Pharmacokinetics and metabolism of H3B-6545, a selective estrogen receptor covalent antagonist, in dog plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry

The present study aimed at developing a simple and sensitive LC-MS/MS assay for the determination of H3B-6545 in dog plasma. The plasma samples were processed with acetonitrile and subsequently separated on a ZORBAX SB-C₁₈ column (50 mm × 4.6 mm, 5 μm), using 0.1% formic acid in water and acetonitrile as mobile phase, at a flow rate of 0.4 mL/min. Mass detection was obtained using selected reaction monitoring mode with precursor-to-product ion transitions at m/z 568.2→155.2 for H3B-6545 and m/z 304.1→161.1 for IS. The assay showed excellent linearity over the concentration range of 0.1-1000 ng/mL, with correlation coefficient >0.9987. The assay was demonstrated to be precise, accurate, and free of matrix effect. The sample preparation procedure offered a high extraction recovery (> 88.67%). H3B-6545 was stable in dog plasma after storage at different conditions. The newly established assay was further applied to the pharmacokinetic study of H3B-6545 in dog plasma after intravenous and oral administration, and the results revealed that H3B-6545 showed dose-independent pharmacokinetic characteristics over the dose range of 1-10 mg/kg with oral bioavailability being ˜50%. Additionally, the metabolites present in dog plasma were identified by LC-ESI-Q-Exactive-Orbitrap-MS and their identities were characterized by retention times, accurate masses and fragment ions. The metabolic pathways referred to N-demethylation, N-dealkylation, deamination, aldehyde oxidation, aldehyde reduction and formation of amide.