Ergocalciferol Capsules
»Ergocalciferol Capsules usually consist of an edible vegetable oil solution of Ergocalciferol,encapsulated with Gelatin.Ergocalciferol Capsules contain not less than 100.0percent and not more than 120.0percent 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 40USP Vitamin D Units =1µg.
USP Reference standards á11ñ— USP Ergocalciferol RS.USP Vitamin D Assay System Suitability RS.
Disintegration á701ñ: 45minutes,determined using 0.05Macetate buffer,prepared by mixing 2.99g of sodium acetate and 1.66mLof glacial acetic acid with water to obtain 1000mLof solution having a pHof 4.5±0.05,maintained at 37±2as the immersion fluid.
Uniformity of dosage units á905ñ: meet the requirements.
Assay— [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.]
Ether— Use ethyl ether.Use within 24hours after opening container.
Dehydrated hexane— Prepare a chromatographic column by packing a chromatographic tube,60cm ×8cm in diameter,with 500g of 50-to 250-µm chromatographic siliceous earth,activated by drying at 150for 4hours (see Column adsorption chromatographyunder Chromatography á621ñ).Pass 500mLof 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 10mg per mL.
Aqueous potassium hydroxide solution— Dissolve 500g of potassium hydroxide in 500mLof freshly boiled water,mix,and cool.Prepare this solution fresh daily.
Alcoholic potassium hydroxide solution— Dissolve 3g of potassium hydroxide in 50mLof freshly boiled water,add 10mLof alcohol,dilute with freshly boiled water to 100mL,and mix.Prepare this solution fresh daily.
Sodium ascorbate solution— Dissolve 3.5g of ascorbic acid in 20mLof 1Nsodium hydroxide.Prepare this solution fresh daily.
Sodium sulfide solution— Dissolve 12g of sodium sulfide in 20mLof water,dilute with glycerin to 100mL,and mix.
Mobile phase— Prepare a 3in 1000mixture 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 25mg of USP Ergocalciferol RS,accurately weighed,to a 50-mLvolumetric flask,dissolve without heat in toluene,add toluene to volume,and mix.Prepare stock solution fresh daily.
Assay preparation— Reflux not less than 10Capsules with a mixture of 10mLof Sodium ascorbate solutionand 2drops of Sodium sulfide solutionon a steam bath for 10minutes,crush any remaining solids with a blunt glass rod,and continue heating for 5minutes.Cool,add 25mLof alcohol and 3mLof Aqueous potassium hydroxide solution,and mix.
Reflux the mixture on a steam bath for 30minutes.Cool rapidly under running water,and transfer the saponified mixture to a conical separator,rinsing the saponification flask with two 15-mLportions of water,10mLof alcohol,and two 50-mLportions of ether.Shake the combined saponified mixture and rinsings vigorously for 30seconds,and allow to stand until both layers are clear.Transfer the aqueous phase to a second conical separator,add a mixture of 10mLof alcohol and 50mLof 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-mLportions of solvent hexane,adding the rinsings to the first separator.Shake the aqueous phase in the third separator with 50mLof solvent hexane,and add the hexane phase to the first separator.Wash the combined ether-hexane extracts by shaking vigorously with three 50-mLportions of Alcoholic potassium hydroxide solution,and wash with 50-mLportions of water vigorously until the last washing is neutral to phenolphthalein.Drain any remaining drops of water from the combined ether-hexane extracts,add 2sheets 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 100µLof 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 1in 5mixture of toluene and Mobile phase,until the concentration of vitamin Dis about 25µg per mL,to obtain the Assay preparation.
Chromatographic system— Use a chromatograph,operated at room temperature,fitted with an UVdetector that monitors absorption at 254nm,and a 25-cm ×4.6-mm stainless steel column packed with column packing L3.
System suitability test— Transfer about 100mg of USP Vitamin D Assay System Suitability RSto a 10-mLvolumetric flask,add a 1in 5mixture of toluene and Mobile phaseto volume,and mix.Heat a portion of this solution under reflux,at 90for 45minutes,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.[NOTE—Chromatograms obtained as directed for this test exhibit relative retention times of approximately 0.4for pre-cholecalciferol,0.5for trans-cholecalciferol,and 1.0for cholecalciferol.]
Vitamin Dresponse factor— Transfer 4.0mLof the Standard preparationto a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix to obtain the Working standard preparation.Store this Working standard preparationat a temperature not above 0,retaining the unused portion for the Procedure.Inject 20µLof the Working standard preparationinto the column,and measure the peak response for vitamin D.Calculate the response factor,FD,by the formula:
CS/rS,
in which CSis the concentration,in µg per mL,of ergocalciferol in the Working standard preparation,and rSis the peak response of ergocalciferol.
Pre-vitamin Dresponse factor— Pipet 4mLof the Standard preparationinto a round-bottom flask fitted with a reflux condenser,and add 2or 3crystals of butylated hydroxytoluene.Displace the air with nitrogen,and heat in a water bath maintained at a temperature of 90in subdued light under a nitrogen atmosphere for 45minutes,to obtain a solution containing vitamin Dand pre-vitamin D.Cool,transfer with the aid of several portions of Mobile phaseto a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix to obtain the Working mixture.Inject 20µLof this Working mixtureinto the analytical column,and measure the peak responses for vitamin Dand pre-vitamin D.Calculate the concentration,C¢S,in µg per mL,of vitamin Din the (heated)Working mixturetaken by the formula:
FDr¢S,
in which r¢Sis the peak response for vitamin D.Calculate the concentration,C¢pre,in µg per mL,of pre-vitamin D,in the Working mixturetaken by the formula:
C¢pre=CS-C¢S.
Calculate the response factor,Fpre,for pre-vitamin Dby the formula C¢pre/r¢pre,in which r¢preis the peak response of pre-vitamin D.[NOTE—The value of Fpredetermined in duplicate,on different days,can be used during the entire procedure.]
Procedure— Inject 20µLof the Assay preparationinto the column,and measure the peak responses for vitamin Dand pre-vitamin D.Calculate the concentration,in µg per mL,of C28H44Oin the Assay preparationtaken by the formula:
r¢¢DFD+r¢¢preFpre,
in which r¢¢Dand r¢¢preare the peak responses of vitamin Dand pre-vitamin D,respectively.
Auxiliary Information— Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist
Expert Committee:(DSN)Dietary Supplements:Non-Botanicals
USP28–NF23Page 746
Phone Number:1-301-816-8389