National Food Safety Standard
Determination of Vitamin B6 in Foods
食品安全国家标准
食品中维生素B6的测定
1 Scope
This standard specifies the determination methods of Vitamin B6 in foods.
Method I of this standard is high performance liquid chromatography, which is applicable to the determination of foods added with Vitamin B6; Method II is microbiological method, which is applicable to the determination of Vitamin B6 in various foods.
Method I High Performance Liquid Chromatography
2 Principle
Specimen is subjected to pretreatments such as extraction and then separated via C18 chromatographic column, detected with fluorescence detector and quantified by external standard method for determining the content of Vitamin B6 (pyridoxine, pyridoxal and pyridoxamine).
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 Sodium 1-octanesulfonate (C8H17NaO3S).
3.1.2 Glacial acetic acid (C2H4O2).
3.1.3 Triethylamine (C6H15N): chromatographically pure.
3.1.4 Methanol (CH4O): chromatographically pure.
3.1.5 Hydrochloric acid (HCl).
3.1.6 Sodium hydroxide (NaOH).
3.1.7 Amylase: enzyme activity≥1.5U/mg.
3.2 Reagents preparation
3.2.1 Hydrochloric acid solution (5.0mol/L): measure 45mL of hydrochloric acid, dilute and bring the volume to 100mL with water.
3.2.2 Hydrochloric acid solution (0.1mol/L): pipet 9mL of hydrochloric acid, dilute and bring the volume to 1 000mL with water.
3.2.3 Sodium hydroxide solution (5.0mol/L): weigh 20g of sodium hydroxide, dissolve it with 50mL of water, after cooling, bring the volume to 100mL with water.
3.2.4 Sodium hydroxide solution (0.1mol/L): weigh 0.4g of sodium hydroxide, dissolve it with 50mL of water, after cooling, bring the volume to 100mL with water.
3.3 Standards
3.3.1 Pyridoxine-hydrochloride (C8H12ClNO3, CAS No.: 58-56-0): purity≥98% or the standard material approved and awarded with reference material certificate by the nation.
3.3.2 Pyridoxal-hydrochloride (C8H10ClNO3, CAS No.: 65-22-5): purity≥99% or the standard material approved and awarded with reference material certificate by the nation.
3.3.3 Pyridoxamine-dihydrochloride (C8H14Cl2N2O3, CAS No.: 524-36-7): purity≥99% or the standard material approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solution
3.4.1 Pyridoxine standard stock solution (1mg/mL): accurately weigh 60.8mg of pyridoxine-hydrochloride standard, dissolve with 0.1mol/L hydrochloric acid solution, bring the volume to 50mL, protect it from light at -20℃ for a validity period of one month.
3.4.2 Pyridoxal standard stock solution (1mg/mL): accurately weigh 60.9mg of pyridoxal-hydrochloride standard, dissolve with 0.1mol/L hydrochloric acid solution, bring the volume to 50mL, protect it from light at -20℃ for a validity period of one month.
3.4.3 Pyridoxamine standard stock solution (1mg/mL): accurately weigh 71.7mg of pyridoxamine-dihydrochloride standard, dissolve with 0.1mol/L hydrochloric acid solution, bring the volume to 50mL, protect it from light at -20℃ for a validity period of one month.
3.4.4 Mixing standard intermediate solution of Vitamin B6 (20μg/mL): accurately pipet 1.00mL of standard stock solution of pyridoxine, pyridoxal and pyridoxamine respectively, dilute with 0.1mol/L hydrochloric acid solution and bring the volume to 50mL. Prepare this solution immediately before use.
3.4.5 Mixing standard series working solutions of Vitamin B6: accurately pipet 0.5mL, 1.0mL, 2.0mL, 3.0mL and 5.0mL of mixing standard intermediate solution of Vitamin B6 into 100mL volumetric flasks and scale the volumes with water. The concentration of this standard series is 0.10μg/mL, 0.20μg/mL, 0.40μg/mL, 0.60μg/mL and 1.00μg/mL respectively. Prepare this solution immediately before use.
Note: Carry out concentration calibration before using the standard stock solution and see Appendix A for the calibration method.
4 Apparatuses
4.1 High performance liquid chromatograph: equipped with fluorescence detector.
4.2 Balance: with sensitivity of 1mg and 0.1mg.
4.3 pH meter: with accuracy of 0.01.
4.4 Vortex mixer.
4.5 Ultrasonic vibrator.
4.6 Spectrophotometer.
4.7 Thermostatic incubator or that with equivalent property.
5 Analysis Procedures
5.1 Specimen preparation
5.1.1 Starch-containing specimen
a) Solid specimen: weigh about 5g (accurate to 0.01g) of well-mixed solid specimen into a 150mL conical flask, add into about 25mL of 45℃~50℃ water and mix well. Add into about 0.5g of amylase, fill nitrogen into the conical flask after mixing well, put on the flask stopper and place the flask into the incubator of 50℃~60℃ for about 30min. Take it out and cool to room temperature.
b) Liquid specimen: weigh about 20g (accurate to 0.01g) of well-mixed liquid specimen into a 150mL conical flask and mix well. Add into about 0.5g of amylase, fill nitrogen into the conical flask after mixing well, put on the flask stopper and place the flask into the incubator of 50℃~60℃ for about 30min. Take it out and cool to room temperature.
5.1.2 Starch-free specimen
a) Solid specimen: weigh about 5g (accurate to 0.01g) of well-mixed solid specimen into a 150mL conical flask, add into about 25mL of 45℃~50℃ water and mix well. Keep it still for 5min~10min and cool to room temperature.
b) Liquid specimen: weigh about 20g (accurate to 0.01g) of well-mixed liquid specimen into a 150mL conical flask. Keep it still for 5min~10min.
5.1.3 Preparation of to-be-determined solution
Use the hydrochloric acid solution to adjust the pH value of the above-mentioned specimen solution to 1.7±0.1 and place the solution for about 1min. Then use the sodium hydroxide solution to adjust the pH value of the specimen solution to 4.5±0.1. Put the above-mentioned conical flask into ultrasonic vibrator to oscillate for about 10min. Transfer the specimen solution into a 50ml volumetric flask and wash the conical flask with water. Combine the washing solution into a 50ml volumetric flask and bring the volume to 50ml with water. Take another 50mL conical flask, put funnel and filter paper on top of it, pour the specimen solution with constant volume in the flask for filtering naturally. Then filter the filtrate through 0.45μm microfiltration membrane, collect it with test tube, and transfer 1mL of filtrate into the sample-injecting bottle to serve as the to-be-determined solution of specimen.
Note: The operation shall be protected against bright light.
5.2 Reference conditions of apparatus
Reference conditions of apparatus are as follows:
a) Chromatographic column: C18 column (150mm in column length, 4.6mm in inner diameter and 5μm in particle size of column fillers) or equivalent;
b) Mobile phase: 50mL of methanol, 2.0g of sodium 1-octanesulfonate and 2.5mL of triethylamine, dissolve them and bring the volume to 1 000ml with water, use glacial acetic acid to adjust the pH to 3.0±0.1, and filter with a 0.45m microfiltration membrane.
c) Flow rate: 1mL/min;
d) Column temperature: 30℃;
e) Detection wavelength: 293nm in excitation wavelength and 395nm in emission wavelength.
f) Sample injection volume: 10μL.
5.3 Plotting of standard curve
Inject the mixed standard series working solution of Vitamin B6 into the high performance liquid chromatograph, determine the corresponding peak area of each component, and plot the standard curve by taking the concentration of corresponding standard working solution as horizontal coordinate and peak area as longitudinal coordinate.
5.4 Determination of specimen solution
Inject the specimen solution into the high performance liquid chromatograph to obtain the corresponding peak area of each component, and obtain the concentration of each component of Vitamin B6 in to-be-determined solution according to standard curve.
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 Each Component Standard Solution of Vitamin B
Appendix B Liquid chromatogram of Vitamin B
Appendix C Medium Component and Preparation Method
