INTRODUCTION:

Desonide is a synthetic nonfluorinated corticosteroid for topical dermatologic use. Chemically Desonide¹ is [(pregna-1,4-diene-3,20-dione,11,21-dihydroxy-16,17-[(1-methylethylidene)bis (oxy)]-,(11β,16α)] (Fig. 1). Desonide is indicated for the treatment of mild to moderate atopic dermatitis^2,3. Desonide is used to treat the inflammation and itching caused by a number of skin conditions such as allergic reactions, eczema, and psoriasis^4-6. The mechanism of action of Desonide is unknown. Corticosteroids are thought to act by the induction of phospholipase A2 inhibitory proteins, collectively called lipocortins. It is thought that these proteins control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of their common precursor arachidonic acid. Arachidonic acid is released from membrane phospholipids by phospholipase A2. Once absorbed through the skin, topical corticosteroids are handled through pharmacokinetic pathways similar to systemically administered corticosteroids^7,8.

Literature survey revealed that few analytical methods such as spectrophotometric⁹, HPLC^10-15 and LC-MS^16-18 methods have been reported for the determination of Desonide. Therefore it was felt necessary to develop and validate an rapid, accurate, specific and precise stability-indicating HPLC method as per ICH guidelines^19,20 for the determination of Desonide in pharmaceutical formulation.

Fig. 1: Molecular structure of Desonide

MATERIALS AND METHODS:

Instrumentation:

Waters Alliance HPLC system equipped with auto sampler, binary gradient pump and dual wavelength UV-Visible detector was used to for the determination. An analytical column Altima C18, (100 x 4.6 mm, 5m); was used in the analysis. Chromatographic software Empower 2 was used for data collection and processing. Elico-SL159 double beam UV-Visible spectrophotometer is used for measuring absorption spectrum.

Chemicals and solvents:

The working standard of Desonide was provided as gift sample from Spectrum Labs, Hyderabad, India. The market formulation DESOWEN cream (Desonide 0.05%) was procured from local market. Acetonitrile of HPLC grade was obtained from E.Merck. (India) Ltd., Mumbai, India. HPLC grade water and methanol were purchased from E.Merck (India) Ltd., Mumbai, India. Potassium dihydrogen phosphate, orthophosphoric acid and glacial acetic acid of AR grade were obtained from Qualigens Fine Chemicals Ltd., Mumbai, India.

Preparation of phosphate buffer pH 4.8:

1.36 grams of potassium dihydrogen phosphate was weighed accurately, transferred into a 1000 ml beaker and dissolved in 500 ml of HPLC grade water. The solution was sonicated for 30 min., degassed and then made to total volume with water. The pH of the resulting solution was adjusted to 4.8 with orthophosphoric acid and filtered through 0.45 μm membrane filter.

Preparation of mobile phase and diluent:

The mobile phase was prepared by mixing 450 ml of the phosphate buffer pH 4.8 with 550 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µm filter under vacuum. The solvent methanol was used as diluent.

Preparation of standard solution:

10 mg of Desonide was accurately weighed, transferred to 10 ml volumetric flask and is dissolved in 7 ml of the diluent. Sonicated the solution for few minutes and dissolved the drug completely. Then it is filtered through 0.45 μm filter and the volume is made up to 10 ml with diluent to get a concentration of 1 mg/ml stock solution. Further pipette 1 ml of the above stock solution into a 10 ml volumetric flask and diluted up to the mark with diluent to obtain required concentration of 10 µg/ml of Desonide.

Preparation of sample solution:

10 g of Desonide cream with label claim of 0.05% was weighed and the cream was extracted with 50 ml of 0.1% glacial acetic acid in methanol in a 50 ml glass centrifugation tube. The sample mixture was heated at 75⁰C for 10 min and mixed occasionally during the heating process. After heating, the sample was allowed to cool for 1 min and 2 ml of water was added into the centrifuge tube, then cooled in an ice bath for 20 min and centrifuged at 3000 RPM for 10 min. After centrifugation, the supernatant liquid layer, which contains the analyte of interest, was separated by allowing mobile phase at a flow rate of 1.0 ml/min prior to the analysis. Samples were prepared from the extracted Desonide solution with 10 µg/ml concentration.

Calibration plot:

About 10 mg of Desonide was weighed accurately, transferred into a 10 ml volumetric flask and dissolved in 7 ml of a 45:55 v/v mixture of phosphate buffer and acetonitrile. The solution was sonicated for 15 min and the volume made up to the mark with a further quantity of the diluent to get a 1000 μg/ml solution. From this, a working standard solution of the drug (100 μg/ml) was prepared by diluting with the above solution to 10 ml in a volumetric flask. Further dilutions ranging from 2.5-15 μg/ml were prepared from the solution in 10 ml volumetric flasks using the above diluent. 10 μl of each dilution was injected six times into the column at a flow rate of 1.0 ml/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The calibration graph constructed by plotting concentration of the drug against peak area (Fig. 2) was found to be linear in the concentration range of 2.5-15 μg/ml of the drug. The relevant data are furnished in Table 1.The regression equation of this curve was computed. This regression equation was later used to estimate the amount of Desonide in pharmaceutical dosage forms.

Fig. 2: Calibration curve of Desonide

Table 1. Optimized chromatographic conditions of Desonide

Parameter	Condition
Mobile phase	Phosphate buffer pH 4.8:acetonitrile (45:55 v/v)
pH	4.8
Diluent	Methanol
Column	Altima C18 column (100 mm x 4.6 mm, 5 µm)
Column temperature	30^◦C
Wave length	240 nm
Injection volume	10 µl
Flow rate	1.0 ml/min
Run time	6 min
Retention time	3.555 min

Procedure:

A mixture of phosphate buffer and acetonitrile in the ratio of 45:55 v/v was found to be the most suitable mobile phase for ideal separation of Desonide. The solvent mixture was filtered through 0.45 μm membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 ml/min. The column was maintained at a temperature of 30^◦C. The pump pressure was set at 800 psi. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. Inject 10 μl of the standard, sample solutions into the chromatographic system and measure the area for the Desonide peak. The detection of the drug was monitored at 240 nm. The run time was set at 6 min. Under these optimized chromatographic conditions the retention time obtained for the drug Desonide standard was 3.555 minutes. The results are furnished in Table 1. Typical chromatograms for the drug Desonide in standard and in sample were given in Fig. 3 and Fig. 4.

Fig. 3: Typical chromatogram of Desonide standard

Fig. 4: Typical chromatogram of Desonide formulation

Validation of the proposed method:

The linearity, limit of detection, limit of quantification, precision, accuracy, system suitability parameters, ruggedness and robustness were studied systematically to validate the proposed HPLC method as per the ICH guidelines for the estimation of Desonide.

Linearity:

Several aliquots of standard solution of Desonide was taken in different 10 ml volumetric flasks and diluted up to the mark with diluent such that the final concentrations of Desonide were in the range of 2.5 to 15 µg/ml. The drug was eluted with UV detector at 240 nm, peak area was recorded for all the peaks. The results are shown in Table 2.

Table 2. Calibration data of the proposed method

Concentration (μg/ml)	Mean peak area
2.5	216217
5	444364
7.5	664881
10	890559
12.5	1105912
15	1365372

Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solution using the developed HPLC method.

Precision:

The precision was determined for Desonide in terms of method precision and intermediate precision and the results are furnished in Table 3.

Table 3. Precision data of the proposed HPLC method

Concentration of Desonide (10 μg/ml)	Peak area
Concentration of Desonide (10 μg/ml)	Method precision	Intermediate precision
Injection-1	720065	723591
Injection-2	715690	718081
Injection-3	715879	727077
Injection-4	717511	712647
Injection-5	722863	725956
Injection-6	717773	716956
Mean	718296	720718
Standard deviation	2739.0	5698.7
% RSD	0.38	0.80

Accuracy:

The accuracy of the method was assessed by recovery study of Desonide in the dosage form at three concentration levels. A fixed amount of pre-analyzed sample was taken and standard drug was added at 50%, 100% and 150% levels. Each level was repeated three times. The content of Desonide was calculated and the results are furnished in Table 4.

Table 4. Accuracy studies

% Concentration at specification level	Standard conc. (μg)	Conc. added (μg)	Conc. found (μg)	% Recovery	% Mean recovery
50 %	10	5	5	100.0%	100.0%
100%	10	10	10.06	100.6%
150%	10	15	14.95	99.66%

System suitability:

System suitability parameters like retention time, theoretical plates and tailing factor were calculated and compared with standard values and the results are shown in Table 5.

Table 5. System suitability parameters

System suitability	Results
Linearity range (μg/ml)	2.5-15
Correlation coefficient	0.999
Theoretical plates (N)	3367
Tailing factor	1.19
LOD (μg/ml)	0.040
LOQ (μg/ml)	0.121

Ruggedness and robustness:

The ruggedness of the method was determined by carrying out the experiment on different instruments by different operators using different columns of similar types. The robustness of the method was determined by influence of small and premeditated alteration of analytical parameters such as flow rate and percent of composition of the mobile phase on the quantification of the drug substance and selectivity was studied.

Assay:

10 µl of sample solution was injected and from the peak area of Desonide, amount of each drug in the sample were computed. The results were compared with the label claim of Desonide. The assay results are shown in Table 6.

Table 6. Assay results of Desonide

Formulation	Label claim	Amount found	%Assay
DESOWEN	5 mg	4.99 mg	99.80%

Degradation studies:

Acid degradation studies:

To 1 ml of stock solution of Desonide, 1 ml of 2N hydrochloric acid was added and refluxed for 30 min at 60⁰C. The resultant solution was diluted to obtain 10 µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Alkali degradation studies:

To 1 ml of stock solution of Desonide, 1 ml of 2N sodium hydroxide was added and refluxed for 30 min at 60⁰C. The resultant solution was diluted to obtain 10 µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Oxidative degradation studies:

To 1 ml of stock solution of Desonide, 1 ml of 20% hydrogen peroxide (H₂O₂) was added and refluxed for 30 min at 60⁰C. The resultant solution was diluted to obtain 10 µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Dry heat degradation studies:

The standard drug solution was placed in an oven at 105⁰C for 6 hours. The resultant solution was diluted to obtain 10 µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Photostability degradation studies:

The standard drug solution was exposed to UV light by keeping the beaker in UV chamber for 7 days or 200 Watt hours/m²in photo stability chamber. The resultant solution was diluted to obtain 10 µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Neutral degradation studies:

The standard drug solution was refluxed in water bath for 6 hours at 60⁰C. The resultant solution was diluted to obtain 10 µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

The percent of drug degraded in the presence of acidic, alkali, oxidative, dry heat, photostability and neutral conditions were studied. The amount of drug recovered or degraded is calculated by comparing the area of the standard with that of the area of the degraded sample. The results are furnished in Table 7.

RESULTS AND DISCUSSION:

In the proposed method, the retention time of Desonide was found to be 3.555 min. Quantification was linear in the concentration range of 2.5-15 μg/ml. The regression equation of the linearity plot of concentration of Desonide over its peak area was found to be (y=90198x-6860, where x is the concentration of Desonide (μg/ml) and y is the corresponding peak area. The correlation coefficient value of Desonide was 0.999. The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range indicated. The limit of detection and limit of quantification for Desonide were found to be 0.040 µg/ml and 0.121 µg/ml respectively, which indicate the sensitivity of the method. The %RSD for method precision and intermediate precision for Desonide were found to be 0.38 and 0.80 respectively (limit %RSD < 2.0%). The mean recovery of the drug Desonide was 100%. The high percentage of recovery indicates that the proposed method is highly accurate. The retention time for the drug Desonide was 3.555 minutes. The use of phosphate buffer and acetonitrile in the ratio of 45:55 v/v resulted in peak with good shape and resolution. The number of theoretical plates calculated was 3367, tailing factor was 1.19, which indicates efficient performance of the column. The robustness studies indicated that no considerable effect on the determination of the drug. Therefore the test method is robust for the quantification of the drug. The %assay of Desonide in commercial formulations was 99.80%. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in ointment formulation did not interfere with the estimation of the drug Desonide by the proposed HPLC method. A study on degradation of the drug was conducted and found to be negligible.

CONCLUSION:

The newly developed RP-HPLC method for determination of Desonide in bulk sample and in pharmaceutical formulation was found to be specific, precise, accurate and robust. The proposed method was completely validated as per ICH guidelines. The method validation data showing satisfactory results for all the method parameters tested. The stability-indicating nature of the proposed method was established by performing forced degradation, which provided degradation behaviour of Desonide under various conditions. Hence the developed HPLC method is stability-indicating and can be used for routine analysis of production samples and also to check the stability of bulk samples of Desonide.

Table 7. Degradation results of Desonide

Degradation Parameter	Peak area of sample	Peak area of standard	% Recovery	% Degradation
Acid degradation	647407	716461	90.36	9.63
Alkali degradation	659694	716461	92.07	7.92
Oxidative degradation	648860	716461	90.56	9.43
Dry heat degradation	679694	716461	94.86	5.13
Photostability degradation	696084	716461	97.15	2.84
Neutral degradation	704854	716461	98.37	1.62

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Received on 16.06.2014 Modified on 26.06.2014

Asian J. Research Chem. 7(9): September 2014; Page 805-809