National Standard of Food Safety
Determination of Nucleotide in Foods and Milk Products for Infants and Young Children
食品安全国家标准
婴幼儿食品和乳品中核苷酸的测定
1 Scope
This standard specifies the method for determining the total amount of free nucleotide through liquid chromatography.
This standard is applicable to the determination of total amount of free nucleotide (including cytidylic acid, uridylic acid, hypoxanthine nucleotide, guanylic acid, adenylic acid) in foods and milk products for infants and young children.
2 Principle
The specimen shall be subject to water extraction; precipitant is used to precipitate the protein; separate the specimen with high performance liquid chromatography; determine the nucleotide content in the specimen by adopting external standard method with ultraviolet detector.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents, Grade III water (defined in GB/T 6682) and Grade I water (defined in GB/T 6682 and used for mobile phase of liquid chromatography) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Amylase: enzyme activity ≥1.5U/mg.
3.1.2 Glacial acetic acid (CH3COOH).
3.1.3 Tetrabutylammonium hydrogen sulfate [(C4H9)4NHSO4].
3.1.4 Methanol (CH3OH).
3.1.5 Dipotassium phosphate (K2HPO4).
3.1.6 Potassium dihydrogen phosphate (KH2PO4).
3.1.7 Phosphoric acid (H3PO4).
3.2 Reagent preparation
3.2.1 Acetic acid solution (100mL/L): pipet 10mL of glacial acetic acid, and scale the volume with water to 100mL.
3.2.2 Dipotassium phosphate solution (0.1 mol/L): weigh 2.28g of dipotassium phosphate, dissolve it and scale the volume to 100mL with ultrapure water.
3.2.3 Phosphate buffer (1.40mmol/L tetrabutylammonium hydrogen sulfate, 0.01 mol/L potassium dihydrogen phosphate): weigh 1.360g of potassium dihydrogen phosphate, add 0.475 3g of tetrabutylammonium hydrogen sulfate, dissolve them with 900mL of water, adjust the pH to 3.2 with dipotassium phosphate solution, and scale the volume with water to 1,000mL. This solution shall be used within a week.
3.3 Nucleotide standard product
3.3.1 Cytidylic acid (CMP) (C9H14N3O8P): purity≥99%.
3.3.2 Adenylic acid (AMP) (C10H14N5O7P): purity≥99%.
3.3.3 Uridylic acid (UMP) (C9H13N2O9P): purity≥99%.
3.3.4 Guanylic acid (GMP) (C10H14N5O8P): purity≥99%.
3.3.5 Hypoxanthine nucleotide (IMP) (C10H13N4O8P): purity≥99%.
3.4 Standard solution preparation
Standard mixed solution of nucleotide (prepared on the application day): weigh nucleotide standard products: 10mg of CMP, AMP and UMP respectively, 5mg of GMP and IMP respectively (to the nearest of 0.1 mg); dissolve with ultrapure water and transfer to the same 100mL volumetric flask and scale the volume with water to 100mL. As for the concentration of this standard solution: it is 100 μg/mL for CMP, AMP and UMP and 50 μg/mL for GMP and IMP. As for the mass weighed of each component, the moisture and sodium salt contents shall be calibrated (counted by acid type).
4 Instruments and Apparatus
4.1 High-performance liquid chromatograph: equipped with ultraviolet detector or diode array detector.
4.2 Balance: with the sensibility of 0.1mg.
4.3 pH meter: with the precision of 0.01.
4.4 Constant temperature incubator: ±2℃.
5 Analysis Steps
5.1 Specimen preparation
5.1.1 Specimen pretreatment
5.1.1.1 Starch-contained specimen
Weigh about 5g of well-mixed solid specimen (to the nearest of 0.1mg), and put it in a 100mL conical flask, add into about 0.2g of amylase, add into 20mL of hot water (30℃~40℃) to sufficiently dissolve the specimen, or weigh about 20g of well-mixed liquid specimen (to the nearest of 1mg) and put it in a 100mL conical flask, add into about 0.2g of amylase and shake well, put it in 37℃±2℃ incubator for enzymolysis for 30 min. Take it out and cool to ambient temperature.
5.1.1.2 Starch-free specimen
Weigh about 5g of well-mixed solid specimen (to the nearest of 0.1mg), and put it in a 100mL conical flask, add into 20mL hot water (50 ℃~60℃) to sufficiently dissolve the specimen, and cool it to ambient temperature, or weigh about 20g of well-mixed liquid specimen (to the nearest of 1mg) and put it in a 100mL conical flask.
5.1.2 Preparation of the to-be-determined solution
Adjust the pH value of specimen solution to 4.1 with acetate solution, transfer the solution into 50mL volumetric flask, scale the volume, filter the solution with filter paper, and filter the obtained filtrate with 0.45 μm micro-membrane for standby.
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Result
7 Precision
8 Others
Appendix A High Performance Liquid Chromatogram for the Nucleotide Standard Product and Specimen
