Cholecalciferol Solution 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). ASSAY • 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. [NotePrepare this solution fresh daily. ] Standard solution A: 5 µg/mL from Standard stock solution in Mobile phase. [NoteStore 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 Chromatographic system Mode: LC Detector: UV 254 nm Column: 4.6-mm × 25-cm; packing L3 Flow rate: 2 mL/min Injection size: 10 µL System suitability Sample: Standard solution B [NoteThe 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% Analysis Samples: Standard solution A, Standard solution B, and Sample solution Cholecalciferol response factor Calculate the cholecalciferol response factor, FC: FC = CS/rS
Pre-cholecalciferol response factor Calculate the concentration, C¢S, in µg/mL, of cholecalciferol in Standard solution B: C¢S = FC × r¢S
Calculate the concentration, C¢pre, in µg/mL, of pre-cholecalciferol: C¢pre = CS C¢S
Calculate the response factor, Fpre, for pre-cholecalciferol: Fpre = C¢pre/rp
Content of vitamin D Calculate the percentage of the labeled amount of vitamin D as cholecalciferol (C27H44O) in the portion of the Cholecalciferol Solution taken: Result = {[(FC × rC) + (Fpre × rpre)]/CU} × 100
Acceptance criteria: 90.0%120.0% 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. Auxiliary Information Please check for your question in the FAQs before contacting USP.
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