B. Determination of vitamin A in fo

B. Determination of vitamin A in foods by high-performance liquid
chromatography
I. References
Horwitz W and GW Latimer (Eds.). AOAC Official Method 2001.13.Vitamin A (Retinol) in
Foods.Vitamins and Other Nutrients.Chapter 45, p.53-56.Official Methods of Analysis of AOAC
International. 18thed, Revision 2, 2007. Maryland.
II. Principle
Standards and samples are saponified in basic ethanol-water solution, neutralized, and diluted. This
process converts fats to fatty acids, and retinyl esters to retinol and the corresponding fatty acids.
Extract clean-up is carried out with a C18 cartridge and vitamin A is concentrated eluting with a
smaller volume of isopropanol than the aliquot taken to clean. Retinol is quantified in an LC system,
using UV detection at 326 nm. Concentration is calculated by comparison of peak heights or peak
areas of retinol in test samples with those of standards.
Recovery of vitamin A in Infant Formula 1849 (NIST) was 99% as measured in the CPHL Laboratory.
III.Critical points and cautions
Due to the labile nature of retinol, it is important to saponify the samples under a nitrogen atmosphere
and in the presence of pyrogallic acid.
Potassium hydroxide is extremely caustic and it can cause severe burns. Protect skin and eyes while
performing this method. This method involves the use of flammable liquids. Perform behind a
barrier when using hot water, steam or an electric heating mantle. Use an effective fume removal
device to remove flammable vapors produced. Leave ample headroom in flask.
Protect samples from light by covering the glassware containing the sample extracts with aluminum
foil or a piece of black cloth, and work under subdue light.
IV. Equipment and materials
− HPLC system
Pump operating continuously at 1.0-2.0 mL/min with a flow precision of ± 1% or better
Injector. A manual injector or autosampling injector with a 100 μL fixed loop having a
typical sampling precision of ±0.25% or better
Reverse-phase C18 column, 5 μm (4.6x250 mm) capable of separating cis and trans isomers
of retinol with a resolution of 1.0 or greater.
Photometric detector monitoring absorbance at 326 nm.
Data collection system or integrator
− Sep-pak Cartridges C18 Vac 3cc (500 mg). Waters.or equivalent.
− Erlenmeyer flasks (125 mL) with neck adapted for connecting reflux condenser
− Hot plate
− Reflux condensers
- 27 -
− Volumetric flasks (100 and 10 mL)
− Nitrogen blanket apparatus8
V. Reagents
− Certified vitamin A acetate concentrate (USP) or
− Retinyl palmitate, all-trans.
− Acetic acid glacial, AR
− Acetonitrile, AR
− Isopropanol, AR
− Methanol, HPLC grade
− Absolute ethanol AR
− Tetrahydrofuran (THF), AR grade
− Hexane (n-Hexane 95% for HPLC)
− Pyrogallic acid, crystal, AR grade
VI.Solutions
a. Mobile phase: Combine 890 mL methanol and 110 mL distilled water. Mix well. Stir
overnight to degas or prior to use.
b. THF-methanol [50+50]: Combine 500 mL tetrahydrofuran and 500 mL 95% ethanol. Mix well.
c. Potassium hydroxide solution-50%: Slowly add 500 g of KOH pellets to 500 mL water
contained in a 2L thick walled Erlenmeyer flask. The solution gives off substantial heat while
KOH is dissolving. Add the KOH in 100 g portions while the flask is being cooled with cold water.
Swirl the flask gently to aid in dissolution of the KOH. Store in glass container with cork stopper.
d. Washing solution-acetonitrile-20% in water: Combine 80 mL water and 20 mLacetonitrile.
Mix well.
e. Vitamin A working standard (ca 5 µg retinol/mL)
1. Using USP standard: Weigh 50 mg retinyl acetate concentrate into a 100-mL volumetric
flask. Record weight to nearest 0.1 mg. Record concentration in mg/g per USP certification.
Add a small amount of acetone (less than 3 mL) to aid dissolution. Dilute to volume with
absolute ethanol. Store at 4°C in dark. Solution is stable for two weeks.
2. Using retinyl palmitate:
Stock solution: Weigh 55 mg retinyl palmitate into 100-mL volumetric flask. Record weight
to nearest 0.1 mg. Record purity per supplier certification or purity test. Add pea-sized piece of
pyrogallic acid. Dissolve and dilute to volume with hexane.
Working solution: Pipet 2 mL solution to second 100-mL flask and dilute to volume with
absolute ethanol. Store at 4°C in dark. Solution is stable for two weeks.
8 A supply of nitrogen gas with appropriate tubing and connectors to provide a constant nitrogen atmosphere blanket in the reflux apparatus during
saponification.
- 28 -
Check the concentration of retinyl palmitate stock solution every time is used. Pipette 2 mL
stock retinyl palmitate solution into a 100-mL volumetric flask and dilute to volume with hexane.
Read the absorbance at the maximum wavelength (325-328 nm) using a 1-cm pathlength cell
and hexane as blank. Calculate the purity of retinol palmitate for the working day as:
Where w = weight to prepare the stock retinyl palmitate in hexane in mg.
Purity of stock solution when a new standard is opened:
Check purity as follows: Dissolve 50 mg (record to nearest 0.1 mg) of retinyl palmitate standard
in 2-propanol (UV-spectroscopy grade) in a 500-mL flask and dilute to volume. Dilute 10 mL
of this solution to 100 mL with 2-propanol (final concentration is approximately 10 mg per
liter). Measure maximum absorbance obtained at 325-328 nm using a 1-cm pathlength cell and
2-propanol as blank. Calculate purity of retinol palmitate as
where Amax = absorbance maximum; (5x106
) = combined dilution factors, conversion to 1%
equivalent solution, and conversion to percent; 960= absorbance of pure retinyl palmitate (1%
solution in 1-cm cell), and w=weight of retinyl palmitate standard in mg

Kết quả (Việt) 1:

B. xác định của vitamin A trong thực phẩm bằng chất lỏng hiệu năng cao sắc kí I. tài liệu tham khảo Horwitz W và GW Latimer (chủ biên). AOAC phương pháp chính thức 2001.13.Vitamin một (Retinol) trong Foods.Vitamins và khác Nutrients.Chapter 45, p.53-56.Official phương pháp của phân tích của AOAC Quốc tế. 18thed, phiên bản 2, 2007. Maryland. II. nguyên tắc Tiêu chuẩn và các mẫu được saponified trong dung dịch nước ethanol cơ bản, vô hiệu hóa, và pha loãng. Điều này quá trình chuyển đổi chất béo để axit béo và retinyl Este retinol và các axit béo tương ứng. Chiết xuất sạch-up được thực hiện với một mực C18 và vitamin A là tập trung eluting với một nhỏ hơn khối lượng của isopropanol hơn aliquot thực hiện để làm sạch. Retinol định lượng trong một hệ thống LC, bằng cách sử dụng UV phát hiện tại 326 nm. Tập trung được tính bằng cách so sánh của đỉnh heights hoặc cao điểm khu vực của retinol trong mẫu thử nghiệm với tiêu chuẩn. Phục hồi của vitamin A trong năm 1849 công thức trẻ sơ sinh (NIST) là 99% tính tại Trung tâm CPHL. III. Điểm quan trọng và cẩn trọng Do bản chất labile của retinol, nó là quan trọng để saponify mẫu dưới một bầu không khí nitơ và sự hiện diện của pyrogallic acid. Kali hydroxit là cực kỳ ăn da và nó có thể gây bỏng nặng. Bảo vệ da và mắt trong khi thực hiện phương pháp này. Phương pháp này liên quan đến việc sử dụng các chất lỏng dễ cháy. Thực hiện đằng sau một rào cản khi sử dụng nước nóng, hơi nước hoặc một lớp phủ điện sưởi ấm. Sử dụng một loại bỏ hiệu quả khói device to remove flammable vapors produced. Leave ample headroom in flask. Protect samples from light by covering the glassware containing the sample extracts with aluminum foil or a piece of black cloth, and work under subdue light. IV. Equipment and materials− HPLC system Pump operating continuously at 1.0-2.0 mL/min with a flow precision of ± 1% or better Injector. A manual injector or autosampling injector with a 100 μL fixed loop having a typical sampling precision of ±0.25% or better Reverse-phase C18 column, 5 μm (4.6x250 mm) capable of separating cis and trans isomers of retinol with a resolution of 1.0 or greater. Photometric detector monitoring absorbance at 326 nm. Data collection system or integrator− Sep-pak Cartridges C18 Vac 3cc (500 mg). Waters.or equivalent.− Erlenmeyer flasks (125 mL) with neck adapted for connecting reflux condenser− Hot plate− Reflux condensers - 27 -− Volumetric flasks (100 and 10 mL)− Nitrogen blanket apparatus8 V. Reagents− Certified vitamin A acetate concentrate (USP) or− Retinyl palmitate, all-trans.− Acetic acid glacial, AR− Acetonitrile, AR− Isopropanol, AR− Methanol, HPLC grade− Absolute ethanol AR− Tetrahydrofuran (THF), AR grade− Hexane (n-Hexane 95% for HPLC)− Pyrogallic acid, crystal, AR grade VI.Solutions a. Mobile phase: Combine 890 mL methanol and 110 mL distilled water. Mix well. Stir overnight to degas or prior to use. b. THF-methanol [50+50]: Combine 500 mL tetrahydrofuran and 500 mL 95% ethanol. Mix well. c. Potassium hydroxide solution-50%: Slowly add 500 g of KOH pellets to 500 mL water contained in a 2L thick walled Erlenmeyer flask. The solution gives off substantial heat while KOH is dissolving. Add the KOH in 100 g portions while the flask is being cooled with cold water. Swirl the flask gently to aid in dissolution of the KOH. Store in glass container with cork stopper. d. Washing solution-acetonitrile-20% in water: Combine 80 mL water and 20 mLacetonitrile. Mix well. e. Vitamin A working standard (ca 5 µg retinol/mL) 1. Using USP standard: Weigh 50 mg retinyl acetate concentrate into a 100-mL volumetric flask. Record weight to nearest 0.1 mg. Record concentration in mg/g per USP certification. Add a small amount of acetone (less than 3 mL) to aid dissolution. Dilute to volume with absolute ethanol. Store at 4°C in dark. Solution is stable for two weeks. 2. Using retinyl palmitate: Stock solution: Weigh 55 mg retinyl palmitate into 100-mL volumetric flask. Record weight to nearest 0.1 mg. Record purity per supplier certification or purity test. Add pea-sized piece of pyrogallic acid. Dissolve and dilute to volume with hexane. Working solution: Pipet 2 mL solution to second 100-mL flask and dilute to volume with absolute ethanol. Store at 4°C in dark. Solution is stable for two weeks. 8 A supply of nitrogen gas with appropriate tubing and connectors to provide a constant nitrogen atmosphere blanket in the reflux apparatus during
saponification.
- 28 -
Check the concentration of retinyl palmitate stock solution every time is used. Pipette 2 mL
stock retinyl palmitate solution into a 100-mL volumetric flask and dilute to volume with hexane.
Read the absorbance at the maximum wavelength (325-328 nm) using a 1-cm pathlength cell
and hexane as blank. Calculate the purity of retinol palmitate for the working day as:
Where w = weight to prepare the stock retinyl palmitate in hexane in mg.
Purity of stock solution when a new standard is opened:
Check purity as follows: Dissolve 50 mg (record to nearest 0.1 mg) of retinyl palmitate standard
in 2-propanol (UV-spectroscopy grade) in a 500-mL flask and dilute to volume. Dilute 10 mL
of this solution to 100 mL with 2-propanol (final concentration is approximately 10 mg per
liter). Measure maximum absorbance obtained at 325-328 nm using a 1-cm pathlength cell and
2-propanol as blank. Calculate purity of retinol palmitate as
where Amax = absorbance maximum; (5x106
) = combined dilution factors, conversion to 1%
equivalent solution, and conversion to percent; 960= absorbance of pure retinyl palmitate (1%
solution in 1-cm cell), and w=weight of retinyl palmitate standard in mg