Quantification of intracellular and extracellular digoxin and ouabain by liquid chromatography/electrospray ionization tandem mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Dec 1:972:73-80. doi: 10.1016/j.jchromb.2014.09.043. Epub 2014 Oct 5.

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.

Keywords: Digoxin; Extracellular measurement; HK-2 cells; Intracellular measurement; LC/MS/MS; Ouabain.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Cell Line
  • Chromatography, Liquid
  • Digoxin / analysis*
  • Humans
  • Limit of Detection
  • Ouabain / analysis*
  • Reproducibility of Results
  • Solid Phase Extraction
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Tandem Mass Spectrometry / methods*

Substances

  • Ouabain
  • Digoxin