title: High-throughput microsomal stability assay for screening new chemical entities in drug discovery.
abstract: In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities.
authors.affiliation: Department of DMPK, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Merck Sharp and Dohme Research Laboratories, Roma, Italy. massimiliano_fonsi@merck.com
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Fonsi
authors.forename: Maria V
authors.initials: MV
authors.lastname: Orsale
authors.forename: Edith
authors.initials: E
authors.lastname: Monteagudo
mesh_terms: D000818:Animals; D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods; D016503:Drug Delivery Systems; D055808:Drug Discovery; D004867:Equipment Design; D013058:Mass Spectrometry / Q000379:methods; D008862:Microsomes, Liver / Q000378:metabolism*; D009249:NADP / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results; D012371:Robotics; D013997:Time Factors
chemical_list: D009249:NADP