1. Description
White, crystalline powder or free-flowing granules, odorless.2. Solubility
Freely soluble in water. and slightly soluble in pyridine. Very slightly soluble in ethanol (95%), insoluble in chloroform and in the ether.3. Identification
A. A clear solution is obtained which remains clear on further addition of NaOH solution.Reagent required
Ferric chloride TS
Sodium hydroxide solution
Procedure: In a clean and dried test tube take 1 ml of saturated solution add 0.5 ml of ferric chloride solution followed by 0.25 ml of sodium hydroxide solution and shake well. A clear solution is obtained which remains clear on further addition of NaOH solution.
B. A pink color is produced
Reagent requiredConcentrated Sulphuric Acid AR
10% w/v Solution of cachepot
Procedure: (Solution A) In 50 ml clean and dried volumetric flask, dissolve accurately about 5 gm of sample in 25 ml CO2 free water and dilute to 50 ml with the same solvent.
Add 0.3 ml of solution A to 3 ml of the cooled mixture prepared by adding 6 ml of sulphuric acid to 3 ml of freshly prepared 10 % w/v cathecol solution while cooling in ice and heat gently over the naked flame for about 30 seconds. A pink color is produced.
C. Melting point
Limit: Melts between 165°C and 170°CD. Thin Layer Chromatography
Stationary phase: Silica gel GMobile Phase: A mixture of 10 volumes of water, 20 ml of ethyl acetate and 70 ml of Propanol. Mix properly and allow to saturate for half hour.
Test solution: Dissolve 25 mg. of the sample in 5 ml of water and dilute to 10 ml with the same solvent
Reference solution (a): Dissolve 25 mg. of mannitol WS in 5 ml of water and dilute to 10 ml with the same solvent.
Reference solution (b): Dissolve 25 mg of mannitol WS and 25 mg of sorbitol Ws in water and dilute to 10 ml with the same solvent.
Procedure: Apply separately to the plate 2 ml each of the test solution, reference solution and dry the spots. Place the plate in the mobile phase and allow it to travel about 17 cms. Remove the plate and allow drying in air. Spray with 4 amino benzoic acid solutions Dry the plate in the current of cold air until the acetone is removed. Heat at 100°C for 15 min. Allow to cool and spray with 0.2% w/v solution of sodium periodate dry the plate in the current of air heat the plate at 100°C for 15 min.
The principal spot in the chromatogram obtained with the test solution is similar in position, color and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.
E. IR:
Examine by infrared absorption spectrophotometry comparing with the spectrum obtained with mannitol WRS. Examine the substances prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in water, evaporate to dryness and record new spectra using the residues.4. Acidity or Alkalinity
Limit: Not more than 0.3 ml of 0.01M hydrochloric acid solution is required to change the color of the solution to pinkReagent required
Methyl red solution
0.01M Hydrochloride Acid
0.01M Sodium Hydroxide
Phenolphthalein solution
Sample solution: Solution A
Procedure: In a clean and dried 100 ml conical flask, take 5 ml of solution A add 5 ml of carbon dioxide free purified water add 0.05 ml of phenolphthalein solution. Titrate with 0.01M sodium hydroxide VS. Not more than 0.2 ml of 0.01M sodium hydroxide is required to change the colour of the solution to pink. To further 5 ml of solution A add 5 ml of carbon dioxide free purified water add 0.05 ml of methyl red solution. Titrate with 0.01M hydrochloric acid VS. Not more than 0.3 ml of 0.01M hydrochloric acid solution is required to change the color of the solution to pink.
5. Clarity and color of solution
Clarity of solution
Reagent requiredOpalescence standard suspension
Standard of opalescence suspension: Take 1.5 ml of clarity Standard suspension in 100 ml clean and dried volumetric flask, make up to the volume with water, mix well
OS1: Transfer 5 ml of Standard of opalescence suspension into a clean and dried 100 ml volumetric flask and dilute it to 100 ml with water.
Sample solution: Use solution A as sample solution.
Procedure: Into separate three clean and dried Nessler’s cylinder, transfer equal quantity of alcohol, the sample solution and freshly prepared reference suspension such that the Nessler’s cylinders are filled to a depth of 40 mm. After 5 minutes compare the contents of the test tubes against a black background by viewing in diffused daylight.
The sample solution is considered Clear if its clarity is same as that of water or if it is less opalescent than that of the reference suspension OS1.
Color of solution
Reagent required1% w/v Hydrochloric acid
Ferric colorimetric solution (FCS)
Cobaltous chloride colorimetric solution (CCS)
Cupric sulphate colorimetric solution (CSS)
Sample solution: Use sample solution of clarity test.
Reference solution BS8: In a clean and dried 100 ml volumetric flask, transfer 0.4 ml of FCS, 0.4 ml of CCS and 0.2 ml of CSS. Dilute to 100 ml with 1% w/v Hydrochloric acid and mix well.
Procedure: Transfer equal volume of the sample solution and reference solution BS8 in two different clean and dried Nessler’s cylinder such that the Nessler’s cylinder is filled to a depth of 40 mm. Compare the color intensity of the sample solution with that of the reference solution BS8 in diffused daylight against a white background. The sample solution is considered colorless if it has the same appearance as that of water or if it is less intensely colored than reference solution BS8.
6. Conductivity
Limit: Not more than 20 µS.cm-1.Procedure: Dissolve 20.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100.0 ml with the same solvent. Measure the conductivity of the solution while gently stirring with a magnetic stirrer.
7. Arsenic
Limit: Not more than 2 ppmReagent required
Arsenic standard solution (10 ppm As)
Zinc granules AsT
Potassium iodide AsT
Lead acetate cotton
Mercuric chloride paper
Stannated hydrochloric acid
Standard Preparation: Transfer 1.0 ml of arsenic standard solution (10 ppm As) and dilute to 50 ml with water. Add 10 ml of Stannated hydrochloric acid.
Sample Preparation: Dissolve 5gm sample in 50 ml of water. And add 10 ml Stannated Hydrochloric acid. The resulting solution complies with limit test for arsenic. Use the same solution of the sample mentioned above.
Procedure: To each of arsenic test apparatus bottle, insert 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. Add accurately about 5 ml of 1M of potassium iodide AsT and 10.0 g of Zinc granules AsT. Assemble the apparatus and immerse the flask in a water bath at a temperature such that a uniform evolution of gas is maintained. Allow reacting for 40 minutes by keeping it in a water bath. Any stain produced on the mercuric chloride paper with the sample solution is not more than that of standard solution.
8. Sulfate
Limit: Not more than 100 ppmReagents Required
25%w/v Barium chloride
Ethanolic sulfate standard solution (10 ppm SO4)
Sulfate standard solution (10 ppm SO4)
Procedure: Test Solution: Dissolve 1gm of the sample in 10 ml of purified water. To 1.0 ml of a 25.0 % w/v solution of barium chloride in a Nessler cylinder add 1.5 ml of ethanolic sulfate standard solution (10 ppm SO4), mix and allow standing for 1 minute. Add 15 ml of the test solution. Add sufficient water to produce 50 ml, stir immediately with a glass rod and allow standing for 5 minutes.
Standard Solution: To 1.0 ml of a 25.0 % w/v solution of barium chloride in a Nessler cylinder add 1.5 ml of ethanolic sulphate standard solution (10 ppm SO4), mix and allow standing for 1 minute. Then add 15ml of sulphate standard solution (10 ppm SO4). Add sufficient water to produce 50 ml, stir immediately with a glass rod and allow standing for 5 minutes.
When viewed transversely against a black background any opalescence produced by test solution is not more intense than that obtained by standard solution.
9. Specific optical rotation
Limit: Between +23° and + 25 ° calculated with reference to dried sample.Procedure: Weigh about 2.0g. of the sample in a 25 ml volumetric flask. Add 2.6 gm of sodium tetraborate Dissolve in 20 ml of water. (previously heated at 30°C ) shake continuously for 15 t0 30 min. without further heating and Dilute to 25ml with water.
Blank: Take about 10.4 gm of sodium tetraborate 100ml volumetric flask dissolve it and dilute to 100 ml with purified water. .Mix well bring the solution temperature to 25°C and fill Polarimeter tube. Blank take five readings of the solution. Clean the Polarimeter tube and fill with the sample solution.T ake five readings at 25°C.
Calculation
Correction of rotation = Av. Observed reading - Av. Blank reading
100 µ x 100
Specific optical rotation [µ]25°D = -----------------------------
LC x (100 - %LOD )
Where,
µ = Corrected observed rotation
D = D-line of sodium light
L = Length of Polarimeter tube in dm
C = Concentration of the substance (in %)
10. Chloride
Limit: Not more than 50 ppm.Reagents required
Dilute nitric acid
0.1M silver nitrate
Chloride standard solution (25 ppm Cl)
Sample preparation: Transfer accurately about 5gm of the sample to a clean and dried 50 ML Nessler cylinder dissolve with water making up the final volume to 10ml with water.
Standard solution: Transfer 10 ml of the chloride standard solution (25 ppm Cl) to a clean and dried Nessler’s cylinder.
Procedure: Follow the same procedure as given below for both standard and sample solution and compare the opalescence (if any) produced in the sample solution with that of the standard solution. To both standard solution and sample solution add 10 ml of dilute nitric acid, dilute to 50 ml with water and add 1 ml of 0.1M silver nitrate. Stir immediately with the glass rod and allow standing for 5 minutes protected from light. View transversely against a black background. Any opalescence produced in the sample solution is not more intense than that of standard solution.
11. Lead
Limit: Not more than 0.5 ppmReagent required
1M Acetic acid
Saturated solution of ammonium pyrroli-dinedithio carba mate
4 methylpentane –2-one
Lead standard solution (10 ppm)
Test solution: Dissolve 20.0g of the sample in sufficient 1 M acetic acid to produce 150 ml add 2 ml of Saturated solution of ammonium pyrroli- dinedithiocarbamate and 10.0 ml of 4 methylpentane –2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer
Reference solution (1): 20.0g of sample in sufficient 1 M acetic acid to produce 150 ml add 0.5 ml of lead standard (10ppm) 2 ml of Saturated solution of ammonium pyrroli-dinedithiocarbamate and 10.0 ml of 4 methylpentane –2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer
Reference solution (2): 20.0g of sample in sufficient 1 M acetic acid to produce 150 ml add 1.0 ml of lead standard (10ppm) 2 ml of Saturated solution of ammonium pyrroli-dinedithiocarbamate and 10.0 ml of 4 methylpentane –2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer
Reference solution (3): 20.0g of sample in sufficient 1 M acetic acid to produce 150 ml add 1.5 ml of lead standard (10ppm) 2 ml of Saturated solution of ammonium pyrroli-dinedithiocarbamate and 10.0 ml of 4 methylpentane -2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer.
Procedure: Prepare a blank solution in the same manner except sample examine above solution at 283.3 nm using blank to set zero of the instrument to construct a calibration curve and determine the content of lead in the sample.
12. Nickel
Limit: Not more than 1 ppmReagent required
1M Acetic acid
Saturated solution of ammonium pyrroli-dinedithiocarbamate
4 methylpentane –2-one
Nickel standard solution (10 ppm)
Test solution procedure: 20.0g of the sample in sufficient 1 M acetic acid 150 ml add 2 ml of Saturated solution of ammonium pyrroli-dinedithiocarbamate and 10.ml of 4 methylpentane –2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer
Reference solution (1): 20.0g of sample in sufficient 1 M acetic acid to produce 150 ml add 0.5 ml of lead standard (10ppm) 2 ml of Saturated solution of ammonium pyrroli-dinedithiocarbamate and 10.0 ml of 4 methylpentane –2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer
Reference solution (2): 20.0g of sample in sufficient 1 M acetic acid to produce 150 ml add 1.0 ml of lead standard (10ppm) 2 ml of Saturated solution of ammonium pyrroli-dinedithiocarbamate and 10.0 ml of 4 methylpentane –2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer
Reference solution (3): 20.0g of sample in sufficient 1 M acetic acid to produce 150 ml add 1.5 ml of lead standard (10ppm) 2 ml of Saturated solution of ammonium pyrroli-dinedithiocarbamate and 10.0 ml of 4 methylpentane –2-one and shake for 30 sec protect it from bright light allow two layers to separate and use the methylpantanone layer.
Procedure: Prepare a blank solution in the same manner except sample examine above solution at 232.0 nm using blank to set zero of the instrument contract the calibration curve and determine the content of lead in the sample.
13. Reducing sugar
Limit: NMT 12.8 ml of 0.05M Sodium thiosulphate is requiredReagent required
Cupri citric solution
2.4% glacial acetic acid
0.025Miodine
Hydrochloric acid
0.05M Sodium thiosulphate
Procedure: Take a clean and dry conical flask transfer 5.0 gm of the sample and dissolve in 25 ml of water gently heat the solution cool and add 20 ml of Cupri citric solution add few glass bead. Heat the solution so that boiling begins 4 minutes later and continue to boil for 3 min. cool rapidly and add 100 ml of a2.4%v/v solution of glacial acetic acid and 20.0 ml of 0.025M iodine with continuous shaking add 25 ml of a mixture of 6 ml of hydrochloric acid and 94 ml of water. Dissolve ppt titrate excess of iodine with 0.05 M sodium thiosulphate. Add 1 ml of starch as indicator.
Not more than 12.8 ml of 0.05 M sodium thiosulphate
14. Sorbitol
By thin layer chromatographyReagent required
2M NaOH
2 Propanol 0.2% w/v solution of boric acid ethanol (95%)
0.5% w/v solution of potassium permanganate
1M NaOH
Stationary Phase: silica gel H
Mobile Phase: A mixture of 85 volumes of 2–Propanol, 15 volume of 0.2% w/v solution of boric acid. Mix properly and allow saturating for half an hour.
Solution (1): Dissolve 0.5g of the sample in 5 ml of ethanol (95%) and dilute to 10 ml with the same solvent and shake for 30 min and filter
Solution (2): Dissolve 0.1g of Sorbitol WS in 50 ml of ethanol (95%) and dilute to100 ml with the same solvent mixture.
Procedure: Carry out the method for thin layer chromatography using above prepared coating material. Apply separately to the plate 2 ml each of the solution (1), solution (2) and dry the spots. Place the plate in the mobile phase and allow it to travel about 15 cms. Remove w/v solution the plate. Heat at 100°C for 15 min. Allow to cool and spray with 0.5% of potassium permanganate 1 M NaOH and heat at 100° for 2 min
Any spot corresponding to Sorbitol in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (2)
15. Sulfated ash
Limit: Not more than 0.1%Procedure: Heat a silica crucible to redness for 10 min., allow cooling in desiccators and weighing. Place about 1 g of accurately weighed substance being examined in the silica crucible, moisten with sulphuric acid, ignite gently, again moisten with sulphuric acid and ignite at about 800o, cool, weigh again, ignite for 15 min. and repeat this procedure until two successive weighings 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)
16. Loss on drying
Limit: Not more than 0.5%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 the oven and remove the cover and leave it also inside the oven. Dry the sample at 105° C for 2 hr. 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)
17. Related Substances by HPLC
Test solution: Dissolve 5gm of the sample in 25ml of water and dilute to 100.0 ml with the same solvent.Reference solution (a): Dissolve 0.50 g of mannitol CRS in 2.5 ml of water and dilute to 10.0 ml with the same solvent.
Reference solution (b): Dilute 2.0 ml of the test solution to 100.0 ml with water.
Reference solution (c): Dilute 5.0 ml of the reference solution (b) to 100.0 ml with water.
Reference solution (d): Dissolve 0.5 g of Mannitol and 0.5 g of sorbitol in 5 ml of water and dilute to 10.0 ml with the same solvent.
Mobile Phase: Degassed water
Chromatographic Conditions
|
|
Column
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0.3 m X 7.8 mm X 9 µm packed with strong cation exchange resin (calcium form) and maintained at 85°C ±1°C.
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Detector
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Refrectometer
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Flow rate
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0.5 ml/ minute
|
Injection volume
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20µl
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Run time
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22 minute
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18. Assay
Limit: NLT 98.0% and NMT 101.5% of mannitol calculated with reference to the dried substancesProcedure: Mobile phase, reference solution, test solution and chromatographic conditions same as given in the related substances.
Inject 20 µl of the reference solution (d). Continue the chromatography for 3 times the retention time of mannitol.
When the chromatograms are recorded in the prescribed conditions, the retention time of mannitol is about 22 min and the relative retention of sorbitol with reference to mannitol is about 1.25. The test is not valid unless the resolution between the peaks due to mannitol and to sorbitol is at least 2 in the chromatogram obtained with reference solution (d).
Inject 20 µl of the test solution and 20 µl of the reference solution (a). Continue the chromatography for twice the retention time of mannitol.
Calculate the percentage content of D-mannitol from the areas of the peaks and the declared content of mannitol CRS.
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