Wavelength selected for simultaneous estimation of two drugs was 270 nm. The column was saturated with the selleck screening library mobile phase for about an hour at a flow rate of 1.0 ml/min, monitoring the eluent at 270 nm so as to obtain a steady base line. After the chromatographic conditions were set and the instrument was stabilized to obtain a steady baseline, 20 ��L of standard drug solution each of BH (25 ��g/ ml) and TB (25 ��g/ml) made in the mobile phase were loaded into the injection port of the instrument and injected after filtration through a 0.2 ��m membrane filter. The injection was repeated three times. This was done to check retention times of the individual drugs. The mean retention time for BT and TB were found to be 15.50 min and 9.85 min, respectively [Figure 1].

Figure 1 Chromatogram of BT and TB Standard stock solutions of pure drugs were made separately in the mobile phase containing 100 ��g/ml of BH and TB, filtered through a 0.2 ��m membrane filter. In a 10 ml volumetric flask, 2.5 ml standard stock solution of BH with 2.5 ml standard stock solution of TB was taken and volume made to the mark with the mobile phase. This mixed standard solution was loaded in the injector port of the instrument. The solution was injected and a chromatogram was recorded. This was done to check the resolution of two drugs. The two drugs were found to be perfectly resolved. Calibration curve In a series of a 10 ml volumetric flask, several dilutions of BH (15�C55 ��g/ml) and TB (6�C36 ��g/ml) were prepared in the mobile phase. Each solution was injected and a chromatogram was recorded.

The peak area of a drug was calculated for each concentration level of two drugs and a graph was plotted between drug concentrations against the peak area. The linearity was observed in the concentration range of 15�C55 ��g/ml for BH and 6�C36 ��g/ml for TB. Method validation Linearity A series of standard curves were prepared over a concentration range of 15�C55 ��g/ml for BH and 6�C36 ��g/ml for TB from a stock solution of BH Carfilzomib and TB (100 ��g/ml) in the mobile phase. Dilutions were prepared in the mobile phase, phosphate buffer: acetonitrile (70:30% v/v). The data from peak area vs. drug concentration plots were treated by linear least square regression analysis. The standard curves were evaluated for intra-day and inter-day reproducibility. The experiment was performed in triplicate [Table 1]. Table 1 Validation parameters Accuracy Recovery studies by the standard addition method were performed with a view to justify the accuracy of the proposed method. Previously analyzed samples of BH and TB were spiked with known amounts of BH and TB standard drugs and the mixtures were analyzed by the proposed method. The experiment was performed in triplicate.