A: Infrared Absorption 197K (range 2 µm to 12 µm).

B: Ultraviolet Absorption 197U—

C: To a solution of about 0.5 mg in 5 mL of chloroform add 0.3 mL of acetic anhydride and 0.1 mL of 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 1 in 100 solution of squalane in chloroform containing 50 mg of cholecalciferol per mL, and prepare a Standard solution of USP Cholecalciferol RS in the same solvent and having the same concentration. Apply 10 µL of the test solution and 10 µL of the Standard solution on a line parallel to and about 2.5 cm from the bottom edge of a thin-layer chromatographic plate (see Chromatography 621) 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 15 cm 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 1 in 50 solution of acetyl chloride in antimony trichloride TS: the chromatogram obtained from the test solution shows a yellowish orange area (cholecalciferol) having the same RF value as the area of the Standard solution, and may show below the cholecalciferol area a violet area attributed to 7-dehydrocholesterol.

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

Dehydrated hexane— Prepare a chromatographic column by packing a chromatographic tube, 60- × 8-cm in diameter, with 500 g of 50- to 250-µm chromatographic siliceous earth, activated by drying at 150 for 4 hours (see Column adsorption chromatography under Chromatography 621). Pass 500 mL of 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 30 mg of USP Cholecalciferol RS, accurately weighed, to a 50-mL volumetric flask, dissolve without heat in toluene, add toluene to volume, and mix. Pipet 10 mL of this stock solution into a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix to obtain a solution having a known concentration of about 120 µg per mL.

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

Mobile phase— Prepare a 3 in 1000 mixture 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 621)—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 250 mg of USP Vitamin D Assay System Suitability RS in 10 mL of a mixture of equal volumes of toluene and Mobile phase. Heat this solution, under reflux, at 90 for 45 minutes, 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 0.4 for precholecalciferol, 0.5 for trans-cholecalciferol, and 1.0 for cholecalciferol.]

Procedure— Introduce equal volumes (5 to 10 µL) of the Standard preparation and the Assay preparation into the chromatograph (see Chromatography 621) by means of a suitable sampling valve. Measure the peak responses for the major peaks obtained, at corresponding retention times, from the Assay preparation and the Standard preparation. Calculate the quantity, in mg, of C27H44O in the portion of Cholecalciferol taken by the formula: in which C is the concentration, in µg per mL, of USP Cholecalciferol RS in the Standard preparation; and rU and rS are the peak responses for cholecalciferol obtained from the Assay preparation and the Standard preparation, respectively.