A concentration-time curve was plotted and AUC calculated by trapezoidal rule. KRX-0401 mouse In a similar way (AUC0–12) and (AUC0–∞) were

calculated. Time to achieve the maximum concentration (CMAX), tMAX was obtained directly from the concentration time curve without interpolation. All the pharmacokinetic parameters were calculated by using WinNonlin Professional Software (Version 6.3). Liquid–liquid extraction with dichloromethane, diethyl ether, n-hexane, tertbutyl methyl ether and mixtures of these solvents was evaluated. The extraction efficiency of the drug was found to be poor and also interference at the retention time of drug was observed. Poor extraction efficiency was also observed using precipitation. Hence solid phase extraction (SPE) technique was used with Oasis HLB extraction cartridges. Samples were retrieved from the deep freezer then thawed and vortexed. Each 0.2 mL of sample was transferred to pre-labeled tubes which contained extraction buffer. The tubes were vortexed for about 10 s and centrifuged at 4000 rpm and 10 °C for 2 min. HLB extraction cartridges (1 cc, 30 mg) were arranged in solid phase extraction manifolds to condition the cartridge with 1.0 mL of methanol followed by 1.0 mL of water. The conditioned cartridges were loaded with prepared samples and the cartridges were then subjected to positive pressure. The contents

were eluted from the cartridge by the addition of 0.5 mL of mobile phase into Cell Cycle inhibitor pre-labeled tubes then vortexed for 10 s and transferred to the HPLC vials to inject 10 μL of the sample. No significant interfering peaks were observed

at the retention time of either analyte or internal standard in six different lots of drug free Endonuclease human plasma samples. Chromatograms of extracted blank, LLOQ sample and internal standard are shown in Fig. 2. The matrix effect for both amoxicillin and clavulanic acid was calculated as a percentage of the comparison of area response obtained with the post extracted and the aqueous samples and was found to be more than 98.00% at LQC and HQC levels which implies that there is no matrix effect in the extracted samples on comparison with aqueous samples. All calibration curves were found to be linear over the range of 50.43–31500.68 and 25.28–6185.18 ng/mL. The mean correlation coefficient was 0.9998 for AMX and 0.9997 for CLV. The back calculated concentrations of calibration standards for AMX and CLV are presented in Tables 1 and 2 respectively. The inter-batch assay accuracy for amoxicillin and clavulanic acid ranged between 97.29–103.56 and 97.28–101.22% respectively, whereas intra-batch accuracy ranged between 100.38–103.99 and 95.48–102.17%. The inter-batch precision for amoxicillin and clavulanic acid ranged between 2.97–3.55 and 1.73–2.03% and intra-batch precision ranged between 1.06–3.07 and 1.