Ergocalciferol Capsules

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 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 (C₂₂H₄₄O).

2 ASSAY

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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.]

_{(USP 1-Aug-2019)}

Butylated hydroxytoluene solution: 10 mg/mL of butylated hydroxytoluene in n-hexane

Aqueous potassium hydroxide solution: 1 g/mL of potassium hydroxide in freshly boiled water. [NOTE-Prepare 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 solution fresh daily.]

Sodium ascorbate solution: 175 mg/mL of ascorbic acid in 1 N sodium hydroxide. [NOTE-Prepare solution fresh daily.]

Sodium sulfide solution: 12 g of sodium sulfide in 20 mL of water. Dilute with Glycerin to 100 mL.

Mobile phase: -Hexane _{(USP 1-Aug-2019)} and n-amyl alcohol (997:3). The ratio of components and the flow rate may be varied to meet the System suitability requirements.

System suitability solution: Transfer 100 mg of USP Vitamin D Assay System Suitability RS to a 10-mL volumetric flask. Add a mixture of toluene and Mobile phase (1 in 5) to volume, and mix. Heat a portion of this solution under reflux, at 90 for 45 min, and cool.

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 NMT 0^o]

Standard solution B: Pipet 4 mL of the Standard stock solution into a round-bottomed flask fitted with a reflux condenser, and then 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^o 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.

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 under vacuum to dryness by swirling in a water bath maintained at a temperature NMT 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 of toluene and Mobile phase (1 in 5), until the nominal concentration of vitamin D is about 25 µg/mL

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm x 25-cm; packing 13

Injection volume: 20 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 cholecalciferol

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Ergocalciferol response factor

Calculate the Ergocalciferol response factor (F_D ):

F_D = C_S /r_S

C_S = concentration of USP Ergocalciferol RS in Standard solution A (µg/mL)

r_S = peak response of ergocalciferol from Standard solution A

Pre-ergocalciferol response factor

Calculate the concentration of ergocalciferol (C′_S ), in µg/mL, in Standard solution B:

C′_S = F_D × r′_S

F_D = Ergocalciferol response factor, as previously determined

r′_S = peak area of ergocalciferol from Standard solution B

Calculate the concentration of pre-ergocalciferol (C′ _pre), in µg/mL, in Standard solution B:

C′_pre = C_s − C′_s

C_s = concentration of USP Ergocalciferol RS in Standard solution A (µg/mL)

C′_s = concentration of ergocalciferol in Standard solution B (µg/mL)

Calculate the Pre-ergocalciferol response factor (F_pre ):

F_pre = C′_pre /r′_pre

C′_pre = concentration of pre-ergocalciferol in Standard solution B (µg/mL)

r′_pre = peak response of pre-ergocalciferol from Standard solution B

[Note—The value of F 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 (C₂₂H₄₄O) in the portion of Capsules taken:

Result = {[(F_D × r_C ) + (F_pre × r′_pre )]/C_U } × 100

F_D = Ergocalciferol response factor, as previously determined

r_C = peak area of ergocalciferol from the Sample solution

F_pre = Pre-ergocalciferol response factor, as previously determined

r′_pre = peak area of pre-ergocalciferol from the Sample solution

C_U = nominal concentration of ergocalciferol in the Sample solution (µg/mL)

Acceptance criteria: 100.0%–120.0%

3 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 a 1000-mL 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

4 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers.

Change to read:

LABELING: Label the Capsules to indicate the content of ergocalciferol in micrograms. Expression of the amount of ergocalciferol in terms of units may be added in parentheses after the mass units._{(USP 1-Aug-2019)}

USP REFERENCE STANDARDS (11)

USP Ergocalciferol RS

USP Vitamin D Assay System Suitability RS