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GB 5413.40-2016: National Standard of Food Safety - Determination of Nucleotide in Foods and Milk Products for Infants and Young Children
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GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Standard of Food Safety - Determination of Nucleotide in Foods and Milk Products for Infants and Young Children Issued on. AUGUST 31, 2016 Implemented on. MARCH 1, 2017 Issued by. National Health and Family Planning Commission of the People's Republic of China
Table of Contents
1 Scope... 3 2 Principle... 3 3 Reagents and material... 3 4 Apparatus... 4 5 Analysis steps... 5 6 Representation of analysis results... 6 7 Precision... 7 8 Others... 7 Annex A Nucleotide standard product and sample high performance liquid chromatography... 9 Determination of Nucleotide in Foods and Milk Products for Infants and Young Children1 Scope
This Standard specifies the method for the determination of free nucleotide by liquid chromatography. This Standard applies to the determination of total amount of free nucleotides (including cytosine nucleotides, uracil nucleotides, hypoxanthine nucleotides, guanine nucleotides, adenine nucleotides) in infants and young children.2 Principle
The following referenced documents are indispensable for the application of this document.3 Reagents and material
Unless otherwise stated, the reagents used in this method are of pure analytical grade. 3.1 Reagents 3.1.1 Amylase. Enzyme activity ≥1.5 U/mg 3.1.2 Glacial acetic acid (CH3COOH) 3.1.7 Phosphoric acid (H3PO4) 3.2 Preparation of reagents 3.2.1 Acetic acid solution (100 mL/L). PIPET 10 mL of glacial acetic acid; ADD water to a volume of 100 mL. 3.3 Nucleotide standard products 3.3.1 Cytidine (CMP) (C9H14N3O8P). purity ≥ 99% 3.3.2 Adenine nucleotide (AMP) (C10H14N5O7P). purity ≥ 99% 3.3.3 Uracil (UMP) (C9H13N2O9P). purity ≥ 99% 3.3.4 Guanine nucleotide (GMP) (C10H14N5O8P). purity ≥ 99% 3.3.5 Hypoxanthine (IMP) (C10H13N4O8P). purity ≥ 99% 3.4 Preparation of standard solution Nucleotide standard mixture (should be configured that day). respectively WEIGH 10 mg of nucleotide standard products CMP, AMP, UMP, 5 mg of GMP and IMP (to the nearest of 0.1 mg); USE ultra-pure water to dissolve into the same 100 mL volumetric flask; USE water to set the volume to 100 mL.4 Apparatus
4.1 High performance liquid chromatograph. equipped with UV detector or diode array detector 4.4 Constant temperature incubator. ±2°C5 Analysis steps
5.1 Sample preparation 5.1.1 Sample pre-treatment 5.1.2 Preparation of test solution USE acid solution to adjust pH of sample solution to 4.1.MOVE it to a 50 mL measuring flask. SET volume. USE filter paper to filter. USE 0.45 μm micro- membrane to filter the obtained filtrate for use. 5.2 Reference chromatographic conditions 5.2.1 Mobile phase. phosphate buffer + methanol = 1000 + 40 5.2.2 Chromatographic column. C18-T reversed-phase column (250 mm × 4.6 mm, 5 μm) or equivalent column 5.2.3 Flow rate. 1 mL/min 5.2.4 Wavelength. 254 nm 5.3 Standard curve drawing Respectively PIPET 2 mL, 4 mL, 6 mL, 8 mL, 10 mL of standard mixed solution. ADD ultra-pure water to set the volume to 50 mL to prepare nucleotide series of standard working fluid, as shown in Table 1. 5.4 Determination of sample solution Inject the prepared sample solution into the high performance liquid chromatography. Determine the corresponding peak area or peak height.6 Representation of analysis results
6.1 Results calculation Calculate the content of the free nucleotides in the sample Xi (CMP, UMP, GMP, IMP, AMP) according to equation (1). 6.2 Results representation The arithmetic results are expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions. The results shall be reserved for two digits after the decimal point.7 Precision
The absolute difference between the two independent determinations obtained under repeatability shall not exceed 10% of the arithmetic mean.8 Others
The high performance liquid chromatography ultraviolet standard and the sample nucleotide chromatogram are shown in Annex A.Annex A
Nucleotide standard product and sample high performance liquid chromatography A.1 Nucleotide standard chromatogram The nucleotide standard chromatogram is shown as Figure A.1. A.2 Sample chromatogram The sample chromatogram is shown as Figure A.2. ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
