GB/T 30636-2014Determination of sialic acid in swiftlets nest and swiftlets nest products -- Liquid chromatographic method (English PDF)

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.

4.7 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 µm 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

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 µm microporous membrane. Organic phase.

4.13 0.45 µm syringe filter. Organic phase.

5.1 Liquid chromatograph. Equipped with ultraviolet detector.

5.2 Constant temperature water bath.

5.3 Mortar.

5.4 Homogenizer. Rotation speed is 10000 r/min.

5.5 Oven.

6.1 Specimen processing

6.1.1 Dried swiflets nest

Take an appropriate amount of specimen and dry it at 105 °C for 60 minutes.

Cool it in a desiccator.

6.1.2 Swiflets nest products with rock sugar

Take an appropriate amount of specimen in a beaker. Homogenize at a speed

of 10000 r/min for 3 minutes.

6.2 Preparation of sialic acid series standard working solution

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.

7.1 Chromatographic reference conditions

7.1.1 Chromatographic column. 300SCX cation exchange chromatographic

column, 4.6 mm × 250 mm, particle size 5 µm, or equivalent.

7.1.2 Mobile phase. Acetonitrile-0.1% phosphoric acid aqueous solution (90 +

10, volume ratio).

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.

9.1 General provisions

The precision of this standard is determined in accordance with the provisions

of GB/T 6379.2.The repeatability and reproducibility values are calculated with

95% confidence.

9.2 Repeatability

Under repeatability conditions, the absolute difference between two

independent test results as obtained does not exceed the repeatability limit (r).

The content range and repeatability equation of different samples are as shown

in Table 1.

9.3 Reproducibility

Under the condition of reproducibility, the absolute difference between the two

independent test results as obtained does not exceed the reproducibility limit

(R). The content range and reproducibility equation of different samples are as

shown in Table 1.

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