Polymyxin b

Sample pre-treatment: Five microliters of polymyxin B (Toronto Research Chemicals, CAS number 1405-20-5) dissolved in water (420 μg/mL, an internal standard) was added to 100 μL rat plasma sample. After that, 400 μL of acetonitrile and 10% trichloroacetic acid mixed solution (50/50, v/v) was added. The samples were vortex-mixed for 1 min and then centrifuged at 12,000 rpm for 10 min. Following centrifugation, the supernatant was collected and filtered with 0.45 μm PVDF membrane filter for analysis.
Linearity: The standard curve was constructed using seven non-zero plasma standards covering the concentrations expected in the study. The original concentrations were then plotted against the responses to obtain the slope, intercept, and correlation coefficient (r²) by the least-square linear regression methods.
Accuracy and precision: Accuracy was measured as the percent of deviation from the nominal concentration, whereas precision was determined as the relative standard deviation from the mean (RSD, %). The accuracy results should not be less than 85%. The precision results should not deviate by more than 15% (RSD, % ≤ 15%) except at the lower limit of quantification (LLOQ) where the values should not deviate by more than 20%.

Aztreonam, meropenem, and polymyxin B were purchased from Toronto Research Chemicals. Cefepime was purchased from Kemimac(s) Pte Ltd. Piperacillin/tazobactam and tigecycline were purchased from Sigma-Aldrich. Doripenem was obtained from Shionogi and Co. Aliquots of stock solutions of all antibiotics were prepared in sterile water and stored at −80°C. Before each experiment, the aliquots were thawed and diluted to the desired concentrations with cation-adjusted Mueller Hinton broth (Ca-MHB).