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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 5413.9-2010 
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Determination of vitamin A, D, E in foods for infants and
young children, milk and milk products
ISSUED ON. MARCH 26, 2010
IMPLEMENTED ON. JUNE 1, 2010
Issued by. Ministry of Health of the People's Republic of China
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Principle ... 4 
4 Reagents and materials ... 4 
5 Apparatuses and instruments ... 5 
6 Analytical Steps ... 6 
7 Expression of analysis results ... 9 
8 Precision ... 10 
9 Others ... 11 
Annex A ... 12 
Annex B ... 14 
Foreword
This Standard replaces GB/T 5413.9 -1997 Milk powder and formula foods for infant and
young children - Determination of vitamin A, D, E content.
This Standard, compared with GB/T 5413.9 -1997, has been modified as follows.
- The antioxidant reagent has been modified from original pyrogallic acid to ascorbic
acid.
- The heating and refluxing in the pretreatment method has been modified as constant
temperature saponification.
- The correction of standard solution has been added.
- The previous single-point quantitative has been modified as the standard curve
method.
- The determination of vitamin D recovery has been supplemented.
- The calculation formula has been modified.
Annex A of this Standard is normative, and Annex B is informative.
The previous edition replaced by this Standard is as follows.
- GB/T 5413.9 -1997.
   
National food safety standard -
Determination of vitamin A, D, E in foods for infants and
young children, milk and milk products
1 Scope
This Standard specifies the method for determination of vitamin A, D, E in foods for infant
and young children, milk and milk products.
This Standard is applicable to the determination of vitamin A, D, E content in foods for
infant and young children, milk and milk products.
2 Normative references
The following referenced documents are indispensable for the application of this
document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
3 Principle
After saponification and extraction with petroleum ether, vitamin A and E is separated
by reverse phase chromatography method, of which the content is determined by
external standard method; vitamin D is purified by normal phase chromatography
method and separated by reverse phase chromatography method, of which the
quantitative is determined by external standard method.
4 Reagents and materials
Unless otherwise specified, all reagents shall be analytical pure or higher graded, and
all water used for the experiment shall be Grade 1 water specified in GB/T 6682.
4.1 α-Diastase. Enzyme activity ≥ 1.5 U/mg.
4.2 Anhydrous sodium sulfate.
4.3 Isopropyl alcohol. chromatographically pure.
4.4 Alcohol. chromatographically pure.
4.5 Potassium hydroxide solution. weigh 250 g of solid potassium hydroxide, and
dissolve it with 200 mL of water.
5.7 Balance. accuracy is 0.1 mg.
6 Analytical Steps
6.1 Sample pretreatment
6.1.1 Sample containing starch
Weigh about 5 g of solid sample that has been mixed well or about 50 g of liquid sample
(accurate to 0.1 mg), and put it into a 250 mL Erlenmeyer flask. Add 1g of α-amylase
(4.1). For solid sample, about 50 mL of distilled water at 45～50°C shall be added in to
dissolve it. After it is well mixed, use nitrogen gas to exhaust air from the flask. Insert
the stopper. Place it in a 60°C±2°C incubator (5.6) for 30 min.
6.1.2 Sample free of starch
Weigh about 10 g of solid sample that has been mixed well or about 50 g of liquid sample
(accurate to 0.1 mg), and put it into a 250 mL Erlenmeyer flask. For solid sample, about
50 mL of distilled water at 45°C～50°C shall be added in to dissolve it, mix well.
6.2 Recovery test shall be carried out simultaneously for test of sample with
vitamin D.
6.3 Preparation of test solution
6.3.1 Saponification. Add about 100 mL of ethyl alcohol solution of vitamin C (4.10) in
the above-mentioned sample solution that has been pretreated. After it is well mixed,
add 25 mL of the potassium hydroxide solution (4.5). Well mix. Then add magnetic rod
and fill nitrogen gas for protection. Insert the rubber stopper. Add about 300 mL of water
in a 1000 mL beaker, and place the beaker in a constant temperature magnetic stirrer
(5.3); when the temperature is controlled at 53°C ±2°C, place the triangular flask in a
beaker, after saponification under magnetic force stirring for about 45 min, immediately
take it out and cool it down to room temperature.
6.3.2 Extraction. Transfer all the saponified solution into a 500 mL separating funnel.
Add 100 mL of petroleum ether (4.6) in. Shake it gently. Insert the stopper after exhaust.
Severely vibrate it for about 10 min; let stand for demixing. Place the aqueous phase in
another 500 mL separating funnel. Repeat the above extraction process. Merge the
ether solution, and wash it with distilled water until it becomes neutral. Use anhydrous
sodium sulfate for filter and dehydration. Collect the filtrate in a 500 mL flat-bottomed
flask. After it is evaporated under 40°C±2°C nitrogen gas flow on rotator evaporator and
becomes nearly dry (it is not allowed to make it completely dry), use petroleum ether to
transfer it into a 10 mL volumetric flask, and dilute it to fixed-volume.
6.3.3 Take 2.0 mL of petroleum ether solution from the aforesaid volumetric flask and
put it into cuvette A. Then take 7.0 mL of petroleum ether solution and put it into cuvette
B. Blow-dry the petroleum ether in both cuvettes A and B with nitrogen gas respectively
in a 40°C±2°C of nitrogen evaporator (5.4). Add 5.0 mL of methanol (4.7) in cuvette A
to stir and dissolve the residues. Add 2.0 mL of normal hexane (4.8) in cuvette B to stir
and dissolve the residues. Then centrifuge the cuvettes A and B at 5000 r/min for 10
min; take out, let stand until it cools to room temperature before determination. The
cuvette A is used for determination of vitamin A and E, and the cuvette B is used for
determination of vitamin D.
6.4 Determination
6.4.1 Determination of vitamin A and E
6.4.1.1 Reference condition for chromatography
Chromatographic column. C18 column, 250 mm × 4.6 mm, 5 μm, or chromatographic
column with the same performance.
Mobile phase. methanol (4.7).
Flow rate. 1.0 mL/min.
Wavelength. Vitamin A, 325 nm; vitamin E, 294 nm.
Column temperature. 35°C±1°C.
Injection amount. 20 μL.
6.4.1.2 Drawing of standard curve of vitamin A and E
Draw 0.50 mL, 1.00 mL, 1.50 mL, 2.00 mL, and 2.50 mL of the vitamin A standard stock
solution (4.11.1) in 50 mL brown volumetric flasks respectively; dilute them with ethanol
to graduation; mix well. The concentrations of this series of standard working solutions
are 1.00 μg/mL, 2.00 μg/mL, 3.00 μg/mL, 4.00 μg/mL, and 5.00 μg/mL respectively.
Draw 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL and 5.0 mL of the vitamin E standard stock solution
(4.11.2) in 50 mL brown volumetric flasks respectively; dilute them with ethanol to
graduation; mix well. The concentrations of this series of standard working solutions are
10.0μg/mL, 20.0 μg/mL, 30.0 μg/mL, 40.0 μg/mL, and 50.0 μg/mL respectively. Inject
the standard working solutions of vitamin A and E into the liquid chromatograph (the
chromatogram is shown in Annex B) to get the peak heights (or peak areas). Draw the
standard curves of vitamin A and E respectively, in which the y-axis is peak height (or
peak area), and the x-axis is the concentration of standard working solutions of vitamin
A and E.
6.4.1.3 Determination of test sample of vitamin A and E
Column temperature. 35°C ±1°C.
Injection volume. 100 μL.
6.4.2.2.2 Drawing of standard curve
Draw 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, and 1.00 mL of the standard stock solution
(4.11.3 or 4.11.4) of vitamin D2 (or D3) into 100 mL brown volumetric flasks respectively,
dilute to volume with ethanol, and mix well. The concentrations of this series of standard
working solutions are 0.200μg/mL, 0.400μg/mL, 0.600μg/mL, 0.800μg/mL, and
1.000μg/mL respectively.
Inject the standard working solutions of vitamin D2 (or D3) into the liquid chromatograph
to get the peak height (or peak area) (see annex B). Draw the standard curve, in which
the y-axis is peak height (or peak ...
GB 5413.9-2010 
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Determination of vitamin A, D, E in foods for infants and
young children, milk and milk products
ISSUED ON. MARCH 26, 2010
IMPLEMENTED ON. JUNE 1, 2010
Issued by. Ministry of Health of the People's Republic of China
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Principle ... 4 
4 Reagents and materials ... 4 
5 Apparatuses and instruments ... 5 
6 Analytical Steps ... 6 
7 Expression of analysis results ... 9 
8 Precision ... 10 
9 Others ... 11 
Annex A ... 12 
Annex B ... 14 
Foreword
This Standard replaces GB/T 5413.9 -1997 Milk powder and formula foods for infant and
young children - Determination of vitamin A, D, E content.
This Standard, compared with GB/T 5413.9 -1997, has been modified as follows.
- The antioxidant reagent has been modified from original pyrogallic acid to ascorbic
acid.
- The heating and refluxing in the pretreatment method has been modified as constant
temperature saponification.
- The correction of standard solution has been added.
- The previous single-point quantitative has been modified as the standard curve
method.
- The determination of vitamin D recovery has been supplemented.
- The calculation formula has been modified.
Annex A of this Standard is normative, and Annex B is informative.
The previous edition replaced by this Standard is as follows.
- GB/T 5413.9 -1997.
   
National food safety standard -
Determination of vitamin A, D, E in foods for infants and
young children, milk and milk products
1 Scope
This Standard specifies the method for determination of vitamin A, D, E in foods for infant
and young children, milk and milk products.
This Standard is applicable to the determination of vitamin A, D, E content in foods for
infant and young children, milk and milk products.
2 Normative references
The following referenced documents are indispensable for the application of this
document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
3 Principle
After saponification and extraction with petroleum ether, vitamin A and E is separated
by reverse phase chromatography method, of which the content is determined by
external standard method; vitamin D is purified by normal phase chromatography
method and separated by reverse phase chromatography method, of which the
quantitative is determined by external standard method.
4 Reagents and materials
Unless otherwise specified, all reagents shall be analytical pure or higher graded, and
all water used for the experiment shall be Grade 1 water specified in GB/T 6682.
4.1 α-Diastase. Enzyme activity ≥ 1.5 U/mg.
4.2 Anhydrous sodium sulfate.
4.3 Isopropyl alcohol. chromatographically pure.
4.4 Alcohol. chromatographically pure.
4.5 Potassium hydroxide solution. weigh 250 g of solid potassium hydroxide, and
dissolve it with 200 mL of water.
5.7 Balance. accuracy is 0.1 mg.
6 Analytical Steps
6.1 Sample pretreatment
6.1.1 Sample containing starch
Weigh about 5 g of solid sample that has been mixed well or about 50 g of liquid sample
(accurate to 0.1 mg), and put it into a 250 mL Erlenmeyer flask. Add 1g of α-amylase
(4.1). For solid sample, about 50 mL of distilled water at 45～50°C shall be added in to
dissolve it. After it is well mixed, use nitrogen gas to exhaust air from the flask. Insert
the stopper. Place it in a 60°C±2°C incubator (5.6) for 30 min.
6.1.2 Sample free of starch
Weigh about 10 g of solid sample that has been mixed well or about 50 g of liquid sample
(accurate to 0.1 mg), and put it into a 250 mL Erlenmeyer flask. For solid sample, about
50 mL of distilled water at 45°C～50°C shall be added in to dissolve it, mix well.
6.2 Recovery test shall be carried out simultaneously for test of sample with
vitamin D.
6.3 Preparation of test solution
6.3.1 Saponification. Add about 100 mL of ethyl alcohol solution of vitamin C (4.10) in
the above-mentioned sample solution that has been pretreated. After it is well mixed,
add 25 mL of the potassium hydroxide solution (4.5). Well mix. Then add magnetic rod
and fill nitrogen gas for protection. Insert the rubber stopper. Add about 300 mL of water
in a 1000 mL beaker, and place the beaker in a constant temperature magnetic stirrer
(5.3); when the temperature is controlled at 53°C ±2°C, place the triangular flask in a
beaker, after saponification under magnetic force stirring for about 45 min, immediately
take it out and cool it down to room temperature.
6.3.2 Extraction. Transfer all the saponified solution into a 500 mL separating funnel.
Add 100 mL of petroleum ether (4.6) in. Shake it gently. Insert the stopper after exhaust.
Severely vibrate it for about 10 min; let stand for demixing. Place the aqueous phase in
another 500 mL separating funnel. Repeat the above extraction process. Merge the
ether solution, and wash it with distilled water until it becomes neutral. Use anhydrous
sodium sulfate for filter and dehydration. Collect the filtrate in a 500 mL flat-bottomed
flask. After it is evaporated under 40°C±2°C nitrogen gas flow on rotator evaporator and
becomes nearly dry (it is not allowed to make it completely dry), use petroleum ether to
transfer it into a 10 mL volumetric flask, and dilute it to fixed-volume.
6.3.3 Take 2.0 mL of petroleum ether solution from the aforesaid volumetric fl...