GB 5009.82-2016National Food Safety Standard Determination of vitamin A, D, E in food (English PDF)

3.1 Reagents

3.1.1 Absolute ethanol (C2H5OH). it shall not contain aldehydes after inspection,

AND the inspection method is as shown in A.1.

3.1.2 Ascorbic acid (C6H8O6).

3.1.3 Potassium hydroxide (KOH).

3.1.4 Diethyl ether [(CH3CH2)2O]. it shall not contain peroxides after inspection,

AND the inspection method is as shown in A.2.

3.2 Reagent preparation

3.2.1 Potassium hydroxide solution (50 g/100 g). WEIGH 50 g of potassium

hydroxide; ADD 50 mL of water to dissolve it; after cooling it down, STORE it in a polyethylene bottle.

3.2.2 Petroleum ether - ether solution (1 +1). MEASURE 200 mL of petroleum

ether; ADD 200 mL of ether; MIX it uniformly.

3.2.3 Organic filter head (pore size of 0.22 µm).

3.3 Standard substance

3.4 Standard solution preparation

3.4.1 Vitamin A standard stock solution (0.500 mg/mL). accurately WEIGH 25.0

mg vitamin A standard substance; USE absolute ethanol to dissolve it;

TRANSFER it into a 50 mL volumetric flask; MAKE the volume reach to the mark; at this time the solution concentration is about 0.500 mg/mL. TRANSFER the solution into a brown reagent bottle; SEAL it and PRESERVE it at -20 °C in the dark; AND the valid period is 1 month. Before use, INCREASE the temperature of the solution back to 20 °C; and CONDUCT concentration correction (as for the correction method, SEE Appendix B).

4.7 High performance liquid chromatography. equipped with UV detector or

diode array detector or fluorescence detector.

5.1 Preparation of test specimens

A certain amount of samples are divided and crushed homogeneously, stored in the sample bottle under refrigerated conditions in the dark; AND it shall be used for determination as soon as possible.

5.2 Sample treatment

Caution. All vessels used shall not contain oxidizing substances. The surface of the separation funnel piston glass shall not be coated with oil.

During the treatment, AVOID ultraviolet light and AVOID light as far as possible. The extraction process shall be conducted in the fume hood.

5.2.1 Saponification

5.2.1.1 Starch-free samples

WEIGH 2 g ~ 5 g (accurate to 0.01 g) of homogenized solid sample OR 50 g (accurate to 0.01 g) of liquid sample into a 150 mL flat-bottomed flask; as for the solid test specimen, it is required to add about 20 mL of warm water; MIX it uniformly; then ADD 1.0 g of ascorbic acid and 0.1 g of BHT; MIX it uniformly;

ADD 30 mL of absolute ethanol; ADD 10 mL ~ 20 mL potassium hydroxide solution while shaking; after mixing it uniformly, PLACE it in the 80 °C constant temperature water bath for shaking and saponification for 30 min; after saponification, immediately USE cold water to cool it to room temperature.

5.2.2 Extraction

USE 30 mL of water to transfer the saponification solution into a 250 mL separation funnel; ADD 50 mL of petroleum ether-ether mixture; SHAKE it for extraction for 5 min; TRANSFER the lower solution into another 250 mL separation funnel. ADD 50 mL of petroleum ether-ether mixture and CONDUCT extraction again; COMBINE the ether layers.

5.2.3 Washing

USE about 100 mL of water to wash the ether layer; REPEAT washing for about

3 times, until washing the ether layer to neutral (it may use the pH test strips to test the pH value of the lower solution); REMOVE the lower aqueous phase.

5.2.4 Concentration

The washed ether layer is filtered through an anhydrous sodium sulfate (about

3 g) into a 250 mL rotary evaporator or nitrogen concentrator. USE about 15 mL of petroleum ether to rinse the separation funnel and anhydrous sodium sulfate for 2 times; INCLUDE it in the evaporation bottle; and CONNECT it to the rotary evaporator or gas concentration instrument; PLACE it in 40 °C water bath for reduced pressure distillation or air concentration; when the ether solution in the bottle is about 2 mL, TAKE the evaporation bottle off; immediately USE nitrogen to blow it to near dry.

5.3 Chromatographic reference conditions

The chromatographic reference conditions are listed below.

5.4 Production of standard curve

This method uses external standard method for quantitative purposes.

Respectively INJECT the vitamin A and vitamin E standard series working solution into the high performance liquid chromatograph. MEASURE the corresponding peak area. USE the peak area as the ordinate and the concentration of the standard determination solution as the abscissa, DRAW the standard curve, and CALCULATE the linear regression equation.

5.5 Determination of samples

After the sample solution is analyzed by high performance liquid chromatograph, the peak area is measured. USE the external standard method to calculate its concentration based on the aforementioned standard curve.

10.1 Reagents

10.1.1 Absolute ethanol (C2H5OH). Chromatographic purity, it shall not contain aldehydes after inspection, AND the inspection method is as shown in A.1.

10.1.2 Diethyl ether [(CH3CH2)2O]. Analytical grade pure, it shall not contain oxides after inspection, AND the inspection method is as shown in A.1.

10.2 Reagent preparation

10.2.1 Petroleum ether - ether solution (1 +1). MEASURE 200 mL of petroleum

ether; ADD 200 mL of ether; MIX it uniformly; PREPARE it before use.

10.2.2 Mobile phase. n-hexane + [tert-butyl methyl ether - tetrahydrofuran -

methanol mixture (20 + 1 + 0.1)] = 90 +10; PREPARE it before use.

10.3 Standard substance

10.4 Preparation of standard solutions

10.4.1 Vitamin E standard stock solution (1.00 mg/mL). respectively and

accurately WEIGH 50.0 mg of each of alpha-tocopherol, beta-tocopherol gamma-tocopherol and delta-Tocopherol (accurate to 0.1 mg);

11.7 High performance liquid chromatograph with fluorescence detector or UV

detector.

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