A semi-automated method for the integrated evaluation of half-life and metabolic soft spots of discovery compounds.

Mary F Grubb, William G Humphreys, Jonathan L Josephs

Index: Bioanalysis 4(14) , 1747-61, (2012)

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Abstract

An integrated method that provides rates of both parent disappearance and metabolite formation was developed.Buspirone, mirtazapine and verapamil were used as model compounds in developing the method. Incubations were carried out on a robotic platform. Qualitative analysis of metabolites in 30 µM samples was conducted by data-dependent HPLC-MS/MS on a high-resolution instrument. Quantitative analysis of the parent compound and metabolites in 0.5 µM samples was conducted by full-scan MS(2) with product ion extraction using an ion trap mass spectrometer. Data generated for the compounds included half-life and intrinsic clearance of the parent molecule, characterization of metabolites and relative rates of metabolite formation. A correction factor was used to convert MS responses of metabolites in 0.5 µM samples to UV areas in order to compare relative metabolite concentrations.The approach allows for the investigation of a set of six compounds simultaneously, with a turnaround time of 1 week or less.