This standard specifies the determination methods of Vitamin B2 in foods.
Method I (High Performance Liquid Chromatography) and Method II (Fluorescence Spectrophotometry) of this standard are applicable to the determination of Vitamin B2 in various foods.
Method I High Performance Liquid Chromatography
2 Principle
Specimen is hydrolyzed at constant temperature in diluted hydrochloric acid environment, pH is adjusted to 6.0~6.5, enzymolysis is carried out with papain and takadiastase, after being brought to volume and filtered, the filtrate is separated by reversed-phase chromatographic column, detected with high performance liquid chromatography fluorescence detector and quantitated by 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 Hydrochloric acid (HCl).
3.1.2 Glacial acetic acid (CH3COOH).
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Sodium acetate trihydrate (CH3COONa· 3H2O).
3.1.5 Methanol (CH3OH): chromatographically pure.
3.1.6 Papain: activity unit≥10U/mg.
3.1.7 Takadiastase: activity unit≥100U/mg, or that with equivalent performance.
3.2 Reagent preparation
3.2.1 Hydrochloric acid solution (0.1mol/L): pipet 9mL of hydrochloric acid, dilute and bring the volume to 1 000mL with water.
3.2.2 Hydrochloric acid solution (1+1): pour 100mL of hydrochloric acid into 100mL of water slowly and mix well.
3.2.3 Sodium hydroxide solution (1mol/L): accurately weigh 4g of sodium hydroxide, dissolve it with 90mL of water and bring the volume to 100mL after cooling.
3.2.4 Sodium acetate solution (0.1mol/L): accurately weigh 13.60g of sodium acetate trihydrate, dissolve it with 900mL of water and bring the volume with water to 1 000mL.
3.2.5 Sodium acetate solution (0.05mol/L): accurately weigh 6.80g of sodium acetate trihydrate, dissolve it with 900mL of water, adjust the pH value to 4.0~5.0 with glacial acetic acid and bring the volume with water to 1 000mL.
3.2.6 Mixed enzyme solution: accurately weigh 2.345g of papain and 1.175g of takadiastase, dissolve them with water and bring the volume to 50mL. Prepare this solution immediately before use.
3.2.7 Hydrochloric acid solution (0.012mol/L): pipet 1mL of hydrochloric acid, dissolve it with water and bring the volume to 1 000mL.
3.3 Standard
Vitamin B2 (C17H20N4O6, CAS No.: 83-88-5): purity ≥98%.
3.4 Preparation of standard solutions
3.4.1 Vitamin B2 standard stock solution (100μg/mL): place Vitamin B2 standard into vacuum drier or the drier containing phosphorus pentoxide for drying treatment for 24h, accurately weigh 10mg (accurate to 0.1mg) of Vitamin B2 standard, add 2mL of hydrochloric acid solution (1+1), subject to ultrasonic dissolving, then transfer it with water and bring the volume to 100mL. Transfer the well mixed solution into a brown glass vessel, store it in 4℃ refrigerator for a storage time of two months. Carry out concentration calibration before using the standard stock solution and refer to Appendix A for the calibration method.
3.4.2 Vitamin B2 standard intermediate solution (2.00μg/mL): accurately pipet 2.00mL of Vitamin B2 standard stock solution, dilute it and scale the volume to 100mL with water. Prepare this solution immediately before use.
3.4.3 Standard series working solution of Vitamin B2: pipet 0.25mL, 0.50mL, 1.00mL, 2.50mL and 5.00mL of Vitamin B2 standard intermediate solution respectively, bring the volumes to 10mL; the concentration of this standard series is 0.05μg/mL, 0.10μg/mL, 0.20μg/mL, 0.50μg/mL and 1.00μg/mL respectively. Prepare these solutions immediately before use.
4 Apparatuses
4.1 High-performance liquid chromatograph: equipped with fluorescence detector.
4.2 Balance: with sensibility of 1mg and 0.01mg.
4.3 Autoclave.
4.4 pH meter: with an accuracy of 0.01.
4.5 Vortex oscillator.
4.6 Tissue blender.
4.7 Thermostat water bath cauldron.
4.8 Drier.
4.9 Spectrophotometer.
5 Analysis Procedures
5.1 Specimen preparation
Take about 500g of sample, smash and homogenize it sufficiently with tissue blender, and put into clean brown flask with ground mouth, seal the flask and make marking properly, then store it in a dark place for future use.
Weigh 2g~10g (accurate to 0.01g) of homogenized specimen (the Vitamin B2 content in the specimen is greater than 5μg), put it into a 100mL conical flask with stopper, add into 60mL of 0.1mol/L hydrochloric acid solution, shake well and cover the flask stopper. Place the conical flask into autoclave for 30min at 121℃, cool to room temperature and take it out. Adjust the pH value with 1mol/L sodium hydroxide solution to 6.0~6.5, add 2mL of mixed enzyme solution, shake well and place the solution in 37℃ incubator or thermostat water bath cauldron for enzymolysis for the night. Transfer the enzymatic hydrolysis solution into a 100mL volumetric flask, bring the volume to the scale by adding water, filter with filter paper or centrifuge for separation, take filtrate or supernatant to pass 0.45μm water phase filter membrane to serve as to-be-determined solution.
Note: The operation shall be protected against bright light.
Carry out blank test according to the same operating method except for adding specimen.
5.2 Reference conditions of apparatus
a) Chromatographic column: C18 column (150mm in column length, 4.6mm in inner diameter and 5μm in particle size of filler) or equivalent;
b) Mobile phase: sodium acetate solution (0.05mol/L)-methanol (65:35);
c) Flow rate: 1mL/min;
d) Column temperature: 30℃;
e) Detection wavelength: 462nm in excitation wavelength and 522nm in emission wavelength;
f) Injection volume: 20μl.
5.3 Plotting of standard curve
Inject the standard series working solution into high-performance liquid chromatograph respectively, determine the corresponding peak areas, and plot the standard curve by taking the concentration of standard working solution as horizontal coordinate and peak area as longitudinal coordinate.
5.4 Determination of specimen solution
Inject the specimen solution into high-performance liquid chromatograph to obtain the corresponding peak area, and obtain the concentration of Vitamin B2 in to-be-determined solution according to standard curve.
5.5 Blank test requirements
There shall not be to-be-determined component peak or other interference peaks in the chromatogram of blank test solution.
6 Expression of Analysis Results
The content of Vitamin B2 in the specimen is calculated according to Formula (1):
(1)
Where,
X - the content of Vitamin B2 (counted by riboflavin) in the specimen, mg/100g;
ρ - the concentration of Vitamin B2, calculated according to standard curve, in the specimen, μg/mL;
V - the final constant volume of specimen solution, mL;
m - the specimen mass, g;
100 - the conversion coefficient for converting to the content in 100g of sample;
1 000- the conversion coefficient for converting the concentration unit from μg/mL to mg/mL.
Three significant figures are reserved for the result.
7 Accuracy
The absolute difference of the results from two independent determinations under repeatability condition shall not exceed 10% of the arithmetic mean value.
8 Others
Provided that the sampling weight is 10.00g, the method detection limit is 0.02mg/100g and the quantitation limit is 0.05mg/100g.
Method II Fluorescence Spectrophotometry
9 Principle
Vitamin B2 generates yellow-green fluorescence under the irradiation of light of 440nm~500nm wavelength. Its fluorescence intensity is directly proportional to the concentration of Vitamin B2 in dilute solution. Its fluorescence intensity is determined under 525nm wavelength. Then sodium hydrosulfite is added into test solution to resolve Vitamin B2 into non-fluorescence material and the fluorescence intensity of residual fluorescence impurities in test solution is determined, the difference between fluorescence intensity of the initial determination and that of residual fluorescence impurities is the fluorescence intensity generated by Vitamin B2 in the specimen.
Contents Foreword i 1 Scope 2 Principle 3 Reagents and Materials 4 Apparatuses 5 Analysis Procedures 6 Expression of Analysis Results 7 Accuracy 8 Others 9 Principle 10 Reagents and Materials 11 Apparatuses 12 Analysis Procedures 13 Expression of Analysis Results 14 Accuracy 15 Others Appendix A Calibration Method for the Concentration of Vitamin B2 Standard Solution Appendix B Liquid Chromatogram of Vitamin B
1 Scope
This standard specifies the determination methods of Vitamin B2 in foods.
Method I (High Performance Liquid Chromatography) and Method II (Fluorescence Spectrophotometry) of this standard are applicable to the determination of Vitamin B2 in various foods.
Method I High Performance Liquid Chromatography
2 Principle
Specimen is hydrolyzed at constant temperature in diluted hydrochloric acid environment, pH is adjusted to 6.0~6.5, enzymolysis is carried out with papain and takadiastase, after being brought to volume and filtered, the filtrate is separated by reversed-phase chromatographic column, detected with high performance liquid chromatography fluorescence detector and quantitated by 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 Hydrochloric acid (HCl).
3.1.2 Glacial acetic acid (CH3COOH).
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Sodium acetate trihydrate (CH3COONa· 3H2O).
3.1.5 Methanol (CH3OH): chromatographically pure.
3.1.6 Papain: activity unit≥10U/mg.
3.1.7 Takadiastase: activity unit≥100U/mg, or that with equivalent performance.
3.2 Reagent preparation
3.2.1 Hydrochloric acid solution (0.1mol/L): pipet 9mL of hydrochloric acid, dilute and bring the volume to 1 000mL with water.
3.2.2 Hydrochloric acid solution (1+1): pour 100mL of hydrochloric acid into 100mL of water slowly and mix well.
3.2.3 Sodium hydroxide solution (1mol/L): accurately weigh 4g of sodium hydroxide, dissolve it with 90mL of water and bring the volume to 100mL after cooling.
3.2.4 Sodium acetate solution (0.1mol/L): accurately weigh 13.60g of sodium acetate trihydrate, dissolve it with 900mL of water and bring the volume with water to 1 000mL.
3.2.5 Sodium acetate solution (0.05mol/L): accurately weigh 6.80g of sodium acetate trihydrate, dissolve it with 900mL of water, adjust the pH value to 4.0~5.0 with glacial acetic acid and bring the volume with water to 1 000mL.
3.2.6 Mixed enzyme solution: accurately weigh 2.345g of papain and 1.175g of takadiastase, dissolve them with water and bring the volume to 50mL. Prepare this solution immediately before use.
3.2.7 Hydrochloric acid solution (0.012mol/L): pipet 1mL of hydrochloric acid, dissolve it with water and bring the volume to 1 000mL.
3.3 Standard
Vitamin B2 (C17H20N4O6, CAS No.: 83-88-5): purity ≥98%.
3.4 Preparation of standard solutions
3.4.1 Vitamin B2 standard stock solution (100μg/mL): place Vitamin B2 standard into vacuum drier or the drier containing phosphorus pentoxide for drying treatment for 24h, accurately weigh 10mg (accurate to 0.1mg) of Vitamin B2 standard, add 2mL of hydrochloric acid solution (1+1), subject to ultrasonic dissolving, then transfer it with water and bring the volume to 100mL. Transfer the well mixed solution into a brown glass vessel, store it in 4℃ refrigerator for a storage time of two months. Carry out concentration calibration before using the standard stock solution and refer to Appendix A for the calibration method.
3.4.2 Vitamin B2 standard intermediate solution (2.00μg/mL): accurately pipet 2.00mL of Vitamin B2 standard stock solution, dilute it and scale the volume to 100mL with water. Prepare this solution immediately before use.
3.4.3 Standard series working solution of Vitamin B2: pipet 0.25mL, 0.50mL, 1.00mL, 2.50mL and 5.00mL of Vitamin B2 standard intermediate solution respectively, bring the volumes to 10mL; the concentration of this standard series is 0.05μg/mL, 0.10μg/mL, 0.20μg/mL, 0.50μg/mL and 1.00μg/mL respectively. Prepare these solutions immediately before use.
4 Apparatuses
4.1 High-performance liquid chromatograph: equipped with fluorescence detector.
4.2 Balance: with sensibility of 1mg and 0.01mg.
4.3 Autoclave.
4.4 pH meter: with an accuracy of 0.01.
4.5 Vortex oscillator.
4.6 Tissue blender.
4.7 Thermostat water bath cauldron.
4.8 Drier.
4.9 Spectrophotometer.
5 Analysis Procedures
5.1 Specimen preparation
Take about 500g of sample, smash and homogenize it sufficiently with tissue blender, and put into clean brown flask with ground mouth, seal the flask and make marking properly, then store it in a dark place for future use.
Weigh 2g~10g (accurate to 0.01g) of homogenized specimen (the Vitamin B2 content in the specimen is greater than 5μg), put it into a 100mL conical flask with stopper, add into 60mL of 0.1mol/L hydrochloric acid solution, shake well and cover the flask stopper. Place the conical flask into autoclave for 30min at 121℃, cool to room temperature and take it out. Adjust the pH value with 1mol/L sodium hydroxide solution to 6.0~6.5, add 2mL of mixed enzyme solution, shake well and place the solution in 37℃ incubator or thermostat water bath cauldron for enzymolysis for the night. Transfer the enzymatic hydrolysis solution into a 100mL volumetric flask, bring the volume to the scale by adding water, filter with filter paper or centrifuge for separation, take filtrate or supernatant to pass 0.45μm water phase filter membrane to serve as to-be-determined solution.
Note: The operation shall be protected against bright light.
Carry out blank test according to the same operating method except for adding specimen.
5.2 Reference conditions of apparatus
a) Chromatographic column: C18 column (150mm in column length, 4.6mm in inner diameter and 5μm in particle size of filler) or equivalent;
b) Mobile phase: sodium acetate solution (0.05mol/L)-methanol (65:35);
c) Flow rate: 1mL/min;
d) Column temperature: 30℃;
e) Detection wavelength: 462nm in excitation wavelength and 522nm in emission wavelength;
f) Injection volume: 20μl.
5.3 Plotting of standard curve
Inject the standard series working solution into high-performance liquid chromatograph respectively, determine the corresponding peak areas, and plot the standard curve by taking the concentration of standard working solution as horizontal coordinate and peak area as longitudinal coordinate.
5.4 Determination of specimen solution
Inject the specimen solution into high-performance liquid chromatograph to obtain the corresponding peak area, and obtain the concentration of Vitamin B2 in to-be-determined solution according to standard curve.
5.5 Blank test requirements
There shall not be to-be-determined component peak or other interference peaks in the chromatogram of blank test solution.
6 Expression of Analysis Results
The content of Vitamin B2 in the specimen is calculated according to Formula (1):
(1)
Where,
X - the content of Vitamin B2 (counted by riboflavin) in the specimen, mg/100g;
ρ - the concentration of Vitamin B2, calculated according to standard curve, in the specimen, μg/mL;
V - the final constant volume of specimen solution, mL;
m - the specimen mass, g;
100 - the conversion coefficient for converting to the content in 100g of sample;
1 000- the conversion coefficient for converting the concentration unit from μg/mL to mg/mL.
Three significant figures are reserved for the result.
7 Accuracy
The absolute difference of the results from two independent determinations under repeatability condition shall not exceed 10% of the arithmetic mean value.
8 Others
Provided that the sampling weight is 10.00g, the method detection limit is 0.02mg/100g and the quantitation limit is 0.05mg/100g.
Method II Fluorescence Spectrophotometry
9 Principle
Vitamin B2 generates yellow-green fluorescence under the irradiation of light of 440nm~500nm wavelength. Its fluorescence intensity is directly proportional to the concentration of Vitamin B2 in dilute solution. Its fluorescence intensity is determined under 525nm wavelength. Then sodium hydrosulfite is added into test solution to resolve Vitamin B2 into non-fluorescence material and the fluorescence intensity of residual fluorescence impurities in test solution is determined, the difference between fluorescence intensity of the initial determination and that of residual fluorescence impurities is the fluorescence intensity generated by Vitamin B2 in the specimen.
Contents of GB 5009.85-2016
Contents
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
9 Principle
10 Reagents and Materials
11 Apparatuses
12 Analysis Procedures
13 Expression of Analysis Results
14 Accuracy
15 Others
Appendix A Calibration Method for the Concentration of Vitamin B2 Standard Solution
Appendix B Liquid Chromatogram of Vitamin B
