GB 5009.296-2023 PDF English
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National food safety standard - Determination of Vitamin D in foods
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GB 5009.296-2023: National food safety standard - Determination of Vitamin D in foods---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB5009.296-2023
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
Foreword
This Standard replaces Method 3 “Determination of vitamin D in foods - Liquid
chromatography - tandem mass spectrometry” and Method 4 “Determination of vitamin
D in foods - High performance liquid chromatography” in GB 5009.82-2016 National
food safety standard – Determination of vitamins A, D and E in foods.
Compared with GB 5009.82-2016, the major changes of this Standard are as follows.
-- Change the standard name to National food safety standard - Determination of
vitamin D in foods;
-- Add online column switching - reversed-phase liquid chromatography;
-- Add the sample preparation method;
-- Modify the linear range and apparatus reference conditions of the liquid
chromatography-tandem mass spectrometry;
-- Modify the preparation method of the standard calibration solution in the appendix.
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 1 – 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 2 – Online column switching - reversed-phase liquid chromatography applies
to the determination of vitamin D2 and vitamin D3 in foods.
Method 3 – Liquid chromatography - tandem mass spectrometry applies to the
determination of vitamin D2 and vitamin D3 in foods.
Method 1 – 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).
3.1.6 n-hexane (C6H14).
3.1.7 Methanol (CH4O). chromatographic pure.
3.1.8 Anhydrous sodium sulfate (Na2SO4).
3.1.9 Cyclohexane (C6H12).
3.2 Preparation of reagents
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.
3.4.4 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
3.5 Materials
3.5.3 Microporous membrane. organic system, pore size 0.45 μm.
4 Instruments and apparatuses
4.1 Normal-phase high-performance liquid chromatograph. equipped with a UV
detector; the injector is equipped with a 500 μL quantitative loop.
4.2 Reversed-phase high-performance liquid chromatograph. equipped with a UV
detector; the injector is equipped with a 100 μL quantitative loop.
4.3 Balance. The sensitivity is 0.1 mg or 0.01 g.
4.8 Constant-temperature water bath.
4.9 Nitrogen blowing concentrator.
4.10 Ultrasonic cleaner.
4.11 Separating funnel extraction oscillator.
5 Analysis steps
5.1 Sample pretreatment
UV irradiation shall be avoided during processing.
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.
5.1.1.3 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
5.1.3 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;
5.2 Apparatus reference conditions
5.2.1 Purification of vitamin D test solution
5.2.2 Reversed-phase liquid chromatography reference conditions
The reference conditions for reversed-phase liquid chromatography are as follows.
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.
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 working solution.
5.5 Blank test
Without weighing the sample, follow the saponification, extraction and concentration
analysis steps of the sample, it shall not contain substances that interfere with the
components to be tested. The internal standard method requires sample testing without
adding internal standard to confirm the feasibility of adding internal standard.
6 Expression of analysis results
The content of vitamin D2 (or vitamin D3) in the sample is calculated according to
Formula (2) and Formula (3) by the internal standard method, and according to Formula
(4) by the external standard method.
7 Precision
The absolute difference of 2 independent test results obtained under repeatability cannot
exceed 15% of the arithmetic mean value.
8 Others
When the sampling volume of the solid sample is 10.00 g and the normal-phase
injection solution is fixed to a volume of 2 mL, the detection limit of vitamin D2 and
vitamin D3 is 0.6 μg/100 g, and the quantitation-limit is 2 μg/100 g.
9 Principle
After the sample is saponified with potassium hydroxide ethanol solution, purified and
concentrated by liquid-liquid extraction or solid-phase extraction, and vitamin D is
separated from other impurities through the C8 column by the one-dimensional liquid
chromatography, the column switching valve is transferred to the two-dimensional
liquid chromatography, and vitamin D2 and vitamin D3 are separated through the C18
column, detect by the ultraviolet detector, and quantify by the internal standard method
(or external standard method). 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.
10 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical reagents, and
the water is grade-I water specified by GB/T 6682.
10.1 Reagents
10.1.1 Absolute ethanol (C2H6O). chromatographic pure.
10.1.5 Potassium hydroxide (KOH).
10.1.6 n-hexane (C6H14).
10.1.7 Ethyl acetate (C4H8O2).
10.2 Preparation of reagents
10.2.1 Potassium hydroxide solution (50%, mass fraction). same as 3.2.1.
10.2.5 Acetonitrile-methanol solution (3+1). Mix acetonitrile and methanol evenly at a
volume ratio of 3.1 and degas by ultrasonic.
10.2.6 Methanol-water solution (1+19). Mix methanol and water evenly at a volume
ratio of 19.1 and degas by ultrasonic.
10.2.7 Acetonitrile-water solution (19+1). Mix acetonitrile and water evenly at a
volume ratio of 19.1 and degas by ultrasonic.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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