GB 5413.40-2016 English PDF (GB5413.40-2016)

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GB 5413.40-2016: National Food Safety Standard -- Determination of Nucleotide in Foods and Milk Products for Infants and Young Children
GB 5416.40-2016
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 Children
1 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. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any
amendments) applies.
3 Reagents and material
Unless otherwise stated, the reagents used in this method are of pure analytical
grade. The water is the grade three water specified in GB/T 6682. The water
used by liquid chromatography mobile phase is the grade one water specified
in GB/T 6682.
3.1 Reagents
3.1.1 Amylase. Enzyme activity ≥1.5 U/mg
3.1.2 Glacial acetic acid (CH3COOH)
3.1.3 Tetrabutylammonium bisulfate [(C4H9) 4NHSO4]
3.1.4 Methanol (CH3OH)
3.1.5 Potassium hydrogen phosphate (K2HPO4)
3.1.6 Potassium dihydrogen phosphate (KH2PO4)
3.1.7 Phosphoric acid (H3PO4)
5 Analysis steps
5.1 Sample preparation
5.1.1 Sample pre-treatment
5.1.1.1 Sample containing starch
WEIGH about 5 g of well-mixed solid sample (to the nearest of 0.1 mg) into a
100 mL conical flask. ADD about 0.2 g of amylase. ADD 20 mL of hot water
(30°C ~ 40°C) to completely dissolve the sample. OR WEIGH about 20 g of
well-mixed solid sample (to the nearest of 1 mg) into a 100 mL conical flask.
ADD about 0.2 g of amylase and well SHAKE. ENZYMOLYSE in a 37°C±2°C
incubator for 30 min. TAKE it OUT for cooling to room temperature.
5.1.1.2 Starch-free sample
WEIGH about 5 g of well-mixed solid sample (to the nearest of 0.1 mg) into a
100 mL conical flask. ADD 20 mL of hot water (50°C ~ 60°C) to completely
dissolve the sample. COOL to room temperature. OR WEIGH about 20 g of
well-mixed solid sample (to the nearest of 1 mg) into a 100 mL conical flask.
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
5.2.5 Column temperature. 25°C
5.2.6 Injection volume. 10 μL
NOTE If necessary, use phosphoric acid or dipotassium hydrogen phosphate solution to
appropriately adjust the pH of the mobile phase to the best.
5.3 Standard curve drawing