41 into a 50 ml volumetric flask. The filter paper was washed with methanol, adding washings to the volumetric flask, and the volume was made up to the mark with methanol. From the filtrate, an appropriate dilution was prepared in the mobile phase to get this site a solution of 40 ��g/mL of LOR and 20 ��g/mL of THIO, and amount of solutions corresponding to 200 and 400 ng/band, respectively, were applied on the plates and the plates were developed by using optimized chromatographic conditions. These solutions were estimated according to the procedure given above. Method validation As per the ICH guidelines, the method validation parameters checked were linearity, accuracy, precision, limit of detection, limit of quantitation, robustness and specificity.
Linearity Linearity of the method was studied by spotting six concentrations of the drug prepared in the mobile phase in the range of 60�C360 ng/band for LOR and 30�C180 ng/band for THIO, and noting the peak areas. Peak areas of developed spots were measured and used to plot the calibration curve. Accuracy For accuracy of the method, a recovery study was carried out by applying the method to drug samples to which a known amount of LOR and THIO were added at the level of 50%, 100% and 150% of the label claim. At each level of the amount, three determinations were performed and the results obtained were compared with the expected results. Precision The precision of the method was demonstrated by repeatability, intra-day and inter-day variation studies at three different concentration level of analytes covering the concentration range.
In the intra-day studies, three repeated measurements of standard and sample solutions were made in a day and three repeated measurements of standard and sample solutions Cilengitide were made on three consecutive days for inter-day variation studies. Percentages Relative Standard Deviation (RSD) were calculated for intra-day and inter day variation. Limit of detection and limit of quantification LOD and LOQ were calculated using �� (standard deviation of the response) and b (slope of the calibration curve) using the following formulae: LOD = (3.3 �� ��) / b and LOQ = (10 �� ��) / b. Robustness By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different composition like methanol:chloroform:water (9.6:0.2:0.2 v/v/v), (9.6:0.4:0.2 v/v/v), (9.4:0.2:0.2 v/v/v), (9.6:0.2:0.3 v/v/v) and (9.6:0.5:0.2 v/v/v) were tried and chromatograms were run. Time from spotting to chromatography and from chromatography to scanning was varied from 0, 20, 40 and 60 min. The amount of mobile phase was varied by ��5%; development distance was varied by ��5 mm.