GB 5413.10-2010National food safety standard -- Determination of vitamin K1 in foods for infants and young children, milk and milk products (English PDF)

1 Scope

This Standard specifies the method for determination of Vitamin K1 in foods for infants and young children, milk and milk products.

This Standard is applicable to the determination of Vitamin K 1 in foods for infants and young children, milk and milk products.

2 Normative references

The following standards contain provisions which, through the reference in this text, constitute provisions of this Standard. For the dated references, all the amendments or revisions after them, except the corrigenda, are not applicable to this Standard. For the references that are not dated, their most recent editions are applicable to this Standard.

3 Principle

Use lipase to degrade the fats and unsaturated fatty acids in test sample. For the sample containing starch, it needs to use starch amylase to degrade the starch in sample; after saponification by alkali, use normal hexane to extract Vitamin K1 . After separation by liquid chromatography, post-column reduction of Vitamin K1 is carried out. The fluorescence detector is used for detection; and external standard method is used for quantification.

4 Reagents and materials

Unless otherwise specified, all reagents used in this method are analytically pure. Water is the Grade 1 specified in GB/T 6682.

4.1 Sodium hydroxide solution (10 mol/L): Prepare before use.

4.2 95% ethanol.

4.3 Saturated sodium chloride solution.

4.4 Normal hexane: Chromatographically pure.

4.5 Anhydrous sodium sulfate.

4.6 Methanol: Chromatographically pure.

4.7 Dichloromethane: Chromatographically pure.

4.8 Glacial acetic acid.

4.9 Zinc chloride.

4.10 Anhydrous sodium acetate.

4.11 Mobile phase: 900 mL of methanol (4.6), 100 mL of dichloromethane (4.7), 0.3 mL of glacial acetic acid (4.8), 1.5 g of zinc chloride (4.9), and 0.5 g of anhydrous sodium acetate (4.10); after dissolution, use 0.45 µm membrane to filter.

4.12 Amylase: Enzyme activity >= 1.5 U/mg.

4.13 Lipase: Enzyme activity >= 700 U/mg.

4.14 Zinc powder: Particle size: 50µm ~ 70 µm.

4.15 Standard solution of Vitamin K 1 : The calibration method of standard solution concentration is shown in Annex A.

4.15.1 Standard stock solution of Vitamin K1 (2 mg/mL): Weigh 0.05 g of standard substance of Vitamin K 1 (accurate to 0.1 mg). Put into a 25 ml volumetric flask.

Use normal hexane to dissolve and fix the volume.

4.15.2 Standard intermediate solution of Vitamin K (20 µg/ml). Take 1 mL of standard stock solution (4.15.1). Add normal hexane to fix the volume to 100 mL.

5 Instruments and apparatuses

5.1 High-pressure liquid chromatograph: Equipped with fluorescence detector.

5.2 Balance: Sensitivity is 0.1 mg.

5.3 Separating funnel: 250 ml.

5.4 Rotatory evaporator.

5.5 Constant-temperature air bath shaker.

5.6 Centrifuge: Rotary speed >= 3000 rpm.

5.7 Nitrogen evaporator.

6 Analytical procedure