Publications (14) View all
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Article: Detection and characterization of ticlopidine conjugates in rat bile using high-resolution mass spectrometry: applications of various data acquisition and processing tools.
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ABSTRACT: Ticlopidine, an antiplatelet drug, undergoes extensive oxidative metabolism to form S-oxide, N-oxide, hydroxylated and dealkylated metabolites. However, metabolism of ticlopidine via conjugation has not been thoroughly investigated. In this study, multiple data acquisition and processing tools were applied to the detection and characterization of ticlopidine conjugates in rat bile. Accurate full-scan mass spectrometry (MS) and collision-induced dissociation (CID) MS/MS data sets were recorded using isotope pattern-dependent acquisition on an LTQ/Orbitrap system. In addition, mass spectral data from online H/D exchanging and high collision energy dissociation (HCD) were recorded. Data processes were carried out using extracted ion chromatography (EIC), mass defect filter (MDF) and isotope pattern filter (IPF). The total ion chromatogram displayed a few major conjugated metabolites and many endogenous components. Profiles from EIC and IPF processes exhibited multiple conjugates with no or minimal false positives. However, ticlopidine conjugates that were not predictable or lost a chorine atom were not found by EIC or IPF, respectively. MDF was able to detect almost all of ticlopidine conjugates although it led to a few more false positives. In addition to CID spectra, data from HCD, H/D exchanging experiments and isotope pattern simulation facilitated structural characterization of unknown conjugates. Consequently, 20 significant ticlopidine conjugates, including glucuronide, glutathione, cysteinylglycine, cysteine and N-acetylcysteine conjugates, were identified in rat bile, a majority of which are associated with bioactivation and not previously reported. This study demonstrates the utility and limitation of various high-resolution MS-based data acquisition and processing techniques in detection and characterization of conjugated metabolites. Copyright © 2013 John Wiley & Sons, Ltd.Biological Mass Spectrometry 03/2013; 48(3):413-422. · 3.41 Impact Factor -
Article: Strategy and its implications of protein bioanalysis utilizing high-resolution mass spectrometric detection of intact protein.
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ABSTRACT: Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.Analytical Chemistry 12/2011; 83(23):8937-44. · 5.86 Impact Factor -
Article: Development and evaluation of a multiple-plate fraction collector for sample processing: application to radioprofiling in drug metabolism studies.
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ABSTRACT: Microplate scintillation counters are utilized routinely in drug metabolism laboratories for the off-line radioanalysis of fractions collected during HPLC radioprofiling. In this process, the current fraction collection technology is limited by the number of plates that can be used per injection as well as the potential for sample loss due to dripping or spraying as the fraction collector head moves from well to well or between plates. More importantly, sample throughput is limited in the conventional process, since the collection plates must be manually exchanged after each injection. The Collect PAL, an innovative multiple-plate fraction collector, was developed to address these deficiencies and improve overall sample throughput. It employs a zero-loss design and has sub-ambient temperature control. Operation of the system is completely controlled with software and up to 24 (96- or 384-well) fraction collection plates can be loaded in a completely automated run. The system may also be configured for collection into various-sized tubes or vials. At flow rates of 0.5 or 1.0 mL/min and at collection times of 10 or 15s, the system precisely delivered 83-μL fractions (within 4.1% CV) and 250-μL fractions (within 1.4% CV), respectively, of three different mobile phases into 12 mm × 32 mm vials. Similarly, at a flow rate of 1 mL/min and 10s collection times, the system precisely dispensed mobile phase containing a [(14)C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%). Triplicate analyses of metabolism test samples containing [(14)C]buspirone and its metabolites, derived from three different matrices (plasma, urine and bile), indicated that the Collect PAL produced radioprofiles that were reproducible and comparable to the current technology; the % CV for 9 selected peaks in the radioprofiles generated with the Collect PAL were within 9.3%. Radioprofiles generated by collecting into 96- and 384-well plates were qualitatively comparable; however, the peak resolution was greater in the profiles that were collected in 384-well plates due to the collection of a larger number of fractions per minute. In conclusion, this new and innovative fraction collector generated radioprofile results that were comparable to current technology and should provide a major improvement in capacity and throughput for radioprofiling studies.Journal of pharmaceutical and biomedical analysis 04/2011; 54(5):979-86. · 2.45 Impact Factor -
Article: Investigation of bioactivation of ticlopidine using linear ion trap/orbitrap mass spectrometry and an improved mass defect filtering technique.
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ABSTRACT: The bioactivation of ticlopidine, a widely used antiplatelet drug, into reactive metabolites and their subsequent covalent binding to cellular macromolecules are thought to be involved in the occurrence of idiosyncratic hepatotoxicity in patients. In the present study, GSH/stable isotope-labeled GSH was used as the trapping agent to investigate the bioactivation pathways of ticlopidine in rat liver microsomes. The samples were analyzed by high-resolution linear ion trap/Orbitrap followed by multiple mass defect filtering (MDF). In total, 17 GSH adducts were detected, and a comprehensive profile for ticlopidine bioactivation has been proposed. The results show that ticlopidine can be directly bioactivated by rat P450s, forming GSH adducts through two major bioactivation pathways, thiophene-S-oxidation and thiophene epoxidation. These adducts were also formed substantially in human liver microsomes. Moreover, ticlopidine can be metabolized via multiple pathways before giving rise to reactive intermediates. The GSH adducts derived from epoxidation of the chlorophenyl moiety of ticlopide and bioactivation of N-dealkylated metabolites are reported here for the first time. The formation of a number of ticlopidine GSH adducts from diversified metabolic pathways mediated by P450s implies a high potential for protein binding and provides a conceivable link between the high reactivity of ticlopidine after bioactivation and the ticlopidine-induced toxicity. Additionally, the current approach has the following advantages as compared to previous high-resolution LC/MS methodologies. First, novel MDF utilized doubly charged ions as filter templates to detect the GSH adducts, mainly doubly charged in the ion source, resulting in broader detection coverage. Second, multiple mass defect filter templates were for the first time applied to reveal different classes of GSH adducts. Finally, a quick check of isotopic doublets and full examination of isotope fingerprints in the accurate mass were introduced to screen out false positives and enhance the identification of low abundant GSH adducts.Chemical Research in Toxicology 03/2010; 23(5):909-17. · 3.78 Impact Factor -
Article: In vitro screening of 50 highly prescribed drugs for thiol adduct formation--comparison of potential for drug-induced toxicity and extent of adduct formation.
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ABSTRACT: Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.Chemical Research in Toxicology 04/2009; 22(4):690-8. · 3.78 Impact Factor
