Vitamin A or Retinol

Vitamin A is a fat-soluble vitamin and an essential nutrient for animals. Vitamin A it has multiple functions.

Vitamin A BP Ph Eur Grade Specifications:
In the British Pharmacopoeia, the term ‘Retinol’ is used within titles for preparations containing synthetic ester(s) and the term ‘Vitamin A’ within the title for the preparation containing material of natural origin.

DEFINITION
Vitamin A Refers to a number of substances of very similar structure (including (Z)-isomers) found in animal tissues and possessing similar activity. The principal and biologically most active substance is all-(E)-retinol (all-(E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraen-1-ol; C20H30O). Vitamin A is generally used in the form of esters such as the acetate, propionate and palmitate.
Synthetic retinol ester Refers to an ester (acetate, propionate or palmitate) or a mixture of synthetic retinol esters.
The activity of vitamin A is expressed in retinol equivalents (R.E.). 1 mg R.E. corresponds to the activity of 1 mg of all-(E)-retinol. The activity of the other retinol esters is calculated stoichiometrically, so that 1 mg R.E. of vitamin A corresponds to the activity of:
- 1.147 mg of all-(E)-retinol acetate,
- 1.195 mg of all-(E)-retinol propionate,
- 1.832 mg of all-(E)-retinol palmitate.
International Units (IU) are also used to express the activity of vitamin A. 1 IU of vitamin A is equivalent to the activity of 0.300 µg of all-(E)-retinol. The activity of the other retinol esters is calculated stoichiometrically, so that 1 IU of vitamin A is equivalent to the activity of:
- 0.344 µg of all-(E)-retinol acetate,
- 0.359 µg of all-(E)-retinol propionate,
- 0.550 µg of all-(E)-retinol palmitate,
1 mg of retinol equivalent is equivalent to 3333 IU.

CHARACTERS
Appearance: Retinol acetate: pale-yellow crystals (mp: about 60C). Once melted retinol acetate tends to yield a supercooled melt.
Retinol propionate: reddish-brown oily liquid.
Retinol palmitate: a fat-like, light yellow solid or a yellow oily liquid, if melted (mp: about 26C).
Solubility: All retinol esters are practically insoluble in water, soluble or partly soluble in anhydrous ethanol and miscible with organic solvents.Vitamin A and its esters are very sensitive to the action of air, oxidising agents, acids, light and heat.

IDENTIFICATION

A. Thin-layer chromatography: To pass the test.
B. Related substances: To pass the test.
Retinol: To pass the test by thin-layer chromatography
Related substances: To pass the test by Ultraviolet and visible absorption spectrophotometry.

ACTIVITY
To meet the requirements.

STORAGE
In an airtight container, protected from light. Once the container has been opened, its contents are to be used as soon as possible; any part of the contents not used at once should be protected by an atmosphere of inert gas.

Vitamin A USP Grade Specifications:

DEFINITION
Vitamin A contains a suitable form of retinol (C20H30O; vitamin A alcohol) and possesses vitamin A activity equivalent to NLT 95.0% of that declared on the label. It may consist of retinol or esters of retinol formed from edible fatty acids, principally acetic and palmitic acids. It may be diluted with edible oils; or it may be incorporated in solid, edible carriers, or excipients; and it may contain suitable antimicrobial agents, dispersants, and antioxidants.

IDENTIFICATION
A. Color Reaction with Antimony III
Sample solution: Equivalent to 6 µg/mL of retinol in chloroform.
Analysis: To 1 mL of the Sample solution add 10 mL of antimony trichloride.
Acceptance criteria: A transient blue color appears at once.
B. Thin-Layer Chromatography: To pass the test.
Absorbance Ratio: To meet the requirement.
Packaging and Storage: Preserve in tight containers, preferably under an atmosphere of an inert gas, protected from light.

Vitamin A FCC Food Grade Specifications:
All-trans-Retinol

DESCRIPTION
Vitamin A occurs as a light yellow to red oil that may solidify on refrigeration (liquid form). In solid form it may have the appearance of the diluent that has been added to it. It is a suitable form or derivative of retinol (C20H30O; Vitamin A alcohol). It usually consists of retinol or esters of retinol formed from edible fatty acids, principally acetic and palmitic acids, or mixtures of these. It may be diluted with edible oils, or it may be incorporated in solid edible carriers, extenders, or excipients. It may contain suitable preservatives, dispersants, and antioxidants, providing it is not to be used in foods in which such substances are prohibited. In liquid form it is very soluble in chloroform and in ether, it is soluble in absolute alcohol and in vegetable oils, but it is insoluble in glycerin and in water. In solid form it may be dispersible in water. It is unstable in air and light.
Function: Nutrient.
Identification:
A. Dissolve an amount of sample equivalent to about 6 µg of retinol in 1 mL of chloroform, and add 10 mL of antimony trichloride. A transient blue color appears at once.
B. Assemble an apparatus for Thin-Layer Chromatography, using chromatographic silica gel as the adsorbent and a 4:1 mixture of cyclohexane:ether as the solvent system. Prepare a Standard Solution by dissolving the contents of 1 capsule of USP Vitamin A Reference Standard in sufficient chloroform to make 25.0 mL.

If the sample is in liquid form, dissolve a volume representing approximately 15,000 International Units in sufficient chloroform to make 10 mL of Sample Solution. If the sample is in solid form, weigh a quantity representing approximately 15,000 International Units, place in a separator, add 75 mL of water, heat, if necessary, to dissolve the carrier, and cool. Shake vigorously for 1 min, extract with 10 mL of chloroform by shaking for 1 min, and centrifuge to clarify the chloroform extract.
At the starting point of the chromatogram, apply 0.015 mL of the Standard Solution and 0.01 mL of the Sample Solution. Develop the chromatogram in the chromatographic chamber lined with filter paper dipping into the solvent mixture. When the solvent has ascended 10 cm, remove the plate, allow it to dry in air, and spray it with antimony trichloride. The blue spot formed is indicative of the presence of retinol. The approximate Rf values of the predominant spots, corresponding to the different forms of retinol, are 0.1 for the alcohol, 0.45 for the acetate, and 0.7 for the palmitate.

Assay: Not less than 95.0% and not more than 100.5% of the Vitamin A activity declared on the label.
Absorbance Ratio: (corrected/observed at 325 nm) Not less than 0.85.
Lead: Not more than 2 mg/kg.