GB 5009.158-2016National food safety standard - Determination of Vitamin K1 in Foods (English PDF)

3.1 Reagents

3.1.9 Glacial acetic acid (CH3COOH). chromatographic purity.

3.1.10 Zinc chloride (ZnCl2). chromatographic purity.

3.1.11 Anhydrous sodium acetate (CH3COONa).

3.1.12 Potassium hydroxide (KOH).

3.1.13 Lipase. enzyme activity>=700 U/mg.

3.2 Preparation of Reagents

3.2.1 40% potassium hydroxide solution. weigh-take 20 g of potassium hydroxide and

place it in 100 mL beaker; add 20 mL of water to dissolve it; cool it down, then, add

water to 50 mL; store in polyethylene bottle.

3.3 Standards

Vitamin K1 (C31H46O2, CAS No..84-80-0). purity>=99%, or standard substance with a

national-level certificate and a certificate of standard substance.

3.4 Preparation of Standard Solutions

3.4.1 Standard stock solution of vitamin K1 (1 mg/mL). accurately weigh-take 50 mg

(accurate to 0.1 mg) of standard vitamin K1 and place it in 50 mL volumetric flask; use

methanol to dissolve and dilute to the constant volume. Transfer the solution to a

brown glass container, store under -20 °C and keep away from light.

3.4.2 Standard intermediate fluid of vitamin K1 (100 µg/mL). accurately absorb 10.00

mL of standard stock solution of vitamin K1 and place it in 100 mL volumetric flask; add

methanol to the constant volume, mix it up. Transfer the solution to a brown glass

container, store under -20 °C and keep away from light. It can remain valid for 2

months.

3.5 Materials

3.5.1 Neutral alumina. particle size 50 µm~150µm.

3.5.2 Neutral alumina column. 2 g/6 mL, 10% of water is contained in the filling.

Commodity column can be purchased directly, or it can be replaced by filling.

3.5.3 Zinc column. length 50 mm, internal diameter 4.6 mm; zinc column can be

purchased directly, or it can be replaced by filling.

3.5.4 Microporous filter head. equipped with 0.22 µm organic microporous membrane.

4.1 High-performance liquid phase chromatograph. equipped with fluorescence detector.

4.2 Homogenizer.

4.7 pH meter. accuracy. 0.01.

4.8 Balance. division value. 1 mg and 0.1 mg.

4.9 Centrifuge. rotating speed>=6,000 r/min.

4.12 Ultrasonic oscillator.

5.1 Preparation of Samples

After mixing powdered samples like rice flour and flour, directly take the sample; use

sample grinder to grind flaky and granular samples into powder, store it in a sample

bag for later usage;

5.2 Processing of Samples

Note. during the processing, avoid direct UV light irradiation, keep away from light.

5.2.1 Foods for infants and young children, dairy products and vegetable oil

5.2.2 Fruit and vegetable

5.2.2.1 Extraction

Accurately weigh-take 1 g~5 g (accurate to 0.01 g, the content of vitamin K1 shall be

>=0.05 µg) of homogenized sample, place it in 50 mL centrifuge tube. Add 5 mL of

isopropanol, start vortex for 1 min and ultrasonic for 5 min. Add 10 mL of N-hexane,

start vortex or oscillation extraction for 3 min, start centrifugation at 6,000 r/min for 5

min. Remove-take the supernatant in 25 mL brown volumetric flask.

5.2.2.2 Purification and concentration

Use a small amount of N-hexane to transfer 1 mL of the above-mentioned extract to

neutral alumina column that's previously activated with 5 mL of N-hexane. Wait till the

extract is almost drained, use 5 mL of N-hexane to rinse it; use 6 mL of N-hexane -

ethyl acetate mixed solution to elute to 10 mL tube.

5.3 Chromatography Reference Conditions

5.4 Draw a Standard Curve Line

Adopt the external standard curve line to quantify. Respectively inject the standard

series of working fluid of vitamin K1 into high-performance liquid phase

chromatograph, determine the corresponding peak area.

5.5 Determination of Sample Solution

Under the same chromatographic conditions, respectively inject the prepared blank

solution and sample solution for high-performance liquid phase chromatographic

analysis.

10.1 Reagents

10.1.8 Methanol (CH3OH). chromatographic purity.

10.1.9 Ethyl acetate (C4H8O2).

10.2 Preparation of Reagents

10.2.1 40% potassium hydroxide solution. weigh-take 20 g of potassium hydroxide

and place it in 100 mL beaker; add 20 mL of water to dissolve it; cool it down, then,

add water to 50 mL; store in polyethylene bottle.

10.3 Standards

10.3.1 Vitamin K1 (C31H46O2, CAS No..84-80-0). purity>=99.3%.

10.3.2 Vitamin K1-D7 (C31H46O2, CAS No..1233937-39-7). purity>=99.5%.

10.4 Preparation of Standard Solutions

10.4.1 Standard stock solution of vitamin K1 (1 mg/mL). accurately weigh-take 50 mg

(accurate to 0.1 mg) of standard vitamin K1 and place it in 50 mL volumetric flask; use

methanol to dissolve and dilute to the constant volume. Transfer the solution to a

brown glass container, store under -20 °C and keep away from light.

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