Cholecalciferol (koe'' le kal sif' er ol). DEFINITION Cholecalciferol contains NLT 97.0% and NMT 103.0% of cholecalciferol (C27H44O). IDENTIFICATION • A. Infrared Absorption 197K Wavelength range: 212 µm • B. Ultraviolet Absorption 197U 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%. • C. Sample solution: 0.5 mg in 5 mL of chloroform Analysis: Add 0.3 mL of acetic anhydride and 0.1 mL of sulfuric acid to the Sample solution, and shake vigorously. Acceptance criteria: A bright red color is produced, and it rapidly changes through violet and blue to green. • D. Thin-Layer Chromatography [NoteFor 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 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 [NotePerform 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 RF value as the area of the Standard solution and may show below the cholecalciferol area a violet area, attributed to 7-dehydrocholesterol. ASSAY • Procedure Dehydrated hexane: Prepare a chromatographic column by packing a chromatographic tube, 8 × 60 cm, with 500 g of 50- to 250-µm chromatographic siliceous earth, activated by drying at 150 for 4 h. (See Chromatography 621, Column Chromatography.) Pass 500 mL of hexane through the column, and collect the eluate in a glass-stoppered flask. Mobile phase: n-Amyl alcohol in Dehydrated hexane (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. [NoteThis solution contains cholecalciferol, precholecalciferol, and trans-cholecalciferol. ] [NoteFor 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 Chromatographic system Mode: LC Detector: UV 254 nm Column: 4.6-mm × 25-cm; packing L3 Injection size: 510 µL System suitability Sample: System suitability solution [NoteThe 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 Analysis Samples: Standard solution and Sample solution Calculate the percentage of cholecalciferol (C27H44O) in the portion of Cholecalciferol taken: Result = (rU/rS) × (CS/CU) × 100
Acceptance criteria: 97.0%103.0% SPECIFIC TESTS • Optical Rotation, Specific Rotation 781S Sample solution: 5 mg/mL in alcohol. [NotePrepare and use the solution without delay. Use Cholecalciferol from a container opened not longer than 30 min. ] Acceptance criteria: +105 to +112 ADDITIONAL REQUIREMENTS • Packaging and Storage: Preserve in hermetically sealed containers under nitrogen, and store in a cool place protected from light. Auxiliary Information Please check for your question in the FAQs before contacting USP.
USP35NF30 Page 2652 Chromatographic Column Chromatographic columns text is not derived from, and not part of, USP 35 or NF 30. |