1 ml of acetonitrile by vigorous vortex mixing using a remi mixer for 2 min and centrifuged at 5000 rpm at 10 min. The organic phase was recovered and evaporated to dryness on a hot plate. The residual mass was reconstituted with 1 ml of methanol. The analysis was carried on HPTLC. Chromatographic done condition The mobile phase was selected as a mixture of chloroform and methanol (9.0:1.0 v/v) for the development of plates. Time for chamber saturation was optimized to 10 min. The length of the chromatographic development was 70 mm. The densitometric scanning was performed at 240 nm. Method validation The method was validated for sensitivity, selectivity, precision, accuracy, linearity, recovery, and stability. The validation of the method was based on FDA guidelines and on the standard bio-analytical method validation recommendations.

The selectivity of method was investigated by analyzing six blank plasma samples. Each blank sample was tested for interference using a proposed extraction procedure. Five replicates of three QC sample low, mid, and high were used for the determination of precision and accuracy. Intra-day and inter-day precision were carried out. Precision and accuracies showed 15% relative standard deviation from nominal values, at lower limit of quantitation (LLOQ) these were both 20%. The recovery of CEFPO and AMBRO was calculated by a comparison of the peak areas of low, mid, and high QC sample (1000, 2000, 3000 ng/ml and 2000, 4000. 6000 ng/ml, respectively) prepared in plasma (extracted) with unextracted CEFPO and AMBRO, respectively.

Stability studies were performed to detect degradation of CEFPO and AMBRO under certain conditions. Freeze�Cthaw stability was determined at two QC concentrations (low, high) after freezing (�C20��C) and thawing for three cycles and compared with the nominal value. Bench-top stability was assessed for low and high QC samples by comparing with the nominal value which stored at room temperature for 12 h. The effect of storage within the auto-sampler was assessed by comparing the QC samples injected immediately after preparation with those left in the auto-sampler for 48 h. RESULTS AND DISCUSSION Extraction procedure optimization One of the most difficult tasks during the method development was to achieve a reproducible recovery from the solvent which is used for the extraction of the drug.

Different solvents GSK-3 were tried for the extraction of CEFPO and AMBRO from human plasma. Three millilitres of chloroform and 3 ml of ethyl acetate were tried for the precipitation of plasma but the recovery was very less up to 50�C60% because of less precipitation of protein from plasma. Finally methanol was tried and 60�C80% of recovery was obtained. It was found that the addition of acetonitrile (0.1 ml) increases the recovery which is reproducible as compared to other solvents. Therefore, methanol and acetonitrile (3.0:0.