After this, an appropriate dilution was made and 6 000 ng/spot was applied. UV degradation For UV degradation study, 10 mg of drug was weighed accurately and kept inside UV chamber for 40 hours at 254 nm. Then, it was transferred into 10-ml volumetric selleck chem flask and volume was made up to the mark with methanol. After this, an appropriate dilution was made and 6 000 ng/spot was applied. Degradation under elevated temperature and humidity For degradation under elevated temperature and humidity study, 10 mg of drug was weighed accurately and kept inside humidity chamber for 7 days at 40��C and 75% relative humidity. Then, it was transferred into 10-ml volumetric flask and volume was made up to the mark with methanol. After this, an appropriate dilution was made and 6 000 ng/spot was applied.

Thermal degradation For thermal degradation study, 10 mg of drug was weighed accurately and kept inside hot air oven for 3 days at 80��C. Then, it was transferred into 10-ml volumetric flask and volume was made up to the mark with methanol. After this, an appropriate dilution was made and 6 000 ng/spot was applied. Validation of analytical method The developed HPTLC method for the estimation of ITZ was validated for specificity, linearity, precision and accuracy, limit of detection (LOD), and limit of quantitation (LOQ) according to ICH guidelines. It was established that products of degradation do not interfere with the peak response of the drug. Linearity of response To demonstrate the linearity, concentrations of 1 000 to 6 000 ng were applied to TLC plate and densitograms were developed.

The graph was constructed between concentrations vs peak area and treated by regression analysis. Accuracy and precision The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume. Accuracy and precision were determined by replicate analysis of samples containing known amounts of the analyte. For reproducibility and accuracy, standard solution at 50%, 100%, and 150% concentrations were prepared. Intraday precision and accuracy were studied by three replicate measurements at three concentration level in one day.

Interday precision was conducted during routine operation of system over a period of three consecutive days. Statistical evaluation like relative standard deviation of drug at different concentrations Brefeldin_A of three replicates application was calculated to determine intraday and interday variation. Specificity Specificity of a method was determined by analyzing drug and forcefully degraded products.