Metabolite identification by data‐dependent accurate mass spectrometric analysis at resolving power of 60 000 in external calibration mode using an LTQ/Orbitrap

Abstract

Performance evaluation of accurate mass measurement by the LTQ/Orbitrap, at a resolving power of 60,000 and in external calibration mode, indicated that the Orbitrap is capable of providing high mass accuracy of <2 ppm for over 24 h post-calibration. This, together with limited trade-off between sensitivity and resolving power plus a wide dynamic range for mass accuracy, suggested that the LTQ/Orbitrap is an ideal analytical tool for structural elucidation of metabolites. The application of the LTQ/Orbitrap to identification of human liver microsomal metabolites of carvedilol was evaluated, using parent mass list triggered data-dependent multiple-stage accurate mass analysis, at a resolving power of 60,000 in external calibration mode. A metabolite identification workflow was developed to utilize chemical formulas from high-resolution accurate mass measurements to confirm structures of product ions of a drug proposed by Mass Frontier, illustrated by identification of structures used to establish lineage of product ions of carvedilol, which later served as a template for identification of its metabolites. A total of 58 in vitro metabolites of carvedilol were detected using 5-ppm mass tolerance filters for theoretical m/z of protonated molecules of predicted metabolites in addition to product ions and neutral mass losses diagnostic of carvedilol. The chemical formulas with unsaturation numbers calculated from the accurate m/z of precursor and product ions can be used to assign, with a high degree of confidence, the structures of metabolites and the sites of metabolism. The mass accuracies obtained for all full scan MS and MSn spectra were <2 ppm. The majority of the metabolites identified agreed with those previously reported except for those that have not been reported before. For example, several glutathione conjugates of carvedilol were reported for the first time, which may explain the reported hepatotoxicity during clinical trials and recent clinical use.

Orbitrap mass spectrometry: Instrumentation, ion motion and applications

“…The LTQOrbitrap has also been used to analyze intact small (3-10 kDa) (Bredehöft, Schänzer, & Thevis, 2008;Thevis et al, 2006a;Thevis, Thomas, & Schänzer, 2008b), medium (10-25 kDa) (Macek et al, 2006;Waanders, Hanke, & Mann, 2007) and large ($150 kDa) (Zhang & Shah, 2007) proteins where the MS n capability of the LIT coupled with the high performance features of the orbitrap can facilitate unambiguous determination of the charge states of fragment ions, as well as identification of PTMs by database searching. The high performance attributes of this hybrid mass spectrometer have also found applications in environmental chemistry (Barceló & Petrovic, 2007;Krauss & Hollender, 2008), drug and metabolite analysis (Breitling, Pitt, & Barrett, 2006;Cuyckens et al, 2008;Ding et al, 2007;Karu et al, 2007;Li et al, 2007a;Lim et al, 2007Lim et al, , 2008Madalinski et al, 2008;Nielen et al, 2007;Peterman et al, 2006;Ruan et al, 2008;Thevis et al, 2005;Thevis, Kamber, & Schänzer, 2006b;Thevis, Krug, & Schänzer, 2006c;Thevis et al, 2006dThevis et al, ,e, 2007aThevis et al, , 2008aZhu et al, 2007), lipidomics (Davis et al, 2008;Ejsing et al, 2006;Schwudke et al, 2007;Silipo et al, 2008), small-molecule (Chen et al, 2007b) and reaction product (Jamin et al, 2007) identification, as well as molecular structure characterization using hydrogen/deuterium exchange measurements (Chen et al, 2007a;...…”

Accurate mass error correction in liquid chromatography time-of-flight mass spectrometry based metabolomics

“…Technical advances in mass spectrometry, resulting in increased mass resolution and accuracy, enable the (putative) identification of metabolites simply by their accurate mass determinations (Aharoni et al 2002;Lim et al 2007;Murch et al 2004;Thurman et al 2005). For instance, Fourier transform ion cyclotron mass spectrometry (cyclotron FTMS) and Orbitrap FTMS (Olsen et al 2005) typically analyse the masses of ions with an ultra high accuracy of less than 1 ppm deviation from the calculated masses.…”

Assessment of the Occurrence and Potential Risks of Pharmaceuticals and their Metabolites in Fish and Water Using Liquid Chromatography Mass Spectrometry

“…In recent years, high resolution mass spectrometry including time of flight (TOF) and Orbitrap has become a powerful tool at identifying "unknown" metabolites in both metabolomics and environmental studies (Lim et al 2007;Ferrer and Thurman 2010;Calza et al 2012;Thurman and Ferrer 2012). Operated at a resolving power > 40,000, mass accuracy < 5 ppm is normally achievable on modern TOF instruments allowing positive identification based on accurate mass measurements.…”