液合并于50mL容量瓶中,用水定容至50mL。另取50mL锥
形瓶,上面放入漏斗和滤纸,把定容后的试样溶液倒入其中,自然过滤。滤液再经0.45μm微孔滤膜过
滤,用试管收集,转移1mL滤液至进样瓶作为试样待测液。
注:操作过程应避免强光照射。
5.2 仪器参考条件
仪器参考条件列出如下:
a) 色谱柱:C18柱,柱长150mm,柱内径4.6mm,柱填料粒径5μm,或相当者;
b) 流动相:甲醇50mL、辛烷磺酸钠2.0g、三乙胺2.5mL,用水溶解并定容到1000mL后,用冰
乙酸调pH至3.0±0.1,过0.45μm微孔滤膜过滤;
d) 柱温:30℃;
e) 检测波长:激发波长293nm,发射波长395nm;
f) 进样体积:10μL。
5.3 标准曲线的制作
将维生素B6 混合标准系列工作液分别注入高效液相色谱仪中,测定各组分的峰面积,以相应标准
工作液的浓度为横坐标,以峰面积为纵坐标,绘制标准曲线。
5.4 试样溶液的测定
将试样溶液注入高效液相色谱仪中,得到各组分相应的峰面积,根据标准曲线得到待测试样溶液中
维生素B6 各组分的浓度。
试样中维生素B6 各组分的含量按式(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15%。
8 其他
当取样量为5.00g时,方法检出限为:吡哆醇0.02mg/100g,吡哆醛0.02mg/100g,吡哆胺
0.02mg/100g;方法定量限为:吡哆醇0.05mg/100g,吡哆醛0.05mg/100g,吡哆胺0.05mg/100g。
第二法 微生物法
9 原理
酵母菌在有维生素B6 存在的条件下才能生长,在一定条作下维生素B6 的量与其生长呈正比关系。用
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规定的二级水。培养基可使用符合
测试要求的商品化的培养基。
GB 5009.154-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Vitamin B6 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 Chromatography ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Instruments and Equipment ... 6
5 Analytical Procedures ... 6
6 Expression of Analysis Results ... 8
7 Precision ... 9
8 Others ... 9
Method II Microbial Method ... 9
9 Principle ... 9
10 Reagents and Materials ... 9
11 Instruments and Equipment ... 11
12 Analytical Procedures ... 11
13 Expression of Analysis Results ... 13
14 Precision ... 14
15 Others ... 14
Appendix A Method of Concentration Correction of Vitamin B6 in Each
Component of Standard Solution ... 15
Appendix B Liquid Chromatogram of Vitamin B6 ... 17
Appendix C Medium Component and Preparation Method ... 18
National Food Safety Standard -
Determination of Vitamin B6 in Foods
1 Scope
This Standard specifies methods of determining vitamin B6 in foods.
In this Standard, Method I is high-performance liquid chromatography, which is
applicable to the determination of foods that contain vitamin B6; Method II is microbial
method, which is applicable to the determination of vitamin B6 in all kinds of food.
Method I -- High-performance Liquid Chromatography
2 Principle
After pre-processing of the sample, for example, extraction, the sample is separated
with C18 chromatographic column and detected with high-performance liquid
chromatography - fluorescence detector. The content of vitamin B6 (pyridoxine,
pyridoxal and pyridoxamine) is quantitatively determined 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 Sodium octane sulfonate (C8H17NaO3S).
3.1.2 Glacial acetic acid (C2H4O2).
3.1.3 Triethylamine (C6H15N). chromatographic purity.
3.1.4 Methanol (CH4O). chromatographic purity.
3.1.5 Hydrochloric acid (HCl).
3.1.6 Sodium hydroxide (NaOH).
3.1.7 Amylase. enzyme activity ≥1.5 U/mg.
3.2 Preparation of Reagents
then, mix it up. Place it evenly for 5 min~10 min, then, cool it down to the room
temperature.
b) Liquid sample. weigh-take approximately 20 g (accurate to 0.01 g) of thoroughly
mixed liquid sample; place it in 150 mL conical flask. Place it evenly for 5 min~10 min.
5.1.3 Preparation of test solution
Use hydrochloric acid solution to adjust the above-mentioned sample solution to pH
1.7±0.1; place evenly for around 1 min. Use sodium hydroxide solution to adjust the
sample solution to pH 4.5±0.1. Place the above-mentioned conical flask into ultrasonic
oscillator; start ultrasonic oscillation for around 10 min. Transfer the sample solution
50 mL volumetric flask; use water to dilute to the constant volume of 50 mL. Take
another 50 mL conical flask, place a funnel and filter paper on top of it; pour the
constant-volume sample solution into the conical flask, then, naturally filter it. Use 0.45
μm microporous membrane to filter the filtrate, then, use a tube to collect it; transfer 1
mL of filtrate to inlet bottle as the test solution.
Note. avoid strong light exposure during the operation.
5.2 Instrument Reference Conditions
Please see instrument reference conditions below.
a) Chromatographic column. C18 column, length. 150 mm, internal diameter. 4.6
b) Mobile phase. methanol 50 mL, sodium octane sulfonate 2.0 g, triethylamine 2.5
mL; use water to dissolve to the constant volume of 1,000 mL, then, use glacial
acetic acid to adjust to pH 3.0±0.1; use 0.45 μm microporous membrane to filter
it;
c) Flow rate. 1 mL/min;
d) Column temperature. 30 °C;
e) Detection wavelength. excitation wavelength. 293 nm, emission wavelength. 395
nm;
f) Inlet volume. 10 μL.
Respectively inject vitamin B6 mixed standard series working solution into high-
performance liquid chromatograph, then, measure the peak area of each component;
take the concentration of corresponding standard working solution as x-coordinate,
take the peak area as y-coordinate to draw a standard curve line.
10.1.6 Sodium chloride (NaCl).
10.1.7 Bromo cresol green (C21H14Br4O5S).
10.2 Preparation of Reagents
10.2.1 Hydrochloric acid solution (0.01 mol/L). take 0.9 mL of hydrochloric acid; use
water to dilute to the constant volume of 1,000 mL.
acid to 2,000 mL beaker; use water to dilute to 1,000 mL.
10.2.3 Sulfuric acid solution (0.5 mol/L). add 700 mL of water and 28 mL of sulfuric
acid to 2,000 mL beaker; use water to dilute to 1,000 mL.
10.2.4 Sodium hydroxide solution (10 mol/L). weigh-take 40 g of sodium hydroxide,
then, add 40 mL of water to dissolve it; cool it down and use water to dilute to the
constant volume of 100 mL.
10.2.5 Sodium hydroxide solution (0.1 mol/L). transfer-take 1 mL of 10 mol/L sodium
hydroxide solution, then, add water to dilute to the constant volume of 100 mL.
10.2.6 Saline (9 g/L). weigh-take 9 g of sodium chloride, use water to dissolve it to the
cool it down and reserve for later usage.
10.2.7 Bromo cresol green solution (0.4 g/L). accurately weigh-take 0.1 g of bromo
cresol green and place it in a mortar; add 1.4 mL of 0.1 mol/L sodium hydroxide solution
to grind it; add a little water and continue to grind it, till it completely dissolves; use
water to dilute to 250 mL.
10.3 Medium (refer to Appendix C for medium component and
preparation method)
10.3.1 Pyridoxine Y medium.
10.3.2 Pyridoxine Y agar medium.
10.3.4 YM broth medium.
10.3.5 YM broth agar medium.
10.4 Standards
Pyridoxine hydrochloride (C8H12ClNO3, CAS No.. 58-56-0). purity≥99%, or standard
substance with a national-level certificate and a certificate of standard substance.
12.1.2 Monthly-reserved strain preparation. inoculate strain rejuvenation medium on
the bevel of YM broth agar medium (subculture medium) through streak inoculation;
start culture under 30 °C for 20 h~24 h; store in the refrigerator under 2 °C~8 °C. This
strain is the first generation of monthly-reserved strain. Inoculate the previous
(subculture medium) through streak inoculation every month in the following period;
start culture under 30 °C for 20 h~24 h; store in the refrigerator under 2 °C~8 °C. This
monthly-reserved strain can remain valid for 1 month.
12.1.3 Weekly-reserved strain preparation. inoculate monthly-reserved strain on the
bevel of YM broth agar medium (subculture medium) every week; start culture under
30 °C for 20 h~24 h; store in the refrigerator under 2 °C~8 °C. It can remain valid for 7
days. Strain that’s reserved for over several weeks cannot be immediately adopted for
the preparation of inoculum. Before usage, it must be transferred once a day for 2
days~3 days in a row, otherwise, the growth cannot be guaranteed.
B6, transfer weekly-reserved strain in 10 mL of YM broth medium (seed culture
medium). 2 tubes can be prepared simultaneously. Start oscillating culture under 30 °C
for 20 h~24 h, then, obtain seed culture medium for determination. The number of total
generations from monthly-reserved strain to seed culture medium shall be ≤5. Start
centrifugation of the seed culture medium under 3,000 r/min for 10 min, dump the
supernatant; use 10 mL of saline to rinse it, start centrifugation and dump the
supernatant, then, use saline to repeatedly rinse it for 2 times. Add 10 mL of sterilized
saline, then, place centrifuge tube on vortex mixer to thoroughly mix it; turn the strain
into suspension, then, pour the suspension into a sterilized syringe; use it immediately.
12.2.1 Weigh-take 0.5 g~10 g (accurate to 0.01 g, the content of vitamin B6 shall be
≤10 ng), place it in 100 mL conical flask, then, add 72 mL of 0.22 mol/L sulfuric acid
solution. Place it in autoclave under 121 °C and start hydrolysis for 5 h; take it out and
cool it down. Adopt 10.0 mol/L sodium hydroxide solution and 0.5 mol/L sulfuric acid
solution to adjust to pH 4.5; use bromo cresol green as the indicator (indicator turns
from yellow to yellow-green). Transfer the reagent in the conical flask into 100 mL
volumetric flask; use distilled water to dilute to the constant volume of 100 mL; use
filter paper to filter it, store the filtrate in the refrigerator for later usage (the period of
validity shall be ≤36 h).
12.2.2 Preparation of standard curve line. respectively add 0.00 mL, 0.02 mL, 0.04 mL,
0.08 mL, 0.12 mL and 0.16 mL of pyridoxine working solution to 3 groups of tube; add
pyridoxine Y medium to 5.00 mL; mix it up, put on cotton plug.
12.2.3 Preparation of sample tube. respectively add 0.05 mL, 0.10 mL and 0.20 mL of
Appendix C
Medium Component and Preparation Method
C.1 Pyridoxine Y Medium
Solution (per liter) contains. glucose 40.0 g, L-asparagine 4.0 g, ammonium sulfate 4.0
g, potassium dihydrogen phosphate3.0 g, magnesium sulfate 1.0 g, calcium oxide 0.49
40.0 mg, histidine hydrochloride 20.0 mg, riboflavin 20.0 mg, biotin 8.0 mg, inositol 5.0
mg, ferrous sulfate 500.0 μg, thiamine hydrochloride 400.0 μg, calcium pantothenate
400.0 μg, choline acid 400.0 μg, boric acid 200.0 μg, potassium iodide 200.0 μg,
ammonium molybdate 40.0 μg, manganese sulfate 80.0 μg, copper sulfate 90.0 μg,
zinc sulfate 80.0 μg, distilled water 1,000 mL.
Weigh-take 5.3 g of the above-mentioned pyridoxine Y medium, then, dissolve it in 100
mL of distilled water; adjust to pH 4.1±0.05; start sterilization under 121 °C for 15 min;
reserve for later usage.
C.2 Pyridoxine Y Agar Medium
mL of distilled water; adjust to pH 4.1±0.05. Add 1.2 g of agar, heat it up and boil it to
melt the agar. Thoroughly mix it up and divide it into tubes (10 mL in each tube). Start
sterilization under 121 °C for 15 min; place it in a beveled state and reserve for later
usage.
C.3 Malt Extract Powder Agar Medium
Weigh-take 12.75 g of maltose, 2.75 g of dextrin, 2.35 g of glycerol and 0.78 g of
peptone; dissolve it in 1,000 mL of distilled water; adjust to pH 4.7±0.2. Add 15.0 g of
agar, heat it up and boil it to melt the agar. Thoroughly mix it up and divide it into tubes
(10 mL in each tube). Start sterilization under 121 °C for 15 min; place it in a beveled
reserved strain medium of Saccharomyces carlsbergensis.
C.4 YM Broth Medium
Solution (per liter) contains. yeast extract 3.0 g, malt extract 3.0 g, peptone 5.0 g,
glucose 10.0 g and distilled water 1,000 mL. Weigh the medium in accordance with the
proportion of 2.1 g/100 mL water. Add corresponding volume of distilled water, mix it
up; adjust to pH 6.2±0.2, then, divide it into tubes (10 mL in each tube). Start
sterilization under 121 °C for 15 min; cool it down, then, store it in the refrigerator under
4 °C. It shall remain valid for 1 month and serve as rejuvenation culture medium of
Saccharomyces carlsbergensis and seed culture medium for daily detection.
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