An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection

Abstract

A new strategy using a hybrid linear ion trap/Orbitrap mass spectrometer and multiple post-acquisition data mining techniques was evaluated and applied to the detection and characterization of in vitro metabolites of indinavir. Accurate-mass, full-scan MS and MS/MS data sets were acquired with a generic data-dependent method and processed with extracted-ion chromatography (EIC), mass-defect filter (MDF), product-ion filter (PIF), and neutral-loss filter (NLF) techniques. The high-resolution EIC process was shown to be highly effective in the detection of common metabolites with predicted molecular weights. The MDF process, which searched for metabolites based on the similarity of mass defects of metabolites to those of indinavir and its core substructures, was able to find uncommon metabolites not detected by the EIC processing. The high-resolution PIF and NLF processes selectively detected metabolites that underwent fragmentation pathways similar to those of indinavir or its known metabolites. As a result, a total of 15 metabolites including two new indinavir metabolites were detected and characterized in a rat liver S9 incubation sample. Overall, these data mining techniques, which employed distinct metabolite search mechanisms, were complementary and effective in detecting both common and uncommon metabolites. In summary, the results demonstrated that this analytical strategy enables the high-throughput acquisition of accurate-mass LC/MS data sets, comprehensive search of a variety of metabolites through the post-acquisition processes, and effective structural characterization based on elemental compositions of metabolite molecules and their product ions.

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;...…”

Drug Metabolite Profiling and Identification by High-resolution Mass Spectrometry

“…First, PIF and NLF do not require predetermination of the product ion spectrum of the parent drug because the information is recorded along with the acquisition of the initial MS data set (29,34). Also, HR-PIF and HR-NLF are highly selective, so, in some cases, these data mining tools were able to find trace amounts of unexpected metabolites that were not found by MDF (34). The effectiveness of PIF and NLF depends on the availability of high-quality MS/MS spectral data of unknown metabolites.…”

“…Recording of full-scan MS and product ion spectral data sets for profiling of components including metabolites present in a biological sample can be completed in one or a few LC/MS injections ( Table 1). In many cases, data acquisition of multiple samples by HR-MS can be performed continuously (34,38) with off-line data mining (41). HR-MS instruments with data mining techniques have the potential to greatly increase the speed, selectivity, sensitivity, accuracy, and comprehensive nature of metabolite detection and identification and to fundamentally change the way that many drug metabolism and disposition studies are conducted (26,(41)(42)(43).…”

Exposure Marker Discovery of Phthalates Using 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.…”