GB 5009.296-2023 English PDF

GB 5009.296-2023: National food safety standard - Determination of Vitamin D in foods
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GB 5009.296-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of Vitamin D
in foods
ISSUED ON: SEPTEMBER 06, 2023
IMPLEMENTED ON: MARCH 06, 2024
Issued by: National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Co1ntents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and apparatuses ... 7
5 Analysis steps ... 7
6 Expression of analysis results ... 12
7 Precision ... 13
8 Others ... 13
9 Principle ... 13
10 Reagents and materials ... 14
11 Instruments and apparatuses ... 16
12 Analysis steps ... 16
13 Expression of analysis results ... 21
14 Precision ... 22
15 Others ... 22
16 Principle ... 22
17 Reagents and materials ... 22
18 Instruments and apparatuses ... 25
19 Analysis steps ... 25
Appendix A Vitamin D standard solution concentration calibration method ... 32
Appendix B Vitamin D standard solution chromatogram ... 35
Appendix C Online column switching-liquid chromatography system flow path
diagram ... 38
National food safety standard - Determination of Vitamin D
in foods
1 Scope
This Standard specifies methods for the determination of vitamin D in foods.
Method I – Normal phase chromatography purification - reversed-phase liquid
chromatography applies to the determination of vitamin D2 and vitamin D3 in foods
containing ergocalciferol or cholecalciferol.
Method II – Online column switching - reversed-phase liquid chromatography applies
to the determination of vitamin D2 and vitamin D3 in foods.
Method III – Liquid chromatography - tandem mass spectrometry applies to the
determination of vitamin D2 and vitamin D3 in foods.
Method I – Normal phase chromatography purification – reversed-phase liquid
chromatography.
2 Principle
After the sample is saponified with an ethanol solution of potassium hydroxide, purified
and concentrated by the liquid-liquid extraction method, use a normal-phase high-
performance liquid chromatograph to separate vitamin D from other impurities through
a silica gel column. After concentrating the collected fractions, separate vitamin D2 and
vitamin D3 through a reverse-phase chromatography column. Detect with a UV detector
and use the internal standard method (or external standard method) for quantification.
When the sample does not contain vitamin D2, use vitamin D2 as the internal standard
to determine vitamin D3; when the sample does not contain vitamin D3, use vitamin D3
as the internal standard to determine vitamin D2. Otherwise, use the external standard
method for determination.
3 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical reagents, the
water is grade-I water specified by GB/T 6682.
3.1 Reagents
3.1.1 Absolute ethanol (C2H6O): chromatographic pure.
3.1.2 Ascorbic acid (C6H8O6).
3.1.3 2,6-butylated hydroxytoluene (C15H24O): referred to as BHT.
3.1.4 α-amylase (CAS number: 9000-90-2, mesophilic amylase, derived 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): chromatographic pure.
3.1.8 Anhydrous sodium sulfate (Na2SO4).
3.1.9 Cyclohexane (C6H12).
3.1.10 Isopropyl alcohol (C3H8O).
3.1.11 Petroleum ether: boiling range 30 °C ~ 60 °C.
3.2 Preparation of reagents
3.2.1 Potassium hydroxide solution (50%, mass fraction): Weigh 50 g of potassium
hydroxide; dissolve it in 50 g of water; cool and store in a polyethylene bottle.
3.2.2 BHT-ethanol solution (0.2 g/100 mL): Weigh 1.0 g of BHT; dissolve it in 500 mL
of absolute ethanol; prepare it before use.
3.2.3 Isopropyl alcohol-cyclohexane-n-hexane solution (2+125+125): Mix isopropyl
alcohol, cyclohexane and n-hexane at a volume ratio of 2:125:125 and degas by
ultrasonic.
3.2.4 Methanol-water solution (19+1): Mix methanol and water evenly at a volume ratio
of 19:1 and degas by ultrasonic.
3.3 Standard
3.3.1 Vitamin D2 standard substance: ergocalciferol (C28H44O, CAS number: 50-14-6),
purity ≥98%, or a standard substance certified by the state and awarded a standard
substance certificate.
3.3.2 Vitamin D3 standard substance: cholecalciferol (C27H44O, CAS number: 67-97-0),
purity ≥98%, or a standard substance certified by the state and awarded a standard
substance certificate.
3.4 Preparation of standard solution
3.4.1 Vitamin D2 standard stock solution (1 000 mg/L): Accurately weigh 50 mg
(accurate to 0.1 mg) of vitamin D2 standard substance in a small beaker; dissolve it in
absolute ethanol and transfer it to a 50 mL volumetric flask; adjust the volume to the
mark; mix well. Calibrate the concentration of the stock solution according to Appendix
A. Put the stock solution into a brown reagent bottle; seal it and store it in the dark at -
18 °C. The use-by date is 6 months.
3.4.2 Vitamin D3 standard stock solution (1 000 mg/L): Accurately weigh 50 mg
(accurate to 0.1 mg) of vitamin D3 standard substance in a small beaker; dissolve it in
absolute ethanol and transfer it to a 50 mL volumetric flask; adjust the volume to the
mark; mix well. Calibrate the concentration of the stock solution according to Appendix
A. Put the stock solution into a brown reagent bottle; seal it and store it in the dark at -
18 °C. The use-by date is 6 months.
3.4.3 Vitamin D2 standard working solution (10.0 mg/L): Accurately draw 1.00 mL of
vitamin D2 standard stock solution (1 000 mg/L) into a 100 mL volumetric flask; use
absolute ethanol to dilute to the mark; mix well. Store in the dark at -18 °C, with a use-
by day of 3 months.
3.4.4 Vitamin D3 standard working solution (10.0 mg/L): Accurately draw 1.00 mL of
vitamin D3 standard stock solution (1 000 mg/L) into a 100 mL volumetric flask; use
absolute ethanol to dilute to the mark; mix well. Store in the dark at -18 °C, with a use-
by day of 3 months.
3.4.5 Standard series of working solutions
3.4.5.1 When using vitamin D3 as the internal standard to determine vitamin D2:
respectively pipette 0.50 mL, 1.00 mL, 2.00 mL, 4.00 mL, 6.00 mL and 10.00 mL of
vitamin D2 standard working solution (10.0 mg/L) into a 100 mL brown volumetric
flask; respectively add 2.50 mL of vitamin D3 internal standard working solution (10.0
mg/L) to each; add methanol to dilute to the mark; mix well. The mass concentrations
of this standard series of working solutions are 50 μg/L, 100 μg/L, 200 μg/L, 400 μg/L,
600 μg/L, and 1 000 μg/L, respectively; the vitamin D3 internal standard mass
concentration is 250 μg/L. Prepare before use.
3.4.5.2 When using vitamin D2 as the internal standard to determine vitamin D3:
respectively pipette 0.50 mL, 1.00 mL, 2.00 mL, 4.00 mL, 6.00 mL and 10.00 mL of
vitamin D3 standard working solution (10.0 mg/L) into a 100 mL brown volumetric
flask; respectively add 2.50 mL of vitamin D2 internal standard working solution (10.0
mg/L) to each; add methanol to dilute to the mark; mix well. The mass concentrations
of this standard series of working solutions are 50 μg/L, 100 μg/L, 200 μg/L, 400 μg/L,
600 μg/L, and 1 000 μg/L, respectively; the vitamin D2 internal standard mass
concentration is 250 μg/L. Prepare before use.
3.4.5.3 When using the external standard method to simultaneously measure vitamin
D2 and D3: Respectively take 0.50 mL, 1.00 mL, 2.00 mL, 4.00 mL, 6.00 mL and 10.00
5.1.1 Sample preparation
After a certain number of samples are divided, crushed and homogenized as required,
store them in sample bags, protected from light and refrigerated, and measure as soon
as possible.
5.1.1.1 Infant formula foods, formula foods for special medical purposes, milk and
dairy products, frozen drinks and beverage
Solid samples: weigh about 25 g (m1, accurate to 0.01 g) of the homogenized sample
into a 250 mL dry glass bottle; add about 200 mL of warm water (40 °C ~ 45 °C); record
the mass of the solution after adding water (m2, accurate to 0.01 g). Mix thoroughly and
dissolve, place at room temperature in the dark for 15 minutes, and shake for 30 seconds
every 5 minutes. Weigh 20 g ~ 50 g (m3, accurate to 0.01 g) of the prepared slurry into
a 150 mL flat-bottomed flask. Calculate the sample mass (m) according to Formula (1).
Liquid sample: Weigh 20 g ~ 50 g (m, accurate to 0.01 g) of the shaken sample into a
150 mL flat-bottomed flask.
5.1.1.2 Cereals and their products, bakery products, cereal supplementary foods
for infants and young children and other starch-containing samples
Weigh 5 g ~ 10 g (m, accurate to 0.01 g) of the homogenized sample into a 150 mL flat-
bottomed flask; add about 20 mL of warm water (40 °C ~ 45 °C); mix well. For cereal
supplementary foods for infants and young children, refer to 5.1.1.1 to prepare a slurry
first, and then weigh 20 g ~ 50 g (m3, accurate to 0.01 g) of the prepared slurry into a
150 mL flat-bottomed flask.
5.1.1.3 Other foods
Weigh 5 g ~ 20 g (m, accurate to 0.01 g) of the homogenized solid sample or 20 g ~ 50
g (m, accurate to 0.01 g) of the liquid sample into a 150 mL flat-bottomed flask. For
solid samples, add 20 mL ~ 30 mL of warm water (40 °C ~ 45 °C); mix well.
5.1.2 Sample saponification
5.1.2.1 Infant formulas, formulas for special medical purposes, milk and dairy
products, frozen drinks, beverages and other foods
Add 100 μL of internal standard working solution (10.0 mg/ L) to the flat-bottomed
flask as described in 5.1.1.1 and 5.1.1.3; then, add 1.0 g of ascorbic acid and 30 mL ~
50 mL of BHT-ethanol solution (0.2 g/100 mL); mix well; then, add 20 mL ~ 30 mL of
potassium hydroxide solution (50%, mass fraction); shake while adding; mix well and
saponify in a magnetic stirrer or constant temperature oscillator, at a temperature of
80 °C ± 2 °C, for 30 min (or at a temperature of 25 °C ± 5 °C, for 16 h ± 2 h); after
saponification, immediately use cold water to cool to room temperature.
5.1.2.2 Cereals and their products, bakery products, cereal supplementary foods
for infants and young children, and other starch-containing samples
Add 100 μL of internal standard working solution (10.0 mg/L) and 1.0 g of α-amylase
to the flat-bottomed flask as described in 5.1.1.2; put it into a constant temperature
water bath at 60 °C and shake for 30 minutes; add 1.0 g of ascorbic acid and 30 mL of
BHT-ethanol solution (0.2 g/100 mL) to the enzymatic hydrolysis solution; mix well;
then, add 10 mL ~ 20 mL of potassium hydroxide solution (50%, mass fraction); shake
while adding; mix and then saponify in a magnetic stirrer or constant temperature
oscillator, at a temperature of 80 °C ± 2 °C, for 30 min (or at a temperature of 25 °C ±
5 °C, for 16 h ± 2 h); after saponification, immediately use cold water to cool to room
temperature.
Note: If the external standard method is used, no internal standard is added, and the
remaining operations are the same as above. The saponification conditions are
selected at a temperature of 25 °C ± 5 °C and a saponification time of 16 h ± 2 h.
5.1.3 Sample extraction and concentration
5.1.3.1 Sample extraction
Use 30 mL of water to transfer the above saponification solution into a 250 mL
separatory funnel; add 50 mL of petroleum ether; shake and extract for 5 minutes;
transfer the lower solution to another 250 mL separatory funnel; add 50 mL of
petroleum ether and repeat the extraction 1 to 2 times; combine the petroleum ether
layer. Use about 150 mL of water to wash the petroleum ether layer; remove the lower
water phase; repeat washing at least 3 times until the petroleum ether layer is washed
to neutral (a pH test paper universal can be used to detect the lower solution).
5.1.3.2 Sample concentration
Slowly flow the washed petroleum ether layer through a glass chromatography column
pre-filled with anhydrous sodium sulfate powder with a thickness of 3 cm ~ 5 cm; filter
it into a 250 mL rotary evaporating flask or a nitrogen blowing concentration tube; use
15 mL of petroleum ether to rinse the separatory funnel and the anhydrous sodium
sulfate in the glass chromatography column twice; combine them into an evaporating
flask or a nitrogen blowing concentration tube; connect them to a rotary evaporator or
nitrogen blowing concentrator, and concentrate them by rotary evaporation or nitrogen
blowing in a 40 °C water bath; when about 1 mL of petroleum ether remains, remove
the evaporation flask or concentration tube, and use petroleum ether to transfer the
concentrated liquid to a 2 mL volumetric flask; blow nitrogen until it is nearly dry; use
n-hexane to adjust the volume to the mark; pass through a 0.45 μm microporous filter,
for normal phase high performance liquid chromatography purification.
b) mobile phase: methanol-water solution (19+1);
c) flow velocity: 1.0 mL/min;
d) wavelength: 264 nm;
e) column temperature: 35 °C;
f) injection volume: 100 μL.
5.3 Preparation of standard curve
Inject the standard series of working solutions into the reversed-phase liquid
chromatograph respectively. For the internal standard method, use the mass ratio of
vitamin D2 (or vitamin D3) in the standard series of working solutions to its
corresponding internal standard as the abscissa, and the peak area ratio of vitamin D2
(or vitamin D3) to its corresponding internal standard as the ordinate to draw the
standard curve. For the external standard method, use the concentration of vitamin D2
(or vitamin D3) in the standard series of working solutions as the abscissa and the peak
area of vitamin D2 (or vitamin D3) as the ordinate to draw a standard curve. For the
chromatograms of vitamin D2 and vitamin D3 standard solutions, see Figure B.1 in
Appendix B.
5.4 Determination of sample solution
5.4.1 Qualitative analysis
Inject the sample solution into the liquid chromatograph for measurement. Under the
same test conditions, the retention time of vitamin D2 and vitamin D3 in the sample
solution shall deviate by no more than ±2.5% compared with the corresponding
retention time in the standard...