A: Infrared Absorption á197Kñ(range 2µm to 12µm).

B: Ultraviolet Absorption á197Uñ—

C: To a solution of about 0.5mg in 5mLof chloroform add 0.3mLof acetic anhydride and 0.1mLof sulfuric acid,and shake vigorously:a bright red color is produced,and it rapidly changes through violet and blue to green.

D: Prepare without heating,and handle without delay,a 1in 100solution of squalane in chloroform containing 50mg of cholecalciferol per mL,and prepare a Standard solution of USP Cholecalciferol RSin the same solvent and having the same concentration.Apply 10µLof the test solution and 10µLof the Standard solution on a line parallel to and about 2.5cm from the bottom edge of a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Place the plate in a developing chamber containing and equilibrated with a mixture of equal volumes of cyclohexane and diethyl ether.Develop the chromatogram until the solvent front has moved about 15cm above the line of application.Perform the development and subsequent operations in the dark.Remove the plate,allow the solvent to evaporate,and spray with a 1in 50solution of acetyl chloride in antimony trichloride TS:the chromatogram obtained from the test solution shows a yellowish orange area (cholecalciferol)having the same RFvalue as the area of the Standard solution,and may show below the cholecalciferol area a violet area attributed to 7-dehydrocholesterol.

Test solution: 5mg per mL,in alcohol.Prepare the solution without delay,using Cholecalciferol from a container opened not longer than 30minutes,and determine the optical rotation within 30minutes after the solution has been prepared.

Dehydrated hexane— Prepare a chromatographic column by packing a chromatographic tube,60-×8-cm in diameter,with 500g of 50-to 250-µm chromatographic siliceous earth,activated by drying at 150for 4hours (see Column adsorption chromatographyunder Chromatography á621ñ).Pass 500mLof hexanes through the column,and collect the eluate in a glass-stoppered flask.

Standard preparation— [NOTE—Use low-actinic glassware,and prepare solutions fresh daily.]Transfer about 30mg of USP Cholecalciferol RS,accurately weighed,to a 50-mLvolumetric flask,dissolve without heat in toluene,add toluene to volume,and mix.Pipet 10mLof this stock solution into a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix to obtain a solution having a known concentration of about 120µg per mL.

Assay preparation— [NOTE—Use low-actinic glassware,and prepare solutions fresh daily.]Transfer about 30mg of Cholecalciferol,accurately weighed,to a 50-mLvolumetric flask,and proceed as directed for Standard preparation,beginning with “dissolve without heat in toluene,”to obtain a solution having a concentration of about 120µg per mL.

Mobile phase— Prepare a 3in 1000mixture of n-amyl alcohol in Dehydrated hexane.The ratio of components and the flow rate may be varied to meet system suitability requirements.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L3.

System suitability preparation— Dissolve about 250mg of USP Vitamin D Assay System Suitability RSin 10mLof a mixture of equal volumes of toluene and Mobile phase.Heat this solution,under reflux,at 90for 45minutes,and cool.This solution contains cholecalciferol,pre-cholecalciferol,and trans-cholecalciferol.

System suitability test— Chromatograph five injections of the System suitability preparation,and measure the peak responses as directed under Procedure:the relative standard deviation for the peak response for cholecalciferol does not exceed 2.0%,and the resolution between trans-cholecalciferol and precholecalciferol is not less than 1.0.[NOTE—Chromatograms obtained as directed for this test exhibit relative retention times of approximately 0.4for precholecalciferol,0.5for trans-cholecalciferol,and 1.0for cholecalciferol.]

Procedure— Introduce equal volumes (5to 10µL)of the Standard preparationand the Assay preparationinto the chromatograph (see Chromatography á621ñ)by means of a suitable sampling valve.Measure the peak responses for the major peaks obtained,at corresponding retention times,from the Assay preparationand the Standard preparation.Calculate the quantity,in mg,of C27H44Oin the portion of Cholecalciferol taken by the formula: in which Cis the concentration,in µg per mL,of USP Cholecalciferol RSin the Standard preparation,and rUand rSare the peak responses for cholecalciferol obtained from the Assay preparationand the Standard preparation,respectively.