Optimization of on-line solid phase extraction and HPLC conditions using response surface methodology for determination of WM-5 in mouse plasma and its application to pharmacokinetic study.

Response surface methodology (RSM) was utilized for rapid and systematic optimization of on-line solid-phase extraction (SPE) parameters to maximize the response and separation of WM-5. The optimization was performed with Box-Behnken designs. Four major parameters were investigated for their contributions to the response and separation of WM-5, with a total of 29 experiments being performed for each instrument, respectively. Quantitative determination of WM-5 in mouse plasma was performed to evaluate the statistical significance of the parameters on chromatographic response. A fully automated on-line SPE and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for the determination of WM-5 in mouse plasma. Calibration curve with good linearity (r=0.9989) was obtained in the range of 20-4000 ng/mL in mouse plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) of the assay were 6 ng/mL and 20 ng/mL, respectively. The overall intra-day and the inter-day variations were less than 1.90%. The recovery of the method was in the range of 93.74-96.33% with RSD less than 3.06%. The optimized method demonstrated good performance in terms of specificity, LLOQ, linearity, recovery, precision and accuracy, and was successfully applied to quantify WM-5 in mouse plasma to support the pharmacokinetic study.