Minerals Capsules
DEFINITION
Minerals Capsules contain two or more minerals derived from substances generally recognized as safe, furnishing two or more of the following elements in ionizable form: boron, calcium, chromium, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, tin, vanadium, and zinc. Capsules contain NLT 90.0% and NMT 125.0% of the labeled amounts of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), phosphorus (P), potassium (K), and zinc (Zn); and NLT 90.0% and NMT 160.0% of the labeled amounts of boron (B), chromium (Cr), fluorine (F), iodine (I), molybdenum (Mo), nickel (Ni), selenium (Se), tin (Sn), and vanadium (V). They contain no vitamins. They may contain other labeled added substances in amounts that are unobjectionable.
STRENGTH
[Note—In the following assays, where more than one assay method is given for an individual ingredient, the requirements may be met by following any one of the specified methods, the method used being stated in the labeling only if Method 1 is not used. 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 solutions and the Sample solutions may be modified to fit the linear or working range of the instrument. ]
•  Calcium, Method 1
Lanthanum chloride solution:  267 mg/mL of lanthanum chloride heptahydrate in 0.125 N hydrochloric acid
Calcium standard solution:  400 µg/mL of calcium. Dissolve 1.001 g of calcium carbonate, previously dried at 300 for 3 h and cooled in a desiccator for 2 h, in 25 mL of 1 N hydrochloric acid. Boil to expel carbon dioxide, and dilute with water to 1000 mL.
Standard stock solution:  100 µg/mL of calcium from Calcium standard solution diluted with 0.125 N hydrochloric acid
Standard solutions:  Into separate 100-mL volumetric flasks pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Standard stock solution. To each flask add 1.0 mL of Lanthanum chloride solution and dilute with water to volume to obtain concentrations of 1.0, 1.5, 2.0, 2.5, and 3.0 µg/mL of calcium.
Polysorbate 80 solution:  Dilute Polysorbate 80 with alcohol (1 in 10)
Sample solution:  Transfer 5 Capsules to a 100-mL volumetric flask. [Note—For hard gelatin Capsules, weigh NLT 20 Capsules. Open the Capsules, without loss of shell material, and transfer the contents to a suitable container. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and allow the Capsule shells to dry. Weigh the empty Capsule shells, calculate the net weight of the Capsule contents, and transfer a portion of the Capsule contents, equivalent to 5 Capsules, to a 100-mL volumetric flask. ] Add 15 mL of water, 10 mL of 6 N hydrochloric acid, and 1 mL of Polysorbate 80 solution to the flask. Heat on a hot plate or steam bath, with intermittent swirling, until the Capsules are completely disintegrated or the contents are dissolved. Boil gently for an additional 15 min. Cool, dilute with water to volume, and filter, discarding the first 5 mL of the filtrate. Dilute this solution with 0.125 N hydrochloric acid to obtain a concentration of 2 µg/mL of calcium, adding 1 mL of Lanthanum chloride solution per 100 mL of the final volume.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Calcium hollow-cathode
Flame:  Nitrous oxide–acetylene
Analytical wavelength:  Calcium emission line at 422.7 nm
Blank:  0.125 N hydrochloric acid containing 1 mL of Lanthanum chloride solution per 100 mL
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of calcium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of calcium in the Sample solution.
Calculate the percentage of the labeled amount of calcium (Ca) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of calcium in the Sample solution (µg/mL)
CU== nominal concentration of calcium in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of calclium (Ca)
•  Chromium, Method 1
Chromium standard solution:  1000 µg/mL of chromium from potassium dichromate, previously dried at 120 for 4 h, in water. Store in a polyethylene bottle.
Standard stock solution:  10 µg/mL of chromium from Chromium standard solution diluted with 6 N hydrochloric acid and water (1 in 20)
Standard solutions:  Transfer 10.0 and 20.0 mL of the Standard stock solution to separate 100-mL volumetric flasks, and transfer 15.0 and 20.0 mL of the Standard stock solution to separate 50-mL volumetric flasks. Dilute the contents of each of the four flasks with 0.125 N hydrochloric acid to volume to obtain concentrations of 1.0, 2.0, 3.0, and 4.0 µg/mL of chromium.
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to prepare the Sample solution to contain 1 µg/mL of chromium and to omit the use of the Lanthanum chloride solution.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Chromium hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Chromium emission line at 357.9 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of chromium, and draw the straight line best fitting the four plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of chromium in the Sample solution.
Calculate the percentage of the labeled amount of chromium (Cr) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of chromium in the Sample solution (µg/mL)
CU== nominal concentration of chromium in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of chromium (Cr)
•  Copper, Method 1
Copper standard solution:  Dissolve 1.00 g of copper foil in a minimum volume of a 50% (v/v) solution of nitric acid, and dilute with a 1% (v/v) solution of nitric acid to 1000 mL. This solution contains 1000 µg/mL of copper.
Standard stock solution:  100 µg/mL of copper from Copper standard solution diluted with 0.125 N hydrochloric acid
Standard solutions:  To separate 200-mL volumetric flasks transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of the Standard stock solution. Dilute with water to volume to obtain concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 µg/mL of copper.
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to prepare the Sample solution to contain 2 µg/mL of copper and to omit the use of the Lanthanum chloride solution
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Copper hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Copper emission line at 324.7 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/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/mL, of copper in the Sample solution.
Calculate the percentage of the labeled amount of copper (Cu) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of copper in the Sample solution (µg/mL)
CU== nominal concentration of copper in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of copper (Cu)
•  Fluoride, Method 1
[Note—Store all solutions in plastic containers. ]
3 M sodium acetate solution:  Dissolve 408 g of sodium acetate in 600 mL of water contained in a 1000-mL volumetric flask. Allow the solution to equilibrate to room temperature, and dilute with water to volume. Adjust with a few drops of acetic acid to a pH of 7.0.
Sodium citrate solution:  Dissolve 222 g of sodium citrate in 250 mL of water in a 1000-mL volumetric flask. Add 28 mL of perchloric acid, and dilute with water to volume.
Fluoride standard stock solution:  500 µg/mL of fluoride from a quantity of sodium fluoride, previously dried at 100 for 4 h and cooled in a desiccator, in water
Intermediate stock solution A:  100 µg/mL of fluoride from Fluoride standard stock solution diluted with water
Intermediate stock solution B:  10 µg/mL of fluoride from Fluoride standard stock solution diluted with water
Standard solutions:  To five separate 100-mL volumetric flasks transfer 3.0, 5.0, and 10.0 mL of Intermediate stock solution B and 5.0 and 10.0 mL of Intermediate stock solution A. To each flask add 10.0 mL of 1 N hydrochloric acid, 25 mL of 3 M sodium acetate solution, and 25.0 mL of Sodium citrate solution. Dilute the contents of each flask with water to volume to obtain concentrations of 0.3, 0.5, 1.0, 5.0, and 10.0 µg/mL of fluoride.
Sample solution:  Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the mixed Capsule contents, equivalent to 200 µg of fluoride, to a 100-mL volumetric flask. Add 10.0 mL of 1 N hydrochloric acid, 25.0 mL of 3 M sodium acetate solution, and 25.0 mL of Sodium citrate solution. Dilute with water to volume.
Analysis 
Samples:  Standard solutions and Sample solution
To separate plastic beakers, each containing a plastic-coated stirring bar, transfer 50.0 mL each of the Standard solutions and the Sample solution. Measure the potentials (see pH 791), in mV, of the Standard solutions and the Sample solution, with a pH meter capable of a minimum reproducibility of ±0.2 mV and equipped with a fluoride-specific ion-indicating electrode and a calomel-reference electrode. [Note—When taking measurements, immerse the electrodes in the solution, stir on a magnetic stirrer having an insulated top until equilibrium is attained (1–2 min), and record the potential. Rinse and dry the electrodes between measurements, taking care to avoid damaging the crystal of the specific-ion electrode. ]
Plot the logarithms of fluoride concentrations, in µg/mL, of the Standard solutions versus the potential, in mV. From the standard response curve so obtained and the measured potential of the Sample solution, determine the concentration, C, in µg/mL, of fluoride in the Sample solution.
Calculate the percentage of the labeled amount of fluorine (F) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of fluoride in the Sample solution (µg/mL)
CU== nominal concentration of fluorine in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of fluorine (F)
•  Fluoride, Method 2
[Note—Use plastic containers and deionized water throughout this procedure. ]
pH 10.0 buffer:  Add 214 mL of 0.1 N sodium hydroxide to 1000 mL of 0.05 M sodium bicarbonate.
Mobile phase:  Alcohol, 0.1 N sulfuric acid, and water (20:5:175)
Standard stock solution:  220 µg/mL of USP Sodium Fluoride RS in water. This solution contains 100 µg/mL of fluoride.
Standard solution 
[Note—Condition the solid-phase extraction column specified for use in the Standard solution and the Sample solution in the following manner. Using a vacuum at a pressure not exceeding 5 mm of mercury, wash the column with one column volume of methanol followed by one column volume of pH 10.0 buffer. Do not allow the column top to dry. If the top of the column becomes dry, recondition the column. ]
Transfer 10.0 mL of the Standard stock solution to a 100-mL volumetric flask. Add 75 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.4 ± 0.1. Dilute with water to volume. Filter, discarding the first 15 mL of the filtrate. Transfer 25.0 mL of the filtrate to a 50-mL volumetric flask. Add 15.0 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.0. Dilute with pH 10.0 buffer to volume. Elute a portion of this solution through a 3-mL solid-phase extraction column containing L1 packing that is connected through an adaptor to a second solid-phase extraction column containing sulfonylpropyl strong cation-exchange packing. Discard the first 3 mL of the eluate, and collect the rest of the eluate in a suitable flask for injection into the chromatograph.
Sample solution:  Weigh NLT 20 Capsules in a tared weighing bottle. Open the Capsules, without loss of shell material, and transfer the contents to a 100-mL container. If necessary, remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer a portion of the Capsule contents, equivalent to a nominal amount of 1 mg of fluorine, to a 100-mL volumetric flask. Add 15 mL of water, and shake vigorously. Rinse the sides of the flask with 15 mL of water, and allow to stand for 10 min. Dilute with water to 85 mL, adjust with 1 N sodium hydroxide to a pH of 10.4 ± 0.1, and dilute with water to 100 mL. Proceed as directed for the Standard solution, beginning with “Filter, discarding the first 15 mL of the filtrate.”
Chromatographic system  
Mode:  LC
Detector:  Conductivity
Guard column:  4.6-mm × 3-cm; packing L17
Analytical column:  7.8-mm × 30-cm; packing L17
Flow rate:  0.5 mL/min
Injection size:  100 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
Measure the peak areas for fluoride. Calculate the percentage of the labeled amount of fluorine (F) in the portion of Capsules taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area from the Sample solution
rS== peak area from the Standard solution
CS== concentration of fluoride in the Standard solution (µg/mL)
CU== nominal concentration of fluorine in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of fluorine (F)
•  Iodide, Method 1
Bromine water:  To 20 mL of bromine in a glass-stoppered bottle add 100 mL of water. Insert the stopper into the bottle, and shake. Allow to stand for 30 min, and use the supernatant.
Analysis:  Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the contents, equivalent to 3 mg of iodine, to a nickel crucible. Add 5 g of sodium carbonate, 5 mL of 50% (w/v) sodium hydroxide solution, and 10 mL of alcohol, taking care that the entire specimen is moistened. Heat the crucible on a steam bath to evaporate the alcohol, then dry the crucible at 100 for 30 min to prevent spattering upon subsequent heating. Transfer the crucible with its contents to a furnace heated to 500, and heat the crucible for 15 min. [Note—Heating at 500 is necessary to carbonize any organic matter present; a higher temperature may be used, if necessary, to ensure complete carbonization of all organic matter. ] Cool the crucible, add 25 mL of water, cover the crucible with a watchglass, and boil gently for 10 min. Filter the solution, and wash the crucible with boiling water, collecting the filtrate and washings in a beaker. Add phosphoric acid until the solution is neutral to methyl orange, then add 1 mL excess of phosphoric acid. Add excess of Bromine water, and boil the solution gently until colorless and then for 5 min longer. Add a few crystals of salicylic acid, and cool the solution to 20. Add 1 mL of phosphoric acid and 0.5 g of potassium iodide, and titrate the liberated iodine with 0.005 N sodium thiosulfate VS, adding starch TS when the liberated iodine color has nearly disappeared.
Calculate the percentage of the labeled amount of iodine (I) in the portion of Capsules taken:
Result = V × NA × F × Ime × (Aw/W) × (100/L)
V== volume of sodium thiosulfate consumed (mL)
NA== actual normality of the sodium thiosulfate solution used (meq/mL)
F== correction factor to convert mg to µg, 1000 µg/mg
Ime== milliequivalent of I, 21.16 mg/meq
Aw== average weight of the Capsules content
W== weight of the sample of Capsules content taken
L== labeled amount of iodine (µg/Capsule)
Acceptance criteria:  90.0%–160.0% of the labeled amount of iodine (I)
•  Iodide, Method 2: Proceed as directed in Automated Methods of Analysis 16, Assay for Iodide.
Acceptance criteria:  90.0%–160.0% of the labeled amount of iodine (I)
•  Iron, Method 1
Iron standard stock solution:  Transfer 100 mg of iron powder to a 1000-mL volumetric flask. Dissolve in 25 mL of 6 N hydrochloric acid, dilute with water to volume, and mix.
Standard solutions:  To separate 100-mL volumetric flasks transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of Iron standard stock solution. Dilute the contents of each flask with water to volume to obtain concentrations of 2.0, 4.0, 5.0, 6.0, and 8.0 µg/mL of iron.
Polysorbate 80 solution:  Prepare as directed in the assay for Calcium, Method 1.
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to prepare the Sample solution to contain a nominal concentration of 5 µg/mL of iron and to omit the use of the Lanthanum chloride solution.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Iron hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Iron emission line at 248.3 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of iron, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of iron in the Sample solution.
Calculate the percentage of the labeled amount of iron (Fe) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of iron in the Sample solution (µg/mL)
CU== nominal concentration of iron in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of iron (Fe)
•  Magnesium, Method 1
Lanthanum chloride solution:  Prepare as directed in the assay for Calcium, Method 1.
Magnesium standard solution:  Transfer 1.0 g of magnesium ribbon 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 with a concentration of 1000 µg/mL of magnesium.
Standard stock solution:  20 µg/mL of magnesium from Magnesium standard solution diluted with 0.125 N hydrochloric acid
Standard solutions:  To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Standard stock solution. To each flask add 1.0 mL of Lanthanum chloride solution and dilute with 0.125 N hydrochloric acid to volume to obtain concentrations of 0.2, 0.3, 0.4, 0.5, and 0.6 µg/mL of magnesium.
Polysorbate 80 solution:  Prepare as directed in the assay for Calcium, Method 1.
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to prepare the Sample solution to contain 0.4 µg/mL of magnesium.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Magnesium hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Magnesium emission line at 285.2 nm
Blank:  0.125 N hydrochloric acid containing 1 mL of Lanthanum chloride solution per 100 mL
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/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/mL, of magnesium in the Sample solution.
Calculate the percentage of the labeled amount of magnesium (Mg) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of magnesium in the Sample solution (µg/mL)
CU== nominal concentration of magnesium in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of magnesium (Mg)
•  Manganese, Method 1
Manganese standard stock solution:  Transfer 1.00 g of manganese, 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 with a concentration of 1000 µg/mL of manganese.
Standard stock solution:  50 µg/mL of manganese from Manganese standard stock solution diluted with 0.125 N hydrochloric acid
Standard solutions:  To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Standard stock solution. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain solutions with concentrations of 0.5, 0.75, 1.0, 1.5, and 2.0 µg/mL of manganese.
Polysorbate 80 solution:  Prepare as directed in the assay for Calcium, Method 1.
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to prepare the Sample solution to contain 1 µg/mL of manganese and to omit the use of the Lanthanum chloride solution.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Manganese hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Manganese emission line at 279.5 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/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/mL, of manganese in the Sample solution.
Calculate the percentage of the labeled amount of manganese (Mn) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of manganese in the Sample solution (µg/mL)
CU== nominal concentration of manganese in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of manganese (Mn)
•  Molybdenum, Method 1
Diluent:  20 mg/mL of ammonium chloride in water
Molybdenum standard solution:  Transfer 1.0 g of molybdenum wire to a 1000-mL volumetric flask, and dissolve in 50 mL of nitric acid, warming if necessary. Dilute with water to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of molybdenum.
Standard stock solution:  100 µg/mL of molybdenum from Molybdenum standard solution diluted with water
Standard solutions:  To separate 100-mL volumetric flasks transfer 2.0, 10.0, and 25.0 mL of the Standard stock solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solution in each flask for 15 min. Cool to room temperature, and dilute each with Diluent to volume to obtain concentrations of 5.0, 10.0, and 25.0 µg/mL of molybdenum.
Polysorbate 80 solution:  Prepare as directed in the assay for Calcium, Method 1.
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to take a number of Capsules or a portion of Capsule contents, equivalent to 1000 µg of molybdenum, and to make appropriate dilutions to obtain a final concentration of 10 µg/mL of molybdenum, omitting the addition of the Lanthanum chloride solution.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Molybdenum hollow-cathode
Flame:  Nitrous oxide–acetylene
Analytical wavelength:  Molybdenum emission line at 313.3 nm
Blank:  Diluent and perchloric acid (20:1)
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of molybdenum, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of molybdenum in the Sample solution.
Calculate the percentage of the labeled amount of molybdenum (Mo) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of molybdenum in the Sample solution (µg/mL)
CU== nominal concentration of molybdenum in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of molybdenum (Mo)
•  Molybdenum, Method 2
Sodium fluoride solution:  Add 200 mL of water to 10 g of sodium fluoride, stir until the solution is saturated, and filter. Store in a polyethylene bottle.
Ferrous sulfate solution:  4.98 mg/mL of ferrous sulfate in water
Potassium thocyanate solution:  200 mg/mL of potassium thiocyanate in water
20% Stannous chloride solution:  Transfer 40 g of stannous chloride to a beaker, add 20 mL of 6.5 N hydrochloric acid, and heat the solution until the stannous chloride is dissolved. Cool, and dilute with water to 100 mL.
Diluted stannous chloride solution:  20% Stannous chloride solution diluted with water (1 in 25). Prepare this solution fresh at the time of use.
Standard solution:  20 µg/mL of molybdenum from ammonium molybdate, in water
Sample:  Remove the contents of a counted number of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Use a quantity of Capsule contents, equivalent to a nominal amount of 40 µg of molybdenum
Spectrometric conditions 
Mode:  UV-Vis
Cell:  1 cm
Analytical wavelength:  465 nm
Blank:  Amyl alcohol
Analysis 
Samples:  Standard solution and Sample
Transfer the Sample and 2.0 mL of the Standard solution to separate 200-mL beakers. Add 20 mL of nitric acid to each beaker. Cover each beaker with a watchglass, and boil slowly on a hot plate for 45 min. Cool to room temperature. Add 6 mL of perchloric acid, cover the beakers with a watchglass, and continue the heating until digestion is complete, as indicated when the liquid becomes colorless or pale yellow. Evaporate the solutions in the beakers to dryness. Rinse the sides of the beakers and the watchglasses with water, and add more water to each beaker to complete 50 mL in each beaker. Gently boil the water solutions for a few min. Cool to room temperature. Add 2 drops of methyl orange TS and neutralize with ammonium hydroxide. Add 8.2 mL of hydrochloric acid. Quantitatively transfer the contents of the beakers to separate 100-mL volumetric flasks, rinse the beakers with water, transfer the rinsings to the corresponding volumetric flasks, and dilute with water to volume. Transfer 50.0 mL of each solution to separatory funnels. To each separatory funnel add 1.0 mL of Sodium fluoride solution, 0.5 mL of Ferrous sulfate solution, 4.0 mL of Potassium thiocyanate solution, 1.5 mL of 20% Stannous chloride solution, and 15.0 mL of amyl alcohol, and shake the separatory funnel for 1 min. Allow the layers to separate, and discard the aqueous layers. Add 25 mL of Diluted stannous chloride solution to each separatory funnel, and shake gently for 15 s. Allow the layers to separate, and discard the aqueous layers. Transfer the organic layers from each separatory funnel to a centrifuge tube, and centrifuge at 2000 rpm for 10 min. Determine the absorbances of the organic phases obtained from the Standard solution and the Sample, and correct with the Blank.
Calculate the percentage of the labeled amount of molybdenum (Mo) in the portion of Capsules taken:
Result = (AU/AS) × [(V × CS)/MU] × 100
AU== absorbance of the solution from the Sample
AS== absorbance of the solution from the Standard solution
V== volume of the Standard solution analyzed, 2.0 mL
CS== concentration of molybdenum in the Standard solution (µg/mL)
MU== nominal concentration of molybdenum in the Sample (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of molybdenum (Mo)
•  Phosphorus, Method 1
Sulfuric acid solution:  Cautiously add sulfuric acid to water (37.5:100), and mix.
Ammonium molybdate solution:  50 mg/mL of ammonium molybdate in Sulfuric acid solution and water (2:3). [Note—Dissolve in water first, then dilute with Sulfuric acid solution to volume. ]
Hydroquinone solution:  5 mg/mL of hydroquinone in water. Add one drop of sulfuric acid per 100 mL of solution.
Sodium bisulfite solution:  200 mg/mL of sodium bisulfite in water
Phosphorus standard stock solution:  Weigh 4.395 g of monobasic potassium phosphate, previously dried at 105 for 2 h and stored in a desiccator, and transfer to a 1000-mL volumetric flask. Dissolve in water, add 6 mL of sulfuric acid as a preservative, dilute with water to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of phosphorus.
Standard solution:  20 µg/mL of phosphorus from Phosphorus standard stock solution diluted with water
Sample solution:  Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the Capsule contents, equivalent to 100 mg of phosphorus, to 25 mL of nitric acid, and digest on a hot plate for 30 min. Add 15 mL of hydrochloric acid, and continue the digestion to the cessation of brown fumes. Cool, and transfer the contents of the flask to a 500-mL volumetric flask with the aid of small portions of water. Dilute with water to volume. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, and dilute with water to volume.
Spectrometric conditions 
Mode:  UV-Vis
Cell:  1 cm
Analytical wavelength:  650 nm
Analysis 
Samples:  Standard solution and Sample solution
To three separate 25-mL volumetric flasks transfer 5.0 mL each of the Standard solution, the Sample solution, and water to provide the blank. To each of the three flasks add 1.0 mL each of Ammonium molybdate solution, Hydroquinone solution, and Sodium bisulfite solution, and swirl to mix. Dilute the contents of each flask with water to volume, and allow the flasks to stand for 30 min. Determine the absorbances of the solutions, against the blank.
Calculate the percentage of the labeled amount of phosphorus (P) in the portion of Capsules taken:
Result = (AU/AS) × (CS/CU) × 100
AU== absorbance of the Sample solution
AS== absorbance of the Standard solution
CS== concentration of phosphorus in the Standard solution (µg/mL)
CU== nominal concentration of phosphorus in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of phosphorus (P)
•  Potassium
Potassium standard solution:  100 µg/mL of potassium from potassium chloride, previously dried at 105 for 2 h, in water
Standard stock solution:  10 µg/mL of potassium from Potassium standard solution diluted with 0.125 N hydrochloric acid
Standard solutions:  Transfer 5.0, 10.0, 15.0, 20.0, and 25.0 mL of the Standard stock solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain solutions containing 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of potassium.
Polysorbate 80 solution:  Prepare as directed in the assay for Calcium, Method 1
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to prepare the Sample solution to contain 1 µg/mL of potassium and to omit the use of the Lanthanum chloride solution
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Potassium hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Potassium emission line at 766.5 nm
Blank:  Water
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of potassium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of potassium in the Sample solution.
Calculate the percentage of the labeled amount of potassium (K) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of potassium in the Sample solution (µg/mL)
CU== nominal concentration of potassium in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of potassium (K)
•  Selenium, Method 1
Diluent:  Prepare as directed in the assay for Molybdenum, Method 1.
Selenium standard solution:  [Caution—Selenium is toxic; handle it with care. ] Dissolve 1 g of metallic selenium in a minimum volume of nitric acid. Evaporate to dryness. Add 2 mL of water, and evaporate to dryness. Repeat the addition of water and the evaporation to dryness three times. Dissolve the residue in 3 N hydrochloric acid, transfer to a 1000-mL volumetric flask, and dilute with 3 N hydrochloric acid to volume, to obtain a concentration of 1000 µg/mL of selenium.
Standard stock solution:  100 µg/mL of selenium from Selenium standard solution diluted with water
Standard solutions:  To separate 100-mL volumetric flasks transfer 5.0, 10.0, and 25.0 mL of the Standard stock solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solutions for 15 min, cool to room temperature, and dilute each with Diluent to volume to obtain solutions containing 5.0, 10.0, and 25.0 µg/mL of selenium.
Sample solution:  Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the Capsule content, equivalent to 1000 µg of selenium, to 12 mL of nitric acid. [Note—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed. ] Carefully swirl the flask to disperse the sample specimen. Sonicate for 10 min or until the sample specimen is completely dissolved. Gently boil the solution for 15 min, and cool to room temperature. Carefully add 8 mL of perchloric acid to the flask, heat the flask until perchloric acid fumes appear, and swirl the flask to dissipate the fumes. Repeat the heating and swirling until the fumes appear again. Cool to room temperature. Transfer the contents of the flask to a 50-mL volumetric flask with the aid of the Diluent, and dilute with Diluent to volume.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Selenium hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Selenium emission line at 196.0 nm
Blank:  Diluent and perchloric acid (20:1)
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of selenium, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of selenium in the Sample solution.
Calculate the percentage of the labeled amount of selenium (Se) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of selenium in the Sample solution (µg/mL)
CU== nominal concentration of selenium in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of selenium (Se)
•  Selenium, Method 2
Hydrochloric acid solution:  Hydrochloric acid diluted with water (1 in 10)
50% Ammonium hydroxide solution:  Ammonium hydroxide diluted with water (1 in 2)
Reagent A:  9 mg/mL of edetate disodium and 25 mg/mL of hydroxylamine hydrochloride in water. [Note—Dissolve edetate disodium in a portion of water first, add hydroxylamine hydrochloride, then dilute with water to volume. ]
Reagent B:  Transfer 200 mg of 2,3-diaminonaphthalene to a 250-mL separatory funnel, and add 200 mL of 0.1 N hydrochloric acid. Wash the solution with three 40-mL portions of cyclohexane, and discard the cyclohexane layer. Filter the solution into a brown bottle, and cover the solution with a 1-cm layer of cyclohexane. This solution is stable for 1 week if stored in a refrigerator.
Standard stock solution:  [Caution—Selenium is toxic; handle it with care. ] Dissolve 1 g of metallic selenium in a minimum volume of nitric acid. Evaporate to dryness, add 2 mL of water, and evaporate to dryness. Repeat the addition of water and evaporation to dryness three times. Dissolve the residue in 3 N hydrochoric acid, transfer to a 1000-mL volumetric flask, and dilute with 3 N hydrochloric acid to volume to obtain a solution with a concentration of 1000 µg/mL of selenium. Dilute a volume of the solution with 0.125 N hydrochloric acid, to obtain a concentration of 2.0 µg/mL of selenium.
Standard solution:  Transfer 10.0 mL of Standard stock solution to a glass-stoppered flask. Add 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution, and dilute with water to 20 mL.
Sample solution:  Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the Capsule content, equivalent to a nominal amount of 20 µg of selenium, to a suitable flask. Add 10 mL of nitric acid, and warm gently on a hot plate. Continue heating until the initial nitric acid reaction has subsided, then add 3 mL of perchloric acid. [Caution—Exercise care at this stage because perchloric acid reaction becomes vigorous. ] Continue heating on the hot plate until the appearance of white fumes of perchloric acid or until the digest begins to darken. Add 0.5 mL of nitric acid, and resume heating, adding additional amounts of nitric acid if further darkening occurs. Digest for 10 min after the first appearance of perchloric acid fumes or until the digest becomes colorless. Cool the flask. Add 2.5 mL of Hydrochloric acid solution, and return the flask to the hot plate to expel residual nitric acid. Heat the mixture for 3 min after it begins to boil. Cool the flask to room temperature, and dilute with water to 20 mL.
Spectrometric conditions 
Mode:  UV
Cell:  1 cm
Analytical wavelength:  380 nm
Blank:  1 mL of perchloric acid and 1 mL of Hydrochloric acid solution diluted with water to 20 mL
Analysis 
Samples:  Standard solution and Sample solution
Treat the Sample solution, the Standard solution, and the Blank as follows. Add 5 mL of Reagent A to each flask, and swirl gently to mix. Adjust the solution in each flask with 50% Ammonium hydroxide solution to a pH of 1.1 ± 0.1. Add 5 mL of Reagent B to each flask, and swirl gently to mix. Place the flasks in a water bath maintained at 50, and equilibrate for 30 min, taking care that the flasks are covered to protect them from light. Cool to room temperature, and transfer the contents of each flask to separate separatory funnels. Transfer 10.0 mL of cyclohexane to each separatory funnel, and extract vigorously for 1 min. Discard the aqueous layer. Transfer the cyclohexane layer to a centrifuge tube, and centrifuge at 1000 rpm for 1 min to remove any remaining water. Determine the absorbances of the solutions obtained from the Samples against the solution obtained from the Blank.
Calculate the percentage of the labeled amount of selenium (Se) in the portion of Capsules taken:
Result = (AU/AS) × [(V × CS)/MU] × 100
AU== absorbances of the cyclohexane layer from the Sample solution
AS== absorbances of the cyclohexane layer from the Standard solution
V== volume of the Standard stock solution used to prepare the Standard solution, 10 mL
CS== concentration of selenium in the Standard stock solution (µg/mL)
MU== nominal amount of selenium in the Sample solution (µg)
Acceptance criteria:  90.0%–160.0% of the labeled amount of selenium (Se)
•  Zinc, Method 1
Zinc standard solution:  1000 µg/mL of zinc from zinc oxide dissolved in 5 M hydrochloric acid (3.89 mg/mL), and diluted with water to final volume. [Note—Dissolve in 5 M hydrochloric acid by warming, if necessary, cool, and then dilute to final volume. ]
Standard stock solution:  50 µg/mL of zinc from Zinc standard solution diluted with 0.125 N hydrochloric acid
Standard solutions:  Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of Standard stock solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of zinc.
Polysorbate 80 solution:  Prepare as directed in the assay for Calcium, Method 1.
Sample solution:  Proceed as directed for the Sample solution in the assay for Calcium, Method 1, except to prepare the Sample solution to contain a nominal concentration of 2 µg/mL of zinc and to omit the use of the Lanthanum chloride solution.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometry
Lamp:  Zinc hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  Zinc emission line at 213.8 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/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/mL, of zinc in the Sample solution.
Calculate the percentage of the labeled amount of zinc (Zn) in the portion of Capsules taken:
Result = (C/CU) × 100
C== measured concentration of zinc in the Sample solution (µg/mL)
CU== nominal concentration of zinc in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of zinc (Zn)
•  Boron, Nickel, Tin, and Vanadium, Method 1; Calcium, Chromium, Copper, Iron, Magnesium, Manganese, Phosphorus, and Zinc, Method 2; Molybdenum and Selenium, Method 3
Stock aqua regia solution:  Prepare a mixture of hydrochloric acid and nitric acid (3:1) by adding the nitric acid to the hydrochloric acid. [Note—Periodically vent the solution in an appropriate fume hood. ]
Diluent:  Prepare a mixture of Stock aqua regia solution and water (1:9) by adding one volume of Stock aqua regia solution to two volumes of water. Dilute with additional water to volume, and mix well.
System suitability solution:  Prepare a mixture of 1000 mg/L of yttrium in 5% (v/v) nitric acid solution and 1000 mg/L of scandium in 5% (v/v) nitric acid solution with Diluent (1:1:198), and mix.
Standard stock solution 1 (Ca, Cu, Fe, Mg, Mn, P, and Zn) [Note—It is only necessary to include the minerals of interest in the solution. ] Using commercially available element standard (single- or multi-element) solutions in 5% (v/v) nitric acid solution, pipet the appropriate amount of element standard solution into a volumetric flask, and dilute with 5% (v/v) nitric acid solution to obtain a solution with final concentrations of about 1000 mg/L of calcium, 100 mg/L of copper, 250 mg/L of iron, 500 mg/L of magnesium, 100 mg/L of manganese, 800 mg/L of phosphorus, and 250 mg/L of zinc.
Standard stock solution 2 (B, Cr, Mo, Ni, Se, Sn, and V) [Note—It is only necessary to include the minerals of interest in the solution. ] Using commercially available element standard (single- or multi-element) solutions in 20% (v/v) hydrochloric acid solution, pipet the appropriate amount of element standard solution into a volumetric flask, and dilute with 20% (v/v) hydrochloric acid solution to obtain a solution with final concentrations of about 200 mg/L of boron, and 100 mg/L of chromium, molybdenum, nickel, selenium, tin, and vanadium each.
Standard solutions:  Prepare a mixture of Standard stock solution 1 and Standard stock solution 2, as required, in Diluent, to prepare a six-point calibration curve to bracket the concentration range of each mineral of interest.
Sample solution:  Weigh, then transfer 5 Capsules to a 250-mL volumetric flask, and heat gently on a hot plate until the contents begin to release. Cautiously add 25 mL of Stock aqua regia solution in 5-mL increments, and swirl. Heat, continue to swirl until the Capsules dissolve into the acid, immediately remove from the heat source, and add 150 mL of water. Cool, and dilute with water to volume. Filter about 30 mL into a centrifuge tube using a 5-µm pore size nylon syringe filter. If necessary, make any further adjustments using the Diluent.
Spectrometric conditions 
Mode:  Inductively coupled plasma spectrometry using a spectrometer set to measure the emission of each mineral of interest at about the corresponding wavelength. [Note—The operating conditions may be developed and optimized based on the manufacturer's recommendation. The wavelengths selected should be demonstrated experimentally to provide sufficient specificity, sensitivity, linearity, accuracy, and precision. ]
System suitability 
[Note—Analyze the System suitability solution, and obtain the response as directed for Analysis. ]
Suitability requirements 
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solutions and Sample solution
Determine the emission of each mineral of interest in the Standard solutions and Sample solution with an inductively coupled plasma system using the Diluent as the blank. Plot the emission of the Standard solutions versus the concentration, in mg/L, of the minerals of interest, and draw the straight line best fitting the plotted points. From the graph so obtained, determine the concentration, C, in mg/L, for each mineral of interest in the Sample solution.
Calculate the percentage of the labeled amount for each mineral taken:
Result = C × (V/W) × F × (TW/L) × 100
C== measured concentration of the relevant element in the Sample solution (mg/L)
V== volume of the Sample solution (L)
W== sample weight (mg)
F== dilution factor of the Sample solution
TW== average Capsule weight (mg)
L== labeled amount of the relevant element per Capsule (mg/Capsule)
Acceptance criteria:  90%–125% of the labeled amount of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), phosphorus (P), and zinc (Zn); 90%–160% of the labeled amounts of boron (B), chromium (Cr), molybdenum (Mo), nickel (Ni), selenium (Se), tin (Sn), and vanadium (V)
PERFORMANCE TESTS
•  Weight Variation of Dietary Supplements 2091: Meet the requirements
SPECIFIC TESTS
•  Microbial Enumeration Tests—Nutritional and Dietary Supplements 2021: The total aerobic microbial count does not exceed 3000 cfu/g, and the combined molds and yeasts count does not exceed 300 cfu/g.
•  Absence of Specified Microorganisims—Nutritional and Dietary Supplements 2022: Meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers.
•  Labeling: The label states that the product is Minerals Capsules. The label also states the salt form of the mineral used as the source of each element. Where more than one assay method is given for a particular mineral, the labeling states the assay method used only if Method 1 is not used.
•  USP Reference Standards 11
USP Sodium Fluoride RS
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Topic/Question Contact Expert Committee
Monograph Natalia Davydova
Scientific Liaison
1-301-816-8328
(DS2010) Monographs - Dietary Supplements
2021 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
2022 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
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(GCM2010) General Chapters - Microbiology
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 1391
Pharmacopeial Forum: Volume No. 34(6) Page 1493