title: Simultaneous determination of YM-64227, a phosphodiesterase type 4 inhibitor, and its five metabolites in dog plasma by high-performance liquid chromatography with fluorescence detection.
abstract: We developed and validated a reversed-phase high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of YM-64227 [4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one], a novel and selective phosphodiesterase type 4 inhibitor, and its fi ve hydroxylated metabolites in dog plasma. The plasma samples were extracted with tert-butyl methyl ether under alkali conditions. The analytes were well separated on a phenyl ethyl column (5 microm, 250 x 4.6 mm i.d.), opreating at 40 degrees C and using an acetonitrile-acetic acid gradient at a fl ow rate of 1.0 mL/min. The fluorescence signal was monitored at an excitation and emission wavelength of 330 and 400 nm, respectively. No interfering peak was observed at the retention time of YM-64227, its metabolites or the internal standard. The validated quantitation range of the method was 0.4-200 ng/mL for all analytes using 0.5 mL of the plasma sample. The recovery of analytes in the extraction process was more than 65.5%. The intra- and inter-assay precision was less than 5.1 and 12.6%, respectively, and the intra- and inter-assay accuracy ranged from -8.1 to 11.8% and -8.0 to 9.9%, respectively. Using this assay, the plasma concentration of YM-64227 and metabolites can be determined after the oral administration of YM-64227 to beagle dogs.
authors.affiliation: Drug Metabolism Laboratories, Yamanouchi Pharmaceutical Co. Ltd, Tokyo, Japan. tenmizu.daisuke@yamanouchi.co.jp
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tenmizu
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Fukunaga
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Noguchi
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Kamimura
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors*; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003510:Cyclohexanes / Q000097:blood*; D004285:Dogs; D010726:Phosphodiesterase Inhibitors / Q000097:blood*; D011743:Pyrimidines / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: C499820:4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one; D003510:Cyclohexanes; D010726:Phosphodiesterase Inhibitors; D011743:Pyrimidines; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4