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GB/T 30636-2014 English PDF (GBT30636-2014)

GB/T 30636-2014 English PDF (GBT30636-2014)

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GB/T 30636-2014: Determination of sialic acid in swiftlets nest and swiftlets nest products -- Liquid chromatographic method

This standard specifies the method for the determination of sialic acid in swiflets nest and its products by liquid chromatography. This standard applies to the determination of sialic acid in swiflets nest and its products. The detection limit of this standard is 0.3 g/kg for swiflets nest and 0.003 g/kg for swiflets nest products.
GB/T 30636-2014
GB
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
ICS 67.050
X 04
Determination of sialic acid in swiflets nest and
swiflets nest products - Liquid chromatographic
method
ISSUED ON: DECEMBER 31, 2014
IMPLEMENTED ON: MAY 01, 2015
Issued by: General Administration of Quality Supervision Inspection and Quarantine of PRC;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Method summary ... 4
4 Reagents and materials ... 5
5 Instruments ... 5
6 Analytical procedures ... 6
7 Determination ... 7
8 Result calculation ... 7
9 Precision ... 8
Appendix A (Informative) Chromatograms of sialic acid standard solution and swiflets nest sample hydrolysate ... 9
Determination of sialic acid in swiflets nest and
swiflets nest products - Liquid chromatographic
method
1 Scope
This standard specifies the method for the determination of sialic acid in swiflets nest and its products by liquid chromatography.
This standard applies to the determination of sialic acid in swiflets nest and its products.
The detection limit of this standard is 0.3 g/kg for swiflets nest and 0.003 g/kg for swiflets nest products.
2 Normative references
The following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) are applicable to this standard.
GB/T 6379.2 Accuracy (trueness and precision) of measurement methods
and results - Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method
GB/T 6682 Water for analytical laboratory use - Specification and test
methods
3 Method summary
Swiflets nest and swiflets nest products are heated and hydrolyzed in acetic acid solution to release sialic acid. The hydrolysate is separated by a strong cation exchange column, detected by liquid chromatography-UV detector,
quantified by external standard method.
4 Reagents and materials
4.1 Water: Meet the grade-1 water requirements as specified in GB/T 6682. 4.2 Acetonitrile: Chromatographically pure.
4.3 Glacial acetic acid: Analytically pure.
4.4 Phosphoric acid: Analytically pure.
4.5 Sialic acid (N-acetylneuraminic acid, C11H19NO9): The purity is greater than 99%.
4.6 Polyethylene glycol: The average relative molecular mass is not less than 17000.
4.7 0.1% phosphoric acid solution: Take 1 mL of phosphoric acid; add water to make the volume to 1000 mL; mix well; use 0.45 ??m microporous membrane
to filter it.
4.8 50% acetic acid solution: Take 100 mL of glacial acetic acid; add 100 mL of water; mix it uniformly.
4.9 10% polyethylene glycol solution: Weigh 50 g of polyethylene glycol; add 500 mL of water and stir to dissolve it.
4.10 Standard stock solution of sialic acid (1.00 mg/mL): Weigh 100.0 mg of sialic acid; add 20 mL of water to dissolve it; add acetonitrile to make the volume reach to 100 mL. Store in the dark at 4 ??C; the shelf life is one month. 4.11 Dialysis bag: It allows the penetration of compounds which have a
molecular weight of less than 10000; soak in water for 1 h before use.
4.12 0.45 ??m microporous membrane: Organic phase.
4.13 0.45 ??m syringe filter: Organic phase.
4.14 Mobile phase: Take 100 mL of 0.1% phosphoric acid solution; add
acetonitrile to make the volume to 1000 mL; mix it uniformly; use 0.45 ??m organic phase microporous membrane to filter it.
5 Instruments
5.1 Liquid chromatograph: Equipped with ultraviolet detector.
5.2 Constant temperature water bath.
Measure 1.00 mL, 2.00 mL, 4.00 mL, 6.00 mL, 8.00 mL, 10.0 mL of the standard stock solution of sialic acid, respectively. Place them in a 100 mL volumetric flask. Add the mobile phase to the mark. Prepare the sialic acid standard solution which has a concentration of 10.0 ??g/mL, 20.0 ??g/mL, 40.0 ??g/mL, 60.0 ??g/mL, 80.0 ??g/mL, 100.0 ??g/mL. Prepare for testing.
7 Determination
7.1 Chromatographic reference conditions
7.1.1 Chromatographic column: 300SCX cation exchange chromatographic
column, 4.6 mm ?? 250 mm, particle size 5 ??m, or equivalent.
7.1.2 Mobile phase: Acetonitrile-0.1% phosphoric acid aqueous solution (90 + 10, volume ratio).
7.1.3 Column temperature: 30 ??C.
7.1.4 Flow rate: 1.0 mL/min.
7.1.5 Injection volume: 10 ??L.
7.1.6 Detection wavelength: 205 nm.
7.2 Liquid chromatography determination
Take the sialic acid series standard solution and test solution, respectively. Use the peak area of the standard series solution as the ordinate and the
concentration as the abscissa, draw the standard curve. Use the retention time for qualification and use the external standard method of peak area for quantitation. Refer to Appendix A for the chromatograms of the sialic acid standard solution and the hydrolysate of the swiflets nest sample.
8 Result calculation
The measurement results can be automatically calculated by the instrument workstation, or calculated according to formula (1):
Where:
X - The content of sialic acid in the test solution, in grams per kilogram (g/kg); c - The concentration of sialic acid in the test solution as found from the

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