Quantification and Rapid Metabolite Identification in Drug Discovery Using API Time-of-Flight LC/MS

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Abstract
Liquid chromatography/mass spectrometry (LC/MS), utilizing a time-of-flight (TOF) mass analyzer, has been evaluated and applied to problems in bioanalysis for pharmacokinetics and drug metabolism. The data obtained by TOF MS differ from those obtained using quadrupole mass spectrometer instruments in that full-scan spectra can be routinely collected with greater sensitivity and speed. Both quantitative and qualitative information, including compound concentration in rat plasma and full-scan atmospheric pressure ionization mass spectra, are concurrently obtained. This approach has been used to characterize the disposition of several drug compounds that have been simultaneously dosed to rats in a cassette format. Quantitation limits in the 5−25 ng/mL range (∼20 nM) were obtained from nominal mass chromatograms (0.5 Da resolution). A reference lock mass was used to provide accurate mass measurement to reach third decimal place accuracy in the monoisotopic molecular weight. An improvement in quantitation limits was demonstrated after using accurate mass determinations. Several possible preliminary drug metabolites were confirmed or refuted, based on accurate mass. The trend of metabolite formation and clearance was qualitatively evaluated.