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