SPE is further expensive as compared to LLE technique. Various solvents such as ethyl acetate, diethyl ether, 100% t-butyl methyl ether and combinations of t-butyl methyl ether and dichloromethane were used for

extraction. Akt inhibitor The highest recovery from the plasma samples was obtained with a 70:30% v/v of t-butyl methyl ether: dichloromethane. Fig. 3 shows the typical chromatograms of a blank plasma sample (A), a spiked plasma sample with PZA (300.0 ng/ml, LLOQ) and MTZ (200.0 ng/ml) (B), a zero blank sample containing only the internal standard (C) indicating the specificity of the method. The retention times for PZA and MTZ were 6.80 and 2.56 min, respectively. The method was found to have high selectivity for the analyte; since no interfering peaks from endogenous compounds were observed at the retention time for PZA in any one of the six independent blank plasma extracts evaluated (Table 1). Calibration curves for PZA in human plasma were calculated by weighted1/concentration2 quadratic regression, with the r2 values of >0.99 for all curves generated during the validation. The calibration curve accuracy for plasma is presented in Fig. 4 demonstrating that measured concentration is within

±15% of the actual concentration point (20% for the lowest point on the standard curve, the LLOQ). A detailed summary of the intra-day and click here inter-day precision and accuracy data generated for the assay validation

is presented in Table 2 was <5% for all QC concentrations, which was within the general assay acceptability criteria for QC samples according to FDA guidelines.12 Limit of detection, LOD was defined as the lowest concentration that produces a peak distinguishable from background noise (minimum ratio of 3:1). The approximate LOD was 100 ng/ml. The LLOQ has been accepted as the lowest points on the standard curve with a relative standard deviation of less than 20% and signal to noise ratio of 5:1. Results at lowest concentration studies (250 ng/ml) met the criteria for the LLOQ (Table 3). The upper limit of quantification (ULOQ) has been accepted as the highest points on the standard curve with a relative standard deviation of less than 15%.12 A critical unless issue with the analysis of many drugs is their tendency to get adsorbed by reversed phase octadecyl-based chromatographic packing materials, resulting in the carryover effect. However in this analysis no quantifiable carryover effect was obtained when a series of blank (plasma) solutions were injected immediately following the highest calibration standard. The results of auto sampler and freeze–thaw stability are presented in Table 4. Determination of PZA stability following three freeze–thaw cycles showed that for all QC samples there was a minor change in the PZA concentration.