High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs during In Vitro Metabolite Profiling

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Drug metabolism and disposition: the biological fate of chemicals (Impact Factor: 3.25). 07/2012; 40(10):1966-75. DOI: 10.1124/dmd.112.047027
Source: PubMed


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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    • "The first recording allows the m/z ratio of the pseudo-molecular ion [M–H]− to be obtained, while the second recording displays the m/z ratio of the daughter ions. Thus, UHPLC–Q-TOF–MSE is a powerful tool to analyze complex mixtures, especially biological fluids, and enables to obtain the elucidation of elemental composition and fragmentation information [26]. Herein, the composition of mouse-lemur plasma was analyzed with this new approach, which permitted the structures of several endogenous components to be confirmed in one analytical run by the simultaneous acquisition of exact masses of precursor and daughter ions. "
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    ABSTRACT: The grey mouse lemur (Microcebus murinus) is a non-human primate used to study the ageing process. Resveratrol is a polyphenol that may increase lifespan by delaying age-associated pathologies. However, no information about resveratrol absorption and metabolism is available for this primate. Resveratrol and its metabolites were qualitatively and quantitatively analyzed in male mouse-lemur plasma (after 200 of oral resveratrol) by ultra-high performance liquid chromatography (UHPLC), coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer used in full-scan mode. Data analyses showed, in MSE mode, an ion common to resveratrol and all its metabolites: m/z 227.072, and an ion common to dihydro-resveratrol metabolites: m/z 229.08. A semi-targeted study enabled us to identify six hydrophilic resveratrol metabolites (one diglucurono-conjugated, two monoglucurono-conjugated, one monosulfo-conjugated and two both sulfo- and glucurono-conjugated derivatives) and three hydrophilic metabolites of dihydro-resveratrol (one monoglucurono-conjugated, one monosulfo-conjugated, and one both sulfo- and glucurono-conjugated derivatives). The presence of such metabolites has been already detected in the mouse, rat, pig, and humans. Free resveratrol was measurable for several hours in mouse-lemur plasma, and its two main metabolites were trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate. Free dihydro-resveratrol was not measurable whatever the time of plasma collection, while its hydrophilic metabolites were present at 24 h after intake. These data will help us interpret the effect of resveratrol in mouse lemurs and provide further information on the inter-species characteristics of resveratrol metabolism.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "Our preliminary results indicate that semicarbazide could decrease the formation of 49-N-methyl piperazine CN adducts in the HLM incubation of I with KCN and [ 13 C]-labeled potassium carbonate, further supporting the hypothesis of aldehyde formation by piperazine 49-N-carbamic acid reduction. Iminium ions from piperazine a-carbon oxidation (pathway 1) have been considered a bioactivation process and those from pathway 2 as experimental artifact resulting from the potential interaction with formaldehyde in the incubation buffer (Gorrod and Sai, 1997; Li et al., 2006; Rousu and Tolonen, 2011; Barbara et al., 2012). However, the biologic and toxicological implications of iminium ions via pathway 2 require further investigation for the following reasons. "
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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.
    Preview · Article · Feb 2013 · Drug metabolism and disposition: the biological fate of chemicals
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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.
    No preview · Article · Jun 2013 · International Journal for Ion Mobility Spectrometry
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