High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs during In Vitro Metabolite Profiling.
ABSTRACT In vitro metabolite profiling and characterization experiments are widely employed in early drug development to support safety studies. Samples from incubations of investigational drugs with liver microsomes or hepatocytes are commonly analyzed by liquid chromatography/mass spectrometry for detection and structural elucidation of metabolites. Advanced mass spectrometers with accurate mass capabilities are becoming increasingly popular for characterization of drugs and metabolites, spurring changes in the routine workflows applied. In the present study, using a generic full-scan high-resolution data acquisition approach with a time-of-flight mass spectrometer combined with postacquisition data mining, we detected and characterized metabonates (false metabolites) in microsomal incubations of several alkylamine drugs. If a targeted approach to mass spectrometric detection (without full-scan acquisition and appropriate data mining) were employed, the metabonates may not have been detected, hence their formation underappreciated. In the absence of accurate mass data, the metabonate formation would have been incorrectly characterized because the detected metabonates manifested as direct cyanide-trapped conjugates or as cyanide-trapped metabolites formed from the parent drugs by the addition of 14 Da, the mass shift commonly associated with oxidation to yield a carbonyl. This study demonstrates that high-resolution mass spectrometry and the associated workflow is very useful for the detection and characterization of unpredicted sample components and that accurate mass data were critical to assignment of the correct metabonate structures. In addition, for drugs containing an alkylamine moiety, the results suggest that multiple negative controls and chemical trapping agents may be necessary to correctly interpret the results of in vitro experiments.
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ABSTRACT: In the liver microsome cyanide trapping assays piperazine containing compounds were found to form significant N-methyl piperazine cyanide (CN) adducts. Two pathways for the N-methyl piperazine CN-adduct formation were proposed. (1) The α-carbon in the N-methyl piperazine is oxidized to form a reactive iminium ion which can react with cyanide ion. (2) N-dealkylation occurs followed by condensation with formaldehyde and dehydration to produce N-methylenepiperazine iminium ion which then reacts with cyanide ion to form the N-methyl CN-adduct. The CN-adduct from the second pathway was believed an artifact or metabonate. In the present study, a group of 4'-N-alkyl piperazines and 4'-N-[(13)C]methyl labeled piperazines were used to determine which pathway was predominant. Following microsomal incubations in the presence of cyanide ions, a significant percentage of 4'-N-[(13)C]methyl group in the CN-adduct was replaced by an unlabeled natural methyl group suggesting that the second pathway was predominant. For 4'-N-alkyl piperazine, the level of 4'-N-methyl piperazine CN-adduct formation was limited by the extent of prior 4'-N-dealkylation. In a separate study, when 4'-NH-piperaziens were incubated with KCN and [(13)C]-labeled formaldehyde, 4'-N-[(13)C]methyl piperazine CN-adduct was formed without NADPH or liver microsome suggesting a direct Mannich reaction is involved. However, when [(13)C]-labeled methanol or potassium carbonate was used as the one-carbon donor, 4'-N-[(13)C]methyl piperazine CN-adduct was not detected without liver microsome or NADPH present. The biological and toxicological implications of bioactivation via the second pathway necessitate further investigation because these one-carbon donors for the formation of reactive iminium ions could be endogenous and readily available in vivo.Drug metabolism and disposition: the biological fate of chemicals 02/2013; · 3.74 Impact Factor
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ABSTRACT: Recently discovered ionization methods for use in mass spectrometry (MS), are widely applicable to biological materials, robust, and easy to automate. Among these, matrix assisted ionization vacuum (MAIV) is astonishing in that ionization of low and high-mass compounds are converted to gas-phase ions with charge states similar to electrospray ionization simply by exposing a matrix:analyte mixture to the vacuum of a mass spectrometer. Using the matrix compound, 3-nitrobenzonitrile, abundant ions are produced at room temperature without the need of high voltage or a laser. Here we discuss chemical analyses advances using MAIV combined with ion mobility spectrometry (IMS) real time separation, high resolution MS, and mass selected and non-mass selected MS/MS providing rapid analyte characterization. Drugs, their metabolites, lipids, peptides, and proteins can be ionized simultaneously from a variety of different biological matrixes such as urine, plasma, whole blood, and tissue. These complex mixtures are best characterized using a separation step, which is obtained nearly instantaneously with IMS, and together with direct ionization and MS or MS/MS provides a fast analysis method that has considerable potential for non-targeted clinical analyses.International Journal for Ion Mobility Spectrometry 06/2013; 16(2).
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ABSTRACT: Metabolism-dependent inhibition (MDI) of cytochrome P450 (CYP) enzymes has the potential to cause clinically-relevant drug-drug interactions. In the case of several alkylamine drugs, MDI of CYP involves formation of a metabolite that binds quasi-irreversibly to the ferrous heme iron to form a metabolic intermediate (MI) complex. The specific metabolites coordinately bound to ferrous iron and the pathways leading to MI complex formation are the subject of debate. We describe an approach combining heme iron oxidation with potassium ferricyanide and metabolite profiling to probe the mechanism of MI complex-based CYP3A4 inactivation by the secondary alkylamine drug lapatinib. Ten metabolites formed from lapatinib by CYP3A4-mediated heteroatom dealkylation, C-hydroxylation, N-oxygenation with or without further oxidation, or a combination thereof, were detected by accurate mass spectrometry. The abundance of one metabolite, the N-dealkylated nitroso/oxime lapatinib metabolite (M9) correlated directly with the prevalence or the disruption of the MI complex with CYP3A4. Nitroso/oxime metabolite formation from secondary alkylamines has been proposed to occur through two possible pathways: sequential N-dealkylation, N-hydroxylation and dehydrogenation (primary hydroxylamine pathway) or N-hydroxylation with dehydrogenation to yield a nitrone followed by N-dealkylation (secondary hydroxylamine pathway). All intermediates for the secondary hydroxylamine pathway were detected but the primary N-hydroxylamine intermediate of the primary hydroxylamine pathway was not. Our findings support the mechanism of lapatinib CYP3A4 inactivation as MI complex formation with the nitroso metabolite formed through the secondary hydroxylamine and nitrone pathway, rather than by N-dealkylation to the primary amine followed by N-hydroxylation and dehydrogenation as is usually assumed.Drug metabolism and disposition: the biological fate of chemicals 02/2013; 41(5). · 3.74 Impact Factor