1. Description
White or almost white powder.2. Solubility
Freely soluble in water, Practically insoluble in alcohol3. Identification
A. The IR absorption spectrum of the sample is concordant with the Anhydrous Lactose reference spectrum or with the spectrum obtained from Anhydrous Lactose WRS.B. By Thin Layer Chromatography
Stationary phase: Silica gel GDiluent: Prepare a mixture of methanol and water (3:2).
Mobile Phase: Prepare a solution consisting of a mixture of ethylene dichloride, glacial acetic acid, methanol, and water (50:25:15:10).
Standard solution A: Prepare a solution of USP Lactose Anhydrous RS in diluent having a known concentration of 0.5 mg per ml.
Standard solution B: Prepare a solution of USP Dextrose RS, USP Lactose Anhydrous RS, USP Fructose RS, and USP Sucrose RS in diluent having a known concentration of 0.5 mg per ml for each Reference Standard.
Test solution: Transfer about 25 mg of Lactose Anhydrous to a 50-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Spray Solution: 0.5 g of Thymol in a mixture of 95 ml of alcohol and 5 ml of sulfuric acid.
Procedure: Apply separately 2 µL each of Standard solution A, Standard solution B, and the Test solution to the plate. Allow the spots to dry, and develop the plate in a paper-lined chromatographic chamber equilibrated with Developing solvent for about 1 hour prior to use. Allow the chromatogram to develop until the solvent front has moved about three-quarters of the length of the plate. Remove the plate from the chamber, dry in a current of warm air, and redevelop the plate in fresh Developing solvent. Remove the plate from the chamber, mark the solvent front, and dry the plate in a current of warm air. Spray the plate with the spray solution. Heat the plate at 130° for 10 minutes: the principal spot obtained from the Test solution corresponds in appearance and Rf value to that obtained from Standard solution A. The test is not valid unless the chromatogram obtained with Standard solution B shows four clearly discernible spots, disregarding any spots at the origin.
C. Color test
Reagent requiredAmmonium hydroxide
Procedure: Dissolve 250 mg in 5 ml of water. Add 3 ml of ammonium hydroxide, and heat in a water bath at 80° for 10 minutes: a red color develops.
4. Clarity and Color of the Solution
Limit: The absorbance divided by the path length in centimeters in not more than 0.04.Procedure: Take 1gm of the sample and dissolve it in 10ml of boiling water. Determine the absorbance of this solution at a wavelength of 400nm.
5. Acidity or Alkalinity
Limit: NMT 0.4 ml of 0.1M Sodium hydroxide is required.Reagent required
Phenolphthalein solution
0.1M Sodium hydroxide
Procedure: Dissolve 6.0 g. sample in a conical flask, with 25 ml water by boiling. Cool, add 0.3 ml of Phenolphthalein solution, the solution is colorless and NMT 0.4 ml of 0.1M sodium hydroxide is required to change the color to pink.
6. Specific Optical Rotation
Limit: Between + 54.4° and + 55.9°, calculated on anhydrous substanceProcedure: Weigh accurately about 10 g. of the sample in a 100 ml volumetric flask. Dissolve in 80 ml of water by heating at 50°c and allow to cool. Add 0.2 ml of 6M ammonia, allow to stand for 30 min. and dilute to 100 ml with water. Bring the solution temperature to 25°c and fill Polarimeter tube. Take five readings of the solution. Clean the Polarimeter tube and filled with a mixture of 99.8 ml of water and 0.2 ml of 6M ammonia. Take five readings at 25°c.
Calculation
Correction of rotation = Av. Observed reading - Av. Blank reading
100 x µ x 100
[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 %)
7. Protein and Light absorbing Impurity
Limit: Absorbance at about 400 nm is max 0.04. In range 210 nm to 220 nm is NMT 0.25. Absorbance in the range 270 nm to 300 nm is NMT 0.07.Procedure: Measure the absorbance of the solution A at 400 nm using1 cm cell and water as the blank. The absorbance is not more than 0.04. Dilute 1 ml of solution A to 10 ml with water and measure the absorbance in the range 210 nm to 300 nm. The absorbance is not more than 0.25 in the range 210 nm to 220 nm and not more than 0.07 in the range 270 nm to 300 nm.
8. Heavy metals
Limit: Not more than 5 ppmReagent required
Dilute ammonia
Dilute acetic acid
Hydrogen sulfide solution
Acetate buffer pH 3.5
Lead standard solution (20 ppm Pb)
0.1M Hydrochloric acid
Standard Solution: Pipette out 1 ml of Lead standard solution (20 ppm Pb) in a Nessler cylinder. Dilute with water to 25 ml. Adjust the pH between 3.0 and 4.0 with dilute acetic acid or dilute ammonia solution. Dilute with water to 35 ml.
Sample Solution: Weigh 4.0 g. of the sample and transfer to a Nessler cylinder, add 20 ml of warm water and 1 ml of 0.1M hydrochloric acid and sufficient water to produce 25 ml. Adjust the pH between 3.0 and 4.0 with dilute acetic acid or dilute ammonia solution. Dilute with water to 35 ml.
Procedure: To each of the Nessler cylinders, add 10 ml of freshly prepared hydrogen sulfide solution, mix and dilute to 50 ml with water. Allow to stand for 5 min. and view downwards over a white surface. The color produced with test solution is not more intense than produced with the standard solution.
9. Residue on Ignition
Limit: Not more than 0.1%Procedure: Heat a silica crucible to redness for 10 min., allow to cool in a desiccator and weigh. 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 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)
10. Water Content
Limit: Not more than 1.0%Reagent required
Anhydrous methanol AR
KF reagent Pyridine free single solution
Procedure: Take about 40 ml of anhydrous methanol in the titration vessel, neutralize with KF reagent and find out the factor of KF reagent in mg H2O/ 5 ml in triplicate and enter the mean value.
Add accurately about 500 mg. of the sample to the titration vessel. Allow titrating with KF reagent to the electrometric endpoint. Record the percentage of water content obtained by Karl Fischer titrator. Find out water content of the sample in triplicate and take mean value.
11. Loss on Drying
Limit: Not more than 0.5%
Procedure: 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 80° 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)
12. Microbial Limits
Limit:
Total microbial count: Not more than 100/gm
E. Coli and Salmonella: Absent
Procedure: As per the SOP for Microbial Limit Test of Raw material.
E. Coli and Salmonella: Absent
Procedure: As per the SOP for Microbial Limit Test of Raw material.
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