GB 5009.84-2016/XG1-2023 National Food Safety Standard - Determination of Vitamin B1 in Foods, inlcudes Amendment 1
1 Scope
This standard specifies the methods of high performance liquid chromatography and fluorophotometry for determining Vitamin B1 in foods.
This standard is applicable to the determination of Vitamin B1 in foods.
Method I High Performance Liquid Chromatography
2 Principle
Hydrolyze and neutralize the specimen in the medium of diluted hydrochloric acid, carry out enzymolysis for the specimen, derive the hydrolysate with alkaline potassium ferricyanide solution, extract with n-butyl alcohol, separate with C18 reversed-phase chromatographic column, inspect with high performance liquid chromatography-fluorescence detector, and quantify by using external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 n-butyl alcohol (CH3CH2CH2CH2OH).
3.1.2 Potassium ferricyanide [K3Fe(CN)6].
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Hydrochloric acid (HCl).
3.1.5 Sodium acetate (CH3COONa·3H2O).
3.1.6 Glacial acetic acid (CH3COOH).
3.1.7 Methanol (CH3OH): Chromatographically pure.
3.1.8 Phosphorus pentoxide (P2O5) or calcium chloride (CaCl2).
3.1.9 Papain: shall not include Vitamin B1, enzyme activity ≥800 U (activity unit)/mg.
3.1.10 Amylase: shall not include Vitamin B1, enzyme activity ≥3,700 U/g.
3.2 Preparation of reagents
3.2.1 Potassium ferricyanide solution (20g/L): weigh 2g of potassium ferricyanide, dissolve it and scale the volume to 100mL with water, and shake well; prepare this standard solution immediately before use.
3.2.2 Sodium hydroxide solution (100g/L): weigh 25g of sodium hydroxide, dissolve it and scale the volume to 250mL with water, and shake well.
3.2.3 Alkaline potassium ferricyanide solution: mix 5mL of potassium ferricyanide solution and 200 mL of sodium hydroxide solution, and shake well. It shall be prepared immediately before use.
3.2.4 Hydrochloric acid solution (0.1mol/L): transfer 8.5 mL of hydrochloric acid, dilute it to 1,000mL with water, and shake well.
3.2.5 Hydrochloric acid solution (0.01mol/L): take 50mL of hydrochloric acid solution (0.1mol/L), dilute and scale the volume to 500mL with water, and shake well.
3.2.6 Sodium acetate solution (0.05mol/L): weigh 6.80g of sodium acetate, add 900mL of water to dissolve it, adjust the pH value to 4.0~5.0 with glacial acetic acid, add water to scale the volume to 1,000mL; filter the solution with 0.45 μm microfiltration membrane before use.
3.2.7 Sodium acetate solution (2.0mol/L): weigh 27.2g of sodium acetate, dissolve it and scale the volume to 100mL with water, and shake well.
3.2.8 Mixed enzyme solution: weigh 1.76g of papain and 1.27g of amylase, add water to scale the volume to 50mL, eddy so that the solution becomes suspending liquid, and preserve it in cold environment; shake it well immediately before use.
3.3 Standard product
Vitamin B1 standard product: thiamine hydrochloride (C12H17ClN4OS·HCl), CAS: 67-03-8, purity ≥99.0%.
3.4 Preparation of standard solution
3.4.1 Vitamin B1 standard stock solution (500μg/mL): accurately weigh 56.1mg of thiamine hydrochloride standard product (to the nearest of 0.1mg) which has been dried for 24h with phosphorus pentoxide or calcium chloride, which is equivalent to 50 mg of thiamin; dissolve it with 0.01 mol/L hydrochloric acid solution and scale the volume to 100mL, and shake well. Place it in 0℃~4℃ refrigerator with the retention period of 3 months.
3.4.2 Vitamin B1 standard intermediate solution (10.0μg/mL): accurately transfer 2.00mL of standard stock solution, dilute it and scale the volume to 100mL with water, and shake well; prepare immediately before use.
3.4.3 Vitamin B1 standard series working solution: pipet 0μL, 50.0μL, 100μL, 200μL, 400μL, 800μL and 1,000μL of vitamin B1 standard intermediate solutions, scale the volume to 10 mL with water, and the concentrations of Vitamin B1 in the standard series working solutions are 0μg/mL, 0.0500μg/mL, 0.100μg/mL, 0.200μg/mL, 0.400μg/mL, 0.800μg/mL and 1.00μg/mL respectively. Prepare it immediately before use.
4 Instruments and Apparatus
4.1 High-performance liquid chromatograph, equipped with fluorescence detector.
4.2 Analytical balance: with sensibility of 0.01g and 0.1mg.
4.3 Centrifuge: rotation speed ≥4,000 r/min.
4.4 pH meter: with a precision of 0.01.
4.5 Tissue blender (maximum rotation speed not less than 10,000r/min).
4.6 Electrothermal constant-temperature dry oven or autoclave.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Precision
8 Others
9 Principle
10 Reagents and Materials
11 Instruments and Apparatus
12 Analysis Steps
13 Expression of Analysis Results
14 Precision
15 Others
Appendix A High Performance Liquid Chromatogram of Vitamin B1 Standard Derivative
GB 5009.84-2016/XG1-2023 National Food Safety Standard - Determination of Vitamin B1 in Foods, inlcudes Amendment 1
1 Scope
This standard specifies the methods of high performance liquid chromatography and fluorophotometry for determining Vitamin B1 in foods.
This standard is applicable to the determination of Vitamin B1 in foods.
Method I High Performance Liquid Chromatography
2 Principle
Hydrolyze and neutralize the specimen in the medium of diluted hydrochloric acid, carry out enzymolysis for the specimen, derive the hydrolysate with alkaline potassium ferricyanide solution, extract with n-butyl alcohol, separate with C18 reversed-phase chromatographic column, inspect with high performance liquid chromatography-fluorescence detector, and quantify by using external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 n-butyl alcohol (CH3CH2CH2CH2OH).
3.1.2 Potassium ferricyanide [K3Fe(CN)6].
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Hydrochloric acid (HCl).
3.1.5 Sodium acetate (CH3COONa·3H2O).
3.1.6 Glacial acetic acid (CH3COOH).
3.1.7 Methanol (CH3OH): Chromatographically pure.
3.1.8 Phosphorus pentoxide (P2O5) or calcium chloride (CaCl2).
3.1.9 Papain: shall not include Vitamin B1, enzyme activity ≥800 U (activity unit)/mg.
3.1.10 Amylase: shall not include Vitamin B1, enzyme activity ≥3,700 U/g.
3.2 Preparation of reagents
3.2.1 Potassium ferricyanide solution (20g/L): weigh 2g of potassium ferricyanide, dissolve it and scale the volume to 100mL with water, and shake well; prepare this standard solution immediately before use.
3.2.2 Sodium hydroxide solution (100g/L): weigh 25g of sodium hydroxide, dissolve it and scale the volume to 250mL with water, and shake well.
3.2.3 Alkaline potassium ferricyanide solution: mix 5mL of potassium ferricyanide solution and 200 mL of sodium hydroxide solution, and shake well. It shall be prepared immediately before use.
3.2.4 Hydrochloric acid solution (0.1mol/L): transfer 8.5 mL of hydrochloric acid, dilute it to 1,000mL with water, and shake well.
3.2.5 Hydrochloric acid solution (0.01mol/L): take 50mL of hydrochloric acid solution (0.1mol/L), dilute and scale the volume to 500mL with water, and shake well.
3.2.6 Sodium acetate solution (0.05mol/L): weigh 6.80g of sodium acetate, add 900mL of water to dissolve it, adjust the pH value to 4.0~5.0 with glacial acetic acid, add water to scale the volume to 1,000mL; filter the solution with 0.45 μm microfiltration membrane before use.
3.2.7 Sodium acetate solution (2.0mol/L): weigh 27.2g of sodium acetate, dissolve it and scale the volume to 100mL with water, and shake well.
3.2.8 Mixed enzyme solution: weigh 1.76g of papain and 1.27g of amylase, add water to scale the volume to 50mL, eddy so that the solution becomes suspending liquid, and preserve it in cold environment; shake it well immediately before use.
3.3 Standard product
Vitamin B1 standard product: thiamine hydrochloride (C12H17ClN4OS·HCl), CAS: 67-03-8, purity ≥99.0%.
3.4 Preparation of standard solution
3.4.1 Vitamin B1 standard stock solution (500μg/mL): accurately weigh 56.1mg of thiamine hydrochloride standard product (to the nearest of 0.1mg) which has been dried for 24h with phosphorus pentoxide or calcium chloride, which is equivalent to 50 mg of thiamin; dissolve it with 0.01 mol/L hydrochloric acid solution and scale the volume to 100mL, and shake well. Place it in 0℃~4℃ refrigerator with the retention period of 3 months.
3.4.2 Vitamin B1 standard intermediate solution (10.0μg/mL): accurately transfer 2.00mL of standard stock solution, dilute it and scale the volume to 100mL with water, and shake well; prepare immediately before use.
3.4.3 Vitamin B1 standard series working solution: pipet 0μL, 50.0μL, 100μL, 200μL, 400μL, 800μL and 1,000μL of vitamin B1 standard intermediate solutions, scale the volume to 10 mL with water, and the concentrations of Vitamin B1 in the standard series working solutions are 0μg/mL, 0.0500μg/mL, 0.100μg/mL, 0.200μg/mL, 0.400μg/mL, 0.800μg/mL and 1.00μg/mL respectively. Prepare it immediately before use.
4 Instruments and Apparatus
4.1 High-performance liquid chromatograph, equipped with fluorescence detector.
4.2 Analytical balance: with sensibility of 0.01g and 0.1mg.
4.3 Centrifuge: rotation speed ≥4,000 r/min.
4.4 pH meter: with a precision of 0.01.
4.5 Tissue blender (maximum rotation speed not less than 10,000r/min).
4.6 Electrothermal constant-temperature dry oven or autoclave.
Contents of GB 5009.84-2016/XG1-2023
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Precision
8 Others
9 Principle
10 Reagents and Materials
11 Instruments and Apparatus
12 Analysis Steps
13 Expression of Analysis Results
14 Precision
15 Others
Appendix A High Performance Liquid Chromatogram of Vitamin B1 Standard Derivative
