standard testing procedure cyanocobalamin

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standard testing procedure cyanocobalamin

Storage Requirements:
Store protected from light and moisture.
Sampling:
Sample equal quantity from each container / bag. Collect a minimum of 1g from each container/bag sample into individual,

self –sealing clear polythene bag kept in another clear self sealing polythene bag bearing ‘Sample for Analysis lable.

After completion of sampling return rest sample on same container. Collect control sample in Pet Bottle/Glass Bottle.
Quantity of Composite Sample:
1.0g
Quantity of individual Identification
0.0100 g from each and every container /bag.
Quantity of Control Sample:
2 X 1.0 g

Description: A dark red, crystalline powder; very hygroscopic.
Solubility: Sparingly soluble in water and in ethanol (95 per cent); practically insoluble in chloroform, in acetone and in ether.
Identification:
A. By UV: The ratio of the absorbance at about 361 nm to that at about 278 nm is 1.70 to 1.90
B. By TLC: The principal spot in the chromatogram obtained with the test solution corresponds to that in the
chromatogram obtained with the reference solution.
C. By chemically: a blue colour is formed and is taken into the benzyl alcohol layer.
Related substances (Determine by liquid chromatography):
In the chromatogram obtained with the test solution the sum of the areas of any secondary peaks is not greater than the area of
the principal peak in the chromatogram obtained with reference solution (a). Ignore any peak the area of which is less than that of the principal peak in the chromatogram obtained with reference solution (b). The test is not valid unless the chromatogram obtained with reference solution (c) exhibits two principal peaks, the resolution between these peaks is 2.5 or more and the chromatogram obtained with reference solution (a) exhibits one principal peak.
Loss on drying: Not more than 12.0 % w/w, determined on 20.0 mg by drying in an oven at 105° at a pressure of 1.5 to 2.5 kPa for 2 hours.
Assay: Between 96.0% to 102.0%w/w on the dried basis.

Description: A dark red, crystalline powder; very hygroscopic.
Procedure: Take the sample and spread in petridish and examine the substance by visually.
Reporting: Report as Complies / does not complies.
Solubility: Sparingly soluble in water and in ethanol (95 per cent); practically insoluble in chloroform, in acetone and in ether. Perform test as per sop
Reporting: Report as Complies/Does not comply.
Identification:
A. By UV: When examined in the range 260 nm to 610 nm, a 0.0025 % w/v solution shows absorption maxima, at about 278 nm, 361 nm and 547 nm to 559 nm.

The ratio of the absorbance at about 361 rim to that at about 547nm to 559 nm is 3.15 to 3.45 and the ratio of the absorbance
at about 361 nm to that at about 278 nm is 1.70 to 1.90.
Reporting: Report as in value.
B. By TLC: Determine by thin-layer chromatography, protected from light and coating the plate with silica gel G.
Mobile phase: A mixture of 60 volumes of chloroform, 40 volumes of methanol and 12 volumes of 6 M ammonia. Use an unlined tank.
Test solution: Dissolve 20 mg of the substance under examination in 10 ml of ethanol (50 per cent).
Reference solution: A 0.2 per cent w/v solution of Cyanocobalamin RS in ethanol (50 per cent).
Apply to the plate 10µl of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in air and examine in day light. The principal spot in

the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Reporting: Report as Complies/Does not comply.
C. By chemically: Mix about 1 mg with 10 mg of potassium sulphate and 0.1 ml of 0.5 M sulphuric acid and heat carefully to redness.
Allow to cool, add to the residue 0.1 ml of water, 0.5 ml of saturated solution of ammonium thiocyanate and 0.5 ml of benzyl alcohol and shake;

a blue colour is formed and is taken into the benzyl alcohol layer.
Reporting: Report as Complies/Does not comply.
Related substances (Determine by liquid chromatography):
The following solutions should be used within 1 hour of preparation.
Test solution: Dissolve 10 mg of the substance under examination in 10 ml of the mobile phase.
Reference solution (a): A 0.003 % w/v solution of the substance under examination in the mobile phase.
Reference solution (b): A 0.0001 % w/v solution of the substance under examination in the mobile phase.
Reference solution (c): Dissolve 5 mg of the substance under examination in 2 ml of water, warming if necessary, allow to cool, add 1 ml

of a 0.1 % w/v solution of chloramine T and 0.1 ml of 0.05 M hydrochloric acid, dilute to 5 ml with water, shake and allow to stand

for 5 minutes. Dilute 1 ml of this solution to 10 ml with the mobile phase use immediately.
Chromatographic system:
– A stainless steel column 25 cm x 4 mm, packed with octylsilane bonded to porous silica (5 µm),
– Mobile phase: a mixture of 147 volumes of a 1.0 % w/v solution of disodium hydrogen phosphate and 53 volumes of methanol

adjusted to pH 3.5 with phosphoric acid (to be used within 2 days of preparation),
– Flow rate. 0.8 ml per minute,
– Spectrophotometer set at 361 nm,
– Injection volume. 20 µl.
Inject the test solution and reference solutions (a), (b) and (c). Allow the chromatography to proceed for three times

the retention time of the peak due to cyanocobalamin.
In the chromatogram obtained with the test solution the sum of the areas of any secondary peaks is not greater than

the area of the principal peak in the chromatogram obtained with reference solution (a). Ignore any peak

the area of which is less than that of the principal peak in the chromatogram obtained with reference solution (b).

The test is not valid unless the chromatogram obtained with reference solution (c) exhibits two principal peaks,

the resolution between these peaks is 2.5 or more and the chromatogram obtained with reference solution (a) exhibits one principal peak.
Reporting: Report as Complies/Does not comply.
Loss on drying: Not more than 12.0 % w/w, determined on 20.0 mg by drying in an oven at 105° at a pressure of 1.5 to 2.5 kPa for 2 hours.

Reporting: Report in %w/w.
Assay:
Weigh accurately about 25 mg and dissolve in sufficient water to produce 1000.0 ml.
Measure the absorbance of the solution at the maximum at about 361 nm.
Calculate the content of C63H88CoN14O14P, taking 207as the specific absorbance at 361 nm.