Chromatography was performed on 10 × 10 cm2 aluminum HPTLC plates precoated with 0.2 mm layers of silica gel (E. Merck, Darmstadt, Germany; supplied by Merck India, Mumbai, India). Before chromatography, the plates were prewashed with

methanol and dried in an oven at 50 °C for 5 min. Samples were applied as 6-mm wide bands, under a continuous flow of nitrogen, Y-27632 manufacturer by means of a CAMAG (Muttenz, Switzerland) Linomat V sample applicator equipped with an applicator microsyringe (Hamilton, Bonaduz, Switzerland). A constant application rate of 0.1 μL s−1 was used. The plates were then conditioned for 20 min in a presaturated twin-trough glass chamber (10 × 10 cm2) with the mobile phase of chloroform–toluene–methanol–acetic acid (8:1:1:1, v/v/v/v). The plates were then placed in the mobile phase and ascending development was performed to a distance of 70 mm from the point of application at ambient temperature, Saracatinib chemical structure and the development time was 12 min. Subsequent to the development, the plates were dried in a current of air with the help of an air dryer and spots were visualized in CAMAG UV cabinet with dual wavelength UV lamp (254 and 366 nm); densitometric scanning was performed at 365 nm with CAMAG TLC scanner III operated in reflectance–absorbance mode and controlled by Wincats V software. The concentrations of compound were studied from the intensity of diffusely

reflected light. Evaluation was based on linear regression of peak areas. A stock solution containing 1 mg mL−1 LER was prepared in methanol. Calibration solutions were prepared by diluting the stock solution so that application of 1 μL volumes gave a series of spots covering the range 30–210 ng LER. The developed method was validated for linearity and range, specificity, precision, accuracy

and robustness as per ICH guidelines.7 and 8 Each concentration Metalloexopeptidase in the range of 30–210 ng per spot was spotted five times on individual plates and response was measured after scanning. For evaluation of linearity, peak area and concentrations were subjected to least square regression analysis to calculate calibration equation and correlation coefficient. The specificity of the method was ascertained by analyzing LER in presence of excipients of LER tablet formulations. The bands of LER in the sample were confirmed by comparing RF values and respective spectra of the sample with those of the standard. The peak purity of LER was assured by comparing the spectra at three different levels, that is, peak-start (s), peak-apex (m) and peak-end (e) positions. Precision was measured by using standard solutions containing LER at concentrations covering the entire calibration range. The precision of the method was evaluated by calculating the percent relative standard deviation (%RSD) of mean peak areas obtained from each spot of sample. Same procedure was performed at different time intervals on the same day, on different days and by different analysts.