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. [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
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
[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%
Analysis
Samples:
Standard solution A, Standard solution B, and Sample solution
Cholecalciferol response factor
Calculate the cholecalciferol response factor, FC:
FC = CS/rS
| CS | = | = concentration of USP Cholecalciferol RS in Standard solution A (µg/mL) |
| rS | = | = 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 = FC × r¢S
| FC | = | = 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 = CS 
C¢S
C¢S
| CS | = | = 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, Fpre, for pre-cholecalciferol:
Fpre = C¢pre/rp
| C¢pre | = | = concentration of pre-cholecalciferol (µg/mL) |
| rp | = | = 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 (C27H44O) in the portion of the Cholecalciferol Solution taken:
Result = {[(FC × rC) + (Fpre × rpre)]/CU} × 100
| FC | = | = response factor for cholecalciferol |
| rC | = | = peak area of cholecalciferol from the Sample solution |
| Fpre | = | = response factor for pre-cholecalciferol |
| rpre | = | = peak area of pre-cholecalciferol from the Sample solution |
| CU | = | = nominal concentration of cholecalciferol in the Sample solution (µg/mL) |
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.
• USP Reference Standards 
11
11
USP Cholecalciferol RS 
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Natalia Davydova
Scientific Liaison 1-301-816-8328 |
(DS2010) Monographs - Dietary Supplements |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 2653