GB 5009.82-2016_English: PDF (GB5009.82-2016)
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Determination of retinol and tocopherol in foods
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GB 5009.82-2016
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| GB/T 5009.82-2003 | English | 70 |
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Determination of retinol and tocopherol in foods
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GB/T 5009.82-2003
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| Standard ID | GB 5009.82-2016 (GB5009.82-2016) | | Description (Translated English) | Determination of retinol and tocopherol in foods | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 24,283 | | Date of Issue | 2016-12-23 | | Date of Implementation | 2017-06-23 | | Older Standard (superseded by this standard) | GB 5413.9-2010; GB/T 5009.82-2003; GB/T 9695.26-2008; GB/T 9695.30-2008; NY/T 1598-2008 | | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | | Standard ID | GB/T 5009.82-2003 (GB/T 5009.82-2003) | | Sector / Industry | National Standard (Recommended) | | Classification of Chinese Standard | C53 | | Classification of International Standard | 67.040 | | Word Count Estimation | 8,839 | | Date of Issue | 2003-08-11 | | Date of Implementation | 2004-01-01 | | Older Standard (superseded by this standard) | GB/T 12388-1990 | | Adopted Standard | AOAC 992.06 (��); NEQ; AOAC 974.29 (��); NEQ | | Drafting Organization | Chinese Academy of Preventive Medicine, Institute of Nutrition and Food Hygiene | | Administrative Organization | People's Republic of China Ministry of Health | | Proposing organization | Ministry of Health of the People Republic of China | | Issuing agency(ies) | The People Republic of China Ministry of Health, China National Standardization Management Committee | | Summary | This standard specifies: Vitamin A and Vitamin E-determination in foods, This standard applies to: Food in vitamin A and vitamin E-determination, This standard detection limits were |
GB 5009.82-2016
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard
Determination of vitamin A, D, E in food
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People's Republic of China;
China Food and Drug Administration.
Table of contents
Foreword ... 4
1 Scope ... 5
2 Principles ... 5
3 Reagents and materials ... 5
4 Instruments and equipment ... 8
5 Analytical procedures ... 8
6 Expression of analytical results ... 11
7 Precision... 12
8 Others ... 12
9 Principles ... 12
10 Reagents and materials... 12
11 Instruments and equipment ... 14
12 Analytical procedures ... 15
13 Expression of results ... 17
14 Precision ... 18
15 Others ... 18
16 Principles ... 18
17 Reagents and materials... 18
18 Instruments and equipment ... 21
19 Analytical procedures ... 21
20 Presentation of analytical results ... 25
21 Precision ... 25
22 Others ... 26
23 Principles ... 26
24 Reagents and materials... 26
25 Instruments and equipment ... 29
26 Analytical procedures ... 29
27 Presentation of analytical results ... 33
28 Precision ... 33
29 Others ... 33
Appendix A ... 34
Appendix B Vitamin A, D, E standard solution concentration calibration method
... 36
Appendix C Chromatogram ... 38
Foreword
This standard replaces GB/T 5009.82-2003 “Determination of retinol and
tocopherol in food”, GB 5413.9-2010 “National food safety standard -
Determination of vitamin A, D, E in foods for infants and young children, milk
and milk products”, GB/T 9695.26-2008 “Meat and meat products -
Determination of vitamin A content”, GB/T 9695.30-2008 “Meat and meat
products - Determination of vitamin E content”, and NY/T 1598-2008
“Determination of tocopherol content in edible vegetable oils - High
performance liquid chromatography”.
As compared with GB/T 5009.82-2003, the main changes of this standard are
as follows.
- CHANGE the standard name into “National Food Safety Standard
Determination of vitamin A, D, E in food";
- ADD the “Determination of vitamin E in food - Normal phase high
performance liquid chromatography”;
- ADD the “Determination of vitamin D in food - Liquid chromatography -
Tandem mass spectrometry”;
- ADD the “Determination of vitamin D in food - High performance liquid
chromatography";
- MODIFY the “Determination of vitamin A and vitamin E in food - Reversed-
phase high performance liquid chromatography”;
- MODIFY the reverse phase chromatography separation conditions of
vitamin E isomers, which can simultaneously separate and determine 4
tocopherol isomers;
- DELETE the benzopyrene internal standard quantitative method; CHANGE
it to using the external standard method for quantitative;
- DELETE the “colorimetry” to determine vitamin A.
National Food Safety Standard
Determination of vitamin A, D, E in food
1 Scope
This standard specifies the determination of vitamin A, vitamin E and vitamin D
in food.
The first method of this standard is applicable to the determination of vitamin A
and vitamin E in food.
The second method of this standard is applicable to the determination of vitamin
E in edible oil, nuts, beans, pepper powder and other foods.
The third method of this standard is applicable to the determination of vitamin
D2 and vitamin D3 in food.
The fourth method of this standard is applicable to the determination of vitamin
D2 or vitamin D3 in formulated foods.
Method 1 Determination of vitamin A and vitamin E in food - Reversed-
phase high performance liquid chromatography
2 Principles
The vitamin A and vitamin E in the test specimen are saponified (digested by
amylase if containing starch), extracted, purified and concentrated, then
separated by C30 or PFP reversed-phase liquid chromatography column,
detected by ultraviolet detector or fluorescence detector, AND quantitative by
external standard method.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical
grade pure AND water is the primary water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Absolute ethanol (C2H5OH). it shall not contain aldehydes after inspection,
AND the inspection method is as shown in A.1.
3.3.2.3 γ-tocopherol (C28H48O2, CAS No.. 54-28-4). purity ≥ 95%, OR the
standard substance as certified by the state and awarded with the standard
substance certificate.
3.3.2.4 δ-Tocopherol (C27H46O2, CAS No.. 119-13-1). purity ≥ 95%, OR the
standard substance as certified by the state and awarded with the standard
substance certificate.
3.4 Standard solution preparation
3.4.1 Vitamin A standard stock solution (0.500 mg/mL). accurately WEIGH 25.0
mg vitamin A standard substance; USE absolute ethanol to dissolve it;
TRANSFER it into a 50 mL volumetric flask; MAKE the volume reach to the
mark; at this time the solution concentration is about 0.500 mg/mL. TRANSFER
the solution into a brown reagent bottle; SEAL it and PRESERVE it at -20 °C in
the dark; AND the valid period is 1 month. Before use, INCREASE the
temperature of the solution back to 20 °C; and CONDUCT concentration
correction (as for the correction method, SEE Appendix B).
3.4.2 Vitamin E standard stock solution (1.00 mg/mL). respectively and
accurately WEIGH 50.0 mg of each of α-tocopherol, β-tocopherol γ-tocopherol
and δ-Tocopherol; USE absolute ethanol to dissolve it; TRANSFER it into a 50
mL volumetric flask; MAKE the volume reach to the mark; at this time the
solution concentration is about 1.00 mg/mL. TRANSFER the solution into a
brown reagent bottle; SEAL it and PRESERVE it at -20 °C; AND the valid period
is 6 month. Before use, INCREASE the temperature of the solution back to
20 °C; and CONDUCT concentration correction (as for the correction method,
SEE Appendix B).
3.4.3 Intermediate solution of vitamin A and vitamin E mixed standard solution.
Accurately PIPETTE 1.00 mL of vitamin A standard stock solution and 5.00 mL
of vitamin E standard stock solution into one 50 mL volumetric flask; USE
methanol make it reach to the mark; at this time, the vitamin A concentration of
the solution is 10.0 µg/mL, AND each tocopherol concentration of the vitamin E
is 100 µg/mL. PRESERVE it at -20 °C in dark; AND the valid period is half a
month.
3.4.4 Vitamin A and vitamin E standard series working solution. respectively
and accurately PIPETTE 0.20 mL, 0.50 mL, 1.00 mL, 2.00 mL, 4.00 mL, 6.00
mL of the intermediate solution of vitamin A and vitamin E mixed standard
solution into a 10 mL brown volumetric flask; USE methanol to make the volume
reach to the mark; the vitamin A concentration of this standard series is 0.20
μg/mL, 0.50 μg/mL, 1.00 μg/mL, 2.00 μg/mL, 4.00 μg/mL, and 6.00 μg/mL, AND
the vitamin E concentration is 2.00 μg/mL, 5.00 μg/mL, 10.0 μg/mL, 20.0 μg/mL,
40.0 μg/mL, and 60.0 μg/mL. PREPARE it before use.
Note. saponification period is generally 30 min; after the saponification solution
is cooled down, if there is floating oil on the surface, it shall a...
......
GB/T 5009.82-2003
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.040
C 53
Replacing GB/T 12388-1990
Determination of Retinol and Tocopherol in Foods
ISSUED ON. AUGUST 11, 2003
IMPLEMENTED ON. JANUARY 01, 2004
Issued by. Ministry of Health of the People's Republic of China;
Standardization Administration of the People's Republic of
China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents ... 4
4 Instruments and Equipment ... 5
5 Analysis Steps ... 6
6 Calculation Results ... 9
7 Precision ... 9
8 Principle ... 9
9 Reagents ... 9
10 Instruments ... 10
11 Analysis Steps ... 10
12 Determination ... 12
13 Calculation Results ... 12
Foreword
The first method of this Standard corresponds to AOAC.992.06 (II) “Determination of
Vitamin A in Infant Formula Foods - High Performance Liquid Chromatography” (CAC
reference 1994 edition).
The second method of this Standard corresponds to AOAC.974.29 (IV) “Determination of
Vitamin A in Special Foods - Colorimetry” (CAC reference 1994 edition).
The consistency BETWEEN this Standard AND AOAC.992.06 (II) and AOAC.974.29 (IV)
is not equivalent.
This Standard replaces GB/T 12388-1990 “Determination Methods of Vitamin A and
Vitamin E in Foods”.
Compared with GB/T 12388-1990, the major changes of this Standard are as follows.
- The Chinese name of the standard is changed, which is changed into "Determination
of Vitamin A and Vitamin E in Foods";
- According to GB/T 20001.4-2001 “Rules for Drafting Standards - Part 4. Chemical
Analysis Methods”, the structure of the previous standard is changed.
This Standard was proposed by and shall be under the jurisdiction of the Ministry of Health
of the People's Republic of China.
Drafting organization of this Standard. The Institute of Nutrition and Food Hygiene of
Chinese Academy of Preventive Medicine.
The main drafters of this Standard. Wang Guangya, Li Jing and Wang Guodong.
The original standard was first-time released in 1990; and this is the first revision.
Determination of Retinol and Tocopherol in Foods
1 Scope
This Standard specifies the methods for the determination of retinol and tocopherol
[Translator note. hereafter - Vitamin A and Vitamin E] in foods.
This Standard applies to the determination of Vitamin A and Vitamin E in foods.
The detection limits of this Standard are respectively. VA. 0.8 ng; α-E. 91.8 ng; γ-E. 36.6
ng; δ-E. 20.6 ng.
Method I High Performance Liquid Chromatography
2 Principle
After the saponification extraction process of vitamin A and vitamin E in the sample, extract
it to the organic solvent from the unsaponifiable portion. Use HPLC C18 reverse phase
column to separate vitamin A and vitamin E; use UV detector for testing and carry out
quantitative determination with the internal standard method.
3 Reagents
3.1 Anhydrous ether. containing no peroxide.
3.1.1 Inspection method of peroxide. use 5 mL of ether and add 1 mL of 10% potassium
iodide solution; shake for 1 min; if there is any peroxide, then release the free iodine; the
aqueous layer appears in yellow, or add 4 drops of 0.5% starch solution to make the
aqueous layer appears in blue. The ether shall be treated before use.
3.1.2 Peroxide removal method. When redistilling ether, place a little pure iron wire or iron
dust in the bottle. Discard 10% initial distillate and 10% residual distillate.
3.2 Anhydrous ethyl alcohol. containing no aldehydes.
3.2.1 Inspection method. take 2mL of silver ammonia solution to place into a test tube; add
a small amount of ethanol; shake; then add sodium hydroxide solution; heat and allow to
cool; if there is a silver mirror reaction, then it indicates that there is aldehyde in the ethanol.
3.2.2 Aldehyde removal method. take 2g of silver nitrate to dissolve in a little water. Take
4.4.1 Small centrifuge tubes. 1.5mL ~ 3.0mL plastic centrifuge tube with plastic cover
(matching with high-speed centrifuge).
4.5 High-purity nitrogen.
4.6 Constant temperature water bath.
4.7 UV spectrophotometer.
5 Analysis Steps
5.1 Sample Processing
5.1.1 Saponification. weigh accurately sample 1g ~ 10g (containing vitamin A about 3μg
and vitamin E isomers about 40μg) to place in the saponification flask; add absolute
ethanol 30 mL; stir until the particles are evenly dispersed. Add 10% ascorbic acid 5 mL,
benzo [e] pyrene standard solution 2.00 mL, and mix. Add potassium hydroxide (1 + 1) 10
mL, and mix. Reflux in boiling water bath for 30 min to make complete saponification.
Immediately after saponification, place it into ice water to cool.
5.1.2 Extraction. transfer the sample to the separating funnel after saponification; use 50
mL of water to wash the saponification flask for 2 to 3 times; merge the washing into the
separating funnel. Use about 100 mL of diethyl ether to wash the saponification flask and
its residues twice; merge the diethyl ether solution into the separating funnel. If there is any
residue, pass this solution through the funnel with a small amount of absorbent cotton
before filtering into a separating funnel. Gently shake the separating funnel for 2 min; stand
for layering; discard the aqueous layer.
5.1.3 Washing. use about 50 mL of water to wash the ether layer in the separating funnel;
use pH test paper for testing until the aqueous layer shows no alkaline (shake gently during
initial washing, and increase the intensity of shaking successively).
5.1.4 Concentration. filter the ether extraction solution through the anhydrous sodium
sulfate (about 5 g) into the ball-shaped evaporating 250 mL ~ 300 mL flask that matches
with the rotary evaporator; use about 100 mL of ether to wash the separating funnel and
anhydrous sodium sulfate for 3 times; merge into the evaporating flask; connect it to the
rotary evaporator; do reduced-pressure distillation in water bath at 55°C and recover ether;
take down the evaporating flask when there is about 2 mL of ether left in the flask;
immediately use nitrogen to blow off ether. Immediately add 2.00 mL of ether; mix well to
dissolve the extract.
5.1.5 Transfer the ethanol solution to a small plastic centrifuge tube (4.4.1) for
centrifugation for 5 min (5000 r/min). Use the supernatant for chromatography. If the
vitamin content of the sample is too small, use the ethanol once again for constant volume
after using nitrogen to blow off the ethanol solution. And write down the volume ratio.
5.2 Preparation of standard curve
6 Calculation Results
See Formula (2).
Where.
X -- content of vitamin, in milligram per hundred grams (mg/100 g);
c -- content of a certain vitamin found on the standard curve, in microgram per milliliter
(μg/mL);
V -- concentrated constant volume of sample, in milliliter (mL);
M -- mass of sample, in gram (g).
The calculation results shall be expressed to three significant figures.
7 Precision
The absolute difference of two independent determination results under repeatability
conditions shall not exceed 10% of the arithmetic mean.
Method II Colorimetry
8 Principle
Vitamin A interacts with antimony trichloride in chloroform to produce a blue color, whose
shade is proportional to vitamin A content in the solution. The blue substance is not stable,
but its absorbance can be determined with a spectrophotometer at the wavelength 620 nm
within a certain time.
9 Reagents
Unless otherwise indicated, in the analysis, only use the determined analytical grade
reagents and distilled water or water of equivalent purity.
9.1 Anhydrous sodium sulfate.
9.2 Acetic anhydride.
9.3 Ether.
+ 1) and 20 mL ~ 40 mL of ethanol; reflux on a hot plate for 30 min for complete
saponification.
11.1.1.2 Extraction. transfer the mixture in the saponification flask to a separating funnel;
use 30 mL of water to wash the saponification flask; merge the washing into the separating
funnel. If there is any scum, use absorbent cotton funnel to filter into the separating funnel.
Use 50 mL of ether to wash the saponification flask twice; merge the washing into the
separating funnel. Shake and release gas; place the aqueous layer into the second
separating funnel after standing for layering. Use about 30 mL of ether to wash the
saponification flask twice; pour the washing into the second separating funnel. Stand for
layering after shaking; place the aque...
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