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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...

An automated process is described for the detailed assessment of the in vitro metabolic stability properties of drug candidates in support of pharmaceutical property profiling. Compounds are incubated with liver microsomes using a robotic liquid handler. Aliquots are taken at various time points, and the resulting samples are quantitatively analyzed by liquid chromatography-mass spectrometry utilizing ion trap mass spectrometers to determine the amount of compound remaining. From these data metabolism rates can be calculated. A high degree of automation is achieved through custom software, which is employed for instrument setup, data processing, and results reporting. The assay setup is highly configurable, allowing for any combination of up to six user-selected time points, variable substrate concentration, and microsomes or other biologically active media. The data, based on relative substrate depletion, affords an estimate of metabolic stability through the calculation of half-life (t(1/2)) and intrinsic clearance, which are used to differentiate and rank order drug leads. In general, t(1/2) is the time necessary for the metabolism, following first-order kinetics, of 50% of the initial compound. Intrinsic clearance is the proportionality constant between rate of metabolism of a compound and its concentration at the enzyme site. Described here is the setup of the assay, and data from assay test compounds are presented.

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