title: Development of a high-throughput online solid-phase extraction/tandem mass spectrometry method for cytochrome P450 inhibition screening.
abstract: A high-throughput online solid-phase extraction/tandem mass spectrometry (online SPE/MS/MS) system has been developed to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of 8 test concentrations and against a panel of 6 cytochrome P450 (CYP) enzymes, 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. Previously, a postassay pooling and a 2-min gradient LC/MS/MS method had been reported to increase sample throughput, allowing for a 96-well plate of samples to be analyzed in under 4 h. The development of a new online SPE/MS/MS system has reduced the analysis time to less than 15 min per 96-well plate, translating to a 15-fold time savings compared to the 2-min LC/MS/MS method. Sampling precision without internal standard correction ranged from 3.1% to 5.6% relative standard deviation, and the carryover was determined to be between 1.0% and 4.1%. One hundred twenty in-house compounds were assayed and pooled for analyses using both the online SPE/MS/MS and LC/MS/MS, and the correlation coefficients ranged from 0.89 to 1.13, when comparing the IC(50) results obtained from the 2 approaches for each of the CYP enzymes.
authors.affiliation: Takeda San Diego, Inc., San Diego, California 92121, USA. kheng.lim@takedasd.com
authors.forename: Kheng B
authors.initials: KB
authors.lastname: Lim
authors.forename: Can C
authors.initials: CC
authors.lastname: Ozbal
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Kassel
mesh_terms: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D020128:Inhibitory Concentration 50; D009862:Online Systems; D012015:Reference Standards; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors