title: Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P-glycoprotein (P-gp) inhibition screen.
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.
authors.affiliation: Applied Biotechnology, Bristol-Myers Squibb, Wallingford, CT 06492, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Wagner
authors.forename: Janet M
authors.initials: JM
authors.lastname: Kolb
authors.forename: Can C
authors.initials: CC
authors.lastname: Ozbal
authors.forename: John J
authors.initials: JJ
authors.lastname: Herbst
authors.forename: Timothy V
authors.initials: TV
authors.lastname: Olah
authors.forename: Harold N
authors.initials: HN
authors.lastname: Weller
authors.forename: Tatyana A
authors.initials: TA
authors.lastname: Zvyaga
authors.forename: Wilson Z
authors.initials: WZ
authors.lastname: Shou
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018938:Caco-2 Cells; D002853:Chromatography, Liquid / Q000379:methods*; D016572:Cyclosporine / Q000737:chemistry / Q000494:pharmacology; D004077:Digoxin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods / Q000592:standards; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D016014:Linear Models; D008954:Models, Biological; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014316:Tritium
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014316:Tritium; D004077:Digoxin; D016572:Cyclosporine