Detection and Characterization of Metabolites in Biological Matrices Using Mass Defect Filtering of Liquid Chromatography/High Resolution Mass Spectrometry Data

Abstract

ABSTRACT:An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of high resolution liquid chromatography/mass spectrometry (LC/ MS) data for the detection and structural characterization of oxidative metabolites with mass defects similar to or significantly different from those of the parent drugs. The effectiveness of this approach was investigated using nefazodone as a model compound, which is known to undergo multiple common and uncommon oxidative reactions. Through the selective removal of all ions that fall outside of the preset filter windows, the MDF process facilitated the detection of all 14 nefazodone metabolites presented in human liver microsomes in the MDF-filtered chromatograms. The capability of the MDF approach to remove endogenous interferences from more complex biological matrices was examined by analyzing omeprazole metabolites in human plasma. The unprocessed mass chromatogram showed no distinct indication of metabolite peaks; however, after MDF processing, the metabolite peaks were easily identified in the chromatogram. Compared with precursor ion scan and neutral loss scan techniques, the MDF approach was shown to be more effective for the detection of metabolites in a complex matrix. The comprehensive metabolite detection capability of the MDF approach, together with accurate mass determination, makes high resolution LC/MS a useful tool for the screening and identification of both common and uncommon drug metabolites.The identification of drug metabolites, particularly metabolites formed through oxidation, reduction, or hydrolysis reactions, has become an integral part of the drug discovery and development process. These metabolites may have intrinsic pharmacological activity or display specific toxicity (Parkinson, 1996;Guengerich, 2000). In addition, most clinical drug-drug interactions are associated with oxidative biotransformation mediated by cytochrome P450 (Bjornsson et al., 2003). Although analytical sensitivity and the processing of data for liquid chromatography/mass spectrometry (LC/MS) have been tremendously improved in the last decade (Clarke et al., 2001;Kostiainen et al., 2003;Liu and Hop, 2005), the detection and identification of drug metabolites in complex biological matrices continue to be a challenge.Traditionally, detection of common or expected metabolites has been conducted on LC/MS data by generating extracted or reconstructed ion chromatograms corresponding to the expected protonated molecules of drug metabolites (Plumb et al., 2003). Over the last decade, product ion scanning techniques that use rule-based algorithms to generate a list of potential metabolite masses have been developed and continuously improved for rapid screening for common metabolites (Yu et al., 1999;Gangl et al., 2002;Lafaye et al., 2003). The technique employs a survey mode to search for the metabolites that are listed in the acquisition method. Both the detection of expected metabolites and the acquisition of their pr...

LC-MS-Based Metabolomics in Drug Metabolism

“…Recently, accompanying the enhanced resolution of mass spectrometry (MS), several algorithm-based methods, such as mass defect filtering techniques, have been developed in the drug metabolism field (Mortishire-Smith et al, 2005;Zhu et al, 2006). In essence, these metabolite searching methods use the numerical values of mass increase or decrease caused by known metabolic reactions or an artificial mass defect window as the screening filter to identify the metabolites formed by single or even multiple reactions.…”

Exposure Marker Discovery of Phthalates Using Mass Spectrometry

“…is is especially true when signal doublet responses are low. 51) Setting acceptable tolerance for wanted ratios is still empirical, which results in the challenges while tracing one speci c ratio. 52) An additional statistical procedure to assist the conventional isotope-labeling method was reported by Lin et al in 2010.…”

“…Various methods have been developed to perform objective searching/ ltering of accurate-mass-based LC-HRMS data to facilitate metabolite detection, such as isotope pattern lters (IPF), 42) mass defect ltering (MDF), 35,43) XCMS Online, 44) background subtraction, 45,46) and knowledgebased metabolic predictions. 47) A combination of di erent analytical technologies and data mining tools is needed to e ciently and thoroughly identify metabolites for discovering the biomarkers of exposure, toxic e ects, or diseases.…”

“…Recently, a variety of high-resolution mass spectrometers (HRMS), such as the quadrupole-time of ight (Q-TOF), the ion trap-time-of-ight (IT-TOF), the linear trap quadrupole-Fourier transform ion cyclotron resonance-mass spectrometer (LTQ-FTICR-MS), and the LTQOrbitrap have greatly increased the accuracy and selectivity of mass spectrometry. [34][35][36][37][38][39] Because of their advantages, such as fast scan rate, high mass accuracy, excellent resolution, superior sensitivity, and multiple-stage mass spectrometry scanning (MS n ), these mass spectrometers can provide HRMS data for nearly all of the compounds in complex matrix samples with a simple data acquisition method. 39,40) HRMS provides ion measurements with high resolution (>10000 at full-width at half-maximum) and accurate mass (<5 ppm deviation) capabilities.…”

Metabolic Activation of the Antibacterial Agent Triclocarban by Cytochrome P450 1A1 Yielding Glutathione Adducts

“…The data were analyzed for molecules showing the mass defect of TCC (0.0220 6 0.05 Da). The range of 60.05 Da covers most metabolic reactions, but the loss of chlorine or addition of GSH causes a greater increase in mass defect (Zhang et al, 2003;Zhu et al, 2006). Therefore, other filters were used for the mass defects of 29-OH-TCC GSH adduct -0.0411 Da (Baumann et al, 2010) and potential bis-GSH adduct -0.1093 Da, as well as DHC (-0.0119) and its GSH and bis-GSH adducts -0.0801 Da and -0.1482 Da each in a range of 60.05 Da.…”