Separation of Diastereomeric Flubatine Metabolites using SelexION® Technology

Significance of the Topic

The ability to separate diastereomeric monohydroxy metabolites of Flubatine is critical for accurate quantification of PET tracers targeting α4β2 nicotinic acetylcholine receptors, which are biomarkers for neurological disorders such as Alzheimer disease.

Objectives and Study Overview

This study explores the use of SelexION differential mobility separation (DMS) in conjunction with LC-MS/MS to achieve interference-free resolution of Flubatine metabolites M1 and M2 generated in vitro from mouse liver microsomes.

Methodology

Mouse liver microsome incubations were processed by protein precipitation with cold acetonitrile and centrifugation. Samples were reconstituted in 50% acetonitrile and diluted for LC-MS/MS injection. Chromatographic separation employed an Agilent 1290 system and a Kinetex PFP column (50×2.1 mm, 2.6 µm) with a gradient from 95:5 to 5:95 water/acetonitrile (2.5 mM ammonium formate) at 0.5 mL/min and 60 °C. Mass spectrometry was performed on a QTRAP 5500 with a SelexION DMS device and Turbo V source in positive ion mode using MRMs 223.1→110.0 and 223.1→124.1.

Used Instrumentation

• Agilent 1290 HPLC system
• Phenomenex Kinetex PFP column
• Sciex QTRAP 5500 mass spectrometer with SelexION DMS and Turbo V source

Major Results and Discussion

DMS optimization identified an optimal separation voltage of 3000 V and compound-specific compensation voltages. Isopropanol as a gas modifier enhanced peak capacity tenfold, while methanol yielded an optimal COV of –13.6 V for M2 with baseline resolution from isobaric interferences. On-column COV mapping enabled fingerprinting of metabolites in complex matrices, revealing M2 and additional unknown isobaric species with signal-to-noise ratios exceeding 1000 for standards and 2000 in matrix.

Benefits and Practical Applications

SelexION DMS provides an orthogonal dimension of selectivity to LC and MS/MS, reducing false positives and isobaric overlap. The technique supports shortened LC gradients, low sample consumption, and high-throughput profiling of radiometabolites, improving workflow efficiency in PET tracer development and drug metabolism studies.

Future Trends and Opportunities

Advances in gas-phase modifiers, mixed solvent systems, and fine-tuned compensation voltage mapping will further refine DMS selectivity. Integration with automated high-throughput platforms and machine-learning-driven data analysis is expected to broaden applications to other isomeric metabolites and trace-level bioanalysis in clinical research.

Conclusion

The combination of SelexION DMS and LC-MS/MS offers robust separation and quantification of Flubatine diastereomers in biological samples, enhancing accuracy and throughput for PET radiotracer metabolite studies.

Reference

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