Application of Mass Spectrometry for Metabolite Identification

Abstract

Metabolism studies play a pivotal role in drug discovery and development. Characterization of metabolic "hot-spots" as well as reactive and pharmacologically active metabolites is critical to designing new drug candidates with improved metabolic stability, toxicological profile and efficacy. Metabolite identification in the preclinical species used for safety evaluation is required in order to determine whether human metabolites have been adequately tested during non-clinical safety assessment. From an instrumental standpoint, high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) dominates all analytical tools used for metabolite identification. The general strategies employed for metabolite identification in both drug discovery and drug development settings together with sample preparation techniques are reviewed herein. These include a discussion of the various ionization methods, mass analyzers, and tandem mass spectrometry (MS/MS) techniques that are used for structural characterization in a modern drug metabolism laboratory. Mass spectrometry-based techniques, such as stable isotope labeling, on-line H/D exchange, accurate mass measurement to enhance metabolite identification and recent improvements in data acquisition and processing for accelerating metabolite identification are also described. Rounding out this review, we offer additional thoughts about the potential of alternative and less frequently used techniques such as LC-NMR/MS, CRIMS and ICPMS.

Differentiation of Regioisomeric Aromatic Ketocarboxylic Acids by Positive Mode Atmospheric Pressure Chemical Ionization Collision-Activated Dissociation Tandem Mass Spectrometry in a Linear Quadrupole Ion Trap Mass Spectrometer

“…With the onset of many new ionization methods, such as electrospray ionization (ESI), atmospheric pressure photoionization (APPI), and atmospheric pressure chemical ionization (APCI), MS has become a valuable tool for mixture analyses, especially when combined with high-performance liquid chromatography (HPLC) [10,11]. However, this approach often only allows for the determination of molecular weights and elemental compositions (for high resolution mass spectrometers) of unknown analytes.…”

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…A short overview of analytical strategies for identification of metabolites will be provided. More information regarding metabolite identification can be found in following review articles [7,[23][24][25][26][27]. The selection of suitable LC-MS instrumentation is needed for qualitative evaluation of metabolites.…”

“…proteins). Despite the numerous benefits of ESI, it suffers from a shortcoming in that it is susceptible to ion suppression effects from high concentrations of buffer, salts and other endogenous compounds in matrix solutions [23].…”

“…APCI technique is less prone to ion suppression and provides a wider dynamic detection range than ESI due to ionization that occurs mainly in gas phase. Also, typically higher flow rate is used with APCI [1-2 mL/min) then that in conventional ESI (0.1-0.5 mL/min) [23].…”

Future of Liquid Chromatography–Mass Spectrometry in Metabolic Profiling and Metabolomic Studies for Biomarker Discovery

“…These initial applications of LC-MS together with subsequent work in metabolic profiling and metabolomics studies initially raised the hope that this particular analytical platform would be of great utility in the identification of novel biomarkers of disease, particularly through the development of LC-MS-based metabolite libraries. However, the variety of soft ionization techniques [electrospray ionization (ESI), atmospheric chemical ionization (APCI), atmospheric photoionization (APPI); reviewed in [98]] together with the additional variety of LC stationary phases and mobile phases has made the inter-laboratory comparison of metabolic profiling and metabolomics data difficult. For example, both reversed-phase packed LC columns [99] and monolithic silica-based columns have been used in metabolomics applications [100,101].…”