A: Infrared Absorption 197K (range 2 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 rapidly changes through violet and blue to green.

D: Prepare without heating, and handle without delay, a solution of squalane in chloroform (1 in 100) containing 50 mg of Ergocalciferol per mL, and prepare a Standard solution of USP Ergocalciferol RS in the same solvent and of the same concentration. Prepare a solution of squalane in chloroform (1 in 100) containing 100 µg of USP Ergosterol RS per mL. Apply 10 µL of the test solution, 10 µL of the Standard solution, and 10 µL of the ergosterol 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 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 solution of acetyl chloride in antimony trichloride TS (1 in 50). The chromatogram obtained with the test solution shows a yellowish orange area (ergocalciferol) having the same RF value as the area of the Standard solution of ergocalciferol and may show a violet area below the ergocalciferol area. The color of the violet area is not more intense than that of the violet area in the chromatogram obtained from the solution of ergosterol.

Test solution: 15 mg per mL, in alcohol. Prepare the solution without delay, using Ergocalciferol 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 , Mobile phase, Chromatographic system, System suitability preparation, and System suitability test—Proceed as directed in the Assay under Cholecalciferol.

Standard preparation— [note—Use low-actinic glassware, and prepare solutions fresh daily.] Transfer about 30 mg of USP Ergocalciferol 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 Ergocalciferol, 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.

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 responses for the major peaks obtained, at corresponding retention times, with the Assay preparation and the Standard preparation. Calculate the quantity, in mg, of C28H44O in the portion of Ergocalciferol taken by the formula: in which C is the concentration, in µg per mL, of USP Ergocalciferol RS in the Standard preparation; and rU and rS are the peak responses for ergocalciferol obtained from the Assay preparation and the Standard preparation, respectively.