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GB 5009.309-2025: National food safety standard - Determination of asparagine and glutamine in food
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National food safety standard - Determination of asparagine and glutamine in food
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GB 5009.309-2025
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Basic data | Standard ID | GB 5009.309-2025 (GB5009.309-2025) | | Description (Translated English) | National food safety standard - Determination of asparagine and glutamine in food | | Sector / Industry | National Standard | | Classification of Chinese Standard | X04 | | Word Count Estimation | 13,196 | | Date of Issue | 2025-09-02 | | Issuing agency(ies) | National Health Commission; State Administration for Market Regulation | GB 5009.309-2025: National food safety standard - Determination of asparagine and glutamine in food
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National Standards of the People's Republic of China
National Food Safety Standards
Determination of Asparagine and Glutamine in Food
Published on 2025-09-02
Implemented on 2026-03-02
National Health Commission of the People's Republic of China
State Administration for Market Regulation issued
National Food Safety Standards
Determination of Asparagine and Glutamine in Food
1.Scope
This standard specifies the pre-column derivatization-high performance liquid chromatography and liquid chromatography-tandem mass transfer method for the determination of asparagine and glutamine in food.
Spectral method.
The first method, direct extraction, is suitable for the determination of free asparagine and free glutamine in special dietary foods. Enzymatic hydrolysis extraction...
The method is applicable to the determination of total asparagine and total glutamine in special dietary foods.
The second method is applicable to the determination of total asparagine and total glutamine in special dietary foods.
First Method. Pre-column Derivatization - High Performance Liquid Chromatography
2.Principle
The sample was directly extracted or hydrolyzed with Streptomyces griseus protease. Asparagine and glutamine underwent a derivatization reaction with dansyl chloride to produce derivatives.
Separated by C18 column, detected by ultraviolet detector, and quantified by external standard method.
3.Reagents and Materials
Unless otherwise specified, all reagents used in this method are of analytical grade, and the water is Grade I water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH4O). chromatographic grade.
3.1.2 Acetonitrile (C2H3N). chromatographic grade.
3.1.3 Dansyl chloride (C12H12ClNO2S).
3.1.4 Anhydrous sodium carbonate (Na2CO3).
3.1.5 Anhydrous disodium hydrogen phosphate (Na2HPO4).
3.1.6 Methylamine hydrochloride (CH5N·HCl).
3.1.7 Tris-hydroxymethylaminomethane (Tris base, C4H11O3N).
3.1.8 Streptomyces griseus protease (type XIV; derived from Streptomyces griseus). enzyme activity ≥3.5 U/mg.
3.1.9 Phosphoric acid (H3PO4). chromatographic grade, content ≥85%.
3.1.10 Concentrated hydrochloric acid (HCl).
3.2 Reagent Preparation
3.2.1 Dansyl chloride solution (2.0 g/L). Weigh 0.2 g of dansyl chloride, dissolve it in acetonitrile and dilute to 100 mL. Prepare fresh before use.
3.2.2 Hydrochloric acid solution (1.0 mol/L). Take 8.3 mL of concentrated hydrochloric acid and dilute it with water to 100 mL.
3.2.3 Tris buffer (0.05 mol/L). Weigh 6.06 g of tris(hydroxymethyl)aminomethane into a 1000 mL beaker, add 800 mL of water to dissolve.
Solution. Adjust the pH to 8.0 ± 0.1 with 1.0 mol/L hydrochloric acid solution, and dilute with water to 1000 mL. Store at 4℃; shelf life is 1 month.
3.2.4 Protease solution (52.5 U/mL). Weigh 0.15 g of Streptomyces griseus protease, dissolve it in Tris buffer and bring the volume to 10 mL.
Prepare and use immediately.
3.2.5 Sodium carbonate buffer solution (60 mmol/L). Weigh 0.318 g of anhydrous sodium carbonate, dissolve in 40 mL of water, and then use 1.0 mol/L hydrochloric acid.
Adjust the pH of the solution to 9.5 ± 0.1, and bring the volume to 50 mL with water. Prepare and use immediately.
3.2.6 Methylamine hydrochloride solution (20 g/L). Weigh 2.0 g of methylamine hydrochloride, dissolve in water, and dilute to 100 mL. Store at 4℃. Shelf life. [Date missing].
3 months.
3.2.7 Disodium hydrogen phosphate solution (10 mmol/L). Weigh 1.42 g of anhydrous disodium hydrogen phosphate, dissolve it in 800 mL of water, and adjust the pH with phosphoric acid.
Dilute to 6.5 ± 0.1 with water to 1000 mL.
3.2.8 20% methanol solution. Measure 20 mL of methanol and dilute with water to 100 mL.
3.3 Standard Products
3.3.1 Asparagine standard (C4H8N2O3, CAS No.. 70-47-3). purity ≥99%, or certified by the state and granted a standard substance certificate.
Standard edition of the book.
3.3.2 Glutamine standard (C5H10N2O3, CAS No.. 56-85-9). purity ≥99%, or certified by the state and granted a standard substance certificate.
Standard edition of the book.
3.4 Preparation of Standard Solutions
3.4.1 Asparagine Standard Stock Solution (2.00 mg/mL). Accurately weigh 100 mg (accurate to 0.0001 g) of asparagine standard, and use...
Dissolve in 20% methanol solution and bring the volume to 50 mL. Store at 4°C protected from light. Shelf life is 3 months.
3.4.2 Glutamine Standard Stock Solution (2.00 mg/mL). Accurately weigh 100 mg (accurate to 0.0001 g) of glutamine standard, and use...
Dissolve in 20% methanol solution and bring the volume to 50 mL. Store at 4°C protected from light. Shelf life is 3 months.
3.4.3 Mixed standard intermediate solution (20.0 μg/mL). Accurately pipette 1.00 mL each of asparagine and glutamine standard stock solutions into a container.
Dilute to the mark with water in a 100mL volumetric flask and mix well. Prepare and use immediately.
3.4.4 Mixing Standard Series Working Solutions. Accurately pipette an appropriate amount of the mixed standard intermediate solution (20.0 μg/mL) into a 10 mL volumetric flask, and use...
The water was diluted to the mark to obtain concentrations of 0 μg/mL, 0.500 μg/mL, 1.00 μg/mL, 2.00 μg/mL, and 5.00 μg/mL.
Standard working solutions in concentrations of 10.0 μg/mL and 20.0 μg/mL. Prepare fresh before use.
3.5 Materials
3.5.1 Threaded glass bottle with cap. 20mL.
3.5.2 Organic microporous filter membrane. 0.22μm.
4.Instruments and Equipment
4.1 High-performance liquid chromatograph. equipped with an ultraviolet detector or a diode array detector.
4.2 Electronic balance. sensitivity 0.001g and 0.0001g.
4.3 Vortex mixer.
4.4 Constant temperature water bath device. 37℃±1℃.
4.5 pH meter. accuracy 0.01.
4.6 Centrifuge. speed ≥ 5000 r/min.
5.Analysis Steps
5.1 Sample Preparation
Non-powdered solid samples should be crushed and mixed evenly, while powdered solid or liquid samples should be shaken well.
5.2 Sample Extraction
5.2.1 Direct Extraction Method
Weigh 5.0 g (accurate to 0.001 g) of the solid sample into a 50 mL dry beaker, add an appropriate amount of warm water (40℃~45℃) to dissolve, and cool.
After reaching room temperature, transfer and bring the volume to 25 mL. Accurately transfer 1.00 mL to a 150 mL Erlenmeyer flask, add approximately 25 mL of water, vortex to mix, and then...
Adjust the pH to 4.5 ± 0.1 with 1.0 mol/L hydrochloric acid solution, then transfer to a 250 mL volumetric flask, dilute to the mark with water, shake well, and centrifuge or
The sample solution to be derived was obtained by filtration.
Weigh 1.0g to 5.0g (accurate to 0.001g) of liquid sample into a 150mL conical flask, and follow the same procedures as for solid sample.
5.2.2 Enzymatic Extraction Method
Weigh 5.0 g (accurate to 0.001 g) of the solid sample into a 50 mL dry beaker, add an appropriate amount of warm water (40℃~45℃) to dissolve, and cool.
After cooling to room temperature, transfer and bring the volume to 25 mL. Accurately transfer 1.00 mL into a 20 mL screw-top glass bottle, and add 0.5 mL of egg white solution.
Mix the enzyme solution, 3.0 mL Tris buffer, and 0.2 mL methanol by vortexing, and incubate at 37°C for 16 hours. Remove the glass bottle and allow it to cool.
After cooling to room temperature, transfer the entire enzymatic hydrolysate to a 250 mL volumetric flask, dilute to the mark with water, and mix well. Centrifuge or filter to obtain the derivatization reagent.
Sample solution.
Weigh 0.5g to 1.0g (accurate to 0.001g) of the liquid sample into a 20mL screw-top glass bottle. Subsequent procedures are the same as for solid samples.
Sample.
5.3 Derivative Reactions
Accurately pipette 1.00 mL of the sample solution to be derivatized into a 15 mL centrifuge tube, add 1.00 mL of sodium carbonate buffer solution and 1.00 mL of sodium carbonate buffer solution.
Mix dansyl chloride solution thoroughly and react at room temperature in the dark for 2 hours (remove and shake well after about 1 hour of reaction). Add 0.10 mL of methylamine hydrochloride solution.
Vortex mix to terminate the reaction, allow to stand in the dark until precipitation is complete, filter through a 0.22μm filter membrane, and then test.
In addition, accurately pipette 1.00 mL of the mixed standard series working solution and derivatize it simultaneously with the sample solution to be derivatized.
5.4 Instrument Reference Conditions
The chromatographic reference conditions are as follows.
a) Chromatographic column. C18 column (250mm × 4.6mm, 5μm) or equivalent;
b) Mobile phase A was 10 mmol/L disodium hydrogen phosphate solution, and mobile phase B was methanol. The gradient elution conditions are shown in Table 1.
c) Flow rate. 1.0 mL/min;
d) Column temperature. 35℃;
e) Detection wavelength. 247nm;
f) Injection volume. 10 μL.
Table 1 Gradient elution conditions
Time/min Mobile phase A/% Mobile phase B/%
0.0 70 30
17.0 70 30
17.1 20 80
19.0 20 80
19.1 70 30
24.0 70 30
5.5 Construction of Standard Curve
The derived mixed standard series working solutions were injected separately into the high-performance liquid chromatograph (HPLC) to extract asparagine from the mixed standard series working solutions.
Alternatively, plot the graph with the mass concentration of glutamine on the x-axis and the peak area of asparagine derivatives or glutamine derivatives on the y-axis, respectively.
Quasi-curve. The chromatogram of the standard solution derivatized is shown in Figure A.1 in Appendix A.
5.6 Determination of Sample Solution
The derivatized analyte solution was injected into a high-performance liquid chromatograph (HPLC) to obtain the corresponding peak area. The aspartic acid content in the sample solution was then determined based on the standard curve.
The mass concentration of amide or glutamine. The response value of the target compound in the test solution should be within the linear range of the standard curve; exceeding the linear range...
If the sample solution to be derivatized is to be diluted, it should be reprocessed according to the sample derivatization analysis procedure before being injected for analysis.
5.7 Blank Test
Except for not adding a sample, the other operations are the same as those for the sample.
6.Presentation of Analysis Results
6.1 Free asparagine and free glutamine
The contents of free asparagine and free glutamine in the sample obtained by direct extraction are calculated according to formula (1).
X=ρ×
V×f
m×1000
(1)
In the formula.
X --- The content of free asparagine and free glutamine in the sample, expressed in milligrams per gram (mg/g);
ρ --- The mass concentration of asparagine and glutamine in the sample solution obtained from the standard curve, in micrograms per milliliter (μg/mL).
mL);
V --- Final volume of the sample, in milliliters (mL);
f --- dilution factor;
m --- Sample mass, in grams (g);
1000 --- Unit conversion factor.
The calculation result should be rounded to 3 significant figures.
6.2 Total Asparagine and Total Glutamine
The total amount of asparagine and glutamine in the sample obtained by enzymatic extraction is calculated according to formula (2).
X=
(ρ-ρ0)×V×f
m×1000
(2)
In the formula.
X --- Total asparagine and total glutamine in the sample, in milligrams per gram (mg/g);
ρ --- The mass concentration of asparagine and glutamine in the sample solution obtained from the standard curve, in micrograms per milliliter (μg/mL).
mL);
ρ0 --- The mass concentration of asparagine and glutamine in the blank test solution obtained from the standard curve, in micrograms per milliliter.
(μg/mL);
V --- Final volume of the sample, in milliliters (mL);
f --- dilution factor;
m --- Sample mass, in grams (g);
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