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

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 (approximately 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.

A Novel P450-Catalyzed Transformation of the 2,2,6,6-Tetramethyl Piperidine Moiety to a 2,2-Dimethyl Pyrrolidine in Human Liver Microsomes: Characterization by High Resolution Quadrupole-Time-of-Flight Mass Spectrometry and 1H-NMR

“…The accurate mass measurements can then be used to derive possible elemental compositions of these ions with the aid of computer software and known structural features of the molecule. Thus, accurate mass measurements frequently are invaluable in arriving at less ambiguous mass-spectral and structural assignments (Pilard et al, 2000;Zhang et al, 2000;Bobeldijk et al, 2001;Watt et al, 2001;Lopes et al, 2002). In our studies, the measurement of accurate masses of the molecular ions of the parent compounds and metabolites were useful at unambiguously establishing that the 2,2-DMPy metabolites had lost a mass equivalent to a ϪC 3 H 6 moiety relative to the corresponding 2,2,6,6-TMPi analogs.…”

Principles and Applications of LC-MS/MS for the Quantitative Bioanalysis of Analytes in Various Biological Samples

“…The API-TOF mass spectrometry provides high-resolution analysis with a mass accuracy better than 10 ppm, and hence the possibility of the determination of the elemental compositions of metabolites and high specificity in their detection. (Zhang et al, 2000a;Zhang et al, 2000b). Furthermore, the quadrupole-TOF mass spectrometer (Q-TOF) provides high sensitivity for the determination of full-scan product ion spectra of metabolites (Zhang et al, 2000a).…”

“…(Zhang et al, 2000a;Zhang et al, 2000b). Furthermore, the quadrupole-TOF mass spectrometer (Q-TOF) provides high sensitivity for the determination of full-scan product ion spectra of metabolites (Zhang et al, 2000a). The first step in metabolite profiling for dertection of metabolitea and deferentiation of the site of biotransformation is to identify all the possible metabolites.…”

Duty cycle enhancement of an orthogonal acceleration TOF mass spectrometer using an axially-resonant excitation linear ion trap

“…A delay between the ejection timing of the non-mass-selective LIT and the push timing of the oaTOF was swept masssynchronously with the axially-resonant-excitation linear ion trap, so that ions are detected with duty cycle larger than 60% over a wide mass range from m/z 174.1 to 1922.0, which is 3 to 10 times better than conventional oaTOF. A n orthogonal-acceleration time-of-flight mass spectrometer (hereafter oaTOF) is now a powerful tool for proteome, metabolite analyses, and other application fields where high-throughput and high mass-accuracy is required [1][2][3]. In addition, many hybrid oaTOF designs for tandem mass spectrometry, incorporating either a quadrupole filter or an MS n -capable ion trap, have been developed and reported [2][3][4][5][6][7].…”