(Ph. Eur. 11.6 update)
Action and use
Vitamin D analogue.
DEFINITION
Calcipotriol Cream contains either Calcipotriol or Calcipotriol Monohydrate in a suitable basis.
The cream complies with the requirements stated under Topical Semi-solid Preparations and with the following requirements.
Content of calcipotriol, C27H40O3
95.0 to 105.0% of the stated amount.
A reversible isomerisation to pre-calcipotriol takes place in solution, depending on temperature and time. The activity is due to both compounds.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography, Appendix III A, protected from light, using the following solutions.
Solvent A 1 volume of triethylamine and 9 volumes of dichloromethane.
(1) To a quantity of the cream containing the equivalent of 0.5 mg of calcipotriol, add 50 mL of absolute ethanol and shake vigorously. Cool in ice and filter. Evaporate the filtrate to dryness at a temperature not exceeding 30° and dissolve the residue in 1 mL of solvent A.
(2) 0.05% w/v of calcipotriol monohydrate EPCRS in solvent A.
(3) Place 2 mg of calcipotriol monohydrate EPCRS in a vial and dissolve in 200 μL of solvent A. Close the vial and keep it in a water bath at 60° for 2 hours (generation of pre-calcipotriol).
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel F254.
(b) Use the mobile phase as described below.
(c) Apply 50 μL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air and then heat at 140° for 10 minutes. Whilst hot, spray the plate with an alcoholic solution of sulfuric acid, dry at 140° for not more than 1 minute and examine in ultraviolet light (366 nm).
MOBILE PHASE
20 volumes of 2-methylpropanol and 80 volumes of dichloromethane.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with solution (3) shows two clearly separated spots.
CONFIRMATION
The principal spot in the chromatogram obtained with solution (1) corresponds in position and colour to that in the chromatogram obtained with solution (2). A secondary spot in the chromatogram obtained with solution (1) corresponds in position and colour to the pre-calcipotriol spot in solution (3).
B. In the Assay, the chromatogram obtained with solution (1) shows a peak with the same retention time as the peak due to calcipotriol in the chromatogram obtained with solution (2).
TESTS
Related substances
Carry out the method for liquid chromatography, Appendix III D, protected from light, using the following solutions prepared in solvent B.
Solvent B 30 volumes of 0.01M ammonium phosphate and 70 volumes of methanol.
(1) To a quantity of the cream containing the equivalent of 0.5 mg of calcipotriol, add 10 mL of methanol and shake vigorously. Cool in ice, centrifuge and filter. Evaporate 5 mL of the filtrate to dryness at a temperature not exceeding 30° and dissolve the residue in 2 mL of solvent B.
(2) Dilute 0.1 mL of solution (1) to 10 mL.
(3) 0.004% w/v of calcipotriol monohydrate EPCRS.
(4) Dilute 1 volume of solution (2) to 10 volumes.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (10 cm × 4.0 mm) packed with octadecylsilyl silica gel for chromatography (3 μm) (Luna C18 is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1.0 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 264 nm.
(f) Inject 50 μL of each solution.
(g) For solution (1), allow the chromatography to proceed for 2 times the retention time of calcipotriol.
MOBILE PHASE
30 volumes of water and 70 volumes of methanol.
When the chromatograms are recorded under the prescribed conditions the retention times relative to calcipotriol (retention time, about 13.5 minutes) are: pre-calcipotriol, about 0.86; impurity C, about 0.92 and impurity D, about 1.3.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the peak-to-valley ratio between calcipotriol and impurity C is at least 5.
LIMITS
In the chromatogram obtained with solution (1):
the area of any peak corresponding to impurity C is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (1%);
the area of any peak corresponding to impurity D is not greater than twice the area of the principal peak in the chromatogram obtained with solution (2) (2%);
the area of any other secondary peak is not greater than twice the area of the principal peak in the chromatogram obtained with solution (4) (0.2%);
the sum of the areas of any other secondary peak is not greater than half the area of the principal peak in the chromatogram obtained with solution (2) (0.5%).
Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with solution (4) (0.1%).
ASSAY
Carry out the method for liquid chromatography, Appendix III D, protected from light, using the following solutions.
(1) To a quantity of the cream containing the equivalent of 0.25 mg of calcipotriol, add 7 mL of methanol and shake vigorously. Dilute to 20 mL with 0.01M ammonium phosphate. Cool in ice, centrifuge and filter.
(2) 0.0013% w/v of calcipotriol monohydrate EPCRS in solvent B.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related substances may be used with an injection volume of 200 μL.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (2), the peak-to-valley ratio between calcipotriol and impurity C is at least 5.
DETERMINATION OF CONTENT
Calculate the content of C27H40O3 in the cream using the combined areas of the peaks due to calcipotriol and pre-calcipotriol in the chromatograms obtained with solutions (1) and (2) and the declared content of C27H40O3 in calcipotriol monohydrate EPCRS.
STORAGE
Calcipotriol Cream should be protected from light.
LABELLING
The quantity of active ingredient is stated in terms of the equivalent amount of calcipotriol.
