59-05-032 Proceeding

71 Proceedings of the Princess Maha Chakri Sirindhorn Congress E. Method validation The method was validated according to standard criteria of Bioanalytical Method Validation of the US FDA, (2001). 1) Specificity is the ability of an analytical method to differentiate and quantify the analyte in matrix blank plasma samples for detection interfering substance in a biological sample. 2) Limit of detection (LOD) and Limit of quantification (LOQ ) defined as the lowest amount detected and quantified with at least 3:1 S/N ratio (LOD) and 10:1 S/N ratio (LOQ) for six replications. 3) Linearity and range Internal standard and 4 BZDs were spiked into 250µL plasma sample. The final concentration of internal standard was 10 µg/mL and the concentrations of 4 BZDs were ranged from0.0104-52.10 (lorazepam), 0.0103-10.30 (alprazolam), 0.0101-15.10 (diazepam) and 0.0412- 1.50 (midazolam) µg/mL. All solutions were extracted and injected into HPLC system. Linearity was obtained by preparing the standard curve and calculating the coefficient of determination, r 2 which should be more than 0.99. 4) Accuracy and precision were determined using quality control sample (QC) with 4 BZDs at low, medium and high concentrations then internal standard was added. The sample solutions were extracted according to step C and then injected into HPLC system (n=6). The intra-day and inter-day precision were performed for 6 replicates. 5) Stability Freeze-thaw stability was made from preparing the standard solutions of 4 BZDs at low and high concentration (n=3) then determined after three freeze and thaw cycles. Each cycle should be stored at the intend storage. Extraction was performed according to step C and supernatant solutions were injected into HPLC system. Recoveries of the analysis were calculated and compared with freshly prepared samples (time 0). Short-term stability of 4 BZDs and internal standard (10 µg/mL) was determined at low and high concentration.The samples were storage at -20°C for 1week then the storage samples were thaw at room temperature. Extraction was performed according to step C and sample solutions were injected into HPLC system (n=3). Recoveries of the analysis were calculated and compared with freshly samples (time 0). Long-term stability was determined by the same process as short term stability but the samples were storage at -20°C for 2 weeks. III. Results 1) Specificity The chromatographic separation of matrix blank plasma and spiked plasma sample shown no endogenous interferences at the retention times of 4 BZDs and internal standard. The retention times of internal standard, lorazepam, alprazolam, diazepamandmidazolamwere 10.34,