Quantitation of Small Molecules with Simultaneous Characterization of Metabolites using High Resolution Mass Spectrometry to Accelerate Drug Discovery

摘要

Using an Orbitrap high resolution mass spectrometer in the full scan mode, we were able to quantitate reserpine over a dynamic range of 5-5000 ng/mL while simultaneously creating metabolite profiles as a function of time. Accurate mass data were obtained over a mass range of 300-1000 daltons at a resolution of 30,000. The reserpine LLOQ of 5 ng/mL was achieved with good signal-to-noise ratio and peak shape. The calibration curves showed goodness of fit (r~(2) velence 0.99) and acceptable accuracy and precision for calibration curve standards as well as three levels of quality controls. Reserpine was quantitated at m/z 609.2807 with a mass tolerance of 5 ppm. The unknown concentration data for reserpine obtained with the Orbitrap correlated well with the data from a TSQ Quantum Ultra in the MRM mode. In addition, profiles for two metabolites identified from the literature were monitored at m/z 415.2227 and 401.2071 and were confirmed as methyl reserpate and reserpic acid, respectively. Multiple Mass Defect Filtering (mMDF) of the full scan data was used to identify four previously unreported metabolites. The use of accurate mass and mMDFs eliminated the need to establish a priori biotransformation tables to monitor metabolites, which would have otherwise been needed using a linear ion trap mass spectrometer.