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

•We developed a LC/MS/MS method for intracellular amount of digoxin and ouabain.•The validation results showed that an accurate, reproducible and selective assay was achieved.•The developed method was applied to the transport studies using Transwell chambers.

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 (100 mg/1 mL) 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 (2.0 mm × 150 mm, 5 μm). This method covered a linear range of 0.5–1000 ng/mL of concentrations in medium and 0.5–1000 ng 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 16 min. Our method was successfully applied to the transport experiments of digoxin and ouabain by HK-2 cell monolayers.