Vitamin E

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 E is a form of alpha tocopherol (C₂₉H₅₀O₂). It includes the following: RRR- or all-rac-alpha tocopherol (C₂₉H₅₀O₂), RRR- or all-rac-alpha tocopheryl acetate (C₃₁H₅₂O₃), and RRR- or all-rac-alpha tocopheryl acid succinate (C₃₃H₅₄O₅). It contains NLT 96.0% and NMT 102.0% of RRR-or all-rac-alpha tocopherol, RRR- or all-rac-alpha tocopheryl acetate, or RRR- or all-rac-alpha tocopheryl acid succinate.

2 IDENTIFICATION

2.1 A.

[NOTE-Use low-actinic glassware.]

Sample solutions

Alpha tocopherol: 1 mg/mL in dehydrated alcohol

Alpha tocopheryl acetate: Transfer 220 mg of RRR- or all-rac-alpha tocopheryl acetate to a round-bottom, glass-stoppered, 150-mL flask, and dissolve in 25 mL of dehydrated alcohol. Add 20 mL of diluted sulfuric acid in alcohol (1 in 7), and reflux in an all-glass apparatus for 3 h, protected from sunlight. Cool, transfer to a 200-mL volumetric flask, and add diluted sulfuric acid in alcohol (1 in 72) to volume.

Alpha tocopheryl acid succinate: [CAUTION-Wear safety goggles.] Transfer an amount of the sample, equivalent to 200 mg of alpha tocopherol, to a round-bottom, glass-stoppered, 250-mL flask; dissolve in 50 mL of dehydrated alcohol; and reflux for 1 min. While the solution is boiling, add, through the condenser, 1 g of potassium hydroxide pellets one at a time to avoid overheating.

Continue refluxing for 20 min and, without cooling, add 2 mL of hydrochloric acid dropwise through the condenser. [NOTE-This technique is essential to prevent oxidative action by air while the sample is in an alkaline medium.]

Cool, and transfer the contents of the flask to a 500-mL separatory funnel, rinsing the flask with 100 mL each of water and ether, and adding the rinsings to the separatory funnel. Shake vigorously, allow the layers to separate, and collect each of the two layers in individual separators. Extract the aqueous layer with two 50-mL portions of ether and add these extracts to the main ether extract. Wash the combined ether extracts with four 100-ml, portions of water, then evaporate the ether solution on a water bath under reduced pressure or in an atmosphere of nitrogen until about 7-8 mL remain. Complete the evaporation, removing the last traces of ether without the application of heat. Immediately dissolve the residue in diluted sulfuric acid in alcohol (1 in 72), transfer to a 200-mL volumetric flask, and dilute with the alcoholic sulfuric acid to volume.

Analysis

Sample: Use the appropriate Sample solution.

To 10 mL of the Sample solution add 2 mL of nitric acid, with swirling, and heat at about 75° for 15 min.

Acceptance criteria: A bright red or orange color develops.

Change to read:

2.2 B. ▲OPTICAL ROTATION (781S), PROCEDURES, SPECIFIC ROTATION ▲(USP 1-MAY-2024)

Sample solutions

Alpha tocopherol: Dissolve 100 mg of alpha tocopherol in 50 mL of ether.

Alpha tocopheryl acetate: Transfer a volume of the Sample solution for Alpha tocopheryl acetate from Identification A, equivalent to 100 mg of alpha tocopheryl acetate, to a separatory funnel, and add 200 ml of water. Extract with ether, first with 75 mL, then with 25 mL, and combine the ether extracts in another separatory funnel.

Alpha tocopheryl acid succinate: Transfer a volume of the Sample solution for Alpha tocopheryl acid succinate from Identification A, equivalent to 100 mg of alpha tocopheryl acid succinate, to a separatory funnel, and add 200 mL of water. Extract with ether, first with 75 mL, then with 25 mL, and combine the ether extracts in another separatory funnel.

Analysis

Sample: Use the appropriate Sample solution.

To the entire volume of a Sample solution ^▲_{▲(USP 1-May-2024)} add 20 mL of a solution (1 in 10) of potassium ferricvanide in sodium hydroxide solution (1 in 125), and shake for 3 min. Wash the ether solution with four 50-mL portions of water, discard the washings, and dry over anhydrous sodium sulfate. Evaporate the dried ether solution on a water bath under reduced pressure or in an atmosphere of nitrogen until 7-8 mL remain, then complete the evaporation, removing the last traces of ether without the application of heat. Immediately dissolve the residue in 5.0 mL of 2.2.4-trimethylpentane, transfer into a sample cell, and record the observed rotation in degrees (°). For RRR-isomers, calculate the specific rotation using c as the concentration of alpha tocopherol determined in the appropriate Assay._{▲(USP 1-May-2024)}

Acceptance criteria

RRR-Isomers: NLT +24°

all-rac Forms: -0.01° to +0.01

Change to read:

2.3 C.

The retention time of the major peak ^▲_{▲(USP 1-May-2024)} of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 ALPHA TOCOPHEROL

[NOTE-Use low-actinic glassware.]

Internal standard solution: 10 mg/mL of squalane in cyclohexane

System suitability solution: 0.1 mg/mL each of USP Alpha Tocopherol RS and USP Alpha Tocopheryl Acetate RS in cyclohexane

Standard solution: 10 mg/mL of USP Alpha Tocopherol RS in Internal standard solution

Sample solution: 10 mg/mL of Vitamin E (RRR- or all-rac-alpha tocopherol) in Internal standard solution

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 0.25 - mm x 30 - m fused silica capillary; bonded with a 0.25-µm film of phase G2

Temperatures

Injection port: 290°

Column: 280°

Detector: 290°

Carrier gas: Helium

Flow rate: 1 mL/min

Injection type: Split, split ratio 100:1

Injection volume: 1 µL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 3.5 between alpha tocopherol and alpha tocopheryl acetate, System suitability solution

Relative standard deviation: NMT 2.0% for the peak response ratios of alpha tocopherol to the internal standard from replicate injections, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of RRR- or all-rac-alpha tocopherol (C₂₉H₅₀O₂) in the portion of Vitamin E taken:

                         Result = (R_U/R_S) × (C_S/C_U) × 100

R_U = peak response ratio of alpha tocopherol to the internal standard from the Sample solution

R_S = peak response ratio of alpha tocopherol to the internal standard from the Standard solution 

C_S = concentration of USP Alpha Tocopherol RS in the Standard solution (mg/mL)

C_U = concentration of Vitamin E in the Sample solution (mg/mL)

Acceptance criteria: 96.0%-102.0% of RRR- or all-rac-alpha tocopherol (C₂₉H₅₀O) 

3.2 ALPHA TOCOPHERYL ACETATE

[NOTE-Use low-actinic glassware.]

Internal standard solution, System suitability solution, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay for Alpha Tocopherol except as follows. For the Standard solution, Sample solution, and Relative standard deviation, substitute alpha tocopheryl acetate for alpha tocopherol, and substitute USP Alpha Tocopheryl Acetate RS for USP Alpha Tocopherol RS.

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of RRR- or all-rac-alpha tocopheryl acetate (C₃₁H₅₂O₃) in the portion of Vitamin E taken: 31 52

                         Result = (R_U/R_S) × (C_S/C_U) × 100

R_U = peak response ratio of alpha tocopheryl acetate to the internal standard from the Sample solution

R_S = peak response ratio of alpha tocopheryl acetate to the internal standard from the Standard solution

C_S = concentration of USP Alpha Tocopheryl Acetate RS in the Standard solution (mg/mL)

C_U = concentration of Vitamin E in the Sample solution (mg/mL)

Acceptance criteria: 96.0%-102.0% of RRR- or all-rac-alpha tocopheryl acetate (CHO)

3.3 ALPHA TOCOPHERYL ACID SUCCINATE

[NOTE-Use low-actinic glassware.]

Internal standard solution, System suitability solution, Chromatographic system, and System suitability: Proceed as directed in the Assay for Alpha Tocopherol except as follows. For the Relative standard deviation, substitute alpha tocopheryl acid succinate for alpha tocopherol.

Standard solution: Transfer 30.0 mg of USP Alpha Tocopheryl Acid Succinate RS into a 20-mL vial. Add 2.0 mL of methanol, 1.0 mL of 2,2-dimethoxypropane, and 0.1 mL of hydrochloric acid to the vial. Cap tightly and sonicate. Allow to stand in the dark for 1 h ± 5 min. Remove from the dark, uncap, and evaporate just to dryness on a steam bath with the aid of a stream of nitrogen. Add 3.0 mL of the Internal standard solution and mix on a vortex mixer to dissolve.

Sample solution: Transfer 30.0 mg of Vitamin E (RRR- or all-rac-alpha tocopheryl acid succinate) into a 20-mL vial. Add 2.0 mL of methanol, 1.0 mL of 2,2-dimethoxypropane, and 0.1 mL of hydrochloric acid to the vial. Cap tightly and sonicate. Allow to stand in the dark for 1 h ± 5 min. Remove from the dark, uncap, and evaporate just to dryness on a steam bath with the aid of a stream of nitrogen. Add 3.0 mL of the Internal standard solution and mix on a vortex mixer to dissolve.

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of RRR- or all-rac-alpha tocopheryl acid succinate (C₃₃H₅₄O₅) in the portion of  Vitamin E taken:

                         Result = (R_U/R_S) × (C_S/C_U) × 100

R_U =  peak response ratio of alpha tocopheryl acid succinate to the internal standard from the Sample solution

R_S = peak response ratio of alpha tocopheryl acid succinate to the internal standard from the Standard solution S

C_S = concentration of USP Alpha Tocopheryl Acid Succinate RS in the Standard solution (mg/mL) 

C_U = concentration of Vitamin E in the Sample solution (mg/mL)

Acceptance criteria: 96.0%-102.0% of RRR- or all-rac-alpha tocopheryl acid succinate (C₃₃H₅₄O₅)

4 SPECIFIC TESTS

ACIDITY

Diluent: Alcohol and ether (1:1), neutralized to phenolphthalein with 0.1 N sodium hydroxide

Sample: 1.0 g

Analysis: Dissolve the Sample in 25 mL of Diluent, add 0.5 mL of phenolphthalein TS, and titrate with 0.10 N sodium hydroxide until the solution remains faintly pink after shaking for 30 s.

Acceptance criteria: Alpha tocopheryl acid succinate requires 18.0-19.3 mL of 0.10 N sodium hydroxide; the other forms of Vitamin E require NMT 1.0 mL of 0.10 N sodium hydroxide.

5 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers, protected from light. Protect RRR- or all-rac-alpha tocopherol with a blanket of an inert gas, and store at room temperature.

Change to read:

LABELING: Label Vitamin E to indicate the chemical form and to indicate whether it is the RRR or the all-rac form. Express Vitamin E content in terms of alpha tocopherol equivalent in mg/g._{▲(USP 1-MAY-2024)} ¹

USP REFERENCE STANDARDS (11)

USP Alpha Tocopherol RS

USP Alpha Tocopheryl Acetate RS

USP Alpha Tocopheryl Acid Succinate RS

^▲1 1 mg of vitamin E (alpha tocopherol) = 1 mg of RRR-alpha tocopherol = 2 mg of all-rac-alpha tocopherol; 1 mg of RRR-alpha tocopheryl acetate = 0.91 mg of alpha tocopherol equivalent; 1 mg of RRR-alpha tocopheryl acid succinate 0.81 mg of alpha-tocopherol equivalent. To convert IU to mg: 1 IU of RRR-alpha tocopherol = 0.67 mg of alpha tocopherol; 1 IU of all-rac-alpha tocopherol = 0.45 mg of alpha tocopherol._{(USP 1-MAY-2024)​​​​​​​}