Author:

Abstract:

Rapid screening for gastro-intestinal permeation in early drug discovery can provide important information useful to select compounds containing desirable bioavailability properties. As a fast and cost efficient screening tool, the Parallel Artificial Membrane Permeability Assay (PAMPA) has been introduced at J&JPRD to address absorption potential. It measures permeability through an immobilized lipid membrane and can be automated, allowing many compounds to be processed in parallel with high efficiencies. With the rapid development of combinatorial chemistry and increasing amount of compounds with poor solubility properties, analysis of samples from PAMPA studies has become the ‘bottleneck’ of this drug screening process. High throughput quantification has become an essential aspect of the drug discovery process. This presentation describes the development and performance evaluation of a novel generic Acquity UPLC/MS/MS method for high throughput quantification of samples generated by PAMPA. The novel UPLC/MS/MS method consists of two stages. In a first stage, MRM-transitions of all compounds were determined by Quanoptimize, a Masslynx algorithm that automatically optimizes cone voltage and collision energy while running a short isocratic method and performing loop injections. With this automatic optimization process each compound specific MRM method was created in 3 minutes and reproducible results were obtained. In a second stage, full loop (2 μl) injections of PAMPA samples were performed and compounds were separated on an Acquity UPLC BEH C18 column (50 mm x 2.1 mm). Separation was achieved with a generic UPLC method with a total run time of 1.5 minutes and the following gradient conditions: 0 min 35% solvent B, 1.0 min 100% solvent B, 1.4 min 100% solvent B, 1.42 min 35% solvent B and 1.5 min 35% solvent B (solvent A = 0.1% formic acid in water, solvent B = 0.1% formic acid in acetonitrile). Compounds were detected with a Waters Micromass Quattro Premier mass spectrometer operating in positive electrospray ionization using the compound specific MRM methods. The generic UPLC/MS/MS method showed good sensitivity and reproducibility for the evaluated compounds as well as good linearity over a large concentration range. The novel generic Acquity UPLC/MS/MS method with automatic optimization resulted in an analysis time of 1.5 min/sample and was shown to be suitable for > 97% of the tested J&J compounds. Using the Acquity UPLC technology combined with the automated MS/MS analysis, a 4-fold increase of throughput was obtained compared to the traditional LC/MS system as well as a significant increase in sensitivity. It was shown that UPLC/MS/MS could be a useful tool for the analysis of samples from permeability ranking experiments. Because of the improved throughput and sensitivity, all PAMPA samples are now routinely analyzed using the Acquity UPLC/MS/MS technology and efforts are on going to decrease run time and to analyze pooled samples.