Analytical strategies for identifying drug metabolites

Abstract

With the dramatic increase in the number of new chemical entities (NCEs) arising from combinatorial chemistry and modern high-throughput bioassays, novel bioanalytical techniques are required for the rapid determination of the metabolic stability and metabolites of these NCEs. Knowledge of the metabolic site(s) of the NCEs in early drug discovery is essential for selecting compounds with favorable pharmacokinetic credentials and aiding medicinal chemists in modifying metabolic "soft spots". In development, elucidation of biotransformation pathways of a drug candidate by identifying its circulatory and excretory metabolites is vitally important to understand its physiological effects. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) have played an invaluable role in the structural characterization and quantification of drug metabolites. Indeed, liquid chromatography (LC) coupled with atmospheric pressure ionization (API) MS has now become the most powerful tool for the rapid detection, structure elucidation, and quantification of drug-derived material within various biological fluids. Often, however, MS alone is insufficient to identify the exact position of oxidation, to differentiate isomers, or to provide the precise structure of unusual and/or unstable metabolites. In addition, an excess of endogenous material in biological samples often suppress the ionization of drug-related material complicating metabolite identification by MS. In these cases, multiple analytical and wet chemistry techniques, such as LC-NMR, enzymatic hydrolysis, chemical derivatization, and hydrogen/deuterium-exchange (H/D-exchange) combined with MS are used to characterize the novel and isomeric metabolites of drug candidates. This review describes sample preparation and introduction strategies to minimize ion suppression by biological matrices for metabolite identification studies, the application of various LC-tandem MS (LC-MS/MS) techniques for the rapid quantification and identification of drug metabolites, and future trends in this field.

Dealing with the unknown: Metabolomics and Metabolite Atlases

“…However, ESI is often preferred for profiling 'unknown' metabolites since this 'soft' desorption/ionization approach efficiently forms 'intact' molecular ions that aids initial identification of unknowns. This coupled with the high mass accuracy of many instruments (Ͻ5 ppm) facilitates the initial ('putative') identification of metabolites by searching metabolite databases [13]. This approach has been implemented in a wide range of applications primarily related to functional genomics and clinical studies [4,14].…”

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.…”

“…IT provides more sensitivity for structural elucidation than QQQ due to its better sensitivity in full scan mode and efficient dissociation of the precursor ions which allows multiple stages mass spectrometry (MS n ). Recently, to address classical ion traps (called also 3D IT) shortcomings of insufficient ion storage efficiency, capacity and deterioration of the mass spectrum and dynamic response range, linear IT has been developed [25]. The detection sensitivity in linear IT is at least two orders of magnitude higher than that in 3D IT.…”

“…Alternatively, LC-NMR analysis can be performed on biological samples with minimal sample processing but certain limitations occur with this technique, such as lower sensitivity compared with LC-MS and the requirements of relatively expensive deuterated buffers in mobile phase. More recently, LC-NMR has been coupled with MS which enables simultaneous metabolite structure elucidation [25]. Tentatively identified structure of metabolites may also be synthesized and LC-MS data for these compounds are compared with data from the actual metabolites.…”