GB 5009.296-2023National food safety standard - Determination of Vitamin D in foods (English PDF)

3.1 Reagents

3.1.1 Absolute ethanol (C2H6O). chromatographic pure.

3.1.2 Ascorbic acid (C6H8O6).

3.1.6 n-hexane (C6H14).

3.1.7 Methanol (CH4O). chromatographic pure.

3.1.8 Anhydrous sodium sulfate (Na2SO4).

3.1.9 Cyclohexane (C6H12).

3.2 Preparation of reagents

3.3 Standard

3.3.1 Vitamin D2 standard substance. ergocalciferol (C28H44O, CAS number. 50-14-6),

purity >=98%, or a standard substance certified by the state and awarded a standard

substance certificate.

3.3.2 Vitamin D3 standard substance. cholecalciferol (C27H44O, CAS number. 67-97-0),

purity >=98%, or a standard substance certified by the state and awarded a standard

substance certificate.

3.4 Preparation of standard solution

3.4.1 Vitamin D2 standard stock solution (1 000 mg/L). Accurately weigh 50 mg

(accurate to 0.1 mg) of vitamin D2 standard substance in a small beaker; dissolve it in

absolute ethanol and transfer it to a 50 mL volumetric flask; adjust the volume to the

mark; mix well. Calibrate the concentration of the stock solution according to Appendix

A. Put the stock solution into a brown reagent bottle; seal it and store it in the dark at -

3.4.4 Vitamin D3 standard working solution (10.0 mg/L). Accurately draw 1.00 mL of

vitamin D3 standard stock solution (1 000 mg/L) into a 100 mL volumetric flask; use

absolute ethanol to dilute to the mark; mix well. Store in the dark at -18 °C, with a use-

by day of 3 months.

3.4.5 Standard series of working solutions

3.5 Materials

3.5.3 Microporous membrane. organic system, pore size 0.45 µm.

4.1 Normal-phase high-performance liquid chromatograph. equipped with a UV

detector; the injector is equipped with a 500 µL quantitative loop.

4.2 Reversed-phase high-performance liquid chromatograph. equipped with a UV

detector; the injector is equipped with a 100 µL quantitative loop.

4.3 Balance. The sensitivity is 0.1 mg or 0.01 g.

4.8 Constant-temperature water bath.

4.9 Nitrogen blowing concentrator.

4.10 Ultrasonic cleaner.

4.11 Separating funnel extraction oscillator.

5.1 Sample pretreatment

UV irradiation shall be avoided during processing.

5.1.1 Sample preparation

After a certain number of samples are divided, crushed and homogenized as required,

store them in sample bags, protected from light and refrigerated, and measure as soon

as possible.

5.1.1.3 Other foods

Weigh 5 g ~ 20 g (m, accurate to 0.01 g) of the homogenized solid sample or 20 g ~ 50

g (m, accurate to 0.01 g) of the liquid sample into a 150 mL flat-bottomed flask. For

solid samples, add 20 mL ~ 30 mL of warm water (40 °C ~ 45 °C); mix well.

5.1.2 Sample saponification

5.1.3 Sample extraction and concentration

5.1.3.1 Sample extraction

Use 30 mL of water to transfer the above saponification solution into a 250 mL

separatory funnel; add 50 mL of petroleum ether; shake and extract for 5 minutes;

transfer the lower solution to another 250 mL separatory funnel; add 50 mL of

petroleum ether and repeat the extraction 1 to 2 times; combine the petroleum ether

layer. Use about 150 mL of water to wash the petroleum ether layer; remove the lower

water phase; repeat washing at least 3 times until the petroleum ether layer is washed

to neutral (a pH test paper universal can be used to detect the lower solution).

5.1.3.2 Sample concentration

Slowly flow the washed petroleum ether layer through a glass chromatography column

pre-filled with anhydrous sodium sulfate powder with a thickness of 3 cm ~ 5 cm; filter

it into a 250 mL rotary evaporating flask or a nitrogen blowing concentration tube; use

5.2 Apparatus reference conditions

5.2.1 Purification of vitamin D test solution

5.2.2 Reversed-phase liquid chromatography reference conditions

The reference conditions for reversed-phase liquid chromatography are as follows.

5.3 Preparation of standard curve

Inject the standard series of working solutions into the reversed-phase liquid

chromatograph respectively. For the internal standard method, use the mass ratio of

vitamin D2 (or vitamin D3) in the standard series of working solutions to its

corresponding internal standard as the abscissa, and the peak area ratio of vitamin D2

(or vitamin D3) to its corresponding internal standard as the ordinate to draw the

standard curve.

5.4 Determination of sample solution

5.4.1 Qualitative analysis

Inject the sample solution into the liquid chromatograph for measurement. Under the

same test conditions, the retention time of vitamin D2 and vitamin D3 in the sample

solution shall deviate by no more than +/-2.5% compared with the corresponding

retention time in the standard working solution.

5.5 Blank test

Without weighing the sample, follow the saponification, extraction and concentration

analysis steps of the sample, it shall not contain substances that interfere with the

components to be tested. The internal standard method requires sample testing without

adding internal standard to confirm the feasibility of adding internal standard.

10.1 Reagents

10.1.1 Absolute ethanol (C2H6O). chromatographic pure.

10.1.5 Potassium hydroxide (KOH).

10.1.6 n-hexane (C6H14).

10.1.7 Ethyl acetate (C4H8O2).

10.2 Preparation of reagents

10.2.1 Potassium hydroxide solution (50%, mass fraction). same as 3.2.1.

10.2.5 Acetonitrile-methanol solution (3+1). Mix acetonitrile and methanol evenly at a

volume ratio of 3.1 and degas by ultrasonic.

10.2.6 Methanol-water solution (1+19). Mix methanol and water evenly at a volume

ratio of 19.1 and degas by ultrasonic.

10.2.7 Acetonitrile-water solution (19+1). Mix acetonitrile and water evenly at a

volume ratio of 19.1 and degas by ultrasonic.

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