Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 2: 75rpm.

Time: 30minutes.

Procedure— Determine the amount of calcium (Ca)dissolved,employing the procedure set forth in the Assay for calcium,making any necessary volumetric adjustments.

Tolerances— Not less than 75%(Q)of the labeled amount of Ca is dissolved in 30minutes.

Lanthanum chloride solution— Dissolve 26.7g of lanthanum chloride in 0.125Nhydrochloric acid to make 100mL.

Calcium standard stock solution— Weigh accurately 1.001g of calcium carbonate,previously dried at 300for 3hours and cooled in a desiccator for 2hours,and dissolve in 25mLof 1Nhydrochloric acid.Boil to expel carbon dioxide,and dilute with water to 1000mLto obtain a solution having a known concentration of about 400µg of calcium per mL.

Standard preparations— Quantitatively dilute a volume of Calcium standard stock solutionwith 0.125Nhydrochloric acid to obtain a standard solution having a known concentration of about 100µg of calcium per mL.Into separate 100-mLvolumetric flasks,separately pipet 1.0,1.5,2.0,2.5,and 3.0mLof the standard solution.To each flask add 1.0mLof Lanthanum chloride solution,dilute with water to volume,and mix to obtain Standard preparationshaving known concentrations of about 1.0,1.5,2.0,2.5,and 3.0µg,respectively,of calcium per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powdered Tablets,equivalent to about 500mg of calcium,to a 1000-mLvolumetric flask.Add 25mLof concentrated hydrochloric acid to the flask,and heat for 30minutes on a steam bath.Cool,dilute with water to volume,mix,and filter.Quantitatively dilute a volume of this solution with 0.125Nhydrochloric acid to obtain a solution having a concentration of 100µg of calcium per mL.Transfer 2.0mLof this solution to a 100-mLvolumetric flask,add 1.0mLof Lanthanum chloride solution,dilute with water to volume,and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the calcium emission line at 422.7nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-scattering á851ñ)equipped with a calcium hollow-cathode lamp and a nitrous oxide–acetylene flame,using 0.125Nhydrochloric acid containing 0.1%Lanthanum chloride solutionas the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of calcium,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,in µg per mL,of calcium in the Assay preparation.Calculate the quantity,in mg,of calcium (Ca)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of calcium in the Assay preparation,and Dis the dilution factor involved in the Assay preparation.

Mobile phase— Prepare a filtered and degassed mixture of n-hexane and isopropyl alcohol (99:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Ergocalciferol RSor USP Cholecalciferol RSin n-hexane,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 2µg per mL.

System suitability preparation— Heat a volume of Standard preparationat 60for 1hour to partially isomerize vitamin D(ergocalciferol or cholecalciferol)to its corresponding precursor.

Assay preparation— Weigh accurately not less than 20Tablets,and grind the Tablets to a fine powder.Transfer an accurately weighed portion of the powder,equivalent to about 20µg of cholecalciferol or ergocalciferol,to a container having a polyteflined screw cap.Add 8mLof dimethyl sulfoxide and 12mLof n-hexane,and shake for 45minutes on a wrist-action shaker with tubes in a water bath maintained at 60.Centrifuge for 10minutes,withdraw the hexane layer by means of a pipet,and transfer to an evaporation flask.Add 12mLof n-hexane to the dimethyl sulfoxide layer,mix on a vortex mixer for 5minutes,and again withdraw the hexane layer by means of a pipet,and add to the evaporation flask.Repeat this extraction with three additional 12-mLportions of n-hexane,adding the hexane extracts to the evaporation flask.Evaporate the combined hexane extracts in vacuum at room temperature to dryness.Dissolve the residue in a known volume of n-hexane,and dilute quantitatively with n-hexane to obtain a solution having a concentration of about 2µg per mL.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm ×15-cm column that contains 5-µm packing L8.The flow rate is about 1mLper minute.Chromatograph the System suitability preparation,and record the peak reponses as directed for Procedure:the resolution,R,between the vitamin Dform present and its corresponding precursor is not less than 10.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the vitamin Dpeaks.Calculate the quantity,in µg,of ergocalciferol (C27H44O)or cholecalciferol (C28H44O)in the Tablets taken by the formula: in which 1.09is a correction factor to account for the average amount of previtamin Dpresent in the formulation,Cis the concentration,in µg per mL,of USP Ergocalciferol RSor USP Cholecalciferol RSin the Standard preparation;Dis the dilution factor,in mL,used to prepare the Assay preparation;and rUand rSare the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparationand the Standard preparation,respectively.