Vitamin E Preparation

DEFINITION

Vitamin E Preparation is a combination of a single form of Vitamin E with one or more inert substances. It may be in a liquid or solid form. It contains NLT 95.0% and NMT 120.0% of the labeled amount of Vitamin E. Vitamin E Preparation labeled to contain a dl-form of Vitamin E may contain also a small amount of a d-form of Vitamin E, occurring as a minor constituent of an added substance.

IDENTIFICATION

[Note—Use low-actinic glassware. ]

• A.

Sample solution: Transfer an amount of Preparation, 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. [Caution—Wear safety goggles. ]

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 separator, rinsing the flask with 100 mL each of water and ether, and adding the rinsings to the separator. 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 dehydrated alcohol, transfer to a 250-mL volumetric flask, and dilute with dehydrated alcohol to volume.

Analysis: 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.

• B. Optical Rotation

781

Sample solution: A volume of the Sample solution from Identification test A., equivalent to 100 mg of Preparation

Analysis: Transfer the Sample solution to a separator, and add 200 mL of water. Extract first with 75 mL, then with 25 mL, of ether, and combine the ether extracts in another separator. To the combined ether extracts, add 20 mL of a solution (1 in 10) of potassium ferricyanide 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 about 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 isooctane, and determine the optical rotation using c as the number of g of total tocopherols, as determined in the Assay, in each 100 mL of solution used for the test.

Acceptance criteria: The d-isomers have a specific rotation of NLT +24

. The dl-forms show essentially no optical rotation.

• C. The retention time of the major peak of the Sample solution corresponds that of the Standard solution, as obtained in the Assay.

ASSAY

• Alpha Tocopherol

[Note—Use low-actinic glassware. ]

Internal standard solution: 1 mg/mL of hexadecyl hexadecanoate in n-hexane

Standard solution: 1 mg/mL of the USP Alpha Tocopherol RS in Internal standard solution

Sample solution

For the Preparation in liquid form: Transfer a portion of Preparation, equivalent to 50 mg of alpha tocopherol, to a 50-mL volumetric flask, and dilute with Internal standard solution to volume.

For the Preparation in solid form: Transfer a portion of Preparation, equivalent to 50 mg of alpha tocopherol, into a flask suitable for refluxing. Add 5 mL of water, and heat on a water bath at 60

for 10 min. Add 25 mL of alcohol, and reflux for 30 min. Cool, and transfer to a separator with the aid of 50 mL of water and 50 mL of ether. Shake vigorously, allow the layers to separate, and collect each layer in individual separators. Extract the aqueous layer with two 25-mL portions of ether, combining the extracts with the original ether layer. Wash the combined extract with one 25-mL portion of water, filter the ether solutions through 1 g of anhydrous sodium sulfate, and with the aid of a stream of nitrogen evaporate the ether solution on a water bath, controlled at a temperature that will not cause the ether solution to boil over. Remove the container from the water bath when 5 mL remains, and complete the evaporation without the application of heat. Dissolve the residue in 50.0 mL of Internal standard solution.

Interference check solution

For the Preparation in liquid form: Proceed as directed for the liquid form Sample solution, except to dissolve the sample in 50.0 mL of n-hexane.

For the Preparation in solid form: Proceed as directed for the solid form Sample solution, except to dissolve the residue in 50.0 mL of n-hexane.

System suitability solution: 1 mg/mL each of USP Alpha Tocopherol RS and USP Alpha Tocopheryl Acetate RS in n-hexane

Chromatographic system

(See Chromatography

621

, System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 4-mm × 2-m borosilicate glass column packed with 2%–5% liquid phase G2; 80- to 100-mesh support S1AB, utilizing either a glass-lined sample introduction system or on-column injection

Temperature

Column: 245

–265

, isothermal

Injector port: 10

higher than the column temperature

Detector: 10

higher than the column temperature

Flow rate: The flow rate of dry carrier gas is adjusted to obtain a hexadecyl hexadecanoate peak approximately 18–20 min after sample introduction when a 2% liquid phase column is used, or 30–32 min when a 5% liquid phase column is used.

Injection size: 2–5 µL

System suitability

Sample: System suitability solution

Suitability requirements

Resolution: NLT 1.0 between alpha tocopherol and alpha tocopheryl acetate

Relative standard deviation: NMT 2.0%

Interference check

Samples: Interference check solution and Internal standard solution

Chromatograph the Interference check solution to obtain a chromatogram in which the principal peak exhibits NLT 50% of maximum recorder response. Similarly chromatograph the Internal standard solution. If a peak observed in the Interference check solution has the same retention time as that for hexadecyl hexadecanoate, make any necessary correction for factors of dilution or attenuation, and determine the area due to the interfering component that must be subtracted from the area of the internal standard peak observed in the Sample solution.

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of alpha tocopherol in the portion of Vitamin E Preparation taken:

Result = (RU/RS) × (CS/CU) × 100

RU	=	= peak response ratio of alpha tocopherol to the internal standard from the Sample solution
RS	=	= peak response ratio of alpha tocopherol to the internal standard from the Standard solution
CS	=	= concentration of USP Alpha Tocopherol RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of alpha tocopherol in the Sample solution (mg/mL)

Acceptance criteria: 95.0%–120.0%

• Alpha Tocopheryl Acetate

Proceed as directed in the Assay for Alpha Tocopherol. For the Standard solution and Sample solution, substitute alpha tocopheryl acetate for alpha tocopherol, and substitute USP Alpha Tocopheryl Acetate RS for USP Alpha Tocopherol RS.

Acceptance criteria: 95.0%–120.0%

• Alpha Tocopheryl Acid Succinate

Proceed as directed in the Assay for Alpha Tocopherol. For the Standard solution and Sample solution, substitute alpha tocopheryl acid succinate for alpha tocopherol, and substitute USP Alpha Tocopheryl Acid Succinate RS for USP Alpha Tocopherol RS.

Acceptance criteria: 95.0%–120.0%

SPECIFIC TESTS

• Acidity (for liquid forms of Vitamin E Preparation)

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

Sample solution: Dissolve 1 g of Preparation in 25 mL of Diluent.

Analysis: To 25 mL of Sample solution, 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: NMT 1.0 mL of 0.10 N sodium hydroxide is required.

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in tight containers, protected from light. Protect Preparation containing d- or dl-alpha tocopherol with a blanket of inert gas.

• Labeling: Label it to indicate the chemical form of Vitamin E present, and to indicate whether the d- or the dl-form is present, excluding any different forms that may be introduced as a minor constituent of the vehicle. Designate the quantity of Vitamin E present.

• USP Reference Standards

USP Alpha Tocopherol RS

USP Alpha Tocopheryl Acetate RS

USP Alpha Tocopheryl Acid Succinate RS

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Huy T. Dinh, M.S. Scientific Liaison 1-301-816-8594	(DS2010) Monographs - Dietary Supplements
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org

USP35–NF30 Page 5033

Chromatographic Column—

VITAMIN E PREPARATION

Chromatographic columns text is not derived from, and not part of, USP 35 or NF 30.