title: Approach to improve compound recovery in a high-throughput Caco-2 permeability assay supported by liquid chromatography-tandem mass spectrometry.
abstract: The Caco-2 cell culture system is widely employed as an in vitro model for prediction of intestinal absorption of test compounds in early drug discovery. Poor recovery is a commonly encountered issue in Caco-2 assay, which can lead to difficulty in data interpretation and underestimation of the apparent permeability of affected compounds. In this study, we systematically investigated the potential sources of compound loss in our automated, high-throughput Caco-2 assay, sample storage, and analysis processes, and as a result found the nonspecific binding to various plastic surfaces to be the major cause of poor compound recovery. To minimize the nonspecific binding, we implemented a simple and practical approach in our assay automation by preloading collection plates with organic solvent containing internal standard prior to transferring incubations samples. The implementation of this new method has been shown to significantly increase recovery in many compounds previously identified as having poor recovery in the Caco-2 permeability assay. With improved recovery, permeability results were obtained for many compounds that were previously not detected in the basolateral samples. In addition to recovery improvement, this new approach also simplified sample preparation for liquid chromatography-tandem mass spectrometric analysis and therefore achieved time and cost savings for the bioanalyst.
authors.affiliation: Synthesis and Analytical Technology Team, Bristol-Myers Squibb, Wallingford, Connecticut 06492, USA. xianmei.cai@bms.com
authors.forename: Xianmei
authors.initials: X
authors.lastname: Cai
authors.forename: Aaron
authors.initials: A
authors.lastname: Walker
authors.forename: Charles
authors.initials: C
authors.lastname: Cheng
authors.forename: Anthony
authors.initials: A
authors.lastname: Paiva
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Janet
authors.initials: J
authors.lastname: Kolb
authors.forename: John
authors.initials: J
authors.lastname: Herbst
authors.forename: Wilson
authors.initials: W
authors.lastname: Shou
authors.forename: Harold
authors.initials: H
authors.lastname: Weller
mesh_terms: D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D002853:Chromatography, Liquid / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007408:Intestinal Absorption; D010599:Pharmacokinetics; D053719:Tandem Mass Spectrometry / Q000379:methods*