GB 5413.40-2016National Standard of Food Safety - Determination of Nucleotide in Foods and Milk Products for Infants and Young Children (English PDF)

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.

4.1 High performance liquid chromatograph. equipped with UV detector or

diode array detector

4.4 Constant temperature incubator. ±2°C

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.

6.1 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.

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.

......