GB 5009.296-2023 National food safety standard—Determination of Vitamin D in foods
1 Scope
This standard specifies the methods for determination of Vitamin D in foods.
Method I “Purification of normal-phase chromatography—Reversed phase liquid chromatography” is applicable to the determination of Vitamins D2 and D3 in foods with ergocalciferol or cholecalciferol.
Method II “On-line column switching—Reversed phase liquid chromatography” is applicable to determination of Vitamins D2 and D3 in foods.
Method III “Liquid chromatography—Tandem mass spectrometry” is applicable to determination of Vitamins D2 and D3 in foods.
Method I Purification of liquid chromatography—Reversed phase liquid chromatography
2 Principle
After the specimen is saponified by potassium hydroxide ethanol solution, purified by liquid-liquid extraction, concentrated, Vitamin D is separated from other impurities by normal phase high-performance liquid chromatographic instrument through silica gel column. After the collected fraction is concentrated and then separated by reverse phase chromatography column to separate Vitamin D2 and D3, which are tested by ultraviolet detector, and quantified by internal standard method (or external standard method). When the specimen does not contain Vitamin D2, Vitamin D2 may be used as internal standard to determine Vitamin D3; when the specimen does not contain Vitamin D3, Vitamin D3 may be used as internal standard to determine Vitamin D2. Otherwise, it is determined by external standard method.
3 Reagents and materials
Unless otherwise stated, reagents used in this method are analytical pure, and the water is Grade I water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Anhydrous ethanol (C2H6O): chromatographically pure.
3.1.2 L-Ascorbic acid (C6H8O6).
3.1.3 2, 6-Butylated hydroxytoluene (C15H24O): BHT for short.
3.1.4 α-amylase (CAS: 9000-90-2, intermediate temperature amylase, from Bacillus): enzyme activity ≥1.5 U/mg.
3.1.5 Potassium hydroxide (KOH).
3.1.6 N-hexane (C6H14).
3.1.7 Methanol (CH4O): chromatographically pure.
3.1.8 Anhydrous sodium sulfate (Na2SO4).
3.1.9 Cyclohexane (C6H12).
3.1.10 Isopropanol (C3H8O).
3.1.11 Petroleum ether: its boiling range is 30°C~60°C.
3.2 Reagent preparation
3.2.1 Potassium hydroxide solution (50%, mass fraction): weigh 50 g potassium hydroxide, dissolve it in 50 g water, cool and store it in polyethylene bottle.
3.2.2 BHT-ethanol solution (0.2 g/100 mL): weigh 1.0 g BHT, dissolve it in 500 mL absolute ethanol, which is prepared before use.
3.2.3 Isopropanol cyclohexane-n-hexane solution (2+125+125): mix isopropanol, cyclohexane and n-hexane were mixed uniformly at a volume ratio of 2:125:125, and degas it by ultrasound.
3.2.4 Methanol-water solution (19 +1): mix methanol and water evenly at a volume ratio of 19:1, and degas it by ultrasound.
3.3 Standards
3.3.1 Standard of Vitamin D2: Ergocalciferol (C28H44O, CAS No. 50-14-6), with a purity of ≥98%, or a standard certified by the nation and award with the standard certificate by the nation.
3.3.2 Standard of Vitamin D3: (C27H44O, CAS No.: 67-97-0): with purity ≥98%, or standard certified by nation and awarded with the standard certificate by the nation.
3.4 Preparation of standard solution
3.4.1 Standard stock solution (1,000 mg/L) of Vitamin D2: accurately weigh 50 mg (accurate to 0.1 mg) of Vitamin D2 standard into a small beaker, dissolve it with anhydrous ethanol, and transfer it to a 50 mL volumetric flask; scale the volume to scale line and mix it well. Correct the concentration of the reserve solution according to Annex A, put the reserve solution into a brown reagent bottle, seal and protect it from light at -18°C. The storage period is 6 months.
Foreword i
1 Scope
Method I Purification of liquid chromatography—Reversed phase liquid chromatography
2 Principle
3 Reagents and materials
4 Apparatus
5 Analytical procedures
6 Expression of analysis results
7 Accuracy
8 Others
Method II On-line column switching - Reversed phase liquid chromatography
9 Principle
10 Reagents and materials
11 Apparatus
12 Analytical procedures
13 Expression of analysis results
14 Accuracy
15 Others
Method III Liquid chromatography - Tandem mass spectrometry
16 Principle
17 Reagents and materials
18 Apparatus
19 Analytical procedures
Annex A Concentration correction method of Vitamin D standard solution
Annex B Chromatogram of Vitamin D standard solution
Annex C Schematic diagram of flow path of online column switching-liquid chromatography system
