Optimization of chromatographic conditions Initially plane solvents such as methanol, ethyl acetate, chloroform, toluene, and acetone were tried. The spots were developed with chloroform and methanol but no proper resolution newsletter subscribe was observed between CEFPO and AMBRO. Paracetamol shows the tailing. Then chloroform and acetone (8:1, v/v) were tried but again there was no proper resolution. Lastly upon increasing the concentration of chloroform from 8 ml to 9 ml and adding methanol instead of acetone good resolution with symmetrical peaks of CEFPO, AMBRO and paracetamol was obtained. Finally the mobile phase consisting chloroform: methanol (9:1, v/v) gave good resolution of peaks for CEFPO, AMBRO, and paracetamol. The Rf values for CEFPO, AMBRO, and paracetamol were found to be 0.69 �� 0.005, 0.

49 �� 0.0057, and 0.31 �� 0.0054, respectively. Well-defined spots were obtained by an activating plate at 120��C for 20 min. The chamber was saturated with the mobile phase for 10 min at room temperature, which gave reproducible Rf values for cefpodoxime proxeti, AMBRO, and paracetamol, respectively [Figure 2]. Figure 2 Densitogram of cefpodoxime proxetil (Rf, 0.69 �� 0.005), ambroxol hydrochloride (Rf, 0.49 �� 0.0057), and paracetamol (Rf, 0.31 �� 0.00543.3). Calibration curves The six-point calibration curve was constructed by plotting the peak response ratio of CEFPO to IS and AMBRO to IS in plasma. Correlation coefficients were found to be 0.9928 and 0.9941 and linearity was found over the range of 10�C60 ��l/ml and 2�C12 ��l/ml for CEFPO and AMBRO, respectively.

The lower limit of quantification was defined as the lowest concentration in the calibration curve. The CEFPO and AMBRO can be determined at LLOQ. Recovery Absolute recoveries were calculated by comparing peak areas obtained from freshly prepared samples extracted with unextracted standard solutions of the same concentration. Recovery data were determined in triplicate at three concentrations (low, mid, and high) as recommended by FDA guidelines. The percent recovery at three concentrations was found to be 65.72, 74.46, and 83.04% for CEFPO and 97.50, 90.89, and 95.11% for AMBRO [Table 1]. Table 1 Result of recovery of cefpodoxime proxetil (CEFPO) and ambroxol hydrochloride (AMBRO) in human plasma Precision and accuracy Precision of the method was determined by intra-day and inter-day and accuracy for a set of QC samples (low, mid, and high) in replicate (n = 5).

In this assay, the intra-day precision was found to be in the range Drug_discovery of 1.78�C2.68% and 6.38�C10.77% and the inter-day precision was 6.61�C19.73% and 6.38�C7.79%. The accuracy was within 1.15�C2.50%. The above values were within the acceptable range, it shows that the method is accurate and precise. The low percent relative standard deviation (%RSD) and the percent relative error (%RE) were within the acceptable limit. The results of inter-day, intra-day precision, and accuracy for the CEFPO and AMBRO are shown in Table 2.