Method of Analysis for Microcrystalline Cellulose : Pharmaceutical Guidelines

Method of Analysis for Microcrystalline Cellulose

Procedure for analysis for Microcrystalline Cellulose in pharmaceutical quality control laboratory.
1. Description
A fine or granular, white or almost white powder; odorless.

2. Solubility
Insoluble in water but swells producing a white opaque dispersion or gel; slightly soluble in dilute Sodium hydroxide solution; insoluble in dilute acids, in acetone, in ethanol and in toluene.

3. Identification
A. Red color is produced.
Reagent required
Phosphoric acid AR
0.2% w/v solution of catechol in phosphoric acid
Procedure
Take 1 mg sample in 20 ml test tube and add 1 ml of phosphoric acid, heat on a water-bath for 30 minutes, add 4 ml of a 0.2% w/v solution of catechol in phosphoric acid and heat for further 30 minutes; a red color is produced

B. A blue-purple color is produced.
Reagent required
Iodine solution
Sulphuric acid 66% v/v
Procedure
Take 50 mg in a tube and add 2 ml of iodine solution, allow to stand for 5 minutes and remove the excess reagent with the aid of a filter paper and add, 1-2 drops of sulphuric acid (66%v/v), A blue-purple color is produced

C. Dispersion
Procedure
Mix about 30 g sample with 270 ml of water, mix in a blender at 18,000 rpm for 5 minutes, transfer 100 ml of the mixture to a 100-ml graduated cylinder and allow standing for 3 hours. A white, opaque, bubble-free dispersion is obtained that does not produce a supernatant liquid.

D. Color Reaction
Procedure
Place about 10 mg on a watch glass and disperse in 2 ml of iodinated zinc chloride solution R. The substance becomes violet-blue.

E. Degree of Polymerization
Procedure
Transfer 1.300 g to a 125 ml conical flask. Add 25.0 ml of water and 25.0 ml of 1M cupriethylenediamine hydroxide solution. Immediately purge the solution with nitrogen, insert the stopper and shake until completely dissolved. Transfer 7.0 ml of the solution to a suitable capillary viscometer (2.2.9). Equilibrate the solution at 25°C ±0.1°C for not less than 5 min. Record the flow time t1 in seconds, between the 2 marks on the viscometer. Calculate the kinematic viscosity n1 of the solution using the formula:
        = t1 (k1)
Where k1 is the viscometer constant.
Dilute a suitable volume of 1M cupriethylenediamine hydroxide solution with an equal volume of water R and measure the flow time t2 using a suitable capillary viscometer. Calculate the kinematic viscosity n2 of the solvent using the formula:
     = t2 (k2)
           
Where k2 is the viscometer constant.
Determine the relative viscosity hrel of the substance to be examined using the formula:
    = V1 /V2
Determine the intrinsic viscosity [h]c by interpolation, using the Intrinsic Viscosity Table.
Calculate the degree of polymerisation P using the formula:


Where m is the mass in grams, of the substance to be examined and b is the loss on drying as a percentage.
The degree of polymerization is not more than 350

4. pH
Limit: Between 5.0 and 7.5
Procedure
Take 5.0 g of sample in a clean and dried 150 ml beaker, add 40 ml of carbon dioxide-free water, and stir for 5 minutes and centrifuge.  Measure the pH of supernatant liquid.

5. Starch and dextrin
Limit: No blue or brownish-red color is produced.
Reagent required
Iodine solution
Procedure
Mix 0.1 g of sample with 5 ml of water by vigorous shaking and add 2-3 drops of iodine solution, no blue or brownish - red color is produced.

6. Organic impurities
Limit: No red color is produced
Reagent required
Phloroglucinol AR
Hydrochloric acid AR
Procedure
Take 10 mg of sample on a watch glass and add 0.05ml of a freshly prepared solution of 0.1g of Phloroglucinol in 5 ml of hydrochloric acid; no red color is produced.

7. Ether Soluble Substances
Limit: Not more than 0.05%
Procedure
Prepare a column using 10.0 g in a tube about 20 mm in internal diameter. Pass 50 ml of peroxide-free ether through the column. Evaporate the eluate to dryness. The residue weighs not more than 5.0 mg.

8. Water-soluble substances
Limit: Not more than 0.2%
Procedure
Weigh acc about 5.0 g sample in a beaker and add about 80 ml of water, stir for 10 minutes, filter through a filter paper (Whatman No.42 or equivalent) into a previously cleaned, dried & weighed beaker and evaporate the filtrate to dryness on hot plate. When traces of sample remains in the beaker, keep the beaker in oven at 105°C for 1hrs. Cool the beaker in a desiccators and weigh. The residue weighs not more than 10 mg (0.2%)
Calculation:
                 (Wr – Ws) x 100
      =  ------------------------
                           Sw
Where,
Wr = Weight of beaker after drying
Ws = Weight of beaker
Sw = Weight of sample taken

9. Arsenic
Limit: Not more than 2 ppm.
Reagent required
Arsenic standard solution (10 ppm)
Brominated hydrochloric acid 
Stannous Chloride solution 
Zinc granules 
Nitric acid 
Sulphuric acid
1M Potassium iodide solution
Lead acetate cotton
Mercuric chloride paper
Standard Preparation: Transfer 1 ml of arsenic standard solution (10 ppm As) in a wide mouthed bottle and dilute to 50 ml with water.
Sample Preparation: Mix 5 g. of the sample with 3 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite and dissolve the cooled residue in a mixture of 15 ml of hydrochloric acid containing 0.15 ml of bromine solution and 45 ml water. Add 2 ml of stannous chloride solution and transfer to a wide mouthed bottle of arsenic apparatus.
Procedure
To each of arsenic test apparatus bottle, add about 5 ml of 1M potassium iodide solution and 10.0 g of Zinc granules. Assemble the apparatus having inserted lead acetate cotton into the lower tube and place a disc or a small square of mercuric chloride paper enough to cover the orifice of the tube and allow reacting for 40 minutes by keeping it in water bath.
Any stain produced on the mercuric chloride paper with the sample solution is not more than that produced with standard solution.

10. Heavy metals
Limit: Not more than 10 ppm
Reagent required
Nitric acid
Sulphuric acid
Hydrochloric acid
Ammonia solution
Dilute acetic acid solution
Hydrogen sulphide solution (freshly prepared)
Standard preparation: Pipette 2 ml of Lead standard solution (10 ppm Pb) in a silica crucible with 4 ml of a 25% w/v solution of magnesium sulphate in 1M sulphuric acid. Mix using a fine glass rod and heat cautiously. If the mixture is liquid, evaporate gently to dryness on a water bath. Progressively heat to ignition, not allowing the temperature to exceed 800°C, and continue heating until a white or at most greyish residue is produced. Allow to cool, moisten the residue with 0.2 ml of 1M sulphuric acid, evaporate, ignite again and allow cooling. The total period of ignition must not exceed 2 hours. Dissolve the residue using two 5-ml quantities of 2M hydrochloric acid. Add 0.1 ml of phenolphthalein solution and 13.5M ammonia dropwise until a pink color is produced. Cool, add glacial acetic acid until the solution is decolorized and add a further 0.5 ml. Filter if necessary and dilute the solution to 20 ml with water.
Sample preparation: Place 2 g of the substance being examined in a silica crucible with 4 ml of a 25% w/v solution of magnesium sulphate in 1M sulphuric acid. Mix using a fine glass rod and heat cautiously. If the mixture is liquid, evaporate gently to dryness on a water bath. Progressively heat to ignition, not allowing the temperature to exceed 800°, and continue heating until a white or at most greyish residue is produced. Allow to cool, moisten the residue with 0.2 ml of 1M sulphuric acid, evaporate, ignite again and allow to cool. The total period of ignition must not exceed 2 hours. Dissolve the residue using two 5-ml quantities of 2M hydrochloric acid. Add 0.1 ml of phenolphthalein solution and 13.5M ammonia dropwise until a pink color is produced. Cool, add glacial acetic acid until the solution is decolorized and add a further 0.5 ml. Filter if necessary and dilute the solution to 20 ml with water.
Procedure
To each of the cylinder containing the standard solution and the sample solution, add 2 ml of acetate buffer pH 3.5, mix, add to 1.2 ml of thioacetamide reagent, mix immediately and allow standing for 2 minutes. Any brown color produced is not more intense than standard solution.

11. Sulphated ash
Limit: Not more than 0.1%
Reagent required
Sulphuric acid
Procedure
Heat a silica crucible to redness for 10 min., allow cooling in desiccators and weighing. Place about 1.00 g of accurately weighed sample in the silica crucible, moisten with sulphuric acid, ignite gently, again moisten with sulphuric acid and ignite at about 800°C, cool, weigh again, ignite for 15 min and repeat this procedure until two successive weighing do not differ by more than 0.5 mg.
Calculation:
                                      W3 – W1
% Sulphated Ash =   -------------- X 100
                                      W2 – W1
Where,
W1 = Weight of empty platinum crucible
W2 = Weight of crucible + sample
W3 = Weight of crucible + residue (After ignition)

12. 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 100 -105°C for 3 hr. On opening the chamber, immediately close the LOD Bottle, transfer it to desiccators and bring it to room temperature.
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)

13. Assay
Limit: NLT 97.0% and NMT 102.0% calculated on dried basis.
Reagent required
0.083M Potassium dichromate
Sulphuric acid
0.1M ferrous ammonium sulphate VS
Ferrion sulphate solution
Procedure
Weigh accurately about 0.125 g. and transfer to a clean and dried 250 ml conical flask and add 25 ml of water. Add 50.0 ml of 0.083 M potassium dichromate, mix, carefully add 100 ml of sulphuric acid and heat to boiling. Remove from heat, allow standing at room temperature for 15 minutes, cool and transfer to a 250 ml volumetric flask. Dilute with water almost to volume, cool to 25°C, dilute with water to volume and mix. Titrate 50.0 ml of the resulting solution with 0.1M ferrous ammonium sulphate VS using 2-3 drops of Ferrion sulphate solution as indicator (R1).  Repeat the procedure without the substance being examined (R2). The difference between the titrations represents the amount of ferrous ammonium sulphate required. Each ml of 0.1M ferrous ammonium sulphate is equivalent to 0.000675 g of cellulose. Each ml of 0.1M ferrous ammonium sulphate is equivalent to 0.000675 g of cellulose. 
Calculation:

                          V x 0.000675 x M x 100 x 250              100
% Assay = -------------------------------------- x --------------------
                                    0.1 x W x 50                       (100 - % LOD)

Where,
V = Blank reading – Sample reading
M = Molarity of 0.1M ferrous ammonium sulphate
F = Factor
W = Weight of sample

14. Microbial contamination
Limit: Total viable aerobic count: NMT 1000 microbes/gm Fungi: NMT 100/gm
E coli : Absent
Salmonella : Absent
Pseudomonas : Absent
Aeruginosa : Absent  
Staphylococcus aureus : Absent
Total viable aerobic count
Procedure
Dissolve 1.0 of the sample in 9 ml of sterile fluid Soyabean casein digest medium containing 0.5% soyalecithin and 4% of Polysorbate 80. Allow standing for 1 hour at 30°c to 35°c with intermitant shaking. Plate out 1 ml of prepared sample in to 90 mm petriplates 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/petri 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 gm or ml of the sample.
Total combined molds and yeast count
Procedure
Dissolve 1 gm of the sample in 9 ml of sterile Soyabean Casein Digest Medium containing 0.5% soyalecithin and 4% of Polysorbate 80.Allow standing for 1 hour at 30°c to 35°c with intermittent shaking. Plate out 1 ml of prepared sample in to 90 mm petriplates in duplicate by pour plate technique using about 15 ml of melted Sabourad Chloremphenicol agar at about 45°c. or use Petrifilm for yeast and mould count for fungi. Incubate the plates/ Petrifilm at 20°c to 25°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 Petrifilm. Calculate CFU/g /ml of the sample.

E. Coli: Absent
Reagents required
Nutrient broth
Mac Coney’s broth
Peptone water
Kovac’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 of the 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 colour 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.  

Salmonella: Absent
Reagents required
Sterile nutrient broth
Selenite F broth
Tetrathionate bile brilliant green broth                              
Bismuth Sulphite agar                               
Brilliant green agar                            
Triple sugar iron agar                               
Urea broth
Procedure
Transfer 1g of the sample to 100 ml of sterile nutrient broth and shake. Allow to stand for 1 hour and shake again. Incubate at 37°C for 24 hours. Take in two test tubes, 1.0 ml of the enrichment culture obtained from above. To one of the tube add 10 ml of selenite F broth and to the other tube add tetrathionate bile-brilliant green broth and incubate both the tubes at 37°C for 48 hours.  From each of these two culture tubes, sub culture on Brilliant green agar and Bismuth sulfite agar. Incubate the plates at 37°C for 24 hours. Observe the black or green colonies on Bismuth Sulphite agar and small transparent and colorless or opaque red zone colonies on Brilliant green agar. Subculture any colony showing the above characteristics in triple sugar iron agar and at the same time into a tube of Urea broth. Incubate at 37°C for 24 hours.
The formation of acid and gas in stab culture (with or without concomittant blackening) and the absence of acidity from the surface of Triple sugar iron and absence of red color in the urea broth indicates the presence of salmonella.
Carryout the control test using 10 to 50 cells of Salmonella abony (NCTC6017), which was sub-cultured 24 hours before and incubated at 37°C for 24 hours.
The test is invalid if controls do not indicate the presence of salmonella.

Pseudomonas aeruginosa: Absent
Media required
100 ml of Soyabean casein digest medium
Cetrimide agar
Pseudomonas agar
Procedure
Incubate 1.0 g /ml of sample in 100 ml of sterile Soyabean casein digest medium (Double strength) and shake. Allow to stand for one hour and shake again, incubate at 37°c ± 1°c for 24 to 48 hours.
Streak a portion of the enricxhment culture on the Cetrimide Agar and incubate at 35°c to 37°c for 18-24 hours. Observe for the greenish colonies.
If any of the colonies showing above characteristics carry out Oxidase and Pigment test. Streak the representative colonies from the Cetrimide agar on to the Pseudomonas Agar; incubate at 33°c to 37°c for not less than 3 days. Examine the streaked surface under UV light to determine whether colonies conforming to the description given in table.

Medium
Characteristic colonial morphology
Fluorescence in UV light
Oxidase test
Gram stain
Cetrimide Agar
Greenish
Greenish
+ ve
- ve rods
Pseudomonas Agar
Colourless to yellowish or greenish

Yellowish or blue
+ ve
- ve rods


If growth of suspected colonies occurs, place 2 or 3 drops of a freshly prepared 1% w/v solution of N, N,N1,N1 tetra methyl 4-phenylenediamine dihydrochloride on filter paper and smear with colony. If there is no development of pink color changing to purple the sample meets the requirement of the test for the absence of Pseudomonas aeruginosa. Carry out the control test using 10 to 50 cells of Pseudomonas aeruginosa (ATCC 9027) from a 24 hours culture. The test is invalid, if controls do not indicate presence of Pseudomonas aeruginosa.

Staphylococcus aureus: Absent
Media required
100 ml of Soyabean casein digest medium
Mannitol salt agar
Procedure
Inoculate 1.0 g./ml of the sample in 100 ml of Soyabean casein digest medium (Double strength) and shake. Allow standing for one hour and shaking again, incubate at 37°c ± 1°c for 24 to 48 hours. Streak a portion of the enrichment culture on to the Mannitol salt agar and incubate at 33°c to 37°c for 24 hours. Observe for the yellow colonies with yellow zones.

If any of the colonies showing above characteristics, carry out Coagulate test. Transfer representative colonies to the tubes containing 0.5 ml of Mammalian preferably rabbit plasma with or without additives. If no coagulation in any degree is observed the sample meets to the requirement of the absence of S. aureus (ATCC 6538) from a 24 hours culture. The test is invalid if controls do not indicate presence of S.aureus.





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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