GB 5009.294-2023 English PDF

GB 5009.294-2023: National food safety standard - Determination of tryptophan in foods
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GB 5009.294-2023
GB
NATIONAL STANDARD OF THE
PEOPLE'S REPUBLIC OF CHINA
National food safety standard - Determination of tryptophan
in food
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
1 Scope ... 3
Method A - Enzymatic hydrolysis-liquid chromatography ... 3
2 Principle ... 3
3 Reagents and materials ... 3
4 Instruments and equipment ... 5
5 Analysis steps ... 5
6 Presentation of analysis results ... 6
7 Precision ... 7
8 Others ... 7
Method B - Alkaline hydrolysis-liquid chromatography ... 7
9 Principle ... 7
10 Reagents and materials ... 7
11 Instruments and equipment ... 9
12 Analysis steps ... 9
13 Presentation of analysis results ... 11
14 Precision ... 11
15 Others ... 12
Appendix A L-tryptophan standard solution chromatogram ... 13
National food safety standard
Determination of tryptophan in food
1 Scope
This standard specifies the determination method of tryptophan in food.
This standard is applicable to the determination of tryptophan in formula foods for
infants and young children, formula foods for special medical purposes, and cereal
supplementary foods for infants and young children.
Method A - Enzymatic hydrolysis-liquid chromatography
2 Principle
The sample is enzymatically hydrolyzed with protease, diluted with methanol-water,
separated by using a reversed-phase liquid chromatography column, detected with a
fluorescence detector or ultraviolet detector, and quantified with an external standard
method.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical grade and the
water is first-grade water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 Acetic acid (C2H4O2): chromatographically pure.
3.1.3 Ammonium acetate (C2H7NO2): chromatographically pure.
3.1.4 Hydrochloric acid (HCl): 12 mol/L.
3.1.5 2-Amino-2-hydroxymethyl-1,3-propanediol (C4H11NO3, Tris base).
3.1.6 Streptomyces griseus protease (type XIV; derived from Streptomyces griseus):
The activity is ≥3.5 U/mg.
3.2 Reagent preparation
3.2.1 Hydrochloric acid solution (1.0 mol/L): Measure 83 mL of hydrochloric acid, pour
it into 300 mL of water, mix well, and then add water to dilute to 1 L.
3.2.2 Tris buffer (0.1 mol/ L, pH=8.5): Weigh 6.06 g of Tris base into a 1000 mL beaker,
add 500 mL of water to dissolve, and adjust the pH of the solution to 8.5±0.05 with 1.0
mol/L hydrochloric acid solution. Store it at room temperature, and the shelf life is 6
months.
3.2.3 Protease solution (17.5 U/mL): Weigh 0.05 g of Streptomyces griseus protease,
dissolve it in Tris buffer, and make the volume up to 10 mL, so that the protease mass
concentration is 17.5 U/mL. Prepare it fresh just before use.
3.2.4 Ammonium acetate solution (10 mmol/ L, pH=4.0): Weigh 0.77 g of ammonium
acetate, add 900 mL of water to dissolve, adjust the pH of the solution to 4.0±0.1 with
acetic acid, add water to 1000 mL, and filter through a 0.22 μm aqueous phase
microporous filter membrane for later use.
3.3 Standards
L-Tryptophan standard (C11H12N2O2, CAS number: 73-22-3): purity of ≥99%, or a
reference material with national authentication and a Reference Material Certificate.
3.4 Preparation of standard solutions
3.4.1 L-tryptophan standard stock solution (1.00 mg/mL): Accurately weigh 25 mg
(accurate to 0.0001 g) of L-tryptophan standard in a beaker, add 10mL of water to
dissolve and transfer to a 25 mL volumetric flask, dilute to volume with water, and mix
well. Transfer it to a brown glass container and store it in a 4 °C refrigerator. The shelf
life is 6 months.
3.4.2 L-Tryptophan standard intermediate solution (100 μg/mL): Take 1.00 mL of L-
tryptophan standard stock solution into a 10 mL volumetric flask, add water to make
the volume up to the mark, and mix well. Transfer it to a brown glass container and
store it in a 4 °C refrigerator. The shelf life is 6 months.
3.4.3 L-tryptophan standard series working solution: Pipette 20.0 μL, 100 μL, 500 μL,
1.00 mL, 2.00 mL, and 5.00 mL of L-tryptophan standard intermediate solution into 10
mL volumetric flasks respectively, add 2.40 mL of methanol to each, add water to make
the volume up to the mark, and mix well. The mass concentrations of L-tryptophan
standard series working solutions are 0.200 μg/mL, 1.00 μg/mL, 5.00 μg/mL, 10.0
μg/mL, 20.0 μg/mL, and 50.0 μg/mL, respectively. Prepare the solutions fresh just
before use.
3.5 Materials
3.5.1 Threaded centrifuge tubes with caps.
3.5.2 Microporous filter membrane: 0.22 μm, organic phase.
3.5.3 Microporous filter membrane: 0.22 μm, aqueous phase.
4 Instruments and equipment
4.1 High-performance liquid chromatograph: equipped with a fluorescence detector or
ultraviolet detector.
4.2 Analytical balance: The sensitivities are 0.01 g, 0.001 g, and 0.0001g, respectively.
4.3 Constant temperature incubator: 50 ℃±0.5 ℃.
4.4 Ultrasonic generator.
4.5 pH meter: The accuracy is ±0.01.
4.6 Vortex mixer.
5 Analysis steps
5.1 Sample processing
5.1.1 Sample preparation
Formula foods for infants and young children and formula foods for special medical
purposes shall be mixed evenly. Cereal supplementary foods for infants and young
children shall be crushed in advance and mixed.
5.1.2 Sample extraction
Accurately weigh 0.2 g of the sample (accurate to 0.001 g) into a 50 mL threaded
centrifuge tube with a cap, add 0.5 mL of protease solution, 3.0 mL of Tris buffer, and
200 μL of methanol in sequence, vortex to mix, then dissolve with ultrasound, and keep
it in an incubator at a constant temperature of 50 °C to enzymatic hydrolysis for 16
hours.
Take out the centrifuge tube, cool to room temperature, add 12 mL of methanol, transfer
and dilute the sample to the mark with water in a 50 mL volumetric flask, and mix well;
filter through a 0.22 μm organic phase microporous filter membrane, and use the filtrate
for liquid chromatography measurement.
5.2 Liquid chromatography reference conditions
ρ -- the tryptophan content in the sample solution obtained from the standard curve,
in micrograms per milliliter (μg/mL);
ρ0 -- the tryptophan content in the blank solution obtained from the standard curve,
in micrograms per milliliter (μg/mL);
V -- the metered volume of the sample, in milliliters (mL);
100 -- conversion factor;
m -- the mass of the sample, in grams (g);
1000 -- conversion factor.
The calculation result is rounded to 3 significant figures.
7 Precision
The absolute difference between two independent determination results obtained under
repeatability conditions shall not exceed 10% of the arithmetic mean.
8 Others
When the sample amount is 0.2 g, the detection limit of the method is 1.5 mg/100 g,
and the quantitation limit is 5.0 mg/100 g.
Method B - Alkaline hydrolysis-liquid chromatography
9 Principle
The sample is hydrolyzed under alkaline conditions, and the hydrolysate is adjusted to
neutrality with hydrochloric acid, separated with a reversed-phase liquid
chromatography column, detected with a fluorescence detector, and quantified with the
internal standard method.
10 Reagents and materials
Unless otherwise specified, all reagents are analytical grade and the water is first-grade
water specified in GB/T 6682.
10.1 Reagents
10.1.1 Methanol (CH3OH): chromatographically pure.
10.1.2 Acetic acid (C2H4O2): chromatographically pure.
10.1.3 Ammonium acetate (C2H7NO2): chromatographically pure.
10.1.4 Lithium hydroxide (LiOH • H2O).
10.1.5 Hydrochloric acid (HCl): 12 mol/L.
10.2 Reagent preparation
10.2.1 Hydrochloric acid solution (3 mol/L): Measure 100 mL of hydrochloric acid,
pour into 300 mL of water, and mix well.
10.2.2 Lithium hydroxide solution (4 mol/L): Weigh 167.8 g of lithium hydroxide, add
an appropriate amount of water to dissolve, and make the volume up to 1000 mL with
water.
10.2.3 Ammonium acetate solution (10 mmol/L, pH=4.0): Weigh 0.77 g of ammonium
acetate, add 900 mL of water to dissolve, and adjust the pH of the solution to 4.0±0.1
with acetic acid; dilute to 1000 mL with water, shake well, and filter with a 0.22 μm
aqueous phase microporous filter membrane for later use.
10.3 Standards
10.3.1 L-Tryptophan standard (C11H12N2O2, CAS number: 73-22-3): a purity of ≥99%,
or a reference material with national authentication and a Reference Material Certificate.
10.3.2 α-Methyl-D-tryptophan standard (C12H14N2O2, CAS number: 110117-83-4): a
purity of ≥98%.
10.4 Preparation of standard solutions
10.4.1 L-tryptophan standard stock solution (1.00 mg/mL): Accurately weigh 25 mg
(accurate to 0.0001 g) of L-tryptophan standard into a beaker, add 10 mL of water to
dissolve and transfer to a 25 mL volumetric flask, dilute to volume with water, and mix
well. Transfer it to a brown glass container and store it in a 4 °C refrigerator. The shelf
life is 6 months.
10.4.2 L-tryptophan standard intermediate solution (100 μg/mL): Take 1.00 mL of L-
tryptophan standard stock solution into a 10 mL volumetric flask, dilute to volume with
water, and mix well. Transfer it to a brown glass container and store it in a 4 °C
refrigerator. The shelf life is 6 months.
10.4.3 Internal standard working solution (1 mg/mL): Weigh 10 mg (accurate to 0.001
g) of α-methyl-D-tryptophan standard into a beaker, add 5 mL of water to dissolve, and
transfer to a 10 mL volumetric flask; dilute to volume with water and mix well. Transfer
it to a brown glass container and store it in a refrigerator at 4 °C. The shelf life is 6
months.
10.4.4 L-tryptophan standard series working solution: Pipette 20 μL, 100 μL, 500 μL,
1.00 mL, 2.00 mL, and 5.00 mL of L-tryptophan standard intermediate solution into 10
mL volumetric flasks, and add 100 μL internal standard working solution to each; add
water to make it up to the mark and mix well. The mass concentrations of L-tryptophan
standard series working solutions are 0.20 μg/mL, 1.00 μg/mL, 5.00 μg/mL, 10.0
μg/mL, 20.0 μg/mL, and 50.0 μg/mL, respectively. Prepare the solutions fresh just
before use.
10.5 Materials
10.5.1 Hydrolysis tube: It is made of polytetrafluoroethylene, equipped with a threaded
outer cover, and lined with polytetrafluoroethylene sealing gasket.
10.5.2 Microporous filter membrane: 0.22 μm, aqueous phase.
11 Instruments and equipment
11.1 High-performance liquid chromatograph: equipped with a fluorescence detector.
11.2 Analytical balance: The sensitivities are 0.01 g, 0.001 g, and 0.0001 g, respectively.
11.3 Electric drying oven: 110 ℃±0.5 ℃.
11.4 Ultrasonic generator.
11.5 Vortex mixer.
11.6 pH meter: The accuracy is ±0.01.
12 Analysis steps
12.1.1 Sample preparation
Formula foods for infants and young children and formula foods for special medical
purposes shall be mixed evenly. Cereal supplementary foods for infants and young
children shall be crushed in advance and mixed.
12.1.2 Sample extraction
Accurately weigh 0.2 g of the sample (accurate to 0.001 g) into the hydrolysis tube, add
0.5 mL of internal standard working solution and 5 mL of lithium hydroxide solution
GB 5009.294-2023
GB
NATIONAL STANDARD OF THE
PEOPLE'S REPUBLIC OF CHINA
National food safety standard - Determination of tryptophan
in food
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
1 Scope ... 3
Method A - Enzymatic hydrolysis-liquid chromatography ... 3
2 Principle ... 3
3 Reagents and materials ... 3
4 Instruments and equipment ... 5
5 Analysis steps ... 5
6 Presentation of analysis results ... 6
7 Precision ... 7
8 Others ... 7
Method B - Alkaline hydrolysis-liquid chromatography ... 7
9 Principle ... 7
10 Reagents and materials ... 7
11 Instruments and equipment ... 9
12 Analysis steps ... 9
13 Presentation of analysis results ... 11
14 Precision ... 11
15 Others ... 12
Appendix A L-tryptophan standard solution chromatogram ... 13
National food safety standard
Determination of tryptophan in food
1 Scope
This standard specifies the determination method of tryptophan in food.
This standard is applicable to the determination of tryptophan in formula foods for
infants and young children, formula foods for special medical purposes, and cereal
supplementary foods for infants and young children.
Method A - Enzymatic hydrolysis-liquid chromatography
2 Principle
The sample is enzymatically hydrolyzed with protease, diluted with methanol-water,
separated by using a reversed-phase liquid chromatography column, detected with a
fluorescence detector or ultraviolet detector, and quantified with an external standard
method.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical grade and the
water is first-grade water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 Acetic acid (C2H4O2): chromatographically pure.
3.1.3 Ammonium acetate (C2H7NO2): chromatographically pure.
3.1.4 Hydrochloric acid (HCl): 12 mol/L.
3.1.5 2-Amino-2-hydroxymethyl-1,3-propanediol (C4H11NO3, Tris base).
3.1.6 Streptomyces griseus protease (type XIV; derived from Streptomyces griseus):
The activity is ≥3.5 U/mg.
3.2 Reagent preparation
3.2.1 Hydrochloric acid solution (1.0 mol/L): Measure 83 mL of hydrochloric acid, pour
it into 300 mL of water, mix well, and then add water to dilute to 1 L.
3.2.2 Tris buffer (0.1 mol/ L, pH=8.5): Weigh 6.06 g of Tris base into a 1000 mL beaker,
add 500 mL of water to dissolve, and adjust the pH of the solution to 8.5±0.05 with 1.0
mol/L hydrochloric acid solution. Store it at room temperature, and the shelf life is 6
months.
3.2.3 Proteas...