Method of Analysis for Magnesium Stearate : Pharmaceutical Guidelines

Method of Analysis for Magnesium Stearate

Procedure for analysis of Magnesium Stearate in pharmaceutical quality control laboratory.

1. Description
Very fine, light white powder greasy to the touch, odorless or with a very faint odor of stearic acid, unctuous and free from grittiness.

2. Solubility
Practically insoluble in water, in ethanol and ether.

3. Identification
A. Freezing Point
Limit: The freezing point of the residue is not less than 53°C
Preparation of Solution A: To 5.0 g of the sample, add 50 ml of ether, 20 ml of 2M nitric acid and 20 ml of distilled water and heat under a reflux condenser until dissolution is complete.  Allow to cool, separate the aqueous layer and shake the ether layer with two 4-ml quantities of distilled water. Combine the aqueous layers, wash with 15 ml of ether and dilute to 50 ml with distilled water (solution A). Evaporate the ether layer to dryness and dry the residue at 100°C to 105°C.
Procedure
Place sufficient of the residue obtained white “Preparation of Solution A”, previously melted, in the inner tube such that the thermometer bulb is well covered and determine the approximate freezing point by cooling rapidly. Place the inner tube in a bath about 60°C (5°C above the approximate freezing point) until all but the last traces of crystals are melted. Fill the beaker with water or a saturated solution of sodium chloride at a temperature about 50°C (5° lower than the approximate freezing point), insert the inner tube into the outer tube, ensuring that some seed crystals are present, and stir thoroughly until solidification takes place. The highest temperature observed during solidification of the substance is regarded as the freezing point of the substance.  

B. Acid value of fatty acids
Limit: Between 195 and 210
Procedure
Weigh accurately about 0.2 g of the residue obtained while “Preparation of solution A” and transfer to a round bottom flask, add 25 ml of a mixture of equal volumes of ethanol (96%) and ether that has been neutralized with 0.1M potassium hydroxide using 0.5 ml of phenolphthalein solution as indicator. Shake well to dissolve, reflux if necessary to effect solution. When the substance has completely dissolved, titrate with 0.1M potassium hydroxide, shaking constantly until a pink colour that persists for at least 15 seconds is produced. Calculate the acid value from the expression 5.610 v/w, where v is the volume, in ml, of 0.1 M potassium hydroxide solution required and w is the weight, in g, of residue taken.

C. Gas chromatography
The retention times of the principal peaks in the chromatogram obtained with the test solution are approximately the same as those of principal peaks in the chromatogram obtained with the reference solution.

D. Reaction A of magnesium
Reagent required
Dilute ammonia
2M Ammonium chloride solution
0.25M Disodium hydrogen phosphate solution
Procedure
Transfer 1 ml of solution A to a test-tube, add 1 ml of dilute ammonia; a white precipitate is produced which is dissolved by adding 1 ml of 2M ammonium chloride solution.  Add 1 ml of 0.25M Disodium hydrogen phosphate solution; a white crystalline precipitate is produced.

4. Acidity or alkalinity
Limit: NMT 0.5 ml of 0.01M HCl or 0.01M NaOH is required
Reagent required
Bromothymol blue solution
0.1M hydrochloric acid.
0.1M sodium hydroxide
Procedure
Take 1 g sample in a 250 ml conical flask, add 20 ml water, boil for 1 min. with continuous shaking, cool and filter. To the 10 ml of filtrate add 0.05 ml of bromothymol blue solution. NMT 0.5 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the color.

5. Color of solution
Limit: The solution is not more colored than reference solution YS6.
Reagent required 
Ferric chloride colorimetric solution (FCS)
Cobaltous chloride colorimetric solution (CCS)
1% w/v Hydrochloric acid
Reference solution YS6: Transfer 1.6 ml of FCS and 0.4 ml of CCS. Make up the volume to 100 ml with 1% w/v hydrochloric acid.
Procedure
Transfer to a separate Nessler cylinder suitable volume of the sample preparation and reference solution YS6 such that the cylinders are filled to a depth of 40 mm. Examine the columns of the liquid in diffused light by viewing down the vertical axis of the cylinders against a white background.
The color intensity of sample preparation is not greater than that of reference solution YS6.

6. Clarity and color of solution of fatty acids
A) Color of solution
Reagent required
Ferric chloride colorimetric solution (FCS)
Cobaltous chloride colorimetric solution (CCS)
1% w/v HHHydrochloric acid
Reference color solution YS5: In a 100 ml volumetric flask, transfer 3.2 ml of FCS and 0.8 ml of CCS.  Make up the volume to 100 ml with 1% w/v of hydrochloric acid.
Sample preparation: Dissolve 0.5 g of the residue obtained in the solution preparation of Solution A in 10 ml of chloroform.
Procedure
Transfer to a separate Nessler cylinder preparation and reference solution YS5 such that the cylinders are filled to a depth of 40 mm. Examine the columns of the liquid in diffused light by viewing down the vertical axis of the cylinders against a white background.  The color intensity of sample preparation is not greater than that of the reference solution.

B) Clarity of solution
Reagent required
10% w/v solution of hexamine
Hydrazine sulphate
Reference suspensions: Dissolve 1.0 g of Hydrazine sulphate with 100 ml water and allow to stand for 4 to 6 hrs. Add 25 ml of this solution to 25 ml of 10% w/v solution of hexamine mix well and allow standing for 24 hrs. Dilute 15 ml to 1000 ml with water. Dilute 5.0 ml of Standard of opalescence to 100 ml with water.
Sample preparation: Dissolve 0.5 g of the residue obtained in the solution preparation of Solution A in 10 ml of chloroform.
Procedure
Into separate Nessler cylinder, place sufficient of the sample preparation, chloroform and of the reference suspension such that the test tubes are filled to a depth of 40 mm. After five minutes, compare the contents of the cylinder against a black background by viewing in diffused daylight down the vertical axes of the cylinder.  The clarity of the sample preparation. is not less than that of chloroform or reference suspension.

7. Acid value of the fatty acids
Limit: Between 195 and 210
Reagent required  
Ethanol (96%)
Ether
0.1M Potassium hydroxide
Phenolphthalein solution
Procedure
Weigh accurately about 0.2 g of the residue obtained while “Preparation of solution A” and transfer to a round bottom flask, add 25 ml of a mixture of equal volumes of ethanol (96%) and ether that has been neutralized with 0.1M potassium hydroxide using 0.5 ml of phenolphthalein solution as indicator. Shake well to dissolve, reflux if necessary to effect solution. When the substance has completely dissolved, titrate with 0.1M potassium hydroxide, shaking constantly until a pink color that persists for at least 15 seconds is produced. Calculate the acid value from the expression 5.610 v/w, where v is the volume, in ml, of 0.1 M potassium hydroxide solution required and w is the weight, in g, of residue taken.

8. Free Stearic acid
Limit: No more than 3%
Reagent required
Chloroform
Ethanol (95%)
Phenolphthalein solution
0.1M Sodium hydroxide
Procedure
Weigh 1 g of sample into a stoppered flask, add 50 ml chloroform, stoppered the flask and shake well, Filter the above solution using double filter paper (Whatman no. 1) into a beaker taking care to avoid evaporation of the solvent. Wash the filter with 10 ml of chloroform and collect the washing in beaker. Evaporate the chloroform by keeping the beaker on water bath and dissolve the residue in about 10 ml of ethanol (95%) previously neutralized with phenolphthalein solution and titrate with 0.1M sodium hydroxide solution. Record the volume consumed of 0.1M Sodium hydroxide solution (V ml). Each ml of 0.1M sodium hydroxide is equivalent to 0.0284 g of Stearic acid. 
Calculation:
% Free Stearic acid =  x 100

9. Zinc Stearate
Reagent required
Dilute sulphuric acid
Ammonium mercuric Thiocyanate Solution
Copper sulfate solution
Procedure
Take 5 g of sample in a 250 ml iodine flask, add the mixture of 50 ml of water and 50 ml of dilute sulphuric acid and heat with continued shaking till fatty acids separate as an oily layer. Cool, filter the aqueous layer and wash the residue with 5 ml of hot water twice and combine the filtrate and washings, dilute to 100 ml with water. Take 5 ml of above solution in a test tube; add 0.5 ml of ammonium mercuric- Thiocyanate solution and 0.05 ml of copper sulphate solution. Scratch the walls of the test tube with a glass rod and allow standing for 15 min. No violet ppt. is formed.

10. Heavy metals
Limit: Not more than 20 ppm
Reagents required
Lead standard solution (10 ppm Pb)
2M Acetic acid.
Acetate buffer pH 3.5
Thioacetamide reagent
Sample preparation: Take 5.0 g of sample in a 100 ml conical flask. Add 40 ml of 2 M acetic acid and heat on a hot plate. Allow to cool filter the solution using whatman 41 filter paper, wash the residue obtained with two qty. each of 5 ml of warm water, combine the filtrate and washing in a 100 ml volumetric flask and make the volume with water, mix well. Transfer 12.0 ml of the solution to a Nessler cylinder.
Standard preparation: Transfer 1.0 ml of lead standard solution (10 ppm Pb) in Nessler cylinder, add 9 ml of water and 2 ml of sample preparation and mix well.
Procedure
To each of Nessler cylinder, add 2 ml of acetate buffer pH 3.5, mix, and 1.2 ml of thioacetamide reagent, mix immediately and allow standing for 2 minutes. Any brown color produced in the sample is not more intense than that produced in the standard.

11. Chloride
Limit: Not more than 0.1 %
Reagent required
Chloride standard solution (25 ppm)
0.1M silver nitrate solution
Standard Preparation: Transfer 10 ml of chloride standard solution (5 ppm Cl) add 5 ml of water.
Sample Preparation: Transfer 0.5 ml of Solution A to a Nessler cylinder diluted to 15 ml with water.
Procedure
Add 1 ml of 2M nitric acid, pour the mixture as a single addition into 1 ml of silver nitrate solution in both sample and standard solution and allow to stand for 5 minutes protected from light. When viewed transversely against a black background any opalescence produced is not more intense than standard solution.

12. Sulphate
Limit: NMT 0.5%
Reagent required
5M acetic acid
Barium chloride solution
Sulphate standard solution (10 ppm SO4)
Sample preparation: Transfer 0.3 ml of Solution A to a Nessler cylinder and dilute to 15 ml with water.
Standard preparation: Transfer 15.0 ml of sulphate standard solution (10 ppm SO4) to a Nessler cylinder.
Procedure
Add 1 ml of a 25% w/v solution of barium chloride to 1.5 ml of sulphate standard solution (10 ppm SO4), shake and allow standing for 1 minute. Add 15 ml of the sample preparation in 15 ml of water and 0.5 ml of 5M acetic acid and allow standing for 5 minutes. Any opalescence produced is not more intense than that of a standard preparation treated in the same manner.

13. Loss on drying
Limit: Not more than 6.0%
Procedure
Weigh 1.000 g of substance in a clean and dried pre-weighed LOD Bottle. Cover the stopper and gently shake to distribute material to not more than 10 MM height. Place the LOD Bottle in oven and remove cover and leave it also inside the oven. Dry the sample at 105° C. On opening the chamber, immediately close the LOD Bottle, transfer it to desiccators and bring it to room temperature. Weigh up to constant weight.    
Calculation:

                                    W2 – W1
% Loss on drying = --------------- X 100
                                   W2 – W3
Where:
W1 = Weight of empty clean and dried LOD Bottle.
W2 = Weight of LOD Bottle + sample.
W3 = Weight of LOD Bottle + sample (After drying)

14. Magnesium
Limit: NMT 4.0% and NLT 5.0% of Mg calculated with reference to the dried substance.
Reagent required
1-Butanol
Ethanol
Strong ammonia solution
0.1M Disodium edetate
Ammonia buffer pH 10.0
Mordant black II mixture
0.1M Zinc sulphate solution
Procedure
Weigh accurately about 0.50 g. of the sample in a 250 ml iodine flask, add 50 ml of a mixture of equal volumes of 1-butanol and ethanol, 5 ml of Strong ammonia solution, 3 ml of ammonium chloride buffer solution pH 10.0, 30 ml of 0.1M Disodium edetate and 15 mg of mordant black 11 mixture, heat to 45° to 50° and titrate with 0.1M zinc sulphate until the color changes from blue to violet.  Carry out a blank titration without sample being examined. Each ml of 0.1M Disodium edetate is equivalent to 2.431 mg. of magnesium.
Calculation:

                                      (Blank reading – test reading) x Fx M x 100 x 100
% Magnesium = --------------------------------------------------------------
                                                           W x (100 - % LOD)

Where,
M = Molarity of 0.1 M Disodium edetate
F = Factor                 
W= Weight of substance

15. Cadmium
Limit: Not more than 3 ppm of Cd
Procedure
Test solution: Place 50.0 mg of the substance to be examined in a polytetrafluoroethylene digestion bomb and add 0.5 ml of a mixture of 1 volume of hydrochloric acid and 5 volumes of cadmium- and lead-free nitric acid R. Allow to digest at 170°C for 5 h. Allow to cool. Dissolve the residue in water and dilute to 5.0 ml with the same solvent.
Reference solutions: Prepare the reference solutions using cadmium standard solution (10 ppm Cd), diluted if necessary with a 1 per cent V/V solution of hydrochloric acid.
Measure the absorbance at 228.8 nm, using a cadmium hollow-cathode lamp as a source of radiation and an air-acetylene flame.

16. Lead
Limit: Not more than 10 ppm of Pb
Procedure
Test solution: Use the solution described in the test for cadmium.
Reference solutions: Prepare the reference solutions using lead standard solution (10 ppm Pb), diluted if necessary with water.
Measure the absorbance at 283.3 nm; using a lead hollow-cathode lamp as a source of radiation and an air-acetylene flame, depending on the apparatus the line at 217.0 nm may be used.

17. Nickel
Limit: Not more than 5 ppm of Ni
Test solution: Use the solution described in the test for cadmium.
Reference solutions: Prepare the reference solutions using nickel standard solution (10 ppm Ni), diluted if necessary with water.
Measure the absorbance at 232.0 nm, using a nickel hollow-cathode lamp as a source of radiation and an air-acetylene flame.

18. Microbial contamination
Limit:
Total viable aerobic count: Not more than 1000/gm
Escherichia coil: It complies with the test
For aerobic bacteria: Dissolve or dilute 1 or 10 g. /ml of the sample in 9 or 90 g./ml of sterile fluid Soyabean casein digest medium containing 0.5% soyalecithin and 4% of Polysorbate 80. Allow to stand for 1 hour. (4 hrs. for gelatin) at 30°c to 35°c with intermitant shaking. Plate out 1 ml of prepared sample in to 90 mm petriplate in duplicate by pour plate technique using about 15 ml of melted Soyabean casein digest agar at about 45°c. or use petri film for total aerobic count for bacteria. Incubate the plates / films at 30°c to 35°c for 5 days unless a more reliable count is obtained in shorter time. On completion of incubation period, count number of colonies/plates or petri film. Calculate CFU per g. or ml of the sample.
E coli
Reagents required
Nutrient broth
Mac Coney’s broth
Peptone water
Kovak’s reagent
Procedure
Transfer 1 g. of sample to a screw capped container containing 50 ml of sterile nutrient broth and shake. Loosen the cap and allow standing for 1 hrs and again shaking. Loosen the cap and incubate at 37°C for 18 to 24 hrs. This is enrichment culture.
Primary test - Add 1.0 ml enrichment culture to a tube containing 5 ml of MacConkey broth.  Incubate in a water-bath at 36° to 38° for 48 hours.  If the contents of the tube show acid and gas carry out the secondary test.
Secondary test - Add 0.1 ml of the contents of the tubes containing (a) 5 ml of MacConkey broth, and (b) 5 ml of peptone water.  Incubate in a water-bath at 43.5° to 44.5° for 24 hours and examine tube (a) for acid and gas and tube (b) for indole.  To test for indole add 0.5 ml of Kovac’s reagent, shake well, and allow to stand for 1 minute; if a red color is produced in the reagent layer indole is present.  The presence of acid and gas and of indole in the secondary test indicates the presence of Escherichia coli. Carry out a control test by repeating the primary and secondary tests adding 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Escherichia coli (NCTC 9002) organisms, prepared from a 24-hour culture in nutrient broth, to 5 ml of MacConkey broth.  The test is not valid unless the results indicate that the control contains E. coli.

19. Fatty acid composition
Limit: The fatty acid fraction contains NLT 40% Stearic acid and the sum of Stearic acid and palmitic acid is NLT 90%.
Reagent required
Boron trifluoride – methanol solution
Heptane AR grade
Sodium chloride AR grade
Anhydrous sodium sulphate AR grade
Test solution: In a conical flask fitted with a reflux condenser, dissolve 0.10 g of the substance to be examined in 5 ml of boron trifluoride-methanol solution. Boil under a reflux condenser for 10 min. Add 4 ml of heptane through the condenser and boil again under a reflux condenser for 10 min. Allow to cool. Add 20 ml of a saturated solution of sodium chloride. Shake and allow the layers to separate. Remove about 2 ml of the organic layer and dry over 0.2 g of anhydrous sodium sulphate. Dilute 1.0 ml of the solution to 100.0 ml with heptane.
Reference solution: Prepare the reference solution in the same manner as the test solution using 50.0 mg of palmitic acid and 50.0 mg of Stearic acid instead of magnesium stearate.
Chromatographic condition
Column: a fused-silica column 30 m long and 0.32 mm in internal diameter coated with macrogol 20,000 R (film thickness 0.5 µm),
Flow rate: helium for chromatography R as the carrier gas at a flow rate of 2.4 ml/min,
Detector: a flame-ionization detector

Temperature program

Time (Min)
Temp. (°C)
Rate (°C/min)
Comment
Column
0 – 2
2 – 36
36 – 41
70
240
240
-
5
-
Isothermal linear gradient
Isothermal
Injection port Detector

220
260



Procedure
Inject 1 µl of reference solution. When the chromatograms are recorded in the prescribed conditions, the retention time of methyl palmitate relative to that of methyl stearate is about 0.88. The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution between the peaks corresponding to methyl stearate and methyl palmitate is at least 5.0. Inject 1 µl of the test solution. Calculate the percentage content of Stearic acid and palmitic acid from the areas of the peaks in the chromatogram obtained with the test solution by the normalization procedure, disregarding the peak due to the solvent.



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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