Clotrimazole was obtained from Glenmark Laboratories Ltd, Mumbai, India. HPLC-grade acetonitrile LiChrosolv, water LiChrosolv, and methanol were procured from Merk Pvt. Ltd., Mumbai. Gelatin and sodium lauryl sulfate were procured from S D Fine-Chem Ltd, Mumbai. Gelatin films (3% w/w of clotrimazole) were prepared in the laboratory.
The Younglin Acme 9000 HPLC system consisting of the components, 4 Channel Vacuum Degasser and Mixer SDV50A, Gradient Pump SP930D, Rheodyne Injector, Phenomenex Luna C18(2) (150 × 4.6 mm, 5 μm) column with Phenomenex security guard cartridge C18 (4 × 3 mm), and UV–VIS Detector UV730D, was used. The equipment was controlled by PC installed with the Younglin Autochro 3000 Chromatograph data system software. Shimadzu UV-Vis Spectrophotometer 1800, Shimadzu Electronic balance AY220, Borosil All Glass Filter with Nylon 6,6 membrane 0.45 μm 47 mm filters, and Phenomenex RC membrane 0.45 μm 15 mm syringe filters were used.
Preparation of standard solution
Ten milligrams of clotrimazole was weighed and transferred into 10 ml volumetric flask. 5 ml of the methanol was added to clotrimazole and sonicated for 10 min; the volume was made up to the mark with methanol (flask A). An aliquot of 5 ml from flask A was transferred into 50 ml volumetric flask, and the volume was made up of the mark with methanol (flask B) to obtain a solution of clotrimazole (100 μg/ml).
Preparation of sample solution
Film equivalent to 10 mg of clotrimazole (332 mg) was taken in a 50 ml volumetric flask and dissolved in 40 ml of 1% sodium lauryl sulfate (SLS) solution with stirring at 50 °C. The volume was made up to the mark with SLS solution (flask C). An aliquot of 1 ml from flask C was transferred into a 10 ml volumetric flask and a mobile phase was added up to the mark to obtain a sample solution (flask D).
Selection of UV wavelength
A solution of clotrimazole in the mobile phase (10 μg/ml) was scanned in the range of 200–400 nm in a UV-VIS spectrophotometer, and λmax was selected from the UV spectrum.
Method development and optimization
To achieve satisfactory separation of clotrimazole, solvents of different proportions were tried as binary eluents on the C18 column. However, ACN achieve good satisfactory results. The optimized chromatogram and optimized conditions are decided on the basis of system suitability parameters.
The chromatographic separation of components was achieved on a Phenomenex Luna C18(2) (150 × 4.6 mm, 5 μm) stationary phase with Phenomenex Security Guard Cartridge C18 (4 × 3 mm) using acetonitrile and water (70:30% v/v) as mobile phase. The flow rate was 1.0 ml/min and the absorbance was monitored at 210 nm. The volume of the sample was 20 μl.
The method has been evaluated for system suitability, specificity, linearity, precision, accuracy, robustness, and stability of solution as per ICH guideline Q2(R1) .
System suitability parameters (capacity factor, tailing factor, number of theoretical plates, and resolution of peak) were assessed by injecting a blank mobile phase followed by clotrimazole solution (20 μg/ml).
The specificity of the method has been established by comparing the chromatograms obtained by injecting blank (mobile phase), solution of clotrimazole (20 μg/ml), and sample solution of clotrimazole gelatin film. The effect of excipients used in film was checked. The parameters retention time, tailing factor, and resolution were calculated in order to prove that the method chosen was specific.
Precision was assessed by injecting six samples of clotrimazole solution (20 μg/ml) on the same day (repeatability) and a different day (intermediate precision). The precision of the method was investigated as %RSD of the response.
Ten, 20, 30, 40, and 50 μg/ml of clotrimazole solutions were prepared by diluting the solution from flask B with mobile phase. Solutions were filtered through 0.45 μm syringe filter and injected in an HPLC system to measure the peak area. The calibration curve (peak area v/s concentration) was plotted. The correlation coefficient of the calibration curve was determined to ensure the linearity of the analytical method.
Recovery studies were conducted by the standard addition (spiking) technique to the matrix of gelatin film. In this method, 80, 100, and 120% of three different levels of pure drug (1.6, 2, and 2.4 ml from flask B) were added to the previously analyzed sample solution (0.5 ml from flask C) in the 10 ml volumetric flask and diluted to 10 ml with mobile phase. Solutions were filtered through 0.45 μm syringe filter and injected in an HPLC system to measure the peak area. Accuracy was evaluated as recovery values of the clotrimazole.
Deliberate minute variations in the chromatographic conditions such as flow rate (±0.1 ml/min), acetonitrile ratio in the mobile phase (± 2%) have been made. After each change, assay results were checked by injecting the clotrimazole solution (20 μg/ml) into the chromatographic system and the results were compared with those under the original chromatographic conditions.
The stability of the clotrimazole solution (20 μg/ml) was assessed over 24 h at room temperature. The peak area and retention time were evaluated after injecting clotrimazole solution (20 μg/ml) into the system after every 8 h.
Analysis of clotrimazole film
Sample solution (flask D) was filtered through 0.45 μm syringe filter and injected in an HPLC system to measure the peak area.