The extracted analytes were chromatographed by reverse-phase HPLC and quantified by a Waters Micromass® Quattro Ultima®, Waters Micromass® Micro or Sciex Qtrap® 3200 triple quadrupole mass spectrometer system using the electrospray interface. Drug and internal standard were detected in positive ionization mode using multiple reaction monitoring (MRM) of the precursor to product ions transition of m/z 626→470. Repeatability (precision), accuracy, assay specificity, stability in plasma, and all solutions

and method robustness were verified and passed FDA guidelines for validated bioanalytical methods (http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf). The validated lower limits of quantitation were 5 ng/mL (lower) and 500 ng/mL (upper), based on calibration curves that ranged from 5 to 500 ng/mL afoxolaner in 0.250 mL plasma. Enzalutamide supplier The limit of detection was 1.0 ng/mL. Dilution quality control samples also passed all guideline criteria, and afoxolaner plasma concentrations above 500 ng/mL were diluted in plasma prior to sample extraction. Selected dog plasma samples were scanned using

LC–MS techniques for metabolites of afoxolaner. The plasma samples from control and treated dogs were prepared via solid phase extraction and protein precipitation. Structural elucidation was accomplished using accurate mass measurements and FRAX597 molecular weight molecular formulae determinations. The molecular ion peak of the metabolite was confirmed by its characteristic 35Cl/37Cl isotope peak clusters representing each mass peak due to the natural relative abundance of each chlorine isotope in the mass spectrum. Elemental composition was determined by MassLynx® 4.1 software based on the accurate mass measurement using a Waters QTof mass spectrometer. The site of hydroxylation was determined based on the daughter ion, which was a result of loss of CF3CHO group from the hydroxylated metabolite. HPLC-photodiode array (PDA) and total

ion chromatograms were determined Parvulin also. The UV (312 nm) peak areas of both afoxolaner and the metabolite, for each animal and at each time point, were compared to estimate the relative percent of the metabolites formed. This gave a good estimate because the UV response (absorption maxima) for afoxolaner and the metabolites are approximately the same. Pharmacokinetic parameters were determined using the WinNonlin® software (Pharsight Corp, version 5.0.1). All plasma concentrations from samples taken prior to the first dose were below the LLOQ of the method. Concentrations < LLOQ were not used in PK calculations. All pharmacokinetic parameters were calculated using the individual plasma concentration data. The first order rate constant, λ  z, associated with the terminal log-linear portion of the curve was estimated via linear regression of the log plasma concentration versus time curve. The terminal plasma half-life (T  1/2) was calculated using the natural logarithm (2)/λ  z.