(Ph. Eur. 11.6 update)
Action and use
Vitamin D analogue + glucocorticoid; maintenance and treatment of psoriasis.
DEFINITION
Calcipotriol and Betamethasone Ointment contains Calcipotriol Monohydrate and Betamethasone Dipropionate in a suitable basis.
The ointment complies with the requirements stated under Topical Semi-solid Preparations and with the following requirements.
Content of calcipotriol, C27H40O3
92.0 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.
Content of betamethasone, C22H29FO5
92.0 to 105.0% of the stated amount.
IDENTIFICATION
A. For calcipotriol In the Assay for calcipotriol, record the UV spectrum of the principal peak in the chromatograms obtained with solutions (1) and (2) with a diode array detector in the range of 220 to 360 nm.
The UV spectrum of the first principal peak in the chromatogram obtained with solution (1) is concordant with that of the peak in the chromatogram obtained with solution (2);
the retention time of the first principal peak in the chromatogram obtained with solution (1) is similar to that of the peak in the chromatogram obtained with solution (2).
B. For betamethasone In the Assay for betamethasone record the UV spectrum of the principal peak in the chromatograms obtained with solutions (3) and (4) with a diode array detector in the range 220 to 360 nm.
The UV spectrum of the second principal peak in the chromatogram obtained with solution (3) is concordant with that of the peak in the chromatogram obtained with solution (4);
the retention time of the second principal peak in the chromatogram obtained with solution (3) is similar to that of the peak in the chromatogram obtained with solution (4).
TESTS
Related substances
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
Solution A 0.132% w/v solution of diammonium hydrogen orthophosphate adjusted to pH 6.4 using 1M orthophosphoric acid.
Solution B 40 volumes of solution A and 60 volumes of acetonitrile.
(1) On a water bath, disperse a quantity of ointment containing the equivalent of 90 μg of calcipotriol in 10 mL of n-heptane. Add 8 mL of solution B and shake for 15 minutes. Allow to separate and add 4 mL of the lower (aqueous) layer to 2 mL of solution A, centrifuge and use the clear lower layer.
(2) On a water bath, disperse a quantity of ointment containing the equivalent of 0.9 mg of betamethasone in 10 mL of n- heptane. Add 8 mL of solution B and shake for 15 minutes. Allow to separate and add 4 mL of the lower (aqueous) layer to 2 mL of solution A, centrifuge and use the clear lower layer.
(3) 0.00075% w/v of calcipotriol for system suitability EPCRS and 0.0097% w/v of betamethasone dipropionate EPCRS in mobile phase A.
(4) 0.0097% w/v of betamethasone dipropionate for system suitability A EPCRS in mobile phase A.
(5) Dilute 1 volume of solution (1) to 100 volumes with mobile phase A. Dilute 1 volume of this solution to 20 volumes with the same solvent.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 2.1 mm) packed with octadecylsilyl silica gel for chromatography (1.8 μm) (Zorbax Eclipse Plus RRHD is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 0.4 mL per minute.
(d) Use a column temperature of 40°.
(e) Use a diode array detector (DAD) with a detection wavelength of 264 nm.
(f) Inject 15 μL of each solution.
MOBILE PHASE
Mobile phase A 40 volumes of acetonitrile and 60 volumes of solution A.
Mobile phase B 80 volumes of acetonitrile and 20 volumes of solution A.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comment |
| 0-2 | 100 | 0 | isocratic |
| 2-38 | 100→10 | 0→90 | linear gradient |
| 38-39 | 10→0 | 90→100 | linear gradient |
| 39-43 | 0 | 100 | isocratic |
| 43-44 | 0→100 | 100→0 | linear gradient |
| 44-50 | 100 | 0 | re-equilibration |
When the chromatograms are recorded under the prescribed conditions the retentions relative to calcipotriol (retention
time, about 14 minutes) are about:
| RRT | Impurity | RRT | Impurity |
| 0.91 | Pre-calcipotriol | 1.16 | Betamethasone unknown* |
| 0.96 | Calcipotriol impurity C | 1.19 | Betamethasone unknown* |
| 1.12 | Calcipotriol impurity D | 1.23 | Betamethasone impurity E |
| 0.42 | Betamethasone impurity B | 1.27 | Betamethasone unknown* |
| 0.52 | Betamethasone unknown* | 1.43 | Betamethasone unknown* |
| 0.54 | Betamethasone impurity C | 1.47 | Betamethasone unknown* |
| 0.84 | Betamethasone impurity D | 1.51 | Betamethasone unknown* |
| 1.05 | Betamethasone unknown* | 1.58 | Betamethasone unknown* |
|
* UV scan from a DAD should be used in conjunction with the information above to attribute unknown peaks to the relevant active ingredient. * Where betamethasone impurities are indicated, these relate to the impurities listed in the monograph for Betamethasone Dipropionate. |
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SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the peak-to-valley ratio is at least 1.5, where Hp is the height above the baseline of the peak due to calcipotriol impurity D and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to betamethasone dipropionate.
LIMITS
Identify any secondary peaks and attribute to the relevant active ingredient using solutions (3) and (4) and the information included in the above table.
For calcipotriol
Calculate the results by normalisation, using all peaks attributed to calcipotriol. Any secondary peak that cannot be attributed to an impurity of betamethasone dipropionate should be calculated with respect to calcipotriol.
In the chromatogram obtained with solution (1):
the area of any peak due to calcipotriol impurity C is not more than 1.5%;
the area of any peak due to calcipotriol impurity D is not more than 1.0%;
the area of any other secondary peak is not greater than 0.5%;
the sum of the areas of all secondary peaks is not greater than 3.0%.
Disregard any peak with an area less than 0.05%, and any peak due to betamethasone dipropionate and betamethasone dipropionate related impurities.
For betamethasone dipropionate
Calculate the results by normalisation, using all peaks attributed to betamethasone dipropionate.
In the chromatogram obtained with solution (2):
the area of any peak due to betamethasone impurity B is not more than 0.8%;
the area of any peak due to betamethasone impurity C is not more than 0.8%;
the area of any other secondary peak is not greater than 0.5%;
the sum of the areas of all secondary peaks, excluding impurities B and C, is not greater than 1.0%.
Disregard any peak with an area less than 0.1%, any peak attributed to calcipotriol, pre-calcipotriol and calcipotriol related impurities, and any unattributed secondary peaks.
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
Solution A 0.132% w/v solution of diammonium hydrogen orthophosphate adjusted to pH 6.4 using 1M orthophosphoric acid
Solution B 40 volumes of solution A and 60 volumes of acetonitrile.
(1). On a water bath, disperse a quantity of ointment containing the equivalent of 90 μg of calcipotriol in 10 mL of n-heptane. Add 8 mL of solution B and shake for 15 minutes. Allow to separate and add 4 mL of the lower (aqueous) layer to 2 mL of solution A, centrifuge and use the clear lower layer.
(2) 0.0008% w/v of calcipotriol monohydrate EPCRS in mobile phase A.
(3) On a water bath, disperse a quantity of ointment containing the equivalent of 0.9 mg of betamethasone in 10 mL of n-heptane. Add 8 mL of solution B and shake for 15 minutes. Allow to separate and add 4 mL of the lower (aqueous) layer to 2 mL of solution A, centrifuge and use the clear lower layer.
(4) 0.0097% w/v of betamethasone dipropionate EPCRS in mobile phase A.
(5) 0.0008% w/v of calcipotriol for system suitability EPCRS and 0.0097% w/v of betamethasone dipropionate EPCRS in mobile phase A.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related substances may be used.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (5), the peak-to-valley ratio is at least 1.5, where Hp is the height above the baseline of the peak due to calcipotriol impurity D and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to betamethasone dipropionate.
DETERMINATION OF CONTENT
For calcipotriol
Combine the areas of the peaks due to calcipotriol and pre-calcipotriol. Calculate the content of C27H40O3 in the ointment using the declared content of C27H40O3 in calcipotriol monohydrate EPCRS.
For betamethasone
Calculate the content of betamethasone C22H29FO5 in the ointment using the declared content of betamethasone dipropionate C28H37FO7 in betamethasone dipropionate EPCRS. Each mg of C28H37FO7 is equivalent to 0.7778 mg of C22H29FO5
LABELLING
The quantity of active ingredients are stated in terms of the equivalent amount of calcipotriol and the equivalent amount of betamethasone.
IMPURITIES
The impurities limited by the requirements of this monograph include impurities C and D listed under Calcipotriol Monohydrate and impurities B, C, D and E listed under Betamethasone Dipropionate:
calcipotriol impurity C
calcipotriol impurity D
betamethasone dipropionate impurity B
betamethasone dipropionate impurity C
betamethasone dipropionate impurity D
betamethasone dipropionate impurity E
