Metabolite Identification

Structural elucidation is a critical step in drug metabolism studies, enabling the localization of putative biotransformations in the parent drug. In vivo and in vitro studies are used to identify major metabolites so that metabolic weak spots on the drug candidate molecule can be recognized and protected by changing the compound structure. Key for analysts in metabolite identification is maintaining high sample throughput and providing results to medicinal chemists as soon as they are available.

ACQUITY UPLC^® with MS/MS or MS^E addresses the complex analytical requirements of biomarker discovery by offering unmatched sensitivity, resolution, dynamic range, and mass accuracy. With workflow tools and software for comprehensive statistical processing, Waters solutions provide a turnkey approach to metabolite ID.

• When used in conjunction with UPLC separations, the SYNAPT™ G2 HDMS™ and Xevo™ QTof MS systems provide an information-rich approach for metabolite detection and identification with multiple stages of fragmentation for low metabolite concentrations.
• Taking advantage of this fragment ion data accuracy, MS/MS interpretation software such as the MetaboLynx™ XS Application Manager and MassFragment™ automate data processing to help you move through structural elucidation quickly and confidently.

UPLC/HDMS metabolite identification applications

• Metabolite identification
• Probing mechanisms of toxicity
• Identification of markers of efficacy
• Impurity profiling
• Natural products analysis
• Profiling foods and beverages
• Characterization of industrial processes

Additional dimension of data with HDMS

A typical challenge when running in vivo samples for identification is that, without the use of radiolabeled compounds, there are no reference points to use to look for xenobiotics and detect and identify low-level metabolites from a highly endogenous background.

Ion mobility spectrometry (IMS), a cutting-edge technology available with the Synapt HDMS System, significantly reduces the complexity of identifying metabolites by taking advantage of multidimensional separation techniques:

• SYNAPT HDMS provides an extra dimension of separation capable for deconvoluting endogeonous and exogenous drug metabolite interferences.
• HDMS-enhanced TAP fragmentation data can be analyzed quickly and easily using MetaboLynx with MassFragment software to turn information into answers.

Clear detection with HDMS

UPLC^®/IMS-TOF-MS analysis was conducted on an in vivo ketotifen sample obtained from a rat bile sample. By using DriftScope™ Software, the metabolites from this complex matrix were easily visualized, as the drift time was used to separate background ions from real drug-related metabolites. Further extracted ion chromatograms were also obtained by selecting specific ions with the software. As a result, the extracted ion chromatogram and MS spectrum for each metabolite were attained without interference from the endogenous compounds.

UPLC/HDMS System:

• ACQUITY UPLC^® System
• ACQUITY UPLC BEH C₈ Column, 2.1 x 100 mm, 1.7 µm
• Synapt HDMS System

Driftscope plot.

DriftScope Software’s dt (msec)/rt (min) plots for rat bile dosed with ketotifen. Regions are easily visualized from the plots: (A) metabolites and the parent drug, (B) PEG, (C) bile salts, and (D) solvent ions.

TIC comparison.

The total ion chromatograms (TICs) for ketotifen rat bile sample:

• (A) TIC without the use of drift time to extract the information
• (B) TIC with the use of drift time to extract the information

Easily visible metabolites in the spectra.

Spectra obtained with the Synapt HDMS System for a rat bile sample dosed with ketotifen, with combined total ion chromatograms (TICs) from the IMS-based acquision using drift time to extract data, top, vs. those from the non-IMS acquisition, bottom. Metabolites are more visible when using drift time.