note—Commercially available atomic absorption standard solutions for the minerals, where applicable, may be used where preparation of a standard stock solution is described in the following Assays. Use deionized water where water is specified. Where atomic absorption spectrophotometry is specified in the Assay, the concentrations of the Standard preparations and the Assay preparation may be modified to fit the linear or working range of the instrument.

Copper standard stock solution— Dissolve 1.00 g of copper foil, accurately weighed, in a minimum volume of a 50% (v/v) nitric acid solution, and dilute with a 1% (v/v) nitric acid solution to 1000 mL to obtain a solution having a known concentration of about 1000 µg of copper per mL.

Standard preparations— Transfer 10.0 mL of Copper standard stock solution to a 100-mL volumetric flask, and quantitatively dilute with 0.125 N hydrochloric acid to volume to obtain a standard solution having a known concentration of about 100 µg of copper per mL. To separate 200-mL volumetric flasks, transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of the standard solution, dilute with water to volume, and mix to obtain solutions having known concentrations of about 0.5, 1.0, 2.0, 3.0, and 4.0 µg of copper per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 5 mg of copper, to a porcelain crucible. Proceed as directed for Assay preparation in the Assay for calcium under Calcium with Vitamin D Tablets, except to prepare the Assay preparation to contain about 2 µg of copper per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the copper emission line at 324.7 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a copper hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of copper, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of copper in the Assay preparation. Calculate the quantity, in mg, of copper (Cu) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

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

Magnesium standard stock solution— Transfer 1.00 g of magnesium, accurately weighed, to a 1000-mL volumetric flask, dissolve in 50 mL of 6 N hydrochloric acid, dilute with water to volume, and mix to obtain a solution having a known concentration of about 1000 µg of magnesium per mL.

Standard preparations— Quantitatively dilute a volume of the Magnesium standard stock solution with 0.125 N hydrochloric acid to obtain a standard solution having a concentration of about 20 µg of magnesium per mL. To separate 100-mL volumetric flasks, transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of standard solution. To each flask add 1.0 mL of Lanthanum chloride solution, dilute with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.2, 0.3, 0.4, 0.5, and 0.6 µg of magnesium per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 200 mg of magnesium, to a porcelain crucible. Heat for 6 to 12 hours in a muffle furnace maintained at about 550, and cool. Add about 15 mL of hydrochloric acid, and boil gently on a hot plate or a steam bath for about 30 minutes, intermittently rinsing the inner surface of the crucible with 6 N hydrochloric acid. Cool, and quantitatively transfer the contents of the crucible to a 100-mL volumetric flask, rinsing the crucible with portions of 6 N hydrochloric acid. Dilute the contents of the flask with water to volume, mix, and filter, discarding the first 5 mL of the filtrate. Dilute the filtrate quantitatively, and stepwise if necessary, with 0.125 N hydrochloric acid to obtain a solution having a concentration of about 0.4 µg of magnesium per mL, adding 1 mL of Lanthanum chloride solution per 100 mL of the final volume.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the magnesium emission line at 285.2 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a magnesium hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid containing 0.1% of Lanthanum chloride solution as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of magnesium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of magnesium in the Assay preparation. Calculate the quantity, in mg, of magnesium (Mg) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Manganese standard stock solution— Transfer 1.00 g of manganese, accurately weighed, to a 1000-mL volumetric flask, dissolve in 20 mL of nitric acid, dilute with 6 N hydrochloric acid to volume, and mix to obtain a solution having a known concentration of about 1000 µg of manganese per mL.

Standard preparations— Quantitatively dilute 10.0 mL of the Manganese standard stock solution with 0.125 N hydrochloric acid to 200.0 mL to obtain a standard solution having a concentration of about 50 µg of manganese per mL. To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the standard solution, dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.5, 0.75, 1.0, 1.5, and 2.0 µg of manganese per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 9 mg of manganese, to a porcelain crucible. Proceed as directed for Assay preparation in the Assay for calcium under Calcium with Vitamin D Tablets, except to prepare the Assay preparation to contain 1 µg of manganese per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the manganese emission line at 279.5 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a manganese hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of manganese, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of manganese in the Assay preparation. Calculate the weight, in mg, of manganese (Mn) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Zinc standard stock solution— Transfer about 311 mg of zinc oxide, accurately weighed, to a 250-mL volumetric flask, add 80 mL of 5 M hydrochloric acid, warm, if necessary, to dissolve, and cool. Dilute with water to volume, and mix to obtain a solution having a known concentration of about 1000 µg of zinc per mL.

Standard preparations— Dilute a volume of the Zinc standard stock solution quantitatively, and stepwise if necessary, with 0.125 N hydrochloric acid to obtain a standard solution having a known concentration of about 50 µg of zinc per mL. To separate 100-mL volumetric flasks transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of this solution. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.5, 1.0, 1.5, 2.0, and 2.5 µg of zinc per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 40 mg of zinc, to a porcelain crucible. Proceed as directed for Assay preparation in the Assay for calcium under Calcium with Vitamin D Tablets, except to prepare the Assay preparation to contain 2 µg of zinc per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the zinc emission line at 213.8 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a zinc hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of zinc, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of zinc in the Assay preparation. Calculate the quantity, in mg, of zinc (Zn) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.