» Ergocalciferol Capsules usually consist of an edible vegetable oil solution of Ergocalciferol, encapsulated with Gelatin. Ergocalciferol Capsules contain not less than 100.0 percent and not more than 120.0 percent of the labeled amount of C28H44O.
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.
Disintegration 701: 45 minutes, determined using 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, maintained at 37 ± 2 as the immersion fluid.
Uniformity of dosage units 905: meet the requirements.
Assay [noteThroughout 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.]
Ether Use ethyl ether. Use within 24 hours after opening container.
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 hours (see Column adsorption chromatography under Chromatography 621). Pass 500 mL of hexanes through the column, and collect the eluate in a glass-stoppered flask.
Butylated hydroxytoluene solution Dissolve a quantity of butylated hydroxytoluene in chromatographic hexane to obtain a solution containing 10 mg per mL.
Aqueous potassium hydroxide solution Dissolve 500 g of potassium hydroxide in 500 mL of freshly boiled water, mix, and cool. Prepare this solution fresh daily.
Alcoholic potassium hydroxide solution Dissolve 3 g of potassium hydroxide in 50 mL of freshly boiled water, add 10 mL of alcohol, dilute with freshly boiled water to 100 mL, and mix. Prepare this solution fresh daily.
Sodium ascorbate solution Dissolve 3.5 g of ascorbic acid in 20 mL of 1 N sodium hydroxide. Prepare this solution fresh daily.
Sodium sulfide solution Dissolve 12 g of sodium sulfide in 20 mL of water, dilute with glycerin to 100 mL, and mix.
Mobile phase Prepare a 3 in 1000 mixture of n-amyl alcohol in Dehydrated hexane. The ratio of components and the flow rate may be varied to meet system suitability requirements.
Standard preparation Transfer about 25 mg of USP Ergocalciferol RS, accurately weighed, to a 50-mL volumetric flask, dissolve without heat in toluene, add toluene to volume, and mix. Prepare stock solution fresh daily.
Assay preparation Reflux not less than 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 minutes, crush any remaining solids with a blunt glass rod, and continue heating for 5 minutes. Cool, add 25 mL of alcohol and 3 mL of Aqueous potassium hydroxide solution, and mix.
Reflux the mixture on a steam bath for 30 minutes. 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. Shake the combined saponified mixture and rinsings vigorously for 30 seconds, 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 hexane phase to the first separator, rinsing the second separator with two 10-mL portions of solvent hexane, adding the rinsings to the first separator. Shake the aqueous phase in the third separator with 50 mL of solvent hexane, and add the hexane phase to the first separator. Wash the combined ether-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-hexane extracts, add 2 sheets of 9-cm filter paper, in strips, to the separator, and shake. Transfer the washed ether-hexane extracts to a round-bottom flask, rinsing the separator and paper with solvent hexane. Combine the hexane rinsings with the ether-hexane extracts, add 100 µL of Butylated hydroxytoluene solution, and mix. Evaporate in 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 the residue in a measured volume of a 1 in 5 mixture of toluene and Mobile phase, until the concentration of vitamin D is about 25 µg per mL, to obtain the Assay preparation.
Chromatographic system Use a chromatograph, operated at room temperature, fitted with an UV detector that monitors absorption at 254 nm, and a 25-cm × 4.6-mm stainless steel column packed with column packing L3.
System suitability test Transfer about 100 mg of USP Vitamin D Assay System Suitability RS to a 10-mL volumetric flask, add a 1 in 5 mixture of toluene and Mobile phase to volume, and mix. Heat a portion of this solution under reflux, at 90 for 45 minutes, and cool. Chromatograph five injections of this solution, and measure the peak responses as directed under Procedure: the relative standard deviation for the cholecalciferol peak response does not exceed 2.0%, and the resolution between trans-cholecalciferol and pre-cholecalciferol is not less than 1.0. [noteChromatograms obtained as directed for this test exhibit relative retention times of approximately 0.4 for pre-cholecalciferol, 0.5 for trans-cholecalciferol, and 1.0 for cholecalciferol.]
Vitamin D response factor Transfer 4.0 mL of the Standard preparation to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix to obtain the Working standard preparation. Store this Working standard preparation at a temperature not above 0, retaining the unused portion for the Procedure. Inject 20 µL of the Working standard preparation into the column, and measure the peak response for vitamin D. Calculate the response factor, FD, by the formula:
CS / rSin which CS is the concentration, in µg per mL, of ergocalciferol in the Working standard preparation, and rS is the peak response of ergocalciferol.
Pre-vitamin D response factor Pipet 4 mL of the Standard preparation into a round-bottom 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 minutes, 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, dilute with Mobile phase to volume, and mix to obtain the Working mixture. Inject 20 µL of this Working mixture into the analytical column, and measure the peak responses for vitamin D and pre-vitamin D. Calculate the concentration, C¢S, in µg per mL, of vitamin D in the (heated) Working mixture taken by the formula:
FDr¢Sin which r¢S is the peak response for vitamin D. Calculate the concentration, C¢pre, in µg per mL, of pre-vitamin D, in the Working mixture taken by the formula:
C¢pre = CS C¢S.Calculate the response factor, Fpre, for pre-vitamin D by the formula C¢pre/r¢pre, in which r¢pre is the peak response of pre-vitamin D. [noteThe value of Fpre determined in duplicate, on different days, can be used during the entire procedure.]
Procedure Inject 20 µL of the Assay preparation into the column, and measure the peak responses for vitamin D and pre-vitamin D. Calculate the concentration, in µg per mL, of C28H44O in the Assay preparation taken by the formula:
r¢¢DFD+ r¢¢preFprein which r¢¢D and r¢¢pre are the peak responses of vitamin D and pre-vitamin D, respectively.
Auxiliary Information Please check for your question in the FAQs before contacting USP.Chromatographic Column
USP32NF27 Page 2269
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.