manual of methods for determining micronutrients in fortified foods

III. Methods to determine vitamin A in wheat flour
Vitamin A is the generic name applied to a group of fat soluble compounds that have the biological
activity of all-trans-retinol and includes: retinol (alcohol), retinal (aldehyde), retinoic acid (carboxylic
acid), and pro-vitamin A carotenoids such as ß-carotene. Retinol and its related compounds consist of
four isoprenoid units joined head to tail, and contain five conjugated double bonds.
Pro-vitamin A carotenoids are found in fruits and vegetables, as well as in egg yolk, and they are
transformed in the organism to all-trans-retinol. Retinol is referred to as preformed vitamin A and
it is found in liver, milk, butter, cheese, eggs and fish liver oils, mainly esterified with fatty acids.
Retinyl palmitate and retinyl acetate are the two main retinyl esters used in food fortification. The vit. A
compound for flour fortification is usually embedded into a protecting matrix that contains starches and
antioxidants to provide stability and dispersibility in water.
General properties
Retinol
• Formula: C 20
H 30
O
• Mol wt: mol wt. 286.45 g/mol
E 1% 1cm
1835 ; 2-propanol:
E 1% 1cm
1824
• UV max.: Ethanol: 324-325 nm.
• Solubility: Soluble in absolute ethanol, methanol, chloroform, ether, fats and oils.
• Exhibits a yellow-green fluorescence (emission: 470 nm) when irradiated with UV light at 325 nm
(excitation wavelength)
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Retinyl acetate
• Formula: C 22
H 32
O 2
.
• Mol wt: 328.49 g/mol
• Pale yellow prismatic crystals. Melting point: 57-58 ºC
• UV max: Ethanol 325 nm.
E 1% 1cm
1550 ; 2-propanol:
Retinyl palmitate
• Formula: C 36
H 60
O 2
.
• Mol wt: 524.86 € g/mol.
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• Morphous or crystaline. Melting point: 28-29 ºC.
• UV max: Ethanol, 325-328 nm, E 1% 1cm
975 ; 2-propanol:
E 1% 1cm
1523
E 1% 1cm
953
• All retinoids are susceptible to isomerization and oxidation when exposed to light, oxygen, reactive
metals and elevated temperatures
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1 IU Vitamin A = 0.3 µg retinol
Retinol and its esters react with trifluoroacetic and trichloroacetic acids to form a blue color in anhydrous
solvents such as chloroform, dichloromethane, or heptane. This reaction is the principle of the qualitative
method to determine vitamin A in wheat flour. Quantitative determination of retinol is carried out
injecting the sample extract in a high-performance liquid chromatographer (HPLC) after the sample
has been saponified, and retinol has been extracted from the matrix. The manual presents two methods:
• Qualitative method to determine vitamin A in wheat flour
• Quantitative method to determine vitamin A in foods by high-performance liquid chromatography
(HPLC)
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