Cholecalciferol

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Cholecalciferol contains NLT 97.0% and NMT 103.0% of cholecalciferol (C₂₇H₄₄O).

2 IDENTIFICATION

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A. SPECTROSCOPIC IDENTIFICATION TESTS 〈197〉 , Infrared Spectroscopy : 197K (USP 1-M  -2020)

Wavelength range: 2–12 µm

Acceptance criteria: Meets the requirements in the chapter

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B. SPECTROSCOPIC IDENTIFICATION TESTS 〈197〉 , Ultraviolet-Visible Spectroscopy : 197U (USP 1-MAY-2020)

Analytical wavelength: 265 nm Sample solution: 10 µg/mL in alcohol

Acceptance criteria: Meets the requirements in the chapter. Absorptivities do not differ by more than 3.0%.

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C. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. (USP 1-May-2020)

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D. THIN-LAYER CHROMATOGRAPHY

[NOTE—For the Standard solution and the Sample solution, follow these procedures: use low-actinic glassware, dissolve the samples without heating, and use the solutions immediately.]

Diluent: 10 mg/mL of squalane in chloroform

Standard solution: 50 mg/mL of USP Cholecalciferol RS in Diluent Sample solution: 50 mg/mL of Cholecalciferol in Diluent

Chromatographic system

(See Chromatography 〈621〉, Thin-Layer Chromatography.) Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture Application volume: 10 µL

Developing solvent system: Cyclohexane and diethyl ether (1:1) Spray reagent: 20 mg/mL of acetyl chloride in antimony trichloride TS

Analysis

Samples: Standard solution and Sample solution

[NOTE—Perform the development and subsequent operations in the dark.]

Place the plate in a chamber containing and equilibrated with Developing solvent system. Develop until the solvent front has moved about 15 cm above the line of application. Remove the plate, allow the solvent to evaporate, and spray with Spray reagent.

Acceptance criteria: The Sample solution shows a yellowish-orange area (cholecalciferol) having the same R value as the area of the Standard solution and may show below the cholecalciferol area a violet area, attributed to 7-dehydrocholesterol. (USP 1-May-2020)

3 ASSAY

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3.1 PROCEDURE

(USP 1-May-2020)

Mobile phase: n-Amyl alcohol in hexane, solvent, chromatographic (USP 1-May-2020) (3 in 1000)

System suitability solution: 250 mg of USP Vitamin D Assay System Suitability RS in 10 mL of a mixture of toluene and Mobile phase (1:1). Heat this solution, under reflux, at 90° for 45 min, and cool. [NOTE—This solution contains cholecalciferol, precholecalciferol, and trans- cholecalciferol.]

[NOTE—For the stock solutions, follow these procedures: use low-actinic glassware, dissolve the samples without heating, and prepare the solutions fresh daily.]

Standard stock solution: 0.6 mg/mL of USP Cholecalciferol RS in toluene

Standard solution: 120 µg/mL of USP Cholecalciferol RS in Mobile phase, prepared from Standard stock solution Sample stock solution: 0.6 mg/mL of Cholecalciferol in toluene

Sample solution: 120 µg/mL of Cholecalciferol in Mobile phase, prepared from Sample stock solution

3.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L3

Flow rate: 1.5 mL/min (USP 1-May-2020)

Injection volume: 5–10 µL

3.1.2 System suitability

Sample: System suitability solution

[NOTE—The relative retention times for precholecalciferol, trans-cholecalciferol, and cholecalciferol are 0.4, 0.5, and 1.0, respectively.]

Suitability requirements

Resolution: NLT 1.0 between trans-cholecalciferol and precholecalciferol Relative standard deviation: NMT 2.0% for the peak response of cholecalciferol

3.1.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of cholecalciferol (C₂₇H₄₄O) in the portion of Cholecalciferol taken:

Result = (r_u/r_s) × (C_s/C_u) × 100

r_u = peak response from the Sample solution

r_s = peak response from the Standard solution

C_s = concentration of USP Cholecalciferol RS in the Standard solution (µg/mL)

C_u = concentration of Cholecalciferol in the Sample solution (µg/mL)

Acceptance criteria: 97.0%–103.0%

4 SPECIFIC TESTS

OPTICAL ROTATION 〈781S〉 , Procedures, Specific Rotation

Sample solution: 5 mg/mL in alcohol. [NOTE—Prepare and use the solution without delay. Use Cholecalciferol from a container opened not longer than 30 min.]

Acceptance criteria: +105° to +112°

5 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in hermetically sealed containers under nitrogen, and store in a cool place protected from light.

USP REFERENCE STANDARDS 〈11〉

USP Cholecalciferol RS

USP Vitamin D Assay System Suitability RS