Method C: Absorption corrected method The value of ��max of EM and TE was determined by scanning the drug solution in the range 200- 400 nm at 0.5 band width and 600 nm/min scan speed and was found to be at 293.38 and 270.29 nm, respectively. Wortmannin ATM EM also showed absorbance at 270.29 nm, while TE did not show any interference at 293.38 nm [Figure 4]. To construct Beer’s plot for EM and TE, stock solutions of 1000 ��g/ml of both the drugs were prepared in 0.1N HCl and working standard dilutions were made in 0.1N HCl using stock solution of 1000 ��g/ml. Also Beer’s plot was constructed for EM and TE in solution mixture at different concentration (4:6, 8:12, 12:18, 16:24, 20:30 ��g/ml) levels. Both the drugs followed linearity individually and in mixture within the concentration range 4-20 ��g/ml and 6-30 ��g/ml for EM and TE, respectively.

Figure 4 Simple overlay spectra of EM (4-20 ��g/ml) and TE (6-30 ��g/ml) in 0.1N HCl with formulation (12 + 18 ��g/ml) of EM and TE, respectively. Determination of absorption factor at selected wavelengths EM and TE solution in 0.1N HCl of known concentrations were scanned against blank on spectrophotometer. The value of absorption factor was found to be 0.52. Quantitative estimation of EM and TE was carried out using following equations: Corrected Absorbance of TE at 270.29 nm = Abs270.29 (TE + EM) �C [(abs270.29 (EM)/ abs293.38 (EM)] �� abs293.38 (EM) or Corrected Absorbance of TE at 270.29 nm = abs270.29 (TE + EM) �C 0.52 �� abs293.38 (EM) where; abs: Absorption value at given wavelengths.

Preparation of standard stock solutions and calibration curve Standard stock solutions of pure drug containing 1000 ��g/ml of TE and EM were prepared separately by dissolving 100 mg of pure TE and EM in a 100 ml volumetric flask and volume was made up to the mark with methanol for method A and B and with 0.1N HCl for method C. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in methanol and 0.1N HCl for proposed methods. Derivative amplitudes of spectrum, by using the above mentioned procedures, were used to prepare calibration curves for both the drugs. Beer’s law obeyed in the concentration range of 3-21 ��g/ml for TE and 2-14 ��g/ml for EM by method A and B whereas 6-30 ��g/ml for TE and 4-20 ��g/ml for EM by method C.

Preparation of sample stock solution and formulation analysis To assay by using method A and B, 20 tablets were weighed accurately and a quantity of tablet powder equivalent to 100 mg of TE (66.66 mg of EM) was weighed and dissolved in the 80 ml of methanol with the aid of ultrasonication for 5 min and solution was filtered through Whatman paper No. 41 into a 100 ml Cilengitide volumetric flask. Filter paper was washed with methanol, adding washings to the volumetric flask and volume was made up to the mark with methanol.