Quantification and rapid metabolite identification in drug discovery usingAPI time-of-flight LC/MS

Liquid chromatography/mass spectrometry (LC/MS), utilizing a time-of-night (TOF) mass analyzer, has been evaluated and applied to problems in bioanaly sis for pharmacokinetics and drug metabolism. The data obtained by TOF MS d iffer 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 compou nd concentration in rat plasma and full-scan atmospheric pressure ionizatio n mass spectra, are concurrently obtained. This approach has been used to c haracterize the disposition of several drug compounds that have been simult aneously dosed to rats in a cassette format. Quantitation limits in the 5-2 5 ng/mL range (similar to 20 nM) were obtained from nominal mass chromatogr ams (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 a fter 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.