Development and Validation of UV Spectrophotometric Method for Quantitative Estimation of Clobetasol 17-Propionate

High potency dihalogenated corticosteroid, Clobetasol 17-Propionate (CP) is used for skin diseases such as vitiligo, psoriasis and atopic dermatitis due to its antiinflammatory, vasoconstrictive, antiproliferative and immunosuppressive activities. It has been approved for the topical use in dosage forms like such as gel, cream, ointment, solution and foam1–2. Clobetasol 17-Propionate is used to relieve redness, itching, sweeling, or other discomfort caused by skin conditions. The treatment of more severe skin disorders using CP with or without the inclusion of other drug substances were compared in several clinical studies. CP has demonstrated excellent recovery, rapid relief and reduced relapses of different skin conditions and symptoms3–5. It was also proven to be the first topical corticosteroid that demonstrated satisfactory results in the treatment for psoriasis2,4. Literature search reveals HPLC, RP-HPLC and liquid chromatography methods were reported for determination of various salts of Clobetasol in formulations like ointment, creams and suspensions6–11. Beside these, some simultaneous analytical estimations of Clobetasol 17-Propionate with other drugs have been reported in literature12–16. Till date, no studies have been reported for estimation of CP in bulk and ointment formulation using a validated UV-visible spectrophotometric assay method. Therefore, the aim of the present work is to develop and validate analytical method by UV-Visible spectrophotometer which is simple, rapid and advantageous and in which no complexation agent, extraction, derivatization, or evaporation steps are involved. Abstract


Materials
Clobetasol 17-Propionate (CP) was obtained from Sigma Aldrich, USA. Ethanol was procured from S D Fine-Chem Ltd, Mumbai, India. Formulation (ointment) collected from market with drug equivalent to 0.05% w/w of CP. All the other reagents and chemicals used were of analytical grade.

Instrument
Double beam UV Visible Spectrophotometer (Variance Carry 5000, India)

Preparation of Standard Stock Solution
Accurately weighed 10 mg of standard CP was dissolved in 100 ml of ethanol (standard stock solution). From this standard stock solution, prepare the aliquots of different concentration by suitable dilutions varying in between 2 and 40 µg/ml using ethanol. These diluted solutions were checked for Linearity, Precision, Accuracy, Robustness, Limit of Quantification (LOQ) and Limit of Detection (LOD).

Selection and Optimization of Solvent
As reported in literature, the solvent have a profound influence on the shape and quality of the peak 17 . The choices of solvents for ultra violet method development are: ethanol, methanol, acetone, etc. Various solvents were checked and ethanol was found to fulfill all the conditions relating to quality and non-interference of peak at the specified wavelength.

Selection of Wavelength
In order to determine the wavelength of absorption maxima (ʎ max ) of CP, aliquot of 100 µg/ml solution was prepared by taking weighed amount of drug (10 mg) in 100 ml of ethanol and scanned by UV-Visible spectrophotometer in the wavelength range of 400-200 nm against ethanol as a blank. The resulting spectrum was shown in Figure  2 and absorption curve showed characteristic maximum absorption at 239 nm for Clobetasol 17-propionate. The wavelength at which maximum absorption observed is 239 nm, which is selected for further analysis.

Method Validation
Method validation was performed as per the International Conference on Harmonization (ICH) guidelines Q2 (R1) (ICH, 2005) 18,19 and all the parameters were evaluated.

Linearity
The linearity of this method was checked at concentrations ranging between 2-40 µg/ml. The curve of absorbance v/s concentration ( Figure 3) of CP was found to be linear as given in Table 1. The investigated concentrations followed Beer's Lambert law 20 .

Precision
The precision of the UV method was performed by intermediate precision (inter-day) and repeatability (intra-day).

Repeatability
Repeatability (intra-day) was carried out by analyzing CP having concentration 4 μg/ml, three times a day. To assess the intra-day variation, the % RSD was calculated from absorbance as obtained.

Intermediate Precision
Intermediate precision (inter-day) was assessed by analyzing 4 µg/ml concentration of CP for three different days. The % RSD was calculated for absorbance thus obtained, to measure the interday variation 21 .

Accuracy
Accuracy is defined as closeness between the actual (true) value and value obtained by repeating test method for a number of times. Accuracy may be expressed as % Recovery by the assay of known analyte which is added. It gives exact measure of the analytical method. The preanalyzed samples were spiked with extra 50, 100 and 150% of the standard CP (10 µg/ml) and the mixtures were analyzed using UV visible spectrophotometer. The experiment was performed in triplicate 22 .

LOD and LOQ
The Detection Limit (DL) is the lowest concentration of analyte present in a sample, which can be analyzed but not necessarily quantitated. The Quantitation Limit (QL) is the lowest concentration of analyte present in a sample, which can be quantitatively analyzed with acceptable precision and accuracy. The limit of detection and limit of quantification were assessed based on the technique of signal-to-noise ratio10 using the Equations (1) and (2).
Where, σ is the standard deviation of the intercept of the calibration plot and S is the slope of the calibration curve 23 .

Result and Discussion
The CP was found to be soluble in ethanol. The ʎ max of drug was found to be 239 nm as shown in (Figure 1). From the result obtained from Table 1, it was observed that CP obeys linearity within the concentration range of 2 µg/ml-40 µg/ml and coefficient correlation was 0.9999. The regression value from the curve was y = 0.0353x + 0.0162 as shown in Figure 2. The detection and quantitation limits were calculated as LOD (k = 3.3) and LOQ (k = 10) and these were found to be 0.84 µg/ml and  (Table  2.) significant reproducibility with % RSD below 2.0 observed. This showed that method is highly precise. The percent recovery value (Table 3.), was observed higher than 100%, indicating the accuracy of the method. The estimation of CP in marketed ointment formulation was found to be 98-99%.

Conclusion
The proposed method was observed as a simple, accurate, precise, sensitive, economical, reproducible and rapid for the routinely estimation of CP. The developed method is specific for estimation commercial formulations like ointments without interference of excipients.