This standard specifies the method for the determination of vitamin A, D and E by liquid chromatography. This standard is applicable to the determination of vitamin A, D and E in various infant formula foods and milk powders. GB/T 5413.9-1997 Determination of vitamin A, D and E in infant formula foods and milk powders GB/T5413.9-1997 Standard download decompression password: www.bzxz.net

GB/T 5413.9—1997
There are many methods for determining vitamin A, D and E, but the most widely used method is high pressure liquid chromatography, which is fast, accurate and avoids mutual interference of the three components.
This standard is proposed by China Light Industry General Association.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting unit of this standard: National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestle (China) Investment Service Co., Ltd. The main drafters of this standard: Yang Jinbao and Wang Yun. 255
National Standard of the People's Republic of China
Infant and young children formula foods and milk powder
Determination of vitamin A, D, E
Milk powder and formula foods for infant and young children-Determination of vitamin A, D, E content1 Scope
This standard specifies the method for determining vitamin A, D, E by liquid chromatography. This standard is applicable to the determination of vitamin A, D, E in various infant and young children formula foods and milk powder. 2 Method Summary
GB/T 5413. 9--1997
Fat-soluble vitamins in the sample are separated from fat during saponification, extracted with petroleum ether, and quantitatively determined by high pressure liquid chromatography and ultraviolet detector.
3 Reagents
All reagents, if the specifications are not specified, are analytically pure; all experimental water, if no other requirements are specified, refers to grade tertiary water. 3.1
Taka-Diastase. 3.2Isopropanol: chromatographically pure.
Ethanol solution of pyrogallic acid: 15 g/L. 3.4
Potassium hydroxide solution: mass ratio is 100:50. 3.5
Petroleum ether: boiling range 30-60°C.
Methanol: chromatographically pure.
3.7N-hexane: chromatographically pure.
3.8Cyclohexane: chromatographically pure.
3.9Standard solutions of vitamin A and E
3.9.1Standard stock solution of vitamin A, containing a methanol solution of 100 μg/mL of vitamin A. Weigh 10 mg of vitamin A and dilute it to a 100 mL volumetric flask with methanol. 3.9.2Standard stock solution of vitamin E, containing a methanol solution of 400 μg/mL of vitamin E. Weigh 40 mg of vitamin E and dilute to a 100 mL volumetric flask with methanol. 3.9.3 Vitamin A and E standard working solutions, where the concentration of vitamin A is 2 μg/mL and the concentration of vitamin E is 20 μg/mL. Take 1 mL of vitamin A standard stock solution (3.9.1) and 2.5 mL of vitamin E standard stock solution (3.9.2) in a 50 mL volumetric flask and dissolve with methanol.
3.10 Vitamin D standard solution
3.10. 1 Vitamin D, standard stock solution, containing vitamin D, 100 μg/mL methanol solution. Weigh 10 mg of vitamin Dz and dilute to a 100 mL volumetric flask with methanol. 3. 10.2 Vitamin D, standard stock solution, containing vitamin D: 100 μg/mL methanol solution. Approved by the State Administration of Technical Supervision on May 28, 1997 256
Implemented on September 1, 1998
GB/T 5413.9-1997
Weigh 10 mg of vitamin D3 and dilute it to a 100 mL volumetric flask with methanol. 3. 10. 3 Vitamin D standard working solution, in which the mass concentrations of vitamin Dz and vitamin D3 are both 1 μg/mL. Take 1 mL of vitamin D standard stock solution and 1 mL of vitamin D standard stock solution in a 100 mL volumetric flask and dilute it to a 100 mL volumetric flask with methanol. 4 Instruments
Common laboratory instruments and:
4.1 Flat-bottom flask: 250 mL.
4.2 Separatory funnel: 500 mL.
4.3 Rotary evaporator.
4.4 High-pressure liquid chromatograph: UV detector with variable wavelength, data processing system or recorder. 5 Operating steps
5.1 Sample processing
5.1.1 Samples containing starch
Accurately weigh 10g sample, place it in a 250mL flat-bottom flask, add 1g Taka amylase (3.1), and then add 30mL 45~~50℃ distilled water. After mixing well, use nitrogen to remove the air in the bottle, cover the bottle with a stopper, and place it in a 45℃ oven for 30min. 5.1.2 Samples without starch
Accurately weigh 10g sample, place it in a 250mL flat-bottom flask, and add 30mL distilled water. 5.2 Preparation of test solution
5.2.1 Add 100mL of gallic acid ethanol solution (3.3) to the treated sample solution, mix thoroughly and add 50mL of potassium hydroxide solution (3.4), reflux continuously on a steam bath for 30min, and immediately cool to room temperature. 5.2.2 Transfer the saponified solution to a 500mL separatory funnel, rinse the flat-bottomed flask with 100mL of water several times, and add the washing solution to the separatory funnel.
5.2.3 Add 100mL of petroleum ether (3.5) to the above separatory funnel, cover the bottle stopper, invert the separatory funnel and shake vigorously for 1min. During the shaking process, pay attention to release the pressure in the bottle. Separate the layers and place the aqueous phase into another 500mL separatory funnel. Repeat the above extraction process twice and combine the ether solution into the first separatory funnel. Wash the ether solution with distilled water until it is neutral, filter and dry it through anhydrous sodium sulfate, evaporate it on a rotary evaporator at 40°C under nitrogen flow until it is nearly dry (never allow it to be evaporated to dryness), then transfer it to a 10mL volumetric flask with petroleum ether and make up the volume. 5.2.4 Take 2mL of petroleum ether solution from the above volumetric flask and put it into test tube A, and then take 7mL of petroleum ether solution and put it into another test tube B. Use nitrogen to blow dry the petroleum ether in test tubes A and B respectively. Put the petroleum ether solution in A and add 5mL of methanol to determine vitamins A and E, and add 1mL of n-hexane to test tube B to determine vitamin D. 5.3 Determination
5.3.1 Determination of vitamins A and E
5.3.1.1 Instrument conditions
Chromatographic column: 25cm×4.6mm, ODS column or chromatographic column with equivalent performance. Mobile phase: methanol.
Flow rate: 1.0mL/min
Sensitivity: 0.005AU/MV.
Wavelength: Vitamin A, 325nm; Vitamin E, 290nm. 5.3.1.2 Inject 50μL of vitamin A and E standards (3.9.3) and 50μL of sample solution (in test tube A in 5.2.4) to obtain the peak height or peak area of ​​vitamin A and E in the standard and sample solutions. 5.3.2 Determination of vitamin D
5.3.2.1 Preparation of determination solution
a) Instrument conditions
Chromatographic column; 30cmX4mm, silica gel column. Www.bzxZ.net
GB/T5413.9—1997
Mobile phase: Cyclohexane (3.7) and n-hexane (3.8) are mixed in a volume ratio of 1:1, and isopropanol (3.2) is added at a volume fraction of 0.8%. Flow rate: 1mL/min.
Wavelength: 265nm.
Column temperature: 20℃.
Sensitivity: 0.005AU/MV.
Injection volume: 200μL.
b) Inject 50μL of vitamin D standard and 200μL of sample solution (in test tube B), collect vitamin D in test tube C according to the retention time of vitamin D standard, blow test tube C dry with nitrogen, and accurately add 0.2mL of methanol (3.6) to dissolve. 5.3.2.2 Determination steps
a) Instrument conditions
Chromatographic column: 25cm×4.6mm, Cig or chromatographic column with equivalent performance. Mobile phase: methanol.
Flow rate: 1mL/min.
Column temperature: 20℃.
Wavelength: 265nm.
Sensitivity: 0.005AU/MU.
Injection volume: 50μL.
b) Inject 50μL of vitamin D standard solution (3.10.3) and 50μL of sample solution (in test tube C) to obtain the peak area or peak height of vitamin D in the standard sample and sample solution.
6 Expression of analysis results
Vitamin A in sample (IU /100g) m×10/2×5×3.33×100m
Vitamin E in sample (IU/100g) = ×10/2×5×100m
Vitamin D in sample (IU/100g) = ×10/75×100×40m × 1 000
Where: c.—
Mass concentration of vitamin A, E or D in the injection solution, ug/mL; m
-mass of the sample, g;
-peak height (or peak area) of vitamin A, E or D in the injection solution; A
-peak height (or peak area) of vitamin A, E or D in the standard sample Ad
-mass concentration of vitamin A, E or D in the standard sample, μg/mL. The calculation result is accurate to the decimal place.
7 Allowable difference
(2)
(4)
The difference between the two measured values ​​of the same sample shall not exceed 5% of the average value of the two measurements for vitamin A; 10% of the average value of the two measurements for vitamin D; and 5% of the average value of the two measurements for vitamin E. 258
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