INTRODUCTION:

Calcipotriene is belonging to the chemical class of vitamin D derivatives and chemically, it is (5Z,7E,22E,24S)-24-Cyclopropyl 9,10-secochola-5,7,10(19),22 tetraene-1a,3b,24-triol. with molecular formula C₂₇H₄₀O₃ and CAS number 112828-00-9; Calcipotriene is official in BP 2007^13-16. The precise mechanism of calcipotriol in remitting psoriasis is not well-understood. However, it has been shown to have comparable affinity with calcitriol (Vit D) for the Vitamin D receptor, while being less than 1% as active as the calcitriol in regulating calcium metabolism. The Vitamin D receptor (VDR) belongs to the steroid/thyroid receptor superfamily, and is found on the cells of many different tissues including the thyroid, bone, kidney, and T cells of the immune system. T cells are known to play a role in psoriasis, and it is thought that the binding of calcipotriol to the VDR modulates the T cells gene transcription of cell differentiation and proliferation related genes. Its metabolism is still not so clear.

Betamethasone is belonging to the chemical class of corticosteroids and chemically, it is 9-fluoro-11b-hydroxy-16b-methyl-3, 20-dioxopregna-1,4-diene-17,21-diyl dipropanoate, with molecular formula C₂₈H₃₇FO₇; Betamethasone is official in BP and USP. The precise mechanism of Betamethasone is when it is applied topically, particularly to large areas, when the skin is broken, or under occlusive dressings, or when given intranasally, they may be absorbed in sufficient amounts to cause systemic effects¹⁷.

Recently a combination of Calcipotriene and Betamethasone dipropionate has been launched in market. In this combination, Betamethasone shows a synergistic effect with Calcipotriene. Various analytical works were obtained which was related to ultra-performance liquid chromatography, tandem mass spectrometry, and High performance liquid chromatography. Simultaneous estimation of Betamethasone dipropionate was done with many other drugs. Analytical works related to photo stability were published but the estimation and degradation are not yet reported. Literature survey revealed that no HPLC method has been reported yet for the analysis of these two drugs in combination without preliminary separation that makes it worthwhile to pursue the present work^18-25.

EXPERIMENTAL:

Reagent and Materials:

Calcipotriene working standard and Donovex cream was provided by ZYG Pharma Pvt. Ltd., Pithampur (Label claim: 0.005%; Calcipotriene cream and 0.05% Betamethasone dipropionate). Methanol, and water (HPLC grade), were used. Instrument used was Agilent Technologies 1200 series²⁶.

Methods:

Preparation of Mobile Phase:

Taking into consideration the system suitability parameters like retention time, and resolution of peaks the mobile phase found to be most suitable for analysis was methanol: buffer (45:55v/v) isocratic flow. Flow rate employed for analysis was 1.0 ml/min, as it is most suitable for C₁₈column.

Preparation of laboratory mixture:

About 10.0 mg of Calcipotriene reference standard and 100.0 mg Betamethasone dipropionate, was weighed out accurately transfered into a 200.0 mL volumetric flask. 100.0 mL of methanol was added to it, sonicate for 5 minutes or until dissolved, if necessary. Diluted to volume with methanol and mixed well (stock std). This solution contains about 0.05 mg/mL of Calcipotriene and 0.5 mg/mL Betamethasone dipropionate. 2.0 mL of stock standard was diluted to 100.0 mL volumetric flask with sample solvent and mixed well. A portion of the sample was filtered through the filtration kit into autosampler vials. Minimum 2.0 mL of filtrate was discarded prior to collecting for analysis (std). This solution contains about 1.0 µg/mL of Calcipotriene and 0.01 or 10.0 µg/mL Betamethasone dipropionate.

Preparation of sample solution:

1.0g of the Calcipotriene Cream was weighed out accurately directly into a 50.0 mL volumetric flask. 45.0 mL of Sample Solvent was added and vortexed to disperse the cream. Sonicate for 30 minutes at room temperature. The volume was diluted with diluting solvent. Then it is mixed well by shaking and vortexing. Using a disposable pipette, 10.0 mL of the top solution was discarded and the sample is placed at 2–8°C for 2 hrs to settle the solution. The supernatant clear solution was filtered through 0.2 µm nylon filter discarding the first few mL. This solution contained about 1.0 µg/mL of Calcipotriene and 10.0 µg/mL Betamethasone dipropionate.

Chromatographic condition:

Chromatographic separation was achieved at 25°C on a reverse phase Zorbax SB-300 column²⁷ using mobile phase consisting of methanol: buffer (45:55v/v) isocratic flow. Flow rate employed for analysis was 1.0 ml/min and detection was carried out at 264 nm. The sample was injected using 10 μl fixed loop, and the total run time was 7.99 min for Calcipotriene and 13.99 min for Betamethasone.

System suitability parameters:

Depending on the theoretical plate count, and other parameters as pressure etc system suitability parameters were studied.

Calibration curve:

The linearity of the response for Calcipotriene and Betamethasone dipropionate assay method was determined by preparing and injecting standard solutions with concentrations of 0.8-1.4 ppm Calcipotriene and Betamethasone dipropionate (Figure 1 and 2).

Figure 1 CALIBRATION CURVE OF CALCIPOTRIENE

Figure 2 CALIBRATION CURVE OF BETAMETHASONE DIPROPIONATE

Determination of assay:

The developed method was applied to the analysis of Calcipotriene and Betamethasone dipropionate in marketed product named as DONOVEX (Label claim 0.005% Calcipotriene and 0.05% Betamethasone dipropionate)²⁸. The contents of marketed dosage form were found to be in the range of 98-102% with RSD less than 2 % which indicate suitability for routine analysis of Calcipotriene and Betamethasone dipropionate in dosage form.

The assay of the combination was determined by the formula given below:

% ASSAY = Aspl/Astd x mg Std x f /200 x 2/100 x 50/ g Spl x 100/LC

Where, f = correction factor for potency

Expressed as % of Calcipotriene reference standard x 10^-2 = 98.6 x 10^-2 = 0.986

Expressed as % of Betamethasone dipropionat reference standard x 10^-2 = 100.21 x 10^-2 = 1.0021

LC = Label claim of Calcipotriene 0.005% = 0.05 mg/g

LC = Label claim of Betamethasone 0.05% = 0.5 mg/g

Recovery studies:

Recovery studies were performed by standard addition method to validate the accuracy of developed method to different concentrations of preanalyzed sample solution of cream, varying concentration of standard drug was added and then its recovery was analyzed.

Preparation of Reference Standard: 50.0 mg of Calcipotriene and 500.0 mg Betamethasone dipropionate reference standard was dissolved in 50.0 mL of methanol. 1.0 mL of above solution was taken and diluted to 100.0 mL sample solvent (stock std solution)

Preparation of solution: About 1.0 g of placebo was weighed accurately in a stoppered test tube and 2.5 mL stock std solution was added and heated on a water bath and cooled to room temperature and the volume was make up to 50.0 mL with sample solvent and mixed and filtered (50 %).

About 1.0 g of placebo was weighed accurately in a stoppered test tube and 5.0 mL stock std solution was added and heated on a water bath and cooled to room temperature and the volume was make up to 50.0 mL with sample solvent and mixed and filtered (100 %).

About 1.0 g of placebo was weighed accurately in a stoppered test tube and 7.5 mL stock std solution was added and heated on a water bath and cooled to room temperature and the volume was make up to 50.0 mL with sample solvent and mixed and filtered (150%).

% of Calcipotriene

% Recovery = ---------------------------------------- x 100

% of Calcipotriene added

And,

% of Betamethasone dipropionate

% Recovery = ---------------------------------------------x 100

% of Betamethasone dipropionate added

Validation of the method:

Validation of the developed method was done according to ICH Q2B guideline 1996^29-34.

accuracy:

The accuracy of method is determined by adding known amount of standard to that of sample (above and below the normal level) at 3 different levels to cover both above and below (50% to 150%) the normal levels expected in the sample.

Precision:

Precision was also determined to assure the repeatability of the method. The % RSD was found < 2.0 which lies within the limit. The solutions were prepared using Calcipotriene and Betamethasone dipropionate standard stock solution as follows in the 100% concentration and 80% and 120% solution.

Ruggedness:

Ruggedness was determined by interday, intraday, change in instrument and change in analyst. six replicates of 100% concentration was injected. In interday precision six replicates of 100% are given three times a day and in intraday the same sample is injected with six replicates of 100% concentration three times.

Specificity:

Specificity was carried out to identify the retention time of the drug and the obtained degradation products by thermal, photo, acid, alkali and oxidative degradation.

Linearity:

The linearity of detector response was studied for simultaneous estimation of Calcipotriene and Betamethasone dipropionate by HPLC at the concentration of 60%, 80%, 90%, 100%, 110%, 120%, 140% and 160% by one analyst on one day.

Preparation of reference standard: 50.0 mg of Calcipotriene and 500.0 mg of Betamethasone dipropionate reference standard was dissolved in 50.0 mL of methanol. 1.0 mL of above solution was taken and diluted to 100.0 mL sample solvent. (stock std solution).

Detection limit and Quantification limit:

The results for LOD and LOQ were calculated by the obtained observation from linearity and calibration results. It is based on the standard deviation of the response and the slope of Calcipotriene and Betamethasone dipropionate.

Results and discussions:

Optimization of the mobile phase was performed based on resolution, asymmetric factor and peak area obtained for both Calcipotriene and Betamethasone dipropionate. The mobile phase Methanol: 0.005M (NH₄)₂PO₄(pH 7.4)- Buffer in the ratio of 45:55% v/v was found to be satisfactory and gave two symmetric and well resolved peaks for Calcipotriene and Betamethasone dipropionate. The retention time for Calcipotriene and Betamethasone dipropionate were 7.99 min and 13.99 min, respectively (Figure 3).

The result of the assay of Calcipotriene and Betamethasone dipropionate was found out to be 98.65%+1.4 and 100.43%+1.2 respectively (Table 1).

TABLE 1: ASSAY RESULTS OF COMBINED DOSAGE FORM

Formulation

Label Claim

mg/gm

%Assay*

±RSD*

Calcipotriene

0.005

98.65%+1.4

Betamethasone dipropionate

0.05

100.43%+1.2

* denotes average of two determinations.

The result for the recovery study for Calcipotriene was found to be for 50% it was 101.32+ 0.1, for 100% it was 101.19+ 0.1, and 150% it was 100.97+ 0.4. The average recovery was 101.16%+ 0.6. The result for the recovery study for Betamethasone dipropionate was found to be for 50% it was 100.44+0.1, for 100% it was 100.00+0.1, and 150% it was 100.43+0.2. The average recovery was 100.29%+0.25 (Table 2).

The calibration curve for Calcipotriene was obtained by plotting the peak area of Calcipotriene versus the concentrations of Calcipotriene over the range of 0.8-1.4ppm, and it was found to be linear with r² = 0.9999. Similarly, the calibration curve for Betamethasone dipropionate was obtained over the range of 0.8-1.4ppm and was found to be linear with r² = 1.

The data of regression analysis of the calibration curves and the validation parameters are summarized in Table 2. The linear regression data for the calibration curves of Calcipotriene and Betamethasone dipropionate indicate that the response is linear over the concentration range studied with correlation coefficient, r² value as 0.9999 and 1 respectively. The value of slope was 1.24 and 915.1 respectively. The LOD was found to be 0.03 and 0.0004ppm respectively and LOQ was found to be 0.11 and 0.001ppm respectively.

Accuracy for the developed method was studied and the obtained results indicated that the percent recovery for Calcipotriene was found within the limit of 98 – 102% and Betamethasone dipropionate was 97 – 103% respectively. The % RSD was found to be not more than 2.

The results of precision (Repeatability) showed that three replicates each of 0.8 ppm (80%), 1.0 ppm (100%), 1.2 ppm (120%) gives standard deviation and relative standard deviation within the limit, showing a précised and repeatable result.

Robustness showed various changes in day to day variations and analyst to analyst variations and standard deviation was found not more than 2%. In Interday and intraday precision was carried out to find deviation obtained in results on the same day three times and on two different days three times a day and had again showed reproducible results. Two different analysts have performed the same study and the results found were reproducible and the standard deviation was within the acceptable limit. The same analyst had run the sample on two different instruments and the results obtained were reproducible and standard deviation was within the acceptable limit. All validation results are mentioned in Table 3.

TABLE 3: VALIDATION AND SYSTEM SUITABILITY PARAMETERS

Parameter	Calcipotriene	Betamethasone dipropionate
Calibration range (ppm)	0.8-1.4	0.8-1.4
Correlation Coefficient (r²)	0.9999	1
Retention time (min.)	7.99	13.99
Tailing factor*	0.64	0.68
No. of theoretical plate*	5941	8022
Limit of quantification (ppm)	0.11	0.001
Precision (%RSD)	0.006	0.006
Ruggedness (%RSD)
Day to Day	0.001	0.0001
Analyst to Analyst	0.004	0.0002
Instrument to Instrument	0.0007	0.0001

* Mean of five determinations.

CONCLUSION:

Proposed study describes a new RP-HPLC method for estimation of Calcipotriene and Betamethasone dipropionate combination in mixture using simple mobile phase. The method gives good resolution between both the compounds with a short analysis time.

The method was validated and found to be simple, sensitive, accurate and precise. Percentage recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of Calcipotriene and Betamethasone dipropionate in their combined dosage form.

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Received on 01.12.2010 Modified on 30.12.2010

Asian J. Research Chem. 4(4): April, 2011; Page 602-606

S. No.	% Drug Added	Calcipotriene		Betamethasone dipropionate
S. No.	% Drug Added	% Drug Recovered ± % RSD*	% Recovery	% Drug Recovered ± % RSD*	% Recovery
1	50	50.6886±0.10	101.3266	50.2244±0.0002	100.4489
2	100	101.2364±0.004	101.2364	100.0010±0.0001	100.0010
3	150	151.5261±0.12	100.9732	150.6477±0.003	100.4318