This standard supersedes Determination of Thiamine (Vitamin B1) in Foods (GB/T 5009.84-2003), National Food Safety Standard - Determination of Vitamin B1 in Foods for Infants and Young Children, Milk and Milk Products (GB 5413.11-2010), Determination of Vitamin B1 in Cereals (GB/T 7628-2008) and Meat and Meat Products - Determination of Vitamin B1 Content (GB/T 9695.27-2008).
Compared with GB/T 5009.84-2003, the main changes in this standard are as follows:
——the standard name is revised as "National Food Safety Standard - Determination of Vitamin B1 in Foods";
——the high performance liquid chromatography is added as Method I, and the fluorophotometry is Method II;
——the detection limit expression is modified, and quantitation limit for methods is added;
——the qualitative identification method for chlorine ion in permutite pretreatment is added;
——the solution color change characteristic when bromocresol green is taken as indicator is added;
——the structure drawings of Figure 1 (reaction bulb) and Figure 2 (base exchange column) are cancelled;
——the weighing mass of permutite expressed by wet weight is added.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中华人民共和国国家标准
GB 5009.84-2016
National Standard of Food Safety
Determination of Vitamin B1 in Foods
食品安全国家标准
食品中维生素B1的测定
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.
5 Analysis Steps
5.1 Specimen preparation
5.1.1 Liquid or solid powder specimen: mix the specimens uniformly, and immediately determine or put them in refrigerator for preservation.
5.1.2 Fresh fruits, vegetables and meat: take about 500g of specimen (for meat, 250g), make homogenate with consistent uniformity by using refiner or grinder, and immediately determine or put the homogenate in refrigerator for preservation.
5.1.3 Other solid specimens with low water content: in case of cereal with water content of about 15 %, take about 100g of specimen, mill it with grinder so as to obtain powder with consistent uniformity, and immediately determine or put the powder in refrigerator for preservation.
5.2 Specimen solution preparation
5.2.1 Test solution extraction
Weigh 3g~5g (to the nearest of 0.01g) of solid specimen or 10g~20g of liquid specimen and put it in a 100mL conical flask (with soft plug), add 60mL of 0.1mol/L hydrochloric acid solution, shake it well, cover the soft plug and put it in 121℃ autoclave for 30min. After hydrolysis, cool it down to a temperature below 40℃, take it out, and shake it for several times; adjust the pH value to about 4.0 with 2.0mol/L sodium acetate solution by using PH meter, add 2.0 mL (the amount may be adjusted properly according to enzyme activity) of mixed enzyme solution, shake it well, and put it in 37 ℃ incubator for a night (about 16h); transfer all the enzymatic hydrolysis solution into 100mL volumetric flask, dilute it with water to the scale, shake it well, centrifuge or filter it, and take the supernatant for standby.
5.2.2 Derivatization of test solution
Accurately transfer 2.0 mL of the above-mentioned supernatant or filtrate and pour it into a 10mL test tube, add 1.0 mL of alkaline potassium ferricyanide solution, eddy to mix it uniformly, and accurately add 2.0 mL of n-butyl alcohol; eddy again for 1.5min, keep it still for about 10 min or centrifuge it, after layering sufficiently, pipet n-butyl alcohol phase (upper-layer) and filter it with 0.45 μm organic microfiltration membrane, and pour the filtrate into a 2mL brown specimen-injecting bottle for analysis. In case the concentration of Vitamin B1 in test solution exceeds the maximum concentration within the linear range, dilute it by a proper factor with supernatant, carry out derivation again and then carry out specimen injection.
Take another 2.0 mL of standard series working solution and carry out derivatization synchronously with the test solution.
Notes:
1 The derived product shall be stable within 4h at room temperature.
2 The operation procedures of 5.2.1 and 5.2.2 shall be conducted in an environment without highlighting.
3 As for specimens like dried chilli, if determine the extraction solution directly derived, the recovery of Vitamin B1 is low. After the extraction solution is purified with permutite, the recovery of Vitamin B1 in derivation meets the requirements. Therefore, as for special specimen, in case the recovery is low, specimen extraction solution shall be derived after purification; see 12.1.3 in Method II for the specific operation steps.
5.3 Reference conditions of apparatus
5.3.1 Chromatographic column: C18 reversed-phase chromatographic column (with the particle size of 5μm, 250mm×4.6mm) or equivalent.
5.3.2 Mobile phase: 0.05mol/L sodium acetate solution - methanol (65+35).
5.3.3 Flow velocity: 0.8 mL/min.
5.3.4 Detection wavelength: excitation wavelength is 375nm, and emission wavelength is 435nm.
5.3.5 Injection volume: 20μL.
5.4 Standard curve drawing
Inject the derivative of standard series working solution into high-performance liquid chromatograph, determine the corresponding Vitamin B1 peak area, and draw the standard curve by taking the concentration of standard working solution (μg/mL) as horizontal coordinate and peak area as longitudinal coordinate.
5.5 Determination of specimen solution
Inject the specimen derivative solution into high-performance liquid chromatograph according to the chromatographic conditions specified in 5.3 so as to obtain the peak area of Vitamin B1, and calculate the concentration of vitamin in the obtained to-be-determined solution according to standard curve.
Contents
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 Standard Derivative
This standard supersedes Determination of Thiamine (Vitamin B1) in Foods (GB/T 5009.84-2003), National Food Safety Standard - Determination of Vitamin B1 in Foods for Infants and Young Children, Milk and Milk Products (GB 5413.11-2010), Determination of Vitamin B1 in Cereals (GB/T 7628-2008) and Meat and Meat Products - Determination of Vitamin B1 Content (GB/T 9695.27-2008).
Compared with GB/T 5009.84-2003, the main changes in this standard are as follows:
——the standard name is revised as "National Food Safety Standard - Determination of Vitamin B1 in Foods";
——the high performance liquid chromatography is added as Method I, and the fluorophotometry is Method II;
——the detection limit expression is modified, and quantitation limit for methods is added;
——the qualitative identification method for chlorine ion in permutite pretreatment is added;
——the solution color change characteristic when bromocresol green is taken as indicator is added;
——the structure drawings of Figure 1 (reaction bulb) and Figure 2 (base exchange column) are cancelled;
——the weighing mass of permutite expressed by wet weight is added.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中华人民共和国国家标准
GB 5009.84-2016
National Standard of Food Safety
Determination of Vitamin B1 in Foods
食品安全国家标准
食品中维生素B1的测定
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.
5 Analysis Steps
5.1 Specimen preparation
5.1.1 Liquid or solid powder specimen: mix the specimens uniformly, and immediately determine or put them in refrigerator for preservation.
5.1.2 Fresh fruits, vegetables and meat: take about 500g of specimen (for meat, 250g), make homogenate with consistent uniformity by using refiner or grinder, and immediately determine or put the homogenate in refrigerator for preservation.
5.1.3 Other solid specimens with low water content: in case of cereal with water content of about 15 %, take about 100g of specimen, mill it with grinder so as to obtain powder with consistent uniformity, and immediately determine or put the powder in refrigerator for preservation.
5.2 Specimen solution preparation
5.2.1 Test solution extraction
Weigh 3g~5g (to the nearest of 0.01g) of solid specimen or 10g~20g of liquid specimen and put it in a 100mL conical flask (with soft plug), add 60mL of 0.1mol/L hydrochloric acid solution, shake it well, cover the soft plug and put it in 121℃ autoclave for 30min. After hydrolysis, cool it down to a temperature below 40℃, take it out, and shake it for several times; adjust the pH value to about 4.0 with 2.0mol/L sodium acetate solution by using PH meter, add 2.0 mL (the amount may be adjusted properly according to enzyme activity) of mixed enzyme solution, shake it well, and put it in 37 ℃ incubator for a night (about 16h); transfer all the enzymatic hydrolysis solution into 100mL volumetric flask, dilute it with water to the scale, shake it well, centrifuge or filter it, and take the supernatant for standby.
5.2.2 Derivatization of test solution
Accurately transfer 2.0 mL of the above-mentioned supernatant or filtrate and pour it into a 10mL test tube, add 1.0 mL of alkaline potassium ferricyanide solution, eddy to mix it uniformly, and accurately add 2.0 mL of n-butyl alcohol; eddy again for 1.5min, keep it still for about 10 min or centrifuge it, after layering sufficiently, pipet n-butyl alcohol phase (upper-layer) and filter it with 0.45 μm organic microfiltration membrane, and pour the filtrate into a 2mL brown specimen-injecting bottle for analysis. In case the concentration of Vitamin B1 in test solution exceeds the maximum concentration within the linear range, dilute it by a proper factor with supernatant, carry out derivation again and then carry out specimen injection.
Take another 2.0 mL of standard series working solution and carry out derivatization synchronously with the test solution.
Notes:
1 The derived product shall be stable within 4h at room temperature.
2 The operation procedures of 5.2.1 and 5.2.2 shall be conducted in an environment without highlighting.
3 As for specimens like dried chilli, if determine the extraction solution directly derived, the recovery of Vitamin B1 is low. After the extraction solution is purified with permutite, the recovery of Vitamin B1 in derivation meets the requirements. Therefore, as for special specimen, in case the recovery is low, specimen extraction solution shall be derived after purification; see 12.1.3 in Method II for the specific operation steps.
5.3 Reference conditions of apparatus
5.3.1 Chromatographic column: C18 reversed-phase chromatographic column (with the particle size of 5μm, 250mm×4.6mm) or equivalent.
5.3.2 Mobile phase: 0.05mol/L sodium acetate solution - methanol (65+35).
5.3.3 Flow velocity: 0.8 mL/min.
5.3.4 Detection wavelength: excitation wavelength is 375nm, and emission wavelength is 435nm.
5.3.5 Injection volume: 20μL.
5.4 Standard curve drawing
Inject the derivative of standard series working solution into high-performance liquid chromatograph, determine the corresponding Vitamin B1 peak area, and draw the standard curve by taking the concentration of standard working solution (μg/mL) as horizontal coordinate and peak area as longitudinal coordinate.
5.5 Determination of specimen solution
Inject the specimen derivative solution into high-performance liquid chromatograph according to the chromatographic conditions specified in 5.3 so as to obtain the peak area of Vitamin B1, and calculate the concentration of vitamin in the obtained to-be-determined solution according to standard curve.
Contents of GB 5009.84-2016
Contents
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 Standard Derivative
