Test solution for alpha tocopheryl acetate— [NOTE—Use low-actinic glassware.]Transfer about 220mg of d-or dl-alpha tocopheryl acetate,accurately weighed,to a round-bottom,glass-stoppered,150-mLflask,and dissolve in 25mLof dehydrated alcohol.Add 20mLof dilute sulfuric acid in alcohol (1in 7),and reflux in an all-glass apparatus for 3hours,protected from sunlight.Cool,transfer to a 200-mLvolumetric flask,add dilute sulfuric acid in alcohol (1in 72)to volume,and mix.

Test solution for alpha tocopheryl acid succinate— [NOTE—Use low-actinic glassware.]Transfer an accurately weighed amount of the sample,equivalent to about 200mg of alpha tocopherol,to a round-bottom,glass-stoppered,250-mLflask,dissolve in 50mLof dehydrated alcohol,and reflux for 1minute.While the solution is boiling,add,through the condenser,1g of potassium hydroxide pellets,one at a time to avoid overheating.[Caution—Wear safety goggles. ]Continue refluxing for 20minutes and,without cooling,add 2mLof 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-mLseparator,rinsing the flask with 100mLeach of water and of 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-mLportions of ether,and add these extracts to the main ether extract.Wash the combined ether extracts with four 100-mLportions of water,then evaporate the ether solution on a water bath under reduced pressure or in an atmosphere of nitrogen until about 7or 8mLremain.Complete the evaporation,removing the last traces of ether without the application of heat.Immediately dissolve the residue in dilute sulfuric acid in alcohol (1in 72),transfer to a 200-mLvolumetric flask,dilute with the alcoholic sulfuric acid to volume,and mix.

A: Prepare a solution in dehydrated alcohol containing 10mg of unesterified alpha tocopherol in 10mL,or use 10mLof Test solution for alpha tocopheryl acetateor of Test solution for alpha tocopheryl acid succinate.Add,with swirling,2mLof nitric acid,and heat at about 75for 15minutes:a bright red or orange color develops.

B: Prepare a solution of about 100mg,accurately weighed,of unesterified alpha tocopherol in 50mLof ether,or in the case of esterified d-tocopherols,transfer an accurately measured volume of Test solution for alpha tocopheryl acetateor of Test solution for alpha tocopheryl acid succinate,equivalent to about 100mg of the test specimen,to a separator,and add 200mLof water.Extract first with 75mL,then with 25mL,of ether,and combine the ether extracts in another separator.To the ether solution of unesterified or hydrolyzed alpha tocopherol,add 20mLof a 1in 10solution of potassium ferricyanide in sodium hydroxide solution (1in 125),and shake for 3minutes.Wash the ether solution with four 50-mLportions 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 7or 8mLremain,then complete the evaporation,removing the last traces of ether without the application of heat.Immediately dissolve the residue in 5.0mLof isooctane,and determine the optical rotation.Calculate the specific rotation (see Optical Rotation á781ñ),using as cthe number of g of total tocopherols,determined in the Assay,in each 100mLof solution employed for the test:the d-isomers have a specific rotation of not less than +24.The dl-forms show essentially no optical rotation.

C: The retention time of the major peak in the chromatogram of the Assay preparationis the same as that of the Standard preparation,both relative to the internal standard,as obtained in the Assay.

Internal standard solution— Dissolve an accurately weighed quantity of hexadecyl hexadecanoate in n-hexane to obtain a solution having a known concentration of about 1mg per mL.

Standard preparation— [NOTE—Use low-actinic glassware.]Dissolve in Internal standard solutiona suitable quantity of USP Alpha Tocopherol RS,accurately weighed,to obtain a solution having a known concentration of about 1mg of the Reference Standard in each mL.

Assay preparation— [NOTE—Use low-actinic glassware.]Transfer about 50mg of Vitamin E(d-or dl-alpha tocopherol),accurately weighed,to a 50-mLvolumetric flask,dissolve in Internal standard solution,dilute with Internal standard solutionto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—Under typical conditions,the instrument is equipped with a flame-ionization detector and contains a 4-mm ×2-m borosilicate glass column packed with 2%to 5%liquid phase G2on 80-to 100-mesh support S1AButilizing either a glass-lined sample introduction system or on-column injection.The column is maintained isothermally at a temperature between 245and 265,and the injection port and detector block are maintained at about 10higher than the column temperature;the flow rate of dry carrier gas is adjusted to obtain a hexadecyl hexadecanoate peak approximately 18to 20minutes after sample introduction when a 2%column is used,or 30to 32minutes when a 5%column is used.[NOTE—Cure and condition the column as necessary (see Chromatography á621ñ).]

Interference check— Dissolve an accurately weighed quantity of the specimen in n-hexane to obtain a solution having a known concentration of about 1mg per mL.Chromatograph an accurately measured volume of this solution to obtain a chromatogram in which the principal peak exhibits not less than 50%of maximum recorder response.Similarly chromatograph an accurately measured volume of Internal standard solution.If a peak observed in the chromatogram for the specimen 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 appearing in the chromatogram recorded for the Assay preparationas directed under Procedure.

System suitability— Chromatograph a sufficient number of injections of a mixture,in n-hexane,of 1mg per mLeach of USP Alpha Tocopherol RSand USP Alpha Tocopheryl Acetate RSas directed for Procedureto ensure that the resolution factor,R(see Chromatography á621ñ),is not less than 1.0.

Calibration— Chromatograph a portion of the Standard preparation,and record peak areas as directed under Procedure.Calculate the relative response factor,F,for the Standard preparationtaken by the formula: in which CDand CSare the concentrations,in mg per mL,of hexadecyl hexadecanoate and of USP Alpha Tocopherol RS,respectively,in the Standard preparation.Successively chromatograph a sufficient number of portions of the Standard preparationto ensure that the relative response factor,F,is constant within a range of 2.0%.

Procedure— Inject a suitable portion (2to 5µL)of the Assay preparationinto a suitable gas chromatograph,and record the chromatogram so as to obtain at least 50%of maximum recorder response.Measure the areas under the first (alpha tocopherol)and second major (hexadecyl hexadecanoate)peaks,record the values as aUand aD,respectively.Calculate the quantity,in mg,of alpha tocopherol in the Vitamin Etaken by the formula: in which CDis the concentration,in mg per mL,of hexadecyl hexadecanoate in the Standard preparation;and Fis the relative response factor (see Calibration).

NOTE—Chromatograms obtained as directed in the foregoing Assays exhibit relative retention times of approximately 0.53for alpha tocopherol,0.62for alpha tocopheryl acetate,0.54for alpha tocopheryl acid succinate,and 1.0for hexadecyl hexadecanoate.