A sensitive and specific HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of isoforskolin in beagle dogs

A sensitive and specific high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) method has been developed and validated for the determination of isoforskolin in canine plasma. Liquid–liquid extraction was used to extract isoforskolin and the internal standard (I.S.) eplerenone from canine plasma. The chromatographic separation was performed on an Agela Venusil XBP Phenyl column with an isocratic mobile phase consisting of methanol–2 mM ammonium acetate–formic acid (62:38:0.1, v/v/v), pumped at 0.35 mL/min. Isoforskolin and I.S. were detected at m/z 433.4 → 373.3 and m/z 415.3 → 163.5 in positive ion and multiple reaction monitoring (MRM) mode, respectively. The standard curves were linear over the concentration range of 0.1–200 ng/mL (r > 0.99). The intra- and inter-batch accuracy values for isoforskolin at four concentrations were 90.2–108.3% and 97.8–106.6%, respectively. The RSDs were less than 6.0%. The mean extraction recoveries of isoforskolin and I.S. were 97.0 and 88.4%, respectively. The method was successfully applied to the pharmacokinetic study after an intravenous administration of isoforskolin in beagle dogs.