National food safety standard - Determination of vitamin K2 in foods
1 Scope
This standard specifies the liquid chromatographic method for determining menaquinone-4 (MK-4), menaquinone-7 (MK-7) and menaquinone-9 (MK-9) in foods.
This standard is applicable to the determination of three types of vitamin K2, i.e., MK-4, MK-7 and MK-9, in milk and dairy products, foods for special dietary uses, fermented bean products, meat and meat products.
2 Principle
The specimen is enzymolyzed by lipase, amylase or protease, and MK-4, MK-7 and MK-9 are extracted by n-hexane. The extracts are concentrated and separated by reversed-phase liquid chromatography, derivatized by zinc reduction column, detected by fluorescence detector, and quantified by external standard method.
3 Reagents and materials
Unless otherwise specified, analytically-pure reagents and Class-I water (defined in GB/T 6682) are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 Anhydrous ethanol (C2H6O).
3.1.3 n-hexane (C6H14).
3.1.4 Potassium carbonate (K2CO3).
3.1.5 Dichloromethane (CH2Cl2): chromatographically pure.
3.1.6 Tetrahydrofuran (C4H8O): chromatographically pure.
3.1.7 Glacial acetic acid (C2H4O2).
3.1.8 Zinc chloride (ZnCl2).
3.1.9 Anhydrous sodium acetate (C2H3O2Na).
3.1.10 Potassium dihydrogen phosphate (KH2PO4).
3.1.11 Amylase: (CAS No.: 9000-92-4) enzyme activity ≥ 4,000 U/g.
3.1.12 Lipase: (CAS No.: 9001-62-1) enzyme activity ≥ 40 U/g.
3.1.13 Protease: (CAS No.: 9001-73-4) enzyme activity ≥ 6,000 U/mg.
3.1.14 Potassium hydroxide (KOH).
3.2 Reagent preparation
3.2.1 400 g/L potassium hydroxide solution: Weigh 20.0 g of potassium hydroxide into a 100 mL beaker, dissolve it in 20 mL of water, cool it, transfer it to a 50 mL volumetric flask, add water to dilute it to the scale, mix it well, and store it in a polyethylene bottle.
3.2.2 Phosphate buffer solution (pH 8.0): Weigh 54.0 g of potassium dihydrogen phosphate into a 500 mL beaker, dissolve it in 300 mL of water, adjust the pH to 8.0 ± 0.2 with 400 g/L potassium hydroxide solution (3.2.1), transfer it to a 500 mL volumetric flask, add water to dilute it to the scale, and mix it well.
3.2.3 Mobile phase: Weigh 1.5 g of zinc chloride and 0.5 g of anhydrous sodium acetate into a 1,000 mL beaker, add 500 mL of methanol, 100 mL of dichloromethane (or tetrahydrofuran), and 0.3 mL of glacial acetic acid, transfer it to a 1,000 mL volumetric flask, dissolve it by ultrasound, add methanol to dilute it to the scale, mix it well, and filter it through a 0.22 μm filter membrane.
Note: The retention time may be shortened by adding dichloromethane, which should not exceed 150 mL/L at maximum.
3.2.4 Dichloromethane-methanol solution (by volume ratio of 10 + 90): Accurately pipette 20 mL of dichloromethane into a 200 mL volumetric flask, dilute it with methanol to the scale, and mix it well.
3.3 Standards
3.3.1 MK-4 standard (C31H40O2, CAS No. 863-61-6): standard substance with a purity of more than or equal to 98% or certified by the state with a certificate of standard substance.
3.3.2 MK-7 standard (C46H64O2, CAS No. 2124-57-4): standard substance with a purity of more than or equal to 98% or certified by the state with a certificate of standard substance.
3.3.3 MK-9 standard (C56H80O2, CAS No. 523-39-7): standard substance with a purity of more than or equal to 98% or certified by the state with a certificate of standard substance.
National food safety standard - Determination of vitamin K2 in foods
1 Scope
This standard specifies the liquid chromatographic method for determining menaquinone-4 (MK-4), menaquinone-7 (MK-7) and menaquinone-9 (MK-9) in foods.
This standard is applicable to the determination of three types of vitamin K2, i.e., MK-4, MK-7 and MK-9, in milk and dairy products, foods for special dietary uses, fermented bean products, meat and meat products.
2 Principle
The specimen is enzymolyzed by lipase, amylase or protease, and MK-4, MK-7 and MK-9 are extracted by n-hexane. The extracts are concentrated and separated by reversed-phase liquid chromatography, derivatized by zinc reduction column, detected by fluorescence detector, and quantified by external standard method.
3 Reagents and materials
Unless otherwise specified, analytically-pure reagents and Class-I water (defined in GB/T 6682) are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 Anhydrous ethanol (C2H6O).
3.1.3 n-hexane (C6H14).
3.1.4 Potassium carbonate (K2CO3).
3.1.5 Dichloromethane (CH2Cl2): chromatographically pure.
3.1.6 Tetrahydrofuran (C4H8O): chromatographically pure.
3.1.7 Glacial acetic acid (C2H4O2).
3.1.8 Zinc chloride (ZnCl2).
3.1.9 Anhydrous sodium acetate (C2H3O2Na).
3.1.10 Potassium dihydrogen phosphate (KH2PO4).
3.1.11 Amylase: (CAS No.: 9000-92-4) enzyme activity ≥ 4,000 U/g.
3.1.12 Lipase: (CAS No.: 9001-62-1) enzyme activity ≥ 40 U/g.
3.1.13 Protease: (CAS No.: 9001-73-4) enzyme activity ≥ 6,000 U/mg.
3.1.14 Potassium hydroxide (KOH).
3.2 Reagent preparation
3.2.1 400 g/L potassium hydroxide solution: Weigh 20.0 g of potassium hydroxide into a 100 mL beaker, dissolve it in 20 mL of water, cool it, transfer it to a 50 mL volumetric flask, add water to dilute it to the scale, mix it well, and store it in a polyethylene bottle.
3.2.2 Phosphate buffer solution (pH 8.0): Weigh 54.0 g of potassium dihydrogen phosphate into a 500 mL beaker, dissolve it in 300 mL of water, adjust the pH to 8.0 ± 0.2 with 400 g/L potassium hydroxide solution (3.2.1), transfer it to a 500 mL volumetric flask, add water to dilute it to the scale, and mix it well.
3.2.3 Mobile phase: Weigh 1.5 g of zinc chloride and 0.5 g of anhydrous sodium acetate into a 1,000 mL beaker, add 500 mL of methanol, 100 mL of dichloromethane (or tetrahydrofuran), and 0.3 mL of glacial acetic acid, transfer it to a 1,000 mL volumetric flask, dissolve it by ultrasound, add methanol to dilute it to the scale, mix it well, and filter it through a 0.22 μm filter membrane.
Note: The retention time may be shortened by adding dichloromethane, which should not exceed 150 mL/L at maximum.
3.2.4 Dichloromethane-methanol solution (by volume ratio of 10 + 90): Accurately pipette 20 mL of dichloromethane into a 200 mL volumetric flask, dilute it with methanol to the scale, and mix it well.
3.3 Standards
3.3.1 MK-4 standard (C31H40O2, CAS No. 863-61-6): standard substance with a purity of more than or equal to 98% or certified by the state with a certificate of standard substance.
3.3.2 MK-7 standard (C46H64O2, CAS No. 2124-57-4): standard substance with a purity of more than or equal to 98% or certified by the state with a certificate of standard substance.
3.3.3 MK-9 standard (C56H80O2, CAS No. 523-39-7): standard substance with a purity of more than or equal to 98% or certified by the state with a certificate of standard substance.
