A rapid and efficient analytical method for the quantification of a novel anticholinergic compound, R‐phencynonate, by stable isotope‐dilution LC–MS/MS and its application to bioavailability and dose proportionality studies

A rapid, specific, and high-throughput stable isotope-dilution LC-MS/MS method was developed and validated with high sensitivity for the quantification of R-phencynonate (a eutomer of phencynonate racemate) in rat and dog plasma. Plasma samples were deproteinized using acetonitrile and then separated on a C8 column with an isocratic mobile phase containing acetonitrile-water-formic acid mixture (60:40:0.1, v/v/v) at a flow rate of 0.2 mL/min. Each sample had a total run time of 3 min. Quantification was performed using triple quadrupole mass spectrometry in selected reaction monitoring mode with positive electrospray ionization. The method was shown to be highly linear (r2 > 0.99) and have a wide dynamic range (0.1 − 100 ng/mL) with favorable accuracy and precision. No matrix effects were observed. The detailed pharmacokinetic profiles of R-phencynonate at therapeutic doses in rats and dogs were characterized by rapid oral absorption, quick clearance, high volume of distribution, and poor absolute bioavailability. R-phencynonate lacked dose proportionality over the oral dose range, based on the power model. However, the area under concentration–time curve and the maximum plasma concentration increased linearly in a dose-dependent manner in both animal models. The absolute bioavailability of R-phencynonate was 16.6 ± 2.75% and 4.78 ± 1.26% in dogs and rats, respectively.