standard testing procedure cholecalciferol

Storage Requirements:
Store protected from light and moisture at a temeperature 2 to 8ºC.

Sampling:
Sample from each container / bag and collect a minimum of 0.5g from each into no toxic, self sealing transparent polyethylene bearing ‘Sample

For Analysis’ label kept in another transperent self sealing polythene bag. Afetr completion of sampling return rest sample on the same container.

Collect control sample in Pet bottle/Glass bottle.

Quantity of Composite Sample:

1.0 g

Description: White or almost white crystals; odourless or almost odourless. It is sensitive to air, heat and light. A
reversible isomerisation to precholecalciferol may occur in solution, depending on temperature and time.
Solubility: Freely soluble in ethanol (95 per cent), in acetone, in chloroform and in ether; practically insoluble in water.
It is soluble in fixed oils. Solutions in volatile solvents are unstable and should be used immediately.
Identification:.
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is
carried out.
A. By IR: Determine by infrared absorption spectrophotometer. Compare the spectrum with that obtained with
Cholecalciferol RS.
B. By Chemical: A yellowish-orange colour is produced.
C. By TLC: In the test for 7-Dehydrocholesterol, the principal spot in the chromatogram obtained with the test
solution corresponds to that in the chromatogram obtained with reference solution (b).
D. By Chemical: A bright red colour is produced which rapidly changes through violet and blue to green.
Specific optical rotation: NLT +105.00º and NMT +112.00º
Light absorption: Absorbance of the sample solution at the maximum at about 265 nm is 0.46 to 0.50.
7-Dehydrocholesterol: Determine by thin-layer chromatography.
The principal spot in the chromatogram obtained with the test solution is initially orange-yellow
but becomes brown later. In the chromatogram obtained with the test solution any violet spot with
An Rf value slightly lower than that of the principal spot (due to 7-dehydrocholesterol and
Appearing slowly) is not more intense than the spot in the chromatogram obtained with reference
Solution (a). The test is not valid unless the chromatogram obtained with reference solution (c)
shows two clearly separated principal spots.
Assay: Cholecalciferol contains not less than-97.0 % and not more than 103.0 %.

Description: White or almost white crystals; odourless or almost odourless. It is sensitive to air, heat and light. A
reversible isomerisation to precholecalciferol may occur in solution, depending on temperature and time.

Examine the individual samples by visually.
Reporting: Report as Complies/Does not comply.

Solubility: Use the following quantities:
Freely soluble in ethanol (95%): 1.0 g in 1 to 10ml.
Freely soluble in ether : 1.0 g in 1 to 10ml.
Freely soluble in acetone : 1.0 g in 1 to 10ml.
Freely soluble in chloroform : 1.0 g in 1 to 10ml.
Practically insoluble in water : 0.01 g in 100ml or more.
It is soluble in fixed oils : 1 g in 10 to 30ml.
Practically insoluble in water : 0.01 g in 100ml or more.
Solutions in volatile solvents are unstable and should be used immediately.
Reporting: Report as Complies/Does not comply.

Identification:
(A) Determine by infrared absorption spectrophotometry. Compare the spectrum with that obtained with
Cholecalciferol RS.
Reporting: Report as Complies/Does not comply.
(B) Dissolve 1 mg in 1 ml of 1,2-dichloroethane and 4 ml of antimony trichloride solution; a yellowish-orange
colour is produced.
Reporting: Report as Complies/Does not comply.
(C) In the test for 7-Dehydrocholesterol, the principal spot in the chromatogram obtained with the test solution
corresponds to that in the chromatogram obtained with reference solution (b).
Reporting: Report as Complies/Does not comply.
(D) To a solution of about 0.5 mg in 5 ml of chloroform add 0.3 ml of acetic anhydride and 0.1 ml sulphuric acid
and shake vigorously; a bright red colour is produced which rapidly changes through violet and blue to green.
Reporting: Report as Complies/Does not comply.

Specific optical rotation: +105° to +112°, determined, within 30 minutes of preparation, in a solution prepared by
dissolving 0.2 g rapidly and without heating in sufficient aldehyde-free ethanol (95 per cent) to
produce 25.0 ml.
Reporting: Report as value in º.
Light absorption. Dissolve 10 mg, rapidly and without heating, in sufficient aldehyde-free ethanol (95 per cent) to
Produce 100.0 ml. Dilute 5.0 ml of this solution to 50.0 ml with aldehydefree ethanol (95 per cent).
Absorbance of the resulting solution at the maximum at about 265 nm, measured within 30 minutes of
preparation, 0.46 to 0.50.

Reporting: Report as value.
7-Dehydrocholesterol: Determine by thin-layer chromatography, coating the plate with silica gel G. Mobile phase. A
0.01 per cent w/v solution of butylated hydroxytoluene in a mixture of equal volumes of
Cyclohexane and peroxide-free ether. Prepare the following solutions immediately before use.

Test solution: Dissolve 0.25 g of the substance under examination in sufficient of 1,2-dichloroethane containing 1 per cent w/v of squalane

and 0.01 per cent w/v of butylated hydroxytoluene (solvent A) to produce 5 ml.

Reference solution (a): A solution containing 0.005 per cent w/v of 7-dehydrocholesterol RS in solvent A.

Reference solution (b): A solution containing 2.5 per cent w/v of cholecalciferol RS in solvent A.

Reference solution (c): Mix equal volumes of reference solutions (a) and (b).

Apply to the plate 10 μl of each solution. Develop the chromatograms immediately, protected from light. After
development, dry the plate in air and spray three times with antimony trichloride reagent. Examine the chromatograms
for not more than 4 minutes after spraying. The principal spot in the chromatogram obtained with the test solution is initially

orange-yellow but becomes brown later. In the chromatogram obtained with the test solution any violet spot with an RF value

slightly lower than that of the principal spot (due to 7-dehydrocholesterol and appearing slowly) is not more intense than

the spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained

with reference solution (c) shows two clearly separated principal spots.

Assay: Carry out the following procedure as rapidly as possible in subdued light and protected from air.
Determine by liquid chromatography.

Test solution: Weigh accurately about 50.0 mg of the substance under examination; dissolve in 10 ml of toluene without
heating and dilute to 100.0 ml with the mobile phase; dilute 5.0 ml of this solution to 50.0 ml with the
mobile phase; further dilute 5.0 ml of this solution to 50.0 ml with the mobile phase.

Reference solution (a): Dissolve 50.0 mg of cholecalciferol RS in 10 ml of toluene without heating and dilute to 100.0 ml
with the mobile phase; dilute 5.0 ml of this solution to 50.0 ml with the mobile phase (Solution
A); further dilute 5.0 ml of solution A to 50.0 ml with the mobile phase.

Reference solution (b): Reflux 5.0 ml of solution A, under nitrogen, on a water-bath for 60 minutes to obtain a solution
of cholecalciferol, precholecalciferol and trans-cholecalciferol.
Chromatographic system:
– A stainless steel column 25 cm × 4.6 mm, packed with porous silica particles (5 μm) (such as Nucleosil 50-S),
– Mobile phase: a mixture of 997 volumes of hexane and 3 volumes of 1-pentanol,
– Flow rate. 2 ml per minute,
– Spectrophotometer set at 254 nm,
– Injection volume: 20 µl.
Inject reference solution (b) and record the chromatogram adjusting the sensitivity so that the height of the peak due to
cholecalciferol is more than 50 per cent of full-scale deflection. The approximate relative retention times calculated with
reference to cholecalciferol are 0.4 for precholecalciferol and 0.5 for trans-cholecalciferol. The resolution between
precholecalciferol and trans-cholecalciferol should be not less than 1.0; if necessary adjust the proportions of the
constituents and flow rate of the mobile phase to obtain the required resolution.
Inject reference solution (a) and record the chromatogram adjusting the sensitivity so that the height of the peak due to
cholecalciferol is more than 50 per cent of full-scale deflection. Inject the test solution. Measure the areas for the major peaks.
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