á571ñVITAMIN A ASSAY
The following procedure is provided for the determination of vitamin Aas an ingredient of Pharmacopeial preparations.It conforms to that which was adopted in 1956for international use by the International Union of Pure and Applied Chemistry.
Complete the assay promptly,and exercise care throughout the procedure to keep to a minimum the exposure to actinic light and to atmospheric oxygen and other oxidizing agents,preferably,by the use of low-actinic glassware and an atmosphere of an inert gas.

Special Reagents—
ETHER— Use ethyl ether,and use it within 24hours after opening the container.
ISOPROPYL ALCOHOL— Use spectrophotometric-grade isopropyl alcohol (see Isopropyl Alcoholunder Reagent Specificationsin the section Reagents,Indicators,and Solutions).

Procedure—
Accurately weigh,count,or measure a portion of the test specimen expected to contain the equivalent of not less than 0.15mg of retinol but containing not more than 1g of fat.If in the form of capsules,tablets,or other solid,so that it cannot be saponified efficiently by the ensuing instructions,reflux the portion taken in 10mLof water on a steam bath for about 10minutes,crush the remaining solid with a blunt glass rod,and warm for about 5minutes longer.
Transfer to a suitable borosilicate glass flask,and add 30mLof alcohol,followed by 3mLof potassium hydroxide solution (9in 10).Reflux in an all-borosilicate glass apparatus for 30minutes.Cool the solution,add 30mLof water,and transfer to a conical separator.Add 4g of finely powdered sodium sulfate decahydrate.Extract by shaking with one 150-mLportion of ether for 2minutes,and then,if an emulsion forms,with three 25-mLportions of ether.Combine the ether extracts,if necessary,and wash by swirling gently with 50mLof water.Repeat the washing more vigorously with three additional 50-mLportions of water.Transfer the washed ether extract to a 250-mLvolumetric flask,add ether to volume,and mix.
Evaporate a 25.0-mLportion of the ether extract to about 5mL.Without applying heat and with the aid of a stream of inert gas or vacuum,continue the evaporation to about 3mL.Dissolve the residue in sufficient isopropyl alcohol to give an expected concentration of the equivalent to 3µg to 5µg of vitamin Aper mLor such that it will give an absorbance in the range 0.5to 0.8at 325nm.Determine the absorbances of the resulting solution at the wavelengths 310nm,325nm,and 334nm,with a suitable spectrophotometer fitted with matched quartz cells,using isopropyl alcohol as the blank.
WHEN TOCOPHEROL IS PRESENT— Transfer to a suitable borosilicate glass flask a test specimen,accurately measured,or not less than 5previously crushed capsules or tablets.Reflux in an all-borosilicate glass apparatus with 30mLof alcohol and 3mLof potassium hydroxide solution (9in 10)for 30minutes.Add through the condenser 2.0g of citric acid monohydrate,washing the walls of the condenser with 10mLof water.Cool,and transfer the solution to a conical separator with the aid of 20mLof water.Add 4g of finely powdered sodium sulfate decahydrate.Extract with one 150-mLportion of ether and then,if an emulsion forms,with three 25-mLportions of ether.Combine the ether extracts,if necessary,and wash by swirling gently with 50mLof water.Repeat the washing more vigorously with three additional 50-mLportions of water.Transfer the washed ether extract to a 250-mLvolumetric flask,and add ether to volume.Transfer a 100.0-mLaliquot of the resulting ether solution to a conical separator,and wash once with 50mLof potassium hydroxide solution (1in 33),using alcohol,if necessary,to break any emulsion that forms.Wash by swirling gently with 50mLof water.Repeat the washing more vigorously with three additional 50-mLportions of water.Transfer the washed ether extract to a 100-mLvolumetric flask,add ether to volume,and mix.
Evaporate a 50.0-mLaliquot of the ether solution of the unsaponifiable extract to about 5mL.Without applying heat and with the aid of a stream of inert gas or vacuum,remove the residual ether.Dissolve the residue in 50.0mLof isopropyl alcohol.
Hydrogenated portion— Pipet 15.0mLof the isopropyl alcohol solution into a 50-mLcentrifuge tube,add approximately 200mg of palladium catalyst,stir with a glass rod,and hydrogenate for 10minutes in a Hydrogenatorsuch as is described in the Alpha Tocopherol Assay á551ñ,using isopropyl alcohol in the blank tube.Add about 300mg of chromatographic siliceous earth,stir with a glass rod,and immediately centrifuge until the solution is clear.
Test a 1-mLaliquot of the solution by removing the solvent by evaporation,dissolving the residue in 1mLof chloroform,and adding 10mLof phosphomolybdic acid TS:no detectable blue-green color appears.[NOTE—If a blue-green color appears,repeat the hydrogenation for a longer time period,or with a new lot of catalyst.]
Into two separate flasks pipet equal volumes of the Hydrogenated portionand the untreated isopropyl alcohol solution,respectively,and add sufficient isopropyl alcohol to give an expected concentration of vitamin Aequivalent to 3µg to 5µg per mL.Determine the absorbances of the untreated solution against the solution from the Hydrogenated portionas a blank,at the wavelengths 310nm,325nm,and 334nm,with a suitable spectrophotometer fitted with matched quartz cells.

Calculation—
Calculate the vitamin Acontent as follows:
Content (in mg)=0.549A325/LC,
in which A325is the observed absorbance at 325nm,Lis the length,in cm,of the absorption cell,and Cis the amount of test specimen expressed as g,capsule,or tablet in each 100mLof the final isopropyl alcohol solution,provided that A325has a value not less than [A325]/1.030and not more than [A325]/0.970,where [A325]is the correctedabsorbance at 325nm and is given by the equation:
[A325]=6.815A325–2.555A310–4.260A334,
in which Adesignates the absorbance at the wavelength indicated by the subscript.
Where [A325]has a value less than A325/1.030,apply the following equation:
Content (in mg)=0.549[A325]/LC,
in which the values are as defined herein.Each mg of vitamin A(alcohol)represents 3333USP Units of vitamin A.

Confidence Interval—
The range of the limits of error,indicating the extent of discrepancy to be expected in the results of different laboratories at P=0.05,is approximately ±8%.