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.

A high‐speed liquid chromatography/tandem mass spectrometry platform using multiplexed multiple‐injection chromatography controlled by single software and its application in discovery ADME screening

“…The developed system and methods using multiplexed LC coupled with MIC data acquisition significantly reduced the injection‐to‐injection cycle time (23 s) than our previously reported platform. This cycle time approaches those reported for the new ADDA system and RapidFire system, with both running direct online SPE in MIC mode. In comparison, advantages of this platform reported herein include its relatively lower cost, its compatibility with various LC and MS components from major vendors, and its single point software control by Aria.…”

Coupling Laser Diode Thermal Desorption with Acoustic Sample Deposition to Improve Throughput of Mass Spectrometry–Based Screening

“…13 The RapidFire system is a fully automated front-end platform that couples high-speed online SPE with MS-based detection, and has been successfully used to support various in vitro high-throughput screening and liability assessment assays, which have been described previously. [14][15][16] By combining an ultrafast microfluidic robotic system with custom micro-SPE cartridges, the RapidFire is capable of quickly desalting samples prior to their introduction into the mass spectrometer and is compatible with existing sample extraction procedures. However, sample volume requirements (~40-50 µL) prevent further miniaturization of existing protocols to accommodate increasing throughput demands (as is typical in a lead discovery/HTS environment), and at 10 s/sample, it is not comparable to the analysis speeds of fluorescence-based plate readers.…”

A Comparison of LC-MS/MS and a Fully Integrated Autosampler/Solid-Phase Extraction System for the Analysis of Protein Binding Samples

“…The absence of a chromatographic separation allows for a significant decrease in cycle time per sample compared with traditional LC-MS/MS (e.g., 8-20 s compared with 3-5 min, respectively). RF-MS/MS and similar systems have proven to be successful with in vitro ADME assays, [3][4][5][6][7][8][9][10] but to our knowledge, there have been no peer-reviewed published reports on the application of RF-MS/MS for PPB analysis. This article explores the application of a fully integrated RF-MS/MS system to increase sample throughput, decrease the time burden on existing laboratory instrumentation, and improve turnaround time for PPB results.…”