To all the calibration standards (0.2 mL)

or QC samples (0.2 mL) taken in polypropylene tubes, 50 μL of internal standard was added and vortexed for 30 s. 0.25 mL of 2.00% ortho phosphoric acid in water was added to the plasma samples, vortexed for 30 s. The samples were transferred to a 1 cc/30 mg Oasis HLB SPE column, which had been conditioned with 1.0 mL methanol, followed by 1.0 mL water. After application of the samples, the SPE column was dried for 1.0 min by applying positive pressure at maximum flow rate. The column was eluted with 1.00 mL mobile phase. The SPE eluates were transferred into 1 mL LC vials for injection of 10 μL into the LC system. Validation was carried out according to the US Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance.20 and 21 SB431542 Accuracy, precision and linearity of the calibration curve were determined. Intra- and inter-day precision were carried out on three different days. Each validation run

consisted of a minimum of one set of calibration standards and six sets of QC samples at four concentrations. Recoveries of AMX, CLV, AMX-D4 and AMP in aqueous solutions were determined at lower limit of quantification (LLOQ QC), low QC (LQC), medium QC (MQC) and high QC (HQC) levels. The stabilities of the stock solution, bench top, autosampler solutions, long term and freeze–thaw stability Fluorouracil manufacturer were carried out. For specificity, six different lots of blank plasma were evaluated for any interference at the retention

times of AMX, CLV, AMX-D4 (IS) and AMP (IS). Selectivity was carried out by analyzing the six blank plasma samples spiked with AMX and CLV (LLOQ level) and IS. Matrix effect was assessed by comparing the mean area responses almost of samples spiked after extraction with those of standard solutions in mobile phase at low and high QC levels. The linearity of the method was evaluated using bulk spiked plasma samples in the concentration range as mentioned above using the method of least squares. Five such linearity curves were analyzed. Each calibration curve consisted of a blank sample, a zero sample (blank + IS) and eight concentrations. Samples were quantified using the ratio of peak area of analyte to that of IS. A weighted linear regression (1/concentration) was performed with the nominal concentrations of calibration levels. Peak area ratios were plotted against plasma concentrations. The extraction efficiency of AMX and CLV was evaluated by comparing the mean peak responses of three QC samples 150.30, 9411.75 and 18823.24 ng/mL of AMX and 76.98, 2368.62 and 4737.23 ng/mL of CLV concentrations to the mean peak responses of three standards of equivalent concentration. Similarly, the recovery of IS was evaluated by comparing the mean peak responses in the three quality control samples to mean peak responses of three standards at a concentration of 9411.62 ng/mL of AMX-D4 and 2368.62 ng/mL of AMP.