GB 5009.154-2023 English PDF

GB 5009.154-2023: National food safety standard - Determination of vitamin B6 in foods
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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 refer...