(er'' goe kal sif' er ol).
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C28H44O 396.65
9,10-Secoergosta-5,7,10 (19),22-tetraen-3-ol, (3,5Z,7E,22E)-;    
Ergocalciferol     [50-14-6].
Ergocalciferol contains NLT 97.0% and NMT 103.0% of ergocalciferol (C28H44O).
•  A. Infrared Absorption 197K
Wavelength range:  2–12 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 rapidly changes through violet and blue to green.
•  D. Thin-Layer Chromatography
[Note—For the Standard solutions 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 A:  50 mg/mL of USP Ergocalciferol RS in Diluent
Standard solution B:  100 µg/mL of USP Ergosterol RS in Diluent
Sample solution:  50 mg/mL of Ergocalciferol 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 ether (1:1)
Spray reagent:  20 mg/mL of acetyl chloride in antimony trichloride TS
Samples:  Standard solution A, Standard solution B, 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 (ergocalciferol) having the same RF value as the area of Standard solution A and may show a violet area below the ergocalciferol area. The color of the violet area is not more intense than that of the violet area from Standard solution B.
•  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. [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 Ergocalciferol RS in toluene
Standard solution:  120 µg/mL of USP Ergocalciferol RS in Mobile phase, prepared from Standard stock solution
Sample stock solution:  0.6 mg/mL of Ergocalciferol in toluene
Sample solution:  120 µg/mL of Ergocalciferol 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:  5–10 µL
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
Samples:  Standard solution and Sample solution
Calculate the percentage of ergocalciferol (C28H44O) in the portion of Ergocalciferol taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response from the Sample solution
rS== peak response from the Standard solution
CS== concentration of USP Ergocalciferol RS in the Standard solution (µg/mL)
CU== concentration of Ergocalciferol in the Sample solution (µg/mL)
Acceptance criteria:  97.0%–103.0%
•  Reducing Substances
Standard solution:  0.2 µg/mL of hydroquinone in dehydrated alcohol
Sample solution:  10 mg/mL of Ergocalciferol in dehydrated alcohol
Blank:  dehydrated alcohol
Samples:  Standard solution, Sample solution, and Blank
To 10 mL each of Standard solution, Sample solution, and Blank, add 0.5 mL of 5 mg/mL blue tetrazolium in methanol. Then add 0.5 mL of tetramethylammonium hydroxide TS in dehydrated alcohol (1 in 10). Allow the mixture to stand for 5 min, accurately timed, then add 1 mL of glacial acetic acid. Determine the absorbance of the solution at 525 nm, with a suitable spectrometer, against the Blank.
Acceptance criteria:  The absorbance of the Sample solution is NMT that of the Standard solution.
•  Optical Rotation, Specific Rotation 781S
Sample solution:  15 mg/mL in alcohol. [Note—Prepare and use the solution without delay. Use Ergocalciferol from a container opened not longer than 30 min. ]
Acceptance criteria:  +103 to +106
•  Packaging and Storage: Preserve in hermetically sealed containers under nitrogen, and store in a cool place protected from light.
•  USP Reference Standards 11
USP Ergocalciferol RS Click to View Structure
USP Ergosterol RS Click to View Structure
    C28H44O        396.66
USP Vitamin D Assay System Suitability RS
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(DS2010) Monographs - Dietary Supplements
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USP35–NF30 Page 3062