title: Quantification of intracellular and extracellular digoxin and ouabain by liquid chromatography/electrospray ionization tandem mass spectrometry.
abstract: A liquid chromatography/tandem mass spectrometry method for the determination of intracellular accumulation in addition to transcellular transport of digoxin and ouabain in renal epithelial HK-2 cells was developed. The solid-phase extraction Bond Elut(®) C18 (100mg/1mL) cartridge was used for the extraction of digoxin and ouabain from extracellular (medium) and intracellular (cell lysate) matrices. Chromatographic separation was performed on a CAPCELL PAK C18 MGII column (2.0mm×150mm, 5μm). This method covered a linear range of 0.5-1000ng/mL of concentrations in medium and 0.5-1000ng of concentrations in cell lysate for digoxin and ouabain. The intra-day precision and inter-day precision of analysis were less than 11.9%, and the accuracy was within ±11.6%. The total run time was 16min. Our method was successfully applied to the transport experiments of digoxin and ouabain by HK-2 cell monolayers.
authors.affiliation: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan. Electronic address: yamaguchi@hosp.tohoku.ac.jp.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Yamaguchi
authors.affiliation: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Miyamori
authors.affiliation: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Sato
authors.affiliation: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Ogura
authors.affiliation: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kobayashi
authors.affiliation: Department of Pharmacy, Hokkaido University Hospital, Sapporo, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.
authors.forename: Nariyasu
authors.initials: N
authors.lastname: Mano
authors.affiliation: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; Department of Pharmacy, Hokkaido University Hospital, Sapporo, Japan. Electronic address: ken-i@pharm.hokudai.ac.jp.
authors.forename: Ken
authors.initials: K
authors.lastname: Iseki
mesh_terms: D002460:Cell Line; D002853:Chromatography, Liquid; D004077:Digoxin / Q000032:analysis*; D006801:Humans; D057230:Limit of Detection; D010042:Ouabain / Q000032:analysis*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010042:Ouabain; D004077:Digoxin
keywords: Digoxin; Extracellular measurement; HK-2 cells; Intracellular measurement; LC/MS/MS; Ouabain