GB 5413.9-2010National food safety standard -- Determination of vitamin A, D, E in foods for infants and young children, milk and milk products (English PDF)

4.1 alpha-Diastase. Enzyme activity >= 1.5 U/mg.

4.2 Anhydrous sodium sulfate.

4.3 Isopropyl alcohol. chromatographically pure.

4.4 Alcohol. chromatographically pure.

4.5 Potassium hydroxide solution. weigh 250 g of solid potassium hydroxide, and

dissolve it with 200 mL of water.

5.7 Balance. accuracy is 0.1 mg.

6.1 Sample pretreatment

6.1.1 Sample containing starch

Weigh about 5 g of solid sample that has been mixed well or about 50 g of liquid sample

(accurate to 0.1 mg), and put it into a 250 mL Erlenmeyer flask. Add 1g of alpha-amylase

(4.1). For solid sample, about 50 mL of distilled water at 45 ~ 50°C shall be added in to

dissolve it. After it is well mixed, use nitrogen gas to exhaust air from the flask. Insert

the stopper. Place it in a 60°C±2°C incubator (5.6) for 30 min.

6.1.2 Sample free of starch

Weigh about 10 g of solid sample that has been mixed well or about 50 g of liquid sample

(accurate to 0.1 mg), and put it into a 250 mL Erlenmeyer flask. For solid sample, about

6.2 Recovery test shall be carried out simultaneously for test of sample with

vitamin D.

6.3 Preparation of test solution

6.3.1 Saponification. Add about 100 mL of ethyl alcohol solution of vitamin C (4.10) in

the above-mentioned sample solution that has been pretreated. After it is well mixed,

add 25 mL of the potassium hydroxide solution (4.5). Well mix. Then add magnetic rod

and fill nitrogen gas for protection. Insert the rubber stopper. Add about 300 mL of water

in a 1000 mL beaker, and place the beaker in a constant temperature magnetic stirrer

(5.3); when the temperature is controlled at 53°C ±2°C, place the triangular flask in a

beaker, after saponification under magnetic force stirring for about 45 min, immediately

take it out and cool it down to room temperature.

6.3.2 Extraction. Transfer all the saponified solution into a 500 mL separating funnel.

Add 100 mL of petroleum ether (4.6) in. Shake it gently. Insert the stopper after exhaust.

Severely vibrate it for about 10 min; let stand for demixing. Place the aqueous phase in

another 500 mL separating funnel. Repeat the above extraction process. Merge the

ether solution, and wash it with distilled water until it becomes neutral. Use anhydrous

sodium sulfate for filter and dehydration. Collect the filtrate in a 500 mL flat-bottomed

flask. After it is evaporated under 40°C±2°C nitrogen gas flow on rotator evaporator and

becomes nearly dry (it is not allowed to make it completely dry), use petroleum ether to

transfer it into a 10 mL volumetric flask, and dilute it to fixed-volume.

6.3.3 Take 2.0 mL of petroleum ether solution from the aforesaid volumetric flask and

put it into cuvette A. Then take 7.0 mL of petroleum ether solution and put it into cuvette

B. Blow-dry the petroleum ether in both cuvettes A and B with nitrogen gas respectively

in a 40°C±2°C of nitrogen evaporator (5.4). Add 5.0 mL of methanol (4.7) in cuvette A

to stir and dissolve the residues. Add 2.0 mL of normal hexane (4.8) in cuvette B to stir

and dissolve the residues. Then centrifuge the cuvettes A and B at 5000 r/min for 10

min; take out, let stand until it cools to room temperature before determination. The

cuvette A is used for determination of vitamin A and E, and the cuvette B is used for

determination of vitamin D.

6.4 Determination

6.4.1 Determination of vitamin A and E

6.4.1.1 Reference condition for chromatography

Chromatographic column. C18 column, 250 mm × 4.6 mm, 5 µm, or chromatographic

column with the same performance.

Mobile phase. methanol (4.7).

Flow rate. 1.0 mL/min.

Wavelength. Vitamin A, 325 nm; vitamin E, 294 nm.

Column temperature. 35°C±1°C.

Injection amount. 20 µL.

6.4.1.2 Drawing of standard curve of vitamin A and E

Draw 0.50 mL, 1.00 mL, 1.50 mL, 2.00 mL, and 2.50 mL of the vitamin A standard stock

solution (4.11.1) in 50 mL brown volumetric flasks respectively; dilute them with ethanol

to graduation; mix well. The concentrations of this series of standard working solutions

are 1.00 µg/mL, 2.00 µg/mL, 3.00 µg/mL, 4.00 µg/mL, and 5.00 µg/mL respectively.

Draw 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL and 5.0 mL of the vitamin E standard stock solution

(4.11.2) in 50 mL brown volumetric flasks respectively; dilute them with ethanol to

graduation; mix well. The concentrations of this series of standard working solutions are

10.0µg/mL, 20.0 µg/mL, 30.0 µg/mL, 40.0 µg/mL, and 50.0 µg/mL respectively. Inject

the standard working solutions of vitamin A and E into the liquid chromatograph (the

chromatogram is shown in Annex B) to get the peak heights (or peak areas). Draw the

standard curves of vitamin A and E respectively, in which the y-axis is peak height (or

peak area), and the x-axis is the concentration of standard working solutions of vitamin

A and E.

6.4.1.3 Determination of test sample of vitamin A and E

Column temperature. 35°C ±1°C.

Injection volume. 100 µL.

6.4.2.2.2 Drawing of standard curve

Draw 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, and 1.00 mL of the standard stock solution

(4.11.3 or 4.11.4) of vitamin D2 (or D3) into 100 mL brown volumetric flasks respectively,

dilute to volume with ethanol, and mix well. The concentrations of this series of standard

working solutions are 0.200µg/mL, 0.400µg/mL, 0.600µg/mL, 0.800µg/mL, and

1.000µg/mL respectively.

Inject the standard working solutions of vitamin D2 (or D3) into the liquid chromatograph

to get the peak height (or peak area) (see annex B). Draw the standard curve, in which

the y-axis is peak height (or peak...

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