Title: Raw Material Test Method
Material Name: Vitamin E Acetate 50 % Dry Powder
Generic Name : Vitamin E Acetate
Empirical
Formula : C31H52O3 
Molecular weight : 472.7
Method of Analysis:
1. Appearance
Almost white, yellowish or light brown, crystalline powder.
2. Solubility
Practically insoluble or swells or forms a dispersion in water, depending on the formulation.
3. Identification
A. Thin-layer chromatography
Test solution
To a quantity of concentrate corresponding to 50 mg of a-tocopherol acetate add 5 ml of 0.01 M hydrochloric acid and treat with ultrasound at 60 °C. Add 5 ml of ethanol R and 10 ml of cyclohexane R, shake for 1 min and centrifuge for 5 min. Use the upper layer.
Reference solution
Dissolve 50 mg of a-tocopherol acetate CRS in cyclohexane R and dilute to 10 ml with the same solvent.
Plate TLC silica gel HF254 plate.
Mobile phase Ether R : Cyclohexane (20:80 V/V).
Application 10 µl.
Development Over a path of 15 cm of the plate.
Drying In a current of air.
Detection Examine in ultraviolet light at 254 nm.
Results The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.
B. OPTICAL ROTATION
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 separator, and add 200 mL of water. Extract with ether, first with 75 mL, then with 25 mL, and combine the ether extracts in another separator.
Alpha tocopheryl acid succinate: Transfer a volume of Sample solution for Alpha tocopheryl acid succinate from Identification A, equivalent to 100 mg of alpha tocopheryl acid succinate, to a separator, and add 200 mL of water. Extract with ether, first with 75 mL, then with 25 mL, and combine the ether extracts in another separator.
Analysis
Sample: Use the appropriate Sample solution. To the entire volume of a Sample solution, as prepared
above, 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 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, and determine the optical rotation using c as the number of grams of total tocopherols, determined in the appropriate Assay, in each 100 mL of solution used for the test.
4.0 Related Substance
The thresholds indicated under Related substances (Table 2034.-1) in the general monograph Substances for pharmaceutical use (2034) do not apply.
5. Assay
Chromatographic Method:
Mobile Phase: Prepare a mixture of Methanol and Water (98:2). Filter and degas before use.
Standard preparation
Accurately weigh 0.50 g of vitamin E Acetate WS in 100 ml volumetric flask. Add 60 ml methanol, shake mechanically for 15 minutes and volume with methanol and mix well. Then filter the solution through Whatman No. 1 and finally through 0.2 mm syringe filter.
Sample preparation
Weigh and take about 0.50 g of sample into a 100 ml volumetric flask. Add 60 ml Methanol, shake mechanically for 15 minutes. Then volume with Methanol and mix well. Then filter the solution through Whatman No. 1 and finally through 0.2 mm syringe filter.
Chromatographic system
Column : USP Type L1 (C18, 4.6 x 250 mm)
Temperature : Ambient
Wavelength(l) : 280 nm
Flow rate : 1.5 ml/minute
Injection Vol. : 20 ml
Procedure
Separately inject equal volumes (about 20 µl) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for Vitamin E. Calculate the quantity by using the following formula:
Calculation
Dilution x Std. wt of (g) x Peak area of of sample x Std. Potency (I.U. /g)
Sample wt. (g) x Dilution x Peak area of std.
= I.U/g
Limit: (96.50 – 102.0) % of label claim
1. 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
Procedure:
Dissolve 1.0 g of the test specimen in 25 ml of a mixture of equal volumes of alcohol and ether (which has been neutralized to phenolphtrhalein with 0.1 N Sodium hydroxide), 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 seconds: alpha tocopheryl acid succinate requires between 18.0 and 19.3 ml of 0.10 N Sodium hydroxide; the other forms of vitamin E require not more than 1.0 ml of 0.10 N Sodium hydroxide.
2. Organic volatile impurities
Standard Solution
Prepare a solution, in organic-free water, or the solvent specified in the monograph, containing in each ml, 12.0 µg of methylene chloride, 7.6 µg of 1, 4-dioxane, 1.6 µg of trichloroethylene, and 1.2 µg of chloroform. (Prepare fresh daily) Pipette 5 ml of the solution into a vial fitted with a septum and crimp cap, containing 1 g of anhydrous sodium sulfate, and seal. Heat the sealed vial at 80° for 60 minutes.
Test Solution
Transfer 100 mg, accurately weighed, of the material under test to a vial, add 5.0 ml of water, or the solvent specified in the monograph, and 1 g of anhydrous sodium sulfate, and seal with a septum and crimp cap. Heat the sealed vial at 80° for 60 minutes, or as specified in the individual monograph.
Chromatographic System
Column: 0.53-mm × 30-m fused silica analytical column coated with a 5-µm chemically cross-linked G27 stationary phase a with phenylmethyl siloxane
Carrier gas: Helium
Linear velocity: 35 cm/s
Column oven temperature:
Initially, the column temperature is maintained at 350 C for 5 minutes, and then increased at a rate of 80 C per minute to 1750 C followed by an increase at a rate of 350 C per minute to 2600 C, and maintained at 2600 C for at least 16 minutes.
Injection port temperature: 700C
Detector temperature: 2600C
Detector: Flame ionization detector
Injection volume: 1 µl.
Procedure:
Separately inject equal volumes (about 1 µL) of the Standard Solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses.
Inject the Standard Solution and record the peak responses as directed for Procedure: a suitable system is one that yields chromatograms in which all of the components in the Standard Solution are resolved; the resolution between any two components is not less than 3; and the relative standard deviation of the individual peak responses from replicate injections is not more than 15%.
