Vitamin A

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 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.

2 IDENTIFICATION

2.1 A. COLOR REACTION WITH ANTIMONY III

Sample solution: Vitamin A equivalent to 6 µg/mL of retinol in chloroform

Analysis: To 1 mL of the Sample solution add 10 mL of antimony trichloride TS.

Acceptance criteria: A transient blue color appears at once.

2.2 B. THIN-LAYER CHROMATOGRAPHY

Standard solution: USP Retinyl Acetate RS and USP Retinyl Palmitate RS in methylene chloride, each equivalent to about 0.2 mg/mL of retinol (C₂₀H₃₀O)

Sample solution for the liquid form of Vitamin A: Dissolve a volume, equivalent to about 5 mg of retinol, in methylene chloride to obtain 10.0mL of solution.

Sample solution for the solid form of Vitamin A: Transfer a quantity, equivalent to about 5 mg of retinol, to a separator, add 75 mL of water, and shake vigorously for 1 min. Extract with 10.0 mL of methylene chloride by shaking for 1 min, and centrifuge to clarify the methylene chloride extract.

Chromatographic system

(See Chromatography (621), General Procedures. Thin-Layer Chromatography.)

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture

Application volume

Standard solution: 15 µL

Sample solution: 10 µL

Developing solvent system: Cyclohexane and ether (4:1)

Developing distance: 10 cm

Spray reagent: Phosphomolybdic acid TS

System suitability

Sample: Standard solution

Suitability requirements: The chromatogram shows two main blue-green spots of the corresponding esters. The R_F values are 0.45 ± 0.10 and 0.7 ± 0.1 for retinyl acetate and retinyl palmitate, respectively.

Analysis

Samples: Standard solution and the appropriate Sample solution

Allow the solvent front to move a distance of 10 cm, remove the plate, and air-dry. Spray the plate with Spray reagent.

Acceptance criteria: The appropriate Sample solution exhibits a main blue-green spot at an R, value that corresponds to one of the two main spots from the Standard solution and is consistent with the labeled ester. The appropriate Sample solution of Vitamin A labeled as containing retinol exhibits the main blue-green spot at an approximate R, value of 0.1.

3 ASSAY

VITAMIN A ASSAY (571).

Sample: Use a suitable quantity of Vitamin A.

Analysis: Proceed as directed in Vitamin A Assay (571), Assay, Chemical Methods or Chromatographic Methods, depending on the form of Vitamin A under test.

Acceptance criteria: Equivalent to NLT 95.0% of the labeled amount of vitamin A activity

4 SPECIFIC TESTS

ABSORBANCE RATIO

[NOTE-This test is required only if Vitamin A Assay (571), Assay, Chemical Methods is used for the Assay.]

Sample: Use a suitable quantity of Vitamin A.

Analysis: Proceed as directed in Vitamin A Assay (571), Assay, Chemical Methods. Procedure 1 or Procedure 2.

Acceptance criteria

Procedure 1: The ratio of the corrected absorbance [A₃₂₅] to the observed absorbance A₃₂₅ is NLT 0.85. 

Procedure 2: The absorption maximum is between 325 and 327 nm. The absorbance ratios are as follows: A₃₀₀/A₃₂₆ is NMT 0.60; A₃₅₀/A₃₂₆ is NMT 0.54; and A₃₇₀/A₁₂₆ Is NMT 0.14.

5 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers, preferably under an atmosphere of an inert gas, protected from light.

Change to read:

LABELING: Label it to indicate the form in which the vitamin is present, and to indicate the presence of any antimicrobial agent, dispersant, antioxidant, or other added substance, and to indicate the vitamin A activity in terms of the equivalent amount of retinol, in mg/g.^▲_{▲(USP 1-Aug-2019)}

Add the following:

^▲USP REFERENCE STANDARDS (11)

USP Retinyl Acetate RS

USP Retinyl Palmitate RS _{▲(ERR 1-Jan-2019)}