GB 5009.154-2023 National food safety standard- Determination of vitamin B6 in foods
1 Scope
This standard specifies the method for determination of B6 in foods.
Method I - liquid chromatography-tandem mass spectrometry is applicable to determination of vitamin B6 in foods.
Method II - liquid chromatography-mass spectrometry is applicable to determination of vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in formulated milk powders, foods for special dietary uses, ready-to-eat cereals, baked foods and beverages to which vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) has been added as a nutrient fortifier.
Method III - high performance liquid chromatography-fluorescence detection is applicable to determination of vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in formulated milk powders, foods for special dietary uses (except foods for special medical purposes), ready-to-eat cereals, baked foods and beverages to which vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) has been added as a nutrient fortifier.
Method IV - microbiological analysis is applicable to determination of vitamin B6 in foods.
Method I Liquid chromatography-tandem mass spectrometry
2 Principle
Vitamin B6 (pyridoxine, pyridoxal, pyridoxamine) in foods is subjected to acid hydrolysis and then enzymatically hydrolyzed to pyridoxamine, pyridoxal and pyridoxine, which are then diluted and filtered. Afterwards, they are separated by reversed-phase liquid chromatography, detected by tandem mass spectrometry, and quantified by isotope-labeled internal standards. Then the total content of vitamin B6 is calculated based on the pyridoxine content.
3 Reagents and materials
Unless otherwise specified, only analytically pure reagents and Grade 1 water specified in GB/T 6682 are used for the purpose of this method.
3.1 Reagents
3.1.1 Methanol (CH3OH): Chromatographically pure.
3.1.2 Methanoic acid (HCOOH): Chromatographically pure.
3.1.3 Ammonium formate (HCOONH4): Chromatographically pure.
3.1.4 Sodium hydroxide (NaOH).
3.1.5 Hydrochloric acid (HCl).
3.1.6 α-amylase: With an enzyme activity ≥ 50U/mg.
3.1.7 Acid phosphatase: With an enzyme activity ≥ 0.5U/mg.
3.1.8 Papain: With an enzyme activity ≥ 800U/mg.
3.2 Reagent preparation
3.2.1 Hydrochloric acid solution (0.1mol/L): Accurately pipette 9mL of hydrochloric acid and dilute to 1,000mL with water.
3.2.2 Sodium hydroxide solution (0.1mol/L): Accurately weigh out 0.4g of sodium hydroxide, add 50mL of water to dissolve it, and dilute the solution to 100mL with water after it gets cool.
3.2.3 Mobile phase A (2% formic acid solution with 10mmol/L ammonium formate): Weigh out 0.63g of ammonium formate, add about 950mL of water to dissolve it, and then add 20mL of formic acid; dilute the solution to 1,000mL with water, mix well, and subject it to ultrasonic degas for later use.
3.2.4 Mobile phase B (0.1% formic acid-methanol solution): Pipette 1mL of formic acid, dilute it to 1,000mL with methanol, and ultrasonically mix it well.
3.3 Standard substances
3.3.1 Pyridoxine-hydrochloride (C8H12ClNO3, CAS No.: 58-56-0): With a purity ≥98%, or a standard substance certified by the state and granted a standard substance certificate.
3.3.2 Pyridoxal-hydrochloride (C8H10ClNO3, CAS No.: 65-22-5): With a purity ≥99%, or a standard substance certified by the state and granted a standard substance certificate.
3.3.3 Pyridoxamine-dihydrochloride (C8H14Cl2N2O2, CAS No.: 524-36-7): With a purity ≥99%, or a standard substance certified by the state and granted a standard substance certificate.
3.3.4 13C4-pyridoxine-hydrochloride (13C4C4H12ClNO3): With a purity ≥98%.
3.3.5 D3-pyridoxal (C8D3H6NO3, CAS No.: 1173023-49-8): With a purity ≥98%.
3.3.6 D3-pyridoxamine-dihydrochloride (C8D3H11Cl2N2O2, CAS No.: 1173023-45-4): With a purity ≥98%.
3.4 Preparation of standard solutions
3.4.1 Pyridoxine standard stock solution (1.00mg/mL): Accurately weigh out 60.8mg of pyridoxine-hydrochloride standard substance, dissolve and dilute it with 0.1mol/L hydrochloric acid solution into a 50mL brown volumetric flask, and then transfer the resultant solution to a brown glass reagent bottle. Store it at -20℃ away from light, with a shelf life of 3 months.
3.4.2 Pyridoxal standard stock solution (1.00mg/mL): Accurately weigh out 60.9mg of pyridoxal-hydrochloride standard substance, dissolve and dilute it with 0.1mol/L hydrochloric acid solution into a 50mL brown volumetric flask, and then transfer the resultant solution to a brown glass reagent bottle. Store it at -20℃ away from light, with a shelf life of 3 months.
3.4.3 Pyridoxamine standard stock solution (1.00mg/mL): Accurately weigh out 71.7mg of pyridoxamine-dihydrochloride standard substance, dissolve and dilute it with 0.1mol/L hydrochloric acid solution into a 50mL brown volumetric flask, and then transfer the resultant solution to a brown glass reagent bottle. Store it at -20℃ away from light, with a shelf life of 3 months.
3.4.4 Mixed standard stock solution of vitamin B6 (50.0μg/mL): Accurately pipette 5.00mL of pyridoxamine, pyridoxal and pyridoxine standard stock solutions (1.00mg/mL) respectively, dilute them with 0.1mol/L hydrochloric acid solution into a 100mL brown volumetric flask and then transfer the resultant solution to brown glass reagent bottles. Store it at -20℃ away from light, with a shelf life of 3 months.
3.4.5 Mixed standard working solution of vitamin B6 (5.00μg/mL): Accurately pipette 1.00mL of mixed standard stock solution of vitamin B6 (50.0μg/mL) and dilute it into a 10mL brown volumetric flask with mobile phase A. The solution shall be freshly prepared before use.
3.4.6 Mixed standard working solution of vitamin B6 (500ng/mL): Accurately pipette 1.00mL of mixed standard stock solution of vitamin B6 (5.00μg/mL) and dilute it into a 10mL brown volumetric flask with mobile phase A. The solution shall be freshly prepared before use.
3.4.7 Mixed standard working solution of vitamin B6 (50.0ng/mL): Accurately pipette 1.00mL of mixed standard stock solution of vitamin B6 (500ng/mL) and dilute it into a 10mL brown volumetric flask with mobile phase A. The solution shall be freshly prepared before use.
Note: The frozen stock solutions shall be thawed at room temperature and mixed well before use.
3.5 Preparation of isotope-labeled internal standard solutions
Foreword II
1 Scope
2 Principle
3 Reagents and materials
4 Apparatus
5 Analytical procedures
6 Expression of analysis results
7 Precision
8 Others
9 Principle
10 Reagents and materials
11 Apparatus
12 Analytical procedures
13 Expression of analysis results
14 Precision
15 Others
16 Principle
17 Reagents and materials
18 Apparatus
19 Analytical procedures
20 Expression of analysis results
21 Precision
22 Others
23 Principle
24 Reagents and materials
25 Apparatus
26 Analytical procedures
27 Expression of analysis results
28 Precision
29 Others
Annex A Concentration calibration method for standard solutions of vitamin B6 components
Annex B MRM mass chromatogram
Annex C
Annex D Liquid chromatogram of vitamin B6
Annex E Components and preparation methods of media
