Cholecalciferol Solution

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 Solution is a solution of Cholecalciferol in an edible vegetable oil, in Polysorbate 80, or in Propylene Glycol. It contains NLT 90.0% and NMT 120.0% of the labeled amount of Vitamin D as cholecalciferol (C₂₇H₄₄O).

2 ASSAY

2.1 PROCEDURE

Mobile phase: Hexane and pentanol (997:3)

Standard stock solution: Dissolve USP Cholecalciferol RS in toluene, and dilute with Mobile phase to 50 µg/mL. [NOTE—Prepare this solution fresh daily.]

Standard solution A: 5 µg/mL from Standard stock solution in Mobile phase. [NOTE—Store at a temperature not above 0°.]

Standard solution B: Transfer 5.0 mL of Standard stock solution to a round-bottom flask fitted with a reflux condenser. Displace the air with nitrogen, and reflux for 1 h in a water bath under a nitrogen atmosphere to obtain a solution containing cholecalciferol and precholecalciferol. Cool, transfer the solution with the aid of several portions of Mobile phase to a 50-mL volumetric flask, and dilute with Mobile phase to volume.

Sample solution: Equivalent to 5 µg/mL of cholecalciferol in Mobile phase from an accurately measured volume of Cholecalciferol Solution

2.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L3 Flow rate: 2 mL/min

Injection size: 10 µL

2.1.2 System suitability

Sample: Standard solution B

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

Suitability requirements

Resolution: NLT 1.0 between the precholecalciferol peak and the cholecalciferol peak Relative standard deviation: NMT 2.0%

2.1.3 Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Cholecalciferol response factor

Calculate the cholecalciferol response factor, F_C :

F_C = C_s/r_s

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

r_s = peak area of cholecalciferol from Standard solution A

Pre-cholecalciferol response factor

Calculate the concentration, C'_s , in µg/mL, of cholecalciferol in Standard solution B:

C'_s = F_C × r'_s

F_C = response factor for cholecalciferol

r'_s = peak area of cholecalciferol from Standard solution B

Calculate the concentration, C′_pre  , in µg/mL, of pre-cholecalciferol:

C′_pre  = C_s − C'_s

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

C'_s = concentration of cholecalciferol in Standard solution B (µg/mL)

Calculate the response factor, F_pre , for pre-cholecalciferol:

F_pre = C′_pre/r_p

C′_pre = concentration of pre-cholecalciferol (µg/mL)

r_p = peak response of pre-cholecalciferol from Standard solution B

Content of vitamin D

Calculate the percentage of the labeled amount of vitamin D as cholecalciferol (C H O) in the portion of the Cholecalciferol Solution taken:

Result = {[(F_C × r_C) + (F_pre × r_pre )]/C_U} × 100

F_C = response factor for cholecalciferol

r_C = peak area of cholecalciferol from the Sample solution

F_pre = response factor for pre-cholecalciferol

r_pre = peak area of pre-cholecalciferol from the Sample solution

C_U = nominal concentration of cholecalciferol in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–120.0%

3 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers.

LABELING: Label it to indicate the concentration, in mg/mL, of cholecalciferol. Label it also to state that it is to be used for manufacturing only.

USP REFERENCE STANDARDS 〈11〉

USP Cholecalciferol RS