Ergocalciferol Capsules
DEFINITION
Ergocalciferol Capsules usually consist of an edible vegetable oil solution of Ergocalciferol, encapsulated with Gelatin. Ergocalciferol Capsules contain NLT 100.0% and NMT 120.0% of the labeled amount of vitamin D as ergocalciferol (C28H44O).
ASSAY
•  Procedure
[Note—Throughout this Assay, protect solutions containing, and derived from, the test specimen and the Reference Standard from the atmosphere and light, preferably by the use of a blanket of inert gas and low-actinic glassware. ]
Dehydrated hexane:  Prepare a chromatographic column by packing a chromatographic tube, 60-cm × 8-cm in diameter, with 500 g of 50- to 250-µm chromatographic siliceous earth, activated by drying at 150 for 4 h. (See Chromatography 621, Column Adsorption Chromatography.) Pass 500 mL of hexanes through the column, and collect the eluate in a glass-stoppered flask.
Butylated hydroxytoluene solution:  10 mg/mL of butylated hydroxytoluene in chromatographic hexane
Aqueous potassium hydroxide solution:  1 g/mL of potassium hydroxide in freshly boiled water. [Note—Prepare this solution fresh daily. ]
Alcoholic potassium hydroxide solution:  3 g of potassium hydroxide in 50 mL of freshly boiled water. Add 10 mL of alcohol, and dilute with freshly boiled water to 100 mL. [Note—Prepare this solution fresh daily. ]
Sodium ascorbate solution:  175 mg/mL of ascorbic acid in 1 N sodium hydroxide. [Note—Prepare this solution fresh daily. ]
Sodium sulfide solution:  12 g of sodium sulfide in 20 mL of water. Dilute with glycerin to 100 mL.
Mobile phase:  Dehydrated hexane and n-amyl alcohol (997:3). The ratio of components and the flow rate may be varied to meet the System suitability requirements.
Standard stock solution:  0.5 mg/mL of USP Ergocalciferol RS in toluene. [Note—Prepare solution fresh daily. ]
Standard solution A:  20 µg/mL from the Standard stock solution in Mobile phase. [Note—Store this solution at a temperature not above 0. ]
Standard solution B:  Pipet 4 mL of the Standard stock solution into a round-bottomed flask fitted with a reflux condenser, and add 2 or 3 crystals of butylated hydroxytoluene. Displace the air with nitrogen, and heat in a water bath maintained at a temperature of 90 in subdued light under a nitrogen atmosphere for 45 min to obtain a solution containing vitamin D and pre-vitamin D. Cool, transfer with the aid of several portions of Mobile phase to a 100-mL volumetric flask, and dilute with Mobile phase to volume.
System suitability solution:  100 mg of USP Vitamin D Assay System Suitability RS to a 10-mL volumetric flask. Add a mixture (1 in 5) of toluene and Mobile phase to volume, and mix. Heat a portion of this solution under reflux, at 90 for 45 min, and cool.
Sample solution:  Reflux NLT 10 Capsules with a mixture of 10 mL of Sodium ascorbate solution and 2 drops of Sodium sulfide solution on a steam bath for 10 min, crush any remaining solids with a blunt glass rod, and continue heating for 5 min. Cool, and add 25 mL of alcohol and 3 mL of Aqueous potassium hydroxide solution. Reflux the mixture on a steam bath for 30 min. Cool rapidly under running water, and transfer the saponified mixture to a conical separator, rinsing the saponification flask with two 15-mL portions of water, 10 mL of alcohol, and two 50-mL portions of ether. [Note—Use ether within 24 h after opening the container. ] Shake the combined saponified mixture and rinsings vigorously for 30 s, and allow to stand until both layers are clear. Transfer the aqueous phase to a second conical separator, add a mixture of 10 mL of alcohol and 50 mL of solvent hexane, and shake vigorously. Allow to separate, transfer the aqueous phase to a third conical separator, and transfer the solvent hexane phase to the first separator, rinsing the second separator with two 10-mL portions of solvent hexane and adding the rinsings to the first separator. Shake the aqueous phase in the third separator with 50 mL of solvent hexane, and add the solvent hexane phase to the first separator. Wash the combined ether-solvent hexane extracts by shaking vigorously with three 50-mL portions of Alcoholic potassium hydroxide solution, and wash with 50-mL portions of water vigorously until the last washing is neutral to phenolphthalein. Drain any remaining drops of water from the combined ether-solvent hexane extracts, add 2 sheets of 9-cm filter paper, in strips, to the separator, and shake. Transfer the washed ether-solvent hexane extracts to a round-bottomed flask, rinsing the separator and paper with solvent hexane. Combine the solvent hexane rinsings with the ether-solvent hexane extracts, add 100 µL of Butylated hydroxytoluene solution, and mix. Evaporate in a vacuum to dryness by swirling in a water bath maintained at a temperature not higher than 40. Cool under running water, and introduce nitrogen sufficient to restore atmospheric pressure. Without delay, dissolve and dilute the residue in an accurately measured volume of a mixture (1 in 5) of toluene and Mobile phase, until the concentration of vitamin D is about 25 µg/mL.
Chromatographic system 
Mode:  LC
Detector:  UV 254 nm
Column:  25-cm × 4.6-mm; packing L3
Injection size:  20 µL
System suitability 
Sample:  System suitability solution
[Note—The relative retention times for pre-cholecalciferol, trans-cholecalciferol, and cholecalciferol are 0.4, 0.5, and 1.0, respectively. ]
Suitability requirements 
Resolution:  NLT 1.0 is between trans-cholecalciferol and pre-cholecalciferol
Relative standard deviation:  NMT 2.0% for the cholecalciferol peak
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Ergocalciferol response factor 
Calculate the Ergocalciferol response factor, FD:
FD = CS/rS
CS== concentration of USP Ergocalciferol RS in the Standard solution A (µg/mL)
rS== peak response of ergocalciferol from Standard solution A
Pre-ergocalciferol response factor 
Calculate the concentration of ergocalciferol, C¢S, in µg/mL, in the Standard solution B:
C¢S = FD × r¢s
FD== ergocalciferol response factor
r¢S== peak area of ergocalciferol from Standard solution B
Calculate the concentration of pre-ergocalciferol, C¢pre, in µg/mL, in the Standard solution B:
C¢pre = CS C¢S
CS== concentration of USP Ergocalciferol RS in the Standard solution A (µg/mL)
C¢S== concentration of ergocalciferol in the Standard solution B (µg/mL)
Calculate the response factor, Fpre, for pre-ergocalciferol:
Fpre = C¢pre/r¢pre
C¢pre== concentration of pre-ergocalciferol (µg/mL)
r¢pre== peak response of pre-ergocalciferol from Standard solution B
[Note—The value of Fpre determined in duplicate, on different days, can be used during the entire procedure. ]
Vitamin D content 
Calculate the percentage of the labeled amount of vitamin D as ergocalciferol (C28H44O) in the portion of Capsules taken:
Result = {[(FD × rC) + (Fpre × r¢pre)]/CU} × 100
FD== ergocalciferol response factor
rC== peak area of ergocalciferol from the Sample solution
Fpre== pre-ergocalciferol response factor
r¢pre== peak area of pre-ergocalciferol from the Sample solution
CU== nominal concentration of ergocalciferol in the Sample solution (µg/mL)
Acceptance criteria:  100.0%–120.0%
PERFORMANCE TESTS
•  Disintegration 701
Buffer solution:  0.05 M acetate buffer, prepared by mixing 2.99 g of sodium acetate and 1.66 mL of glacial acetic acid with water to obtain 1000 mL of solution having a pH of 4.5 ± 0.05
Immersion fluid:  Buffer solution
Time:  45 min
Acceptance criteria:  Meet the requirements
•  Uniformity of Dosage Units 905: Meet the requirements
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers.
•  Labeling: Label the Capsules to indicate the content of ergocalciferol in mg. The activity may be expressed also in terms of USP Units, on the basis that 40 USP Vitamin D Units = 1 µg.
•  USP Reference Standards 11
USP Ergocalciferol RS Click to View Structure
USP Vitamin D Assay System Suitability RS
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USP35–NF30 Page 3063