Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P-glycoprotein (P-gp) inhibition screen

Rapid Commun Mass Spectrom. 2011 May 15;25(9):1231-40. doi: 10.1002/rcm.4984.

Abstract

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / antagonists & inhibitors*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Caco-2 Cells
  • Chromatography, Liquid / methods*
  • Cyclosporine / chemistry
  • Cyclosporine / pharmacology
  • Digoxin / analysis*
  • Digoxin / chemistry
  • Digoxin / metabolism
  • Drug Discovery / methods
  • Drug Discovery / standards
  • High-Throughput Screening Assays / methods*
  • Humans
  • Linear Models
  • Models, Biological
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Solid Phase Extraction
  • Tandem Mass Spectrometry / methods*
  • Tritium

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Tritium
  • Digoxin
  • Cyclosporine