Structural identification of trace level enol tautomer impurity by on‐line hydrogen/deuterium exchange HR‐LC/MS in a LTQ‐Orbitrap hybrid mass spectrometer

Abstract

Structural identification of trace level enol tautomer impurity by on-line hydrogen/deuterium exchange HR-LC/MS in a LTQ-Orbitrap hybrid mass spectrometerWith advancements in ionization methods and instrumentation, liquid chromatography/mass spectrometry (LC/MS) has become a powerful technology for the characterization of pharmaceuticals in drug discovery process. 1 -5 As one of the latest MS instrumentation designs, the LTQ-Orbitrap has the capability of performing highresolution LC/MS and LC/MS n experiments with high resolving power (up to 100 000), excellent mass accuracy (<3 ppm with external calibration) and large dynamic range (over 10 3 ). 6 -10 Compared to other high-resolution LC/MS instruments (LTQ-FT, FT-ICR) with similar capabilities, LTQ-Orbitrap is operated as an electrostatic ion trap without the use of superconducting magnet and offers cost-effective solutions to structural identification problems with ease of operations. Several groups have taken advantages of Orbitrap's low-ppm mass accuracy on mixture analysis involving proteins/peptides, 11 -14 and drug-substancesrelated metabolites. 15,16 Studies on structural identification of impurities in drug substances using LTQ-Orbitrap have just started to appear in the literature. 5 Another approach employed in structural characterization of pharmaceuticals is the on-line hydrogen/deuterium (H/D) exchange LC/MS experiment. 17 -22 The exchangeable hydrogen atoms are usually bound to N, O, or S atoms in functional groups such as OH-, NH-, NH 2 -and COOH-. This method provides accurate measurements of exchangeable hydrogen atoms in a molecule to assist structural elucidation. 23,24 In this report, we explore the use of on-line H/D exchange in conjunction with HR-LC/MS and LC/MS n experiments using a LTQ-Orbitrap mass spectrometer to identify a trace level enol tautomer impurity in a potent Hepatitis C virus (HCV) protease inhibitor (structure shown in Scheme 1). 25 -27 This compound is currently undergoing clinical trials for the treatment of HCV infection.

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;...…”

Advances in Bioanalytical LC–MS Using the Orbitrap™ Mass Analyzer

“…In combination with hydrogen/deuterium-exchange chromatography the measurement of isotope abundancies can be used for structural identification of trace levels of impurities [28].…”

Identification of Pharmaceutical Impurities in Formulated Dosage Forms

“…It offers cost-effective solutions to structural elucidation in protein and peptide, [97][98][99][100][101] drug metabolites, [102][103][104][105] and drug substances. [106][107][108] However, there have been few reports so far on the use of this LTQ-Orbitrap to identify drug degradation products in formulated dosage forms.…”

“…It generates valuable information allowing the complete identification of unknown compounds in some studies such as metformin oxidation end products, 109 process impurities, 110 and proteins/peptides. [111][112][113] In some cases in which neither LC/MS n nor TOF-MS can provide a definite structure, H/D exchange experiments may generate a clear answer to the question. The application of H/D exchangeable LC/MS in some case studies has been described in a recent review paper 114 : differentiation between ketone and methylation (MW + 14 Da) (the difference can also be differentiated by a high-resolution MS), differentiation between N-oxide formation and C-hydroxylation (MW + 16 Da), differentiation between desfluoro and dehydration (MW -18 Da), differentiation between alcoholysis and Michael addition (MW + 60 Da), and so on.…”