Simultaneously Assessing Concentration Changes in 17 Biochemical Pathways as a Result of Drug Dosing and Cytochrome P450 and Non-cytochrome P450-Mediated Metabolism: A Quasi-Untargeted Metabolomics LC/MS Assay

From a number of cellular metabolism enzymes, including cytochrome P450 (CYP) and non-CYP enzymes, dozens of intracellular metabolites that are structurally related to a dosed drug molecule are produced. The drug or its metabolites potentially may cause adverse effects on the normal physiological function of one or more cell components, and thus possibly inducing toxicity. A targeted liquid chromatography, mass spectrometry (LC/MS) metabolomics analysis, which allows interrogation of 17 common biochemical pathways of human plasma, is described that allows for the investigation of possible toxicity in hepatic cell culture, animal, or human fluids. The assay utilizes three liquid chromatographic modes of separation, including ion pairing, reversed-phase, and hydrophobic interaction chromatography, to achieve quantitation of 93 metabolites. Plasma samples are prepared using a simple protein precipitation step followed by drying of supernatant and reconstitution in method-specific diluents. Quantitation is performed using both positive and negative ionization electrospray mass spectrometry in the range of endogenous concentrations. Results for six lots of plasma are reported and generally agree with published data. This method can provide rapid evaluation for off-target effects and adverse toxicity events that might be observed in preclinical or clinical studies supporting various therapeutic areas.