GB 5009.309-2025 PDF English
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GB 5009.309-2025: National food safety standard - Determination of asparagine and glutamine in food
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GB NATIONAL STANDARD OF THE PEOPLE'S REPUBLIC OF CHINA National food safety standard - Determination of asparagine and glutamine in food Issued on: SEPTEMBER 2, 2025 Implemented on: MARCH 2, 2026 Issued by. National Health Commission of the People's Republic of China; State Administration for Market Regulation.
Table of Contents
1 Scope... 3 Method I. Pre-column derivatization-high performance liquid chromatography... 3 2 Principle... 3 3 Reagents and materials... 3 4 Instruments and equipment... 5 5 Analysis steps... 6 6 Presentation of analysis results... 8 7 Precision... 9 8 Others... 9 Method II. Liquid Chromatography-Tandem Mass Spectrometry... 9 9 Principle... 9 10 Reagents and materials... 9 11 Instruments and equipment... 11 12 Analysis steps... 12 13 Presentation of analysis results... 14 14 Precision... 15 15 Others... 15 Appendix A Chromatograms... 16 National food safety standard Determination of asparagine and glutamine in food1 Scope
This standard specifies the pre-column derivatization-high performance liquid chromatography and the liquid chromatography-tandem mass spectrometry for the determination of asparagine and glutamine in food. In Method I, the direct extraction is suitable for the determination of free asparagine and free glutamine in special dietary foods, while the enzymatic extraction is suitable for the determination of total asparagine and total glutamine in special dietary foods. Method II is applicable to the determination of total asparagine and total glutamine in special dietary foods. Method I. Pre-column derivatization-high performance liquid chromatography2 Principle
The specimen is directly extracted or digested with Streptomyces griseus protease. Asparagine and glutamine are derivatized with dansyl chloride. The derivatives are separated by a C18 column, detected by an ultraviolet detector, and quantified by the external standard method.3 Reagents and materials
Unless otherwise stated, 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). chromatographically pure. 3.1.2 Acetonitrile (C2H3N). chromatographically pure. 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 Tri(hydroxymethyl)aminomethane (Tris base, C4H11O3N). 3.1.8 Streptomyces griseus protease (type XIV; derived from Streptomyces griseus). with an enzyme activity of ≥3.5 U/mg. 3.1.9 Phosphoric acid (H3PO4). chromatographically pure; the content is ≥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, 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 ℃; the 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 fresh before use. 3.2.5 Sodium carbonate buffer solution (60 mmol/L). Weigh 0.318 g of anhydrous sodium carbonate, dissolve in 40 mL of water, adjust the pH to 9.5±0.1 with 1.0 mol/L hydrochloric acid solution, and dilute to 50 mL with water. Prepare fresh before use. 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 ℃; the shelf life is 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, adjust the pH to 6.5±0.1 with phosphoric acid, and dilute 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 substances 3.3.1 Asparagine standard (C4H8N2O3, CAS No.. 70-47-3). with a purity of ≥99%, or a standard that has been certified by the nation and granted a reference material certificate. 3.3.2 Glutamine standard (C5H10N2O3, CAS No.. 56-85-9). with a purity of ≥99%, or a standard that has been certified by the nation and granted a reference material certificate. 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, dissolve in 20% methanol solution and dilute to 50 mL. Store at 4 °C, being 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, dissolve in 20% methanol solution and dilute to 50 mL. Store at 4 °C, being 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 100 mL volumetric flasks, dilute to the mark with water, and mix well. Prepare fresh before use. 3.4.4 Mixed standard series working solutions. Accurately pipette appropriate amounts of the mixed standard intermediate solution (20.0 μg/mL) respectively into 10 mL volumetric flasks, and dilute to the mark with water to obtain a series of standard working solutions with mass concentrations of 0 μg/mL, 0.500 μg/mL, 1.00 μg/mL, 2.00 μg/mL, 5.00 μg/mL, 10.0 μg/mL, and 20.0 μg/mL. Prepare fresh before use. 3.5 Materials 3.5.1 Screw-top glass bottle with cap. 20 mL. 3.5.2 Organic microporous filter membrane. 0.22 μm.4 Instruments and equipment
4.1 High-performance liquid chromatograph. It is equipped with an ultraviolet detector or a diode array detector. 4.2 Electronic balance. The sensitivities are 0.001 g and 0.0001 g. 4.3 Vortex mixer. 4.4 Constant temperature water bath. 37 ℃±1 ℃. 4.5 pH meter. with an accuracy of 0.01. 4.6 Centrifuge. with a speed of ≥5000 r/min.5 Analysis steps
5.1 Specimen preparation Non-powdered solid specimens shall be crushed and mixed evenly, while powdered solid or liquid specimens shall be shaken well. 5.2 Specimen extraction 5.2.1 Direct extraction method Weigh 5.0 g (accurate to 0.001 g) of the solid specimen into a 50 mL dry beaker, add an appropriate amount of warm water (40 ℃~45 ℃) to dissolve, cool to room temperature, transfer and dilute to 25 mL. Accurately transfer 1.00 mL into a 150 mL Erlenmeyer flask, add about 25 mL of water and vortex to mix well. 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 filter to obtain the sample solution to be derivatized. Weigh 1.0 g~5.0g (accurate to 0.001g) of liquid specimen into a 150 mL conical flask, and follow the same procedures as for the solid specimen. 5.2.2 Enzymatic extraction method Weigh 5.0 g (accurate to 0.001 g) of the solid specimen into a 50 mL dry beaker, dissolve it in an appropriate amount of warm water (40 ℃~45 ℃), cool to room temperature, transfer and dilute to 25 mL. Accurately transfer 1.00 mL into a 20 mL screw-top glass bottle, add 0.5 mL of protease solution, 3.0 mL of Tris buffer, and 0.2 mL of methanol sequentially, vortex to mix, and incubate in a constant temperature water bath at 37 ℃ for enzymolysis of 16 h. Remove the glass bottle, cool to room temperature, transfer all the enzymatic solution to a 250 mL volumetric flask, dilute to the mark with water, and mix well. Centrifuge or filter to obtain the sample solution to be derivatized. Weigh 0.5 g~1.0g (accurate to 0.001 g) of the liquid specimen into a 20 mL screw-top glass bottle, and follow the same procedures as for the solid specimen. 5.3 Derivative reaction 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 dansyl chloride solution, mix thoroughly, and react at room temperature in the dark for 2 h (remove and shake well after about 1 h of reaction). Add 0.10 mL of methylamine hydrochloride solution and vortex to stop the reaction. Let it stand in the dark until precipitation is complete, filter through a 0.22 μm filter membrane, and then test. ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
