Medium: 0.1 N hydrochloric acid; 900 mL.

Apparatus 2: 75 rpm.

Time: 30 minutes.

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 30 minutes.

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

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

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

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

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the calcium emission line at 422.7 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-scattering 851) equipped with a calcium hollow-cathode lamp and a nitrous oxide–acetylene flame, using 0.125 N hydrochloric acid containing 0.1% Lanthanum chloride solution as the blank. Plot the absorbances of the Standard preparations versus 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 C is the concentration, in µg per mL, of calcium in the Assay preparation, and D is 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 Suitability under Chromatography 621).

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

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

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

Chromatographic system (see Chromatography 621)—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 1 mL per minute. Chromatograph the System suitability preparation, and record the peak reponses as directed for Procedure: the resolution, R, between the vitamin D form 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 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the vitamin D peaks. Calculate the quantity, in µg, of ergocalciferol (C27H44O) or cholecalciferol (C28H44O) in the Tablets taken by the formula: in which 1.09 is a correction factor to account for the average amount of previtamin D present in the formulation, C is the concentration, in µg per mL, of USP Ergocalciferol RS or USP Cholecalciferol RS in the Standard preparation; D is the dilution factor, in mL, used to prepare the Assay preparation; and rU and rS are the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparation and the Standard preparation, respectively.