GB 5009.154-2023 (GB5009.154-2023) & related versions
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GB 5009.154-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of Vitamin
B6 in Foods
ISSUED ON: SEPTEMBER 6, 2023
IMPLEMENTED ON: MARCH 6, 2024
Issued by: National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword ... 4
1 Scope ... 5
Method I - Liquid Chromatography - Tandem Mass Spectrometry ... 5
2 Principle ... 5
3 Reagents and Materials ... 5
4 Instruments and Equipment ... 8
5 Analytical Procedures ... 9
6 Expression of Analysis Results ... 13
7 Precision ... 14
8 Others ... 14
Method II - Liquid Chromatography - Mass Spectrometry ... 14
9 Principle ... 14
10 Reagents and Materials ... 14
11 Instruments and Equipment ... 17
12 Analytical Procedures ... 18
13 Expression of Analysis Results ... 21
14 Precision ... 22
15 Others ... 22
Method III - High-performance Liquid Chromatography - Fluorescence Detection
Method ... 22
16 Principle ... 22
17 Reagents and Materials ... 22
18 Instruments and Equipment ... 24
19 Analytical Procedures ... 25
20 Expression of Analysis Results ... 26
21 Precision ... 27
22 Others ... 27
Method IV - Microbiological Method ... 27
23 Principle ... 27
24 Reagents and Materials ... 28
25 Instruments and Equipment ... 29
26 Analytical Procedures ... 30
27 Expression of Analysis Results ... 32
28 Precision ... 32
29 Others ... 33
Appendix A Concentration Correction Method of Standard Solution of Each
Component of Vitamin B6 ... 34
Appendix B MRM Mass Spectrometry Chromatogram ... 36
Appendix C ... 37
Appendix D Liquid Chromatogram of Vitamin B6 ... 38
Appendix E Culture Medium Components and Preparation Methods ... 39
National Food Safety Standard - Determination of Vitamin
B6 in Foods
1 Scope
This Standard specifies the method for the determination of vitamin B6 in foods.
The first method “liquid chromatography - tandem mass spectrometry” is applicable to the
determination of vitamin B6 in foods.
The second method “liquid chromatography - mass spectrometry” is applicable to the
determination of vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in formulated milk
powder, special dietary foods, ready-to-eat cereals, baked goods and beverages, in which,
vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) is added as a nutritional fortifier.
The third method “high-performance liquid chromatography - fluorescence detection method”
is applicable to the determination of vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in
formulated milk powder, special dietary foods (except foods for special medical purposes),
ready-to-eat cereals, baked goods and beverages, in which, vitamin B6 (pyridoxamine,
pyridoxal and pyridoxine) is added as a nutritional fortifier.
The fourth method “microbiological method” is applicable to the determination of vitamin B6
in foods.
Method I - Liquid Chromatography - Tandem Mass
Spectrometry
2 Principle
Vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in foods is firstly hydrolyzed by acid,
then, enzymatically hydrolyzed into pyridoxamine, pyridoxal and pyridoxine. After dilution
and filtration, reversed-phase liquid chromatography separation, tandem mass spectrometry
detection, and isotope internal standard method for quantitative determination, the total vitamin
B6 content is calculated in terms of pyridoxine.
3 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and
the water is Grade-1 water specified in GB/T 6682.
3.3.6 D3-pyridoxamine dihydrochloride (C8D3H11Cl2N2O2, CAS No.: 1173023-45-4): purity
98%.
3.4 Preparation of Standard Solutions
3.4.1 Pyridoxine standard stock solution (1.00 mg/mL): accurately weigh-take 60.8 mg of
pyridoxine hydrochloride reference material, use 0.1 mol/L hydrochloric acid solution to
dissolve it and dilute into a 50 mL brown volumetric flask. Then, transfer it to a brown glass
reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3 months.
3.4.2 Pyridoxal standard stock solution (1.00 mg/mL): accurately weigh-take 60.9 mg of
pyridoxal hydrochloride reference material, use 0.1 mol/L hydrochloric acid solution to
dissolve it and dilute into a 50 mL brown volumetric flask. Then, transfer it to a brown glass
reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3 months.
3.4.3 Pyridoxamine standard stock solution (1.00 mg/mL): accurately weigh-take 71.7 mg of
pyridoxamine dihydrochloride reference material, use 0.1 mol/L hydrochloric acid solution to
dissolve it and dilute into a 50 mL brown volumetric flask. Then, transfer it to a brown glass
reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3 months.
3.4.4 Vitamin B6 standard mixed stock solution (50.0 g/mL): respectively and accurately draw
5.00 mL of pyridoxamine, pyridoxal and pyridoxine standard stock solution (1.00 mg/mL), use
0.1 mol/L hydrochloric acid solution to dilute into a 100 mL brown volumetric flask. Then,
transfer it to a brown glass reagent bottle, and store it in the dark at 20 C. It shall remain valid
for 3 months.
3.4.5 Vitamin B6 standard mixed working solution (5.00 g/mL): accurately draw 1.00 mL of
vitamin B6 standard mixed stock solution (50.0 g/mL), use mobile phase A to dilute into a 10
mL brown volumetric flask. Prepare it right before use.
3.4.6 Vitamin B6 standard mixed working solution (500 ng/mL): accurately draw 1.00 mL of
vitamin B6 standard mixed stock solution (5.00 g/mL), use mobile phase A to dilute into a 10
mL brown volumetric flask. Prepare it right before use.
3.4.7 Vitamin B6 standard mixed working solution (50.0 ng/mL): accurately draw 1.00 mL of
vitamin B6 standard mixed stock solution (500 ng/mL), use mobile phase A to dilute into a 10
mL brown volumetric flask. Prepare it right before use.
NOTE: the frozen stock solution shall be thawed at room temperature and evenly mixed before use.
3.5 Preparation of Isotope Internal Standard Solutions
3.5.1 13C4-pyridoxine isotope internal standard stock solution (100 g/mL): accurately weigh-
take 12.1 mg (accurate to 0.1 mg) of 13C4-pyridoxine hydrochloride, use 0.1 mol/L hydrochloric
acid solution to dilute into a 100 mL brown volumetric flask. Then, transfer it to a brown glass
reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3 months.
4.8 Homogenizer.
4.9 Constant-temperature water bath or autoclave.
5 Analytical Procedures
5.1 Specimen Preparation
For meat, vegetables and fruits, etc., take the edible parts, use water to wash them, and use dry
gauze to wipe off the surface moisture, use a homogenizer to homogenize them and store in
sample bottles for later use. For powdery samples (milk powder and rice noodles, etc.), evenly
mix them and conduct direct sampling. For liquid samples, evenly shake them and reserve them
for later use. For liquid samples with granules, for example, large-grained yogurt, use a
homogenizer to homogenize them and reserve them for later use. For flaky and granular
samples, use a high-speed pulverizer to grind them into powder and seal for later use. The
prepared specimens shall be kept refrigerated in the dark and determined as soon as possible.
5.2 Specimen Treatment
5.2.1 Specimens containing starch
5.2.1.1 Solid specimens: accurately weigh-take 1 g ~ 5 g (accurate to 0.01 g) of evenly mixed
solid specimen into a 150 mL stoppered conical flask (with a soft stopper), and add 500 L of
vitamin B6 isotope internal standard mixed working solution (10.0 g/mL), add 50 mL of 0.1
mol/L HCl solution to disperse the sample, use a soft stopper to plug it. Place it in 100 C water
bath or 121 C autoclave to perform hydrolysis for 30 min; cool to room temperature and take
it out. After using sodium hydroxide solution (0.1 mol/L) to adjust the pH to 4.2 ~ 4.8,
accurately add 20 mg of acid phosphatase, 100 mg of papain and 10 mg of α-amylase. Fill the
conical flask with nitrogen, plug the flask and thoroughly mix it. In a constant-temperature
incubator at 37 C, conduct enzymatic hydrolysis for above 18 h. After the solution cools to
room temperature, transfer it to a 100 mL brown volumetric flask. Use water to dilute to the
scale and evenly mix it.
5.2.1.2 Liquid specimens: accurately weigh-take 5 g ~ 20 g (accurate to 0.01 g) of evenly mixed
liquid specimen into a 150 mL stoppered conical flask (with a soft stopper), and add 500 L of
vitamin B6 isotope internal standard mixed working solution (10.0 g/mL), add 50 mL of 0.1
mol/L HCl solution to disperse the sample, use a soft stopper to plug it. Place it in 100 C water
bath or 121 C autoclave to perform hydrolysis for 30 min; cool to room temperature and take
it out. After using sodium hydroxide solution (0.1 mol/L) to adjust the pH to 4.2 ~ 4.8,
accurately add 20 mg of acid phosphatase, 100 mg of papain and 10 mg of α-amylase. Fill the
conical flask with nitrogen, plug the flask and thoroughly mix it. In a constant-temperature
incubator at 37 C, conduct enzymatic hydrolysis for above 18 h. After the solution cools to
room temperature, transfer it to a 100 mL brown volumetric flask. Use water to dilute to the
scale and evenly mix it.
5.2.2 Starch-free specimens
5.2.2.1 Solid specimens: weigh-take 1 g ~ 5 g (accurate to 0.01 g) of evenly mixed solid
specimen into a 150 mL stoppered conical flask (with a soft stopper), and add 500 L of vitamin
B6 isotope internal standard mixed working solution (10.0 g/mL), add 50 mL of 0.1 mol/L
HCl solution to disperse the sample, use a soft stopper to plug it. Place it in 100 C water bath
or 121 C autoclave to perform hydrolysis for 30 min; cool to room temperature and take it out.
After using sodium hydroxide solution (0.1 mol/L) to adjust the pH to 4.2 ~ 4.8, accurately add
20 mg of acid phosphatase and 100 mg of papain. Fill the conical flask with nitrogen, plug the
flask and thoroughly mix it. In a constant-temperature incubator at 37 C, conduct enzymatic
hydrolysis for above 18 h. After the solution cools to room temperature, transfer it to a 100 mL
brown volumetric flask. Use water to dilute to the scale and evenly mix it.
5.2.2.2 Liquid specimens: weigh-take 5 g ~ 20 g (accurate to 0.01 g) of evenly mixed liquid
specimen into a 150 mL stoppered conical flask (with a soft stopper), and add 500 L of vitamin
B6 isotope internal standard mixed working solution (10.0 g/mL), add 50 mL of 0.1 mol/L
HCl solution to disperse the sample, use a soft stopper to plug it. Place it in 100 C water bath
or 121 C autoclave to perform hydrolysis for 30 min; cool to room temperature and take it out.
After using sodium hydroxide solution (0.1 mol/L) to adjust the pH to 4.2 ~ 4.8, accurately add
20 mg of acid phosphatase and 100 mg of papain. Fill the conical flask with nitrogen, plug the
flask and thoroughly mix it. In a constant-temperature incubator at 37 C, conduct enzymatic
hydrolysis for above 18 h. After the solution cools to room temperature, transfer it to a 100 mL
brown volumetric flask. Use water to dilute to the scale and evenly mix it.
NOTE 1: the operator may adjust the weighing amount of the specimen and the amount of isotope
internal standard added in accordance with the vitamin B6 content of the specimen and
the sensitivity of the mass spectrometer in this laboratory, and under conditions that are
not lower than the determination range requirements of the standard curve.
NOTE 2: to determine the products in which, vitamin B6 (pyridoxamine, pyridoxal and pyridoxine)
is added as a nutritional fortifier, please refer to 12.2 for the pre-treatment method.
5.2.3 Preparation of test solution
Take another 50 mL conical flask, put in a funnel and filter paper, evenly mix the diluted sample
solution and pour the solution into the flask. Naturally filter it to obtain more than 20 mL of
filtrate. Accurately draw 1.00 mL of the filtrate into a 10 mL brown volumetric flask, use mobile
phase A to dilute to the scale. After vortex mixing, use 0.22 m microporous membrane to filter
it, and transfer it to a brown sample injection bottle for later testing.
5.3 Determination Conditions of Instruments
5.3.1 Reference conditions of chromatography
The reference conditions of chromatography are as follows:
a) Chromatographic column: silica gel matrix pentafluorophenyl column (particle size
1.8 m, 3.0 mm 150 mm), or equivalent;
10.1.2 Formic acid (HCOOH): chromatographically pure.
10.1.3 Ammonium formate (HCOONH4): chromatographically pure.
10.1.4 Hydrochloric acid (HCl).
10.1.5 Sodium hydroxide (NaOH).
10.1.6 α-amylase: enzyme activity 50 U/mg.
10.2 Reagent Preparation
10.2.1 Hydrochloric acid solution (0.1 mol/L): accurately draw 9 mL of hydrochloric acid and
use water to dilute to 1,000 mL.
10.2.2 Sodium hydroxide solution (0.1 mol/L): accurately weigh-take 0.4 g of sodium
hydroxide, add 50 mL of water to dissolve it; after cooling, use water to dilute to 100 mL.
10.2.3 Mobile phase A (2% formic acid aqueous solution of 10 mmol/L ammonium formate):
weigh-take 0.63 g of ammonium formate, successively add about 950 mL of water to dissolve
it, accurately add 20 mL of formic acid, and use water to dilute to 1,000 mL. Then, evenly mix
it, conduct ultrasonic degassing and reserve it for later use.
10.2.4 Mobile phase B (0.1% formic acid - methanol solution): draw 1 mL of formic acid, use
methanol to dilute to 1,000 mL, and conduct ultrasonic mixing.
10.3 Reference Materials
10.3.1 Pyridoxine hydrochloride (C8H12ClNO3, CAS No.: 58-56-0): purity 98%, or a standard
substance certified by the state and awarded a reference material certificate.
10.3.2 Pyridoxal hydrochloride (C8H10ClNO3, CAS No.: 65-22-5): purity 99%, or a standard
substance certified by the state and awarded a reference material certificate.
10.3.3 Pyridoxamine dihydrochloride (C8H14Cl2N2O2, CAS No.: 524-36-7): purity 99%, or a
standard substance certified by the state and awarded a reference material certificate.
10.3.4 13C4-pyridoxine hydrochloride (13C4C4H12ClNO3): purity 98%.
10.3.5 D3-pyridoxal (C8D3H6NO3, CAS No.: 1173023-49-8): purity 98%.
10.3.6 D3-pyridoxamine dihydrochloride (C8D3H11Cl2N2O2, CAS No.: 1173023-45-4): purity
98%.
10.4 Preparation of Standard Solutions
10.4.1 Pyridoxine standard stock solution (1.00 mg/mL): accurately weigh-take 60.8 mg of
pyridoxine hydrochloride reference material, use 0.1 mol/L hydrochloric acid solution to
dissolve it, transfer into a 50 mL brown volumetric flask, and dilute to the scale. Then, transfer
it to a brown glass reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3
months.
10.4.2 Pyridoxal standard stock solution (1.00 mg/mL): accurately weigh-take 60.9 mg of
pyridoxal hydrochloride reference material, use 0.1 mol/L hydrochloric acid solution to
dissolve it, transfer into a 50 mL brown volumetric flask, and dilute to the scale. Then, transfer
it to a brown glass reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3
months.
10.4.3 Pyridoxamine standard stock solution (1.00 mg/mL): accurately weigh-take 71.7 mg of
pyridoxamine dihydrochloride reference material, use 0.1 mol/L hydrochloric acid solution to
dissolve it, transfer into a 50 mL brown volumetric flask, and dilute to the scale. Then, transfer
it to a brown glass reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3
months.
10.4.4 Vitamin B6 standard mixed stock solution (50.0 g/mL): respectively and accurately
draw 5.00 mL of pyridoxamine, pyridoxal and pyridoxine standard stock solution (1.00 mg/mL)
into a 100 mL brown volumetric flask, and use 0.1 mol/L hydrochloric acid solution to dilute
to the scale. Then, transfer it to a brown glass reagent bottle, and store it in the dark at 20 C.
It shall remain valid for 3 months.
10.4.5 Vitamin B6 standard mixed working solution (5.00 g/mL): accurately draw 1.00 mL of
vitamin B6 standard mixed stock solution (50.0 g/mL) into a 10 mL brown volumetric flask,
use mobile phase A to dilute to the scale. Prepare it right before use.
10.4.6 Vitamin B6 standard mixed working solution (500 ng/mL): accurately draw 1.00 mL of
vitamin B6 standard mixed working solution (5.00 g/mL) into a 10 mL brown volumetric flask,
use mobile phase A to dilute to the scale. Prepare it right before use.
10.4.7 Vitamin B6 standard mixed working solution (50.0 ng/mL): accurately draw 1.00 mL of
vitamin B6 standard mixed working solution (500 ng/mL) into a 10 mL brown volumetric flask,
use mobile phase A to dilute to the scale. Prepare it right before use.
NOTE: the frozen stock solution shall be thawed at room temperature and evenly mixed before use.
10.5 Preparation of Isotope Internal Standard Solutions
10.5.1 13C4-pyridoxine isotope internal standard stock solution (1.00 mg/mL): weigh-take 12.1
mg (accurate to 0.1 mg) of 13C4-pyridoxine hydrochloride, use 0.1 mol/L hydrochloric acid
solution to dissolve it, transfer into a 10 mL brown volumetric flask and dilute to the scale.
Then, transfer it to a brown glass reagent bottle, and store it in the dark at 20 C. It shall remain
valid for 3 months.
10.5.2 D3-pyridoxal isotope internal standard stock solution (1.00 mg/mL): weigh-take 10.0 mg
(accurate to 0.1 mg) of D3-pyridoxal hydrochloride, use 0.1 mol/L hydrochloric acid solution
to dissolve it, transfer into a 10 mL brown volumetric flask and dilute to the scale. Then, transfer
it to a brown glass reagent bottle, and store it in the dark at 20 C. It shall remain valid for 3
11.9 Constant-temperature water bath.
12 Analytical Procedures
12.1 Specimen Preparation
For powdery samples (milk powder and rice noodles, etc.), evenly mix them and conduct direct
sampling. For liquid samples, evenly shake them and reserve them for later use. For flaky and
granular samples, use a high-speed pulverizer to grind them into powder or use a homogenizer
to homogenize them, and seal for later use. The prepared specimens shall be kept refrigerated
in the dark and determined as soon as possible.
12.2 Specimen Treatment
12.2.1 Specimens containing starch
12.2.1.1 Solid specimens: weigh-take 1 g ~ 5 g (accurate to 0.01 g) of evenly mixed solid
specimen, add 0.4 mL of vitamin B6 isotope internal standard mixed working solution, then,
add about 25 mL of warm water (45 C ~ 50 C) to dissolve it. Use hydrochloric acid solution
or sodium hydroxide solution to adjust the pH to 6.0 ~ 6.5, add 0.1 g of α-amylase into a 150
mL stoppered conical flask. Shake and evenly mix it, then, fill the conical flask with nitrogen,
plug the flask, and place it in an incubator at 50 C ~ 60 C for about 30 min. Take it out and
cool to room temperature.
12.2.1.2 Liquid specimens: weigh-take 5 g ~ 20 g (accurate to 0.01 g) of evenly mixed liquid
specimen, add 0.4 mL of vitamin B6 isotope internal standard mixed working solution, then,
add 20 mL of water. Use hydrochloric acid solution or sodium hydroxide solution to adjust the
pH to 6.0 ~ 6.5, add 0.1 g of α-amylase into a 150 mL stoppered conical flask. Shake and evenly
mix it, then, fill the conical flask with nitrogen, plug the flask, and place it in an incubator at 50
C ~ 60 C for about 30 min. Take it out and cool to room temperature.
12.2.2 Starch-free specimens
12.2.2.1 Solid specimens: weigh-take 1 g ~ 5 g (accurate to 0.01 g) of evenly mixed solid
specimen into a 150 mL stoppered conical flask, accurately add 0.4 mL of vitamin B6 isotope
internal standard mixed working solution, then, add about 25 mL of water to dissolve it, shake
and evenly mix it.
12.2.2.2 Liquid specimens: weigh-take 5 g ~ 20 g (accurate to 0.01 g) of evenly mixed liquid
specimen into a 150 mL stoppered conical flask, accurately add 0.4 mL of vitamin B6 isotope
internal standard mixed working solution, then, add 20 mL of water, and perform vortex mixing.
12.2.3 Preparation of test solution
Use hydrochloric acid solution to adjust the pH of the above-mentioned specimen solution to
1.7 0.1, and let it stand for about 1 min. Then, use sodium hydroxide solution (0.1 mol/L) to
17.4 Preparation of Standard Solutions
17.4.1 Pyridoxine standard stock solution (1.00 mg/mL): accurately weigh-take 60.8 mg
(accurate to 0.1 mg) of pyridoxine hydrochloride reference material, use 0.1 mol/L hydrochloric
acid solution to dissolve it, then, dilute to 50 mL. Store it in the dark in the refrigerator at 20
C. It shall remain valid for 3 months.
17.4.2 Pyridoxal standard stock solution (1.00 mg/mL): accurately weigh-take 60.9 mg
(accurate to 0.1 mg) of pyridoxal hydrochloride reference material, use 0.1 mol/L hydrochloric
acid solution to dissolve it, then dilute to 50 mL. Store it in the dark in the refrigerator at 20
C. It shall remain valid for 3 months.
17.4.3 Pyridoxamine standard stock solution (1.00 mg/mL): accurately weigh-take 71.7 mg
(accurate to 0.1 mg) of pyridoxamine dihydrochloride reference material, use 0.1 mol/L
hydrochloric acid solution to dissolve it, then dilute to 50 mL. Store it in the dark in the
refrigerator at 20 C. It shall remain valid for 3 months.
17.4.4 Vitamin B6 standard mixed working solution (20.0 g/mL): respectively and accurately
draw 1.0 mL of pyridoxine, pyridoxal and pyridoxamine standard stock solutions (1.00 mg/mL).
Use 0.1 mol/L hydrochloric acid solution to dilute to 50 mL. Prepare it right before use.
17.4.5 Vitamin B6 standard mixed working solution (2.00 g/mL): accurately draw 5.00 mL of
pyridoxine, pyridoxal and pyridoxamine standard mixed working solution (20.0 g/mL), use
0.1 mol/L hydrochloric acid solution to dilute to 50 mL. Prepare it right before use.
17.4.6 Vitamin B6 standard mixed series working solutions: respectively and accurately draw
an appropriate amount of vitamin B6 standard mixed working solution (2.00 g/mL) into a 100
mL volumetric flask, use water to dilute them. The concentrations of this standard series are
respectively: 0.04 g/mL, 0.10 g/mL, 0.20 g/mL, 0.40 g/mL, 0.60 g/mL and 1.00 g/mL.
Prepare them right before use.
NOTE 1: please refer to Appendix A for the concentration correction method of standard stock
solution.
NOTE 2: the frozen stock solution shall be thawed at room temperature and evenly mixed before
use.
18 Instruments and Equipment
18.1 High-performance liquid chromatograph: equipped with a fluorescence detector.
18.2 Balance: with a division value of 0.01 g and 0.1 mg.
18.3 pH meter: with an accuracy of 0.01.
18.4 Vortex mixer.
18.5 Ultrasonic oscillator.
18.6 Spectrophotometer.
18.7 Constant-temperature incubator, or one with equivalent performance.
19 Analytical Procedures
19.1 Specimen Preparation
19.1.1 Specimens containing starch
19.1.1.1 Solid specimens: weigh-take 1 g ~ 5 g (accurate to 0.01 g) of evenly mixed solid
specimen into a 150 mL conical flask, add about 25 mL of 45 C ~ 50 C water, and evenly mix
it. Add about 0.5 g of α-amylase. After evenly mixing it, fill the conical flask with nitrogen,
plug the flask and place it in an incubator at 50 C ~ 60 C for about 30 min. Take it out and
cool to room temperature.
19.1.1.2 Liquid specimens: weigh-take 5 g ~ 20 g (accurate to 0.01 g) of evenly mixed liquid
specimen into a 150 mL conical flask, and evenly mix it. Add about 0.5 g of α-amylase. After
evenly mixing it, fill the conical flask with nitrogen, plug the flask and place it in an incubator
at 50 C ~ 60 C for about 30 min. Take it out and cool to room temperature.
19.1.2 Starch-free specimens
19.1.2.1 Solid specimens: weigh-take 1 g ~ 5 g (accurate to 0.01 g) of evenly mixed solid
specimen into a 150 mL conical flask, add about 25 mL of 45 C ~ 50 C water, and evenly mix
it. Let it stand for 5 min ~ 10 min, then, cool to room temperature.
19.1.2.2 Liquid specimens: weigh-take 5 g ~ 20 g (accurate to 0.01 g) of evenly mixed liquid
specimen into a 150 mL conical flask. Let it stand for 5 min ~ 10 min.
19.1.3 Preparation of test solution
Use hydrochloric acid solution to adjust the pH of the above-mentioned specimen solution to
1.7 0.1 and let it stand for about 1 min. Then, use sodium hydroxide solution to adjust the pH
of the specimen solution to 4.5 0.1. Put the above-mentioned conical flask into an ultrasonic
oscillator and perform ultrasonic oscillation for about 10 min. Transfer the specimen solution
to a 50 mL volumetric flask and use water to rinse the conical flask. Combine the washing
solutions in a 50 mL volumetric flask and use water to dilute to 50 mL. Take another 50 mL
conical flask, put in a funnel and filter paper, pour the diluted specimen solution into it, and
naturally filter it. Filter the filtrate through 0.45 m microporous filter membrane, then, transfer
1 mL to a brown sample injection bottle, and use it as the specimen solution to be tested.
NOTE: during the operation, avoid strong light exposure; when extracting gel candies and jelly
specimens, they can be heated and dissolved in 70 C water bath.
......
| Standard ID | GB 5009.154-2023 (GB5009.154-2023) | | Description (Translated English) | (National Food Safety Standard Determination of Vitamin D in Food) | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 26,234 | | Date of Issue | 2023-09-06 | | Date of Implementation | 2024-03-06 | | Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation | | Summary | This standard specifies the method for the determination of vitamin D in food. The first normal-phase chromatography purification-reverse-phase liquid chromatography method is suitable for the determination of vitamin D and vitamin D in foods added with ergocalciferol or cholecalciferol. The second method, online column switching-reverse-phase liquid chromatography, is suitable for the determination of vitamin D2 and vitamin D in food. The third method liquid chromatography-tandem mass spectrometry is suitable for the determination of vitamin D2 and vitamin D3 in food. |
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