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Method of Analysis for Calcium Citrate

Procedure for analysis of Calcium Citrate including Assay and Organic Volatile impurities.
1. Description
White odorless, crystalline powder.

2. Solubility
Slightly soluble in water, freely soluble in diluted 3N hydrochloric acid and in dilute 2N nitric acid, insoluble in alcohol.

3. Identification
A. Limit: A white precipitates are produced.
Reagent required
2N Nitric acid
Mercuric sulfate
Potassium permanganate
Dissolve 0.5gm in a mixture of 10ml of water and 2.5ml of 2N nitric acid. Add 1ml of mercuric sulfate, heat to boiling, and add 1ml of potassium permanganate.
B. Limit: A voluminous white precipitate is formed, and it is soluble in hydrochloric acid.
Reagent required
Glacial acetic acid
Ammonium oxalate
Ignite completely 0.5gm at as low a temperature as possible, cool, and dissolve the residue in a mixture of 10ml of water and 1ml of glacial acetic acid. Filter, and add 10ml of ammonium oxalate to the filtrate.

4. Loss on Drying
Limit: NLT 10.0% and NMT 13.3%
Weigh 1.000 g of substance in a clean and dried preweighed LOD Bottle. Cover the stopper and gently shake to distribute material to not more than 10 MM height. Place the LOD Bottle in the oven and remove the cover and leave it also inside the oven. Dry the sample at 150° C for 4 hr. On opening the chamber, immediately close the LOD Bottle, transfer it to the desiccator and bring it to room temperature. Weigh up to constant weight.

                                     W2 – W1
% Loss on drying = --------------- X 100
                                     W2 – W3

W1 = Weight of empty clean and dried LOD Bottle.
W2 = Weight of LOD Bottle + sample.
W3 = Weight of LOD Bottle + sample. (After drying)

5. Arsenic
Limit: Not more than 3 ppm
Reagents Required:
Brominated Hydrochloric acid
Stannous chloride
Arsenic standard solution (10 ppm As)
1M potassium iodide
3N Hydrochloric acid
7N Sulfuric acid
Stronger acid stannous chloride
Isopropyl alcohol
Saturated lead acetate solution
Silver diethyldithiocarbamate
Test Solution Preparation: Dissolve 1gm of the sample in 5ml of 3N hydrochloric acid, and dilute with water to 35ml.
Standard Solution Preparation: Pipette 3.0ml of Standard Arsenic Solution into a generator flask, and dilute with water to 35ml.
To each test tube of Standard Preparation and the Test Preparation add 20ml of 7N sulfuric acid, 2ml of potassium iodide, 0.5ml of stronger acid stannous chloride, and 1ml of isopropyl alcohol, and mix. Allow standing at room temperature for 30minutes. Pack the scrubber tube with two pledgets of cotton that have been soaked in saturated lead acetate solution, freed from excess solution by the expression, and dried under vacuum at room temperature, leaving a 2-mm space between the two pledgets. 
Lubricate the joints and with a suitable stopcock grease designed for use with organic solvents, and connect the scrubber unit to the absorber tube. Transfer 3.0ml of silver diethyldithiocarbamate to the absorber tube. Add 3.0 g of granular zinc (No. 20 mesh) to the mixture in the flask, immediately connect the assembled scrubber unit, and allow the evolution of hydrogen and the color development to proceed at room temperature for 45 minutes, swirling the flask gently at 10-minute intervals. Disconnect the absorber tube from the generator and scrubber units, and transfer the absorbing solution to a 1-cm absorption cell. Any red color produced by the Test Preparation does not exceed that produced by the Standard Preparation.

6. Limit of Fluorides
Limit: Not more than 0.003%
Standard solution: Dissolve an accurately weighed quantity of USP Sodium Fluoride RS quantitatively in water to obtain a solution containing 2.210mg per ml. On the day of use, transfer 5.0ml of this solution to a 1000ml volumetric flask, dilute with water to volume and mix. Each ml of this solution contains 5 µg of fluoride ion.
Electrode system: Use a fluoride-specific, ion-indicating electrode and a silver –silver chloride reference electrode connected to a pH meter capable of measuring potentials with a minimum reproducibility of ±0.2mV
Standard response line: Transfer 1.0, 5.0, and 10.0ml of the Standard solution to separate 250ml plastic beakers, to each, add 50 ml of water, 5 ml of 1 N hydrochloric acid, 10 ml of 1.0 M sodium citrate, and 10 ml of 0.2 M Edetate Disodium, and mix. If necessary, adjust with 1N sodium hydroxide or 1N hydrochloric acid to a pH of 5.5. Transfer each solution to a separate 100ml volumetric flask, dilute with water to volume, and mix. Transfer 50 ml of each solution to separate 250ml plastic beakers, and measure the potential of each solution with the Electrode system. Between each reading wash the electrodes with water, and absorb any residual water by blotting the electrodes dry. Plot the logarithms of the fluoride concentrations (0.05, 0.25, and 0.50 µg per ml, respectively) versus potential, in mV.
Procedure: Transfer 1.0gm of Calcium Citrate to a 100ml beaker, add 10ml of water and, while stirring, 10ml of 1N hydrochloric acid. When dissolved, boil rapidly for 1 minute, transfer the solution to a 250ml plastic beaker, and cool in ice water. Add 15ml of 1.0 M sodium citrate and 10ml of 0.2 M Edetate Disodium, and adjust with 1N sodium hydroxide or 1N hydrochloric acid to a pH of 5.5. Transfer this solution to a 100ml volumetric flask, dilute with water to volume, and mix. Transfer 50ml of this solution to a 250ml plastic beaker, and measure the potential of this test solution with the Electrode system. From the measured potential and the Standard response, line determines the concentration, C, in µg per ml, of fluoride ion in the test solution. Calculate the percentage of fluoride in the specimen taken by multiplying C by 0.01.

7. Acid Insoluble Substance
Limit: The residue weights not more than 10mg. (0.2%)
Dissolve 5gm by heating with a mixture of 10ml of hydrochloric acid and 50ml of water for 30 minutes: the residue so obtained, filtered, washed, and dried at 105° for 2 hours.

8. Lead
Limit: Not more than 0.001%
Test Preparation: Dissolve 0.5gm in 20ml of 3N hydrochloric acid, evaporating on a steam bath to about 10ml, diluting with water to about 20ml, and cooling.
Standard Preparation: Take 5ml of Diluted Standard Lead Solution (5 µg of Pb).
Transfer the Test Preparation, rinsing with 10ml of water, add 6ml of Ammonium Citrate Solution and 2ml of Hydroxylamine Hydrochloride Solution. Add 2 drops of phenol red, and make the solution just alkaline (red in color) by the addition of ammonium hydroxide. Cool the solution if necessary, and add 2ml of Potassium Cyanide Solution. Immediately extract the solution with 5ml portions of Dithizone Extraction Solution, draining off each extract into another separator, until the dithizone solution retains its green color. Shake the combined dithizone solutions for 30 seconds with 20ml of dilute nitric acid (1 in 100), and discard the chloroform layer. Add to the acid solution 5.0ml of Standard Dithizone Solution and 4ml of Ammonia-Cyanide Solution, and shake for 30seconds: the color of the chloroform layer is no deeper shade of violet than that of a control made with a volume of Diluted Standard Lead Solution equivalent to the amount of lead permitted in the sample under examination, and the same quantities of the same reagents and in the same manner as in the test with the sample.

9. Heavy metals
Limit: Not more than 0.002%
Reagent required
Dilute Hydrochloric acid
Dilute Acetic acid
Lead standard solution (20 ppm Pb)
Dilute Ammonia solution
Hydrogen sulphide
Test Solution Preparation: Dissolve 1gm in a mixture of 20ml of water and 2ml of hydrochloric acid, add 1.5ml of ammonium hydroxide, and dilute with water to 25ml.
Standard solution: Into a 50ml color-comparison tube pipette 2ml of Standard Lead Solution (20 µg of Pb), and dilute with water to 25ml. Adjust with 1N acetic acid or 6N ammonium hydroxide to a pH between 3.0 and 4.0, using short-range pH indicator paper as the external indicator, dilute with water to 40ml, and mix.
To each of tubes containing the Standard Preparation, and the Test Preparation add 2ml of pH 3.5 Acetate Buffer, then add 1.2ml of thioacetamide-glycerin base, dilute with water to 50ml, mix, allow to stand for 2 minutes, and view downward over a white surface. The color of the solution from the Test Preparation is not darker than that of the solution from the Standard Preparation.

10. Organic Volatile Impurity
Limit: Chloroform: Not more than 60 ppm
1,4-Dioxane: Not more than 380 ppm
Methylene chloride: Not more than 600 ppm
Trichloroethylene: Not more than 80 ppm
Standard solution preparation: Prepare a solution in organic free water containing in each ml, 12mg of Methylene chloride, 7.6mg of 1,4-dioxane, 1.6mg of Trichloroethylene, and 1.2mg of chloroform. Pipette 5ml of this solution into a vial fitted with a septum and crimp cap, containing 1g of anhydrous sodium sulfate, and seal. Heat a sealed vial at 80°C for 60 minutes.
Test Solution: Transfer 100mg, accurately weighed sample to a vial, add 5.0ml of water, and 1gm of anhydrous sodium sulfate, and seal with a septum and crimp cap. Heat the sealed vial at 80°C for 60 minutes.
Chromatographic conditions:
Column: 0.53mm X 30m fused silica analytical column coated with a 3.0 mm G43 stationary phase, and a 0.53mm X 5m silica guard column deactivated with phenyl methyl siloxane.

Carrier gas
Carrier flow
Column Int. Temp
Column Pro. Rate
Column Pro. Rate
Injector temp.
Injection volume
Detector temp
Flame ionization detector

40°C for 20 minutes


Procedure: Separately inject equal volumes (about 1ml) of the standard solution and the test solution into the chromatograph, record the chromatograms, and measure the peak responses. Identify based on retention time, any peaks present in the chromatogram of the test solution. The identity and the peak response in the chromatogram may be established as being from any of the organic volatile impurities that are mentioned in the limit.

                                                                     Rt     WS       P
Content of each components (ppm) = ----- x ----- x ----- x D x 106
                                                                     RS     Wt      100

Rt = Ratio of the detector response of the component to IS in test preparation.
RS = Ratio of the detector response of component to IS in std.preparation.
WS= weight of component in standard in gms.
Wt= weight samples taken in gms
P = % purity of component.
D = Dilution factor 0.01 for methanol and THF, 0.001 for n-hexane and toluene).

11. Assay
Limit: NLT97.5% and NMT 100.5% calculated on dried substance
Reagent required
3N Hydrochloric acid
0.05M Disodium Edetate
Sodium hydroxide solution
Hydroxy naphthol blue
Dissolve about 350mg of Calcium Citrate, previously dried at 150° to constant weight and accurately weighed, in a mixture of 10ml of water and 2ml of 3N hydrochloric acid, and dilute with water to about 100ml. While stirring, add about 30ml of 0.05 M Edetate Disodium VS from a 50ml burette. Add 15ml of 1N sodium hydroxide and 300mg of hydroxy naphthol blue, and continue the titration to a blue endpoint. Each mL of 0.05 M Edetate Disodium is equivalent to 8.307 mg of Ca3(C6H5O7)2

                          V x M x F           100
% Assay = ----------------- x-------------
                           0.05 x W        (100-LOD)

V = Blank reading – Sample reading
M = Molarity of 0.05M Disodium Edetate
F = Factor
W = Weight of sample

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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