Determination of Exemestane in bulk and pharmaceutical dosage form by HPTLC

2.1 Materials

The tablet formulation was procured from local pharmacy (Sun Pharmaceuticals Ltd., India). Pure Exemestane were obtained from (Cipla Ltd., Mumbai, India). All chemicals were of analytical grade. All dilutions were performed in standard volumetric flasks.

2.2 Instrumentation

HPTLC system—Desaga HPTLC system with densitometer CD 60 and auto sampler model AS 30 (Desaga, Wiesloch, Germany). Twin tough developing chamber (Desaga) 200 × 100 mm, Mettler analytical balance (Mettler-Toledo, Columbus, Ohio), and model AE 240 (accuracy ±0.01 mg) Ultrasound cleaning bath.

2.3 Stock and working standard solution

Exemestane (25 mg) was accurately weighed into a 25 mL volumetric flask, and dissolved in a minimum volume of water, and diluted to required volume with water to furnish a solution of concentration 1000 μg mL⁻¹. This was used as stock solution.

Calibration standards were prepared over the concentration range 100–500 μg mL⁻¹ for Exemestane by appropriate dilutions of the above mentioned standard stock solution in 10 mL volumetric flask with methanol.

2.4 Sample preparation

Twenty tablets were weighed and the average weight was calculated. The tablets were then powdered and amount equivalent to 25 mg of pure Exemestane tablet was dissolved in a minimum volume of methanol. This was then filtered through Whatman filter paper number 41 and filtrate was collected in a 10 mL volumetric flask and diluted to volume with methanol to get stock solution of concentration of 100 μg mL⁻¹. Similarly various required strength dilutions of the tablet sample solution is done and analyzed by proposed method and concentration was calculated by using the calibration curve.

2.5 Calibration curve

Separate stock standard solutions of Exemestane were used for the preparation of calibration standard solutions. All calibration standards were prepared freshly every day and found to be stable during the analysis time. The plate was developed, dried, and scanned as described above. After densitometric scanning the peak area was recorded for each concentration and a calibration plot was obtained by plotting average peak area against concentration of Exemestane (ng spot⁻¹). The slope and correlation coefficient were also determined.

2.6 Standard chromatographic HPTLC conditions

Analysis was performed on silica gel 60 F₂₅₄ plates of size 200 × 100 mm. Different volumes of the samples (experimental sample solution and reference standard solution) were applied on the plate separately or together with standard drug solution. Samples (5.00 μL) were applied as 5 mm bands by means of Desaga auto sampler automatic applicator (model AS30) equipped with 10 μL syringe at different positions (lane) on the HPTLC plate under N₂ gas stream. Application parameters were the length of the band is 5 mm, distance between bands was 10 mm, the application position along Y axis was 15 mm, and the start position along X axis was 15.0 mm. Each spot was given by a 1 μL/cycle with application rate of 0.1 μL/s. Linear ascending development is carried out in twin trough glass chamber (200 × 100 mm) covered with stainless steel lid. The optimized chamber saturation time for mobile phase was 25 min at room temperature and the plates were developed up to 80 mm from the bottom of the plate using the solvent systems chloroform:methanol 9.2:0.8 (v/v) as a mobile phase. The average development time was 20 min. After development the plate was air-dried for 15 min. Optical Densitometric scanning at λ = 247 nm was performed. After densitometric scanning, chromatograms were evaluated via peak area. Scanned peak areas were recorded for each sample at each concentration level. In assay experiment six samples were weighed separately and analyzed. For each sample, each concentration was applied six times to the plate at each concentration level. These developed plates are scanned by performing six replicate measurements of peak areas of the six bands of same concentration. The average peak areas, variations in peak area obtained were expressed as a coefficient of variation (% CV) and concentration of the drug present were automatically calculated by comparing the peak area values of the sample with that of the reference standard. The plot was obtained by plotting average peak area at each concentration against the corresponding band concentrations of Exemestane (ng spot⁻¹). From the constructed calibration curve slope, correlation coefficient was calculated. The % CV of six independent assays values is calculated. From amount of drug present in sample, % assay of Exemestane was calculated.

2.7 Validation

The proposed method was validated according to the ICH guidelines by carrying out analysis of six replicate samples of the tablets. The method was validated for accuracy, precision, robustness, linearity and range, selectivity, limit of detection, limit of quantification and robustness.

2.8 Analysis of marketed formulation

In assay experiment five samples weighed separately were analyzed. The standard solution of Exemestane of strength (100 μg mL⁻¹) and the experimental solution (extracted from marketed tablet) of strength (100 μg mL⁻¹) were prepared by the above described procedure. For tablet assay linearity study the solutions were prepared containing 100–500 μg mL⁻¹. The standard solution of Exemestane of strength (100 μg mL⁻¹) and the experimental solution of strength (100 μg mL⁻¹) was applied on HPTLC plate together with reference standard by using the proposed method. Then it was developed and scanned under the optimized conditions by the proposed method.