Gas-Phase Separation of Benzocaine Protonation Site Isomers and Phenylalanine Using Travelling-Wave Ion Mobility MS

Importance of Topic

Ion mobility-mass spectrometry provides a gas-phase separation of isomeric and isobaric species, aiding accurate identification of metabolites in complex biological samples. In newborn screening for metabolic disorders such as phenylketonuria (PKU), resolving phenylalanine from interfering isomers like benzocaine is critical to avoid false positives and ensure timely treatment.

Objectives and Study Overview

This study demonstrates the use of traveling-wave ion mobility-MS on a SYNAPT G2-Si HDMS instrument to separate benzocaine protonation site isomers (protomers) from phenylalanine. It evaluates separation efficiency with nitrogen and carbon dioxide drift gases and characterizes fragmentation patterns for unambiguous compound identification.

Použitá instrumentace

• SYNAPT G2-Si HDMS mass spectrometer
• T-Wave ion mobility cell with N₂ or CO₂ drift gas
• Electrospray ionization in positive mode (ESI⁺)

Methodology

Benzocaine and phenylalanine were prepared in methanol and diluted to 0.5 ng/µL. Direct infusion ESI⁺ introduced ions into the mobility cell. For N₂ drift gas, conditions were 90 mL/min flow, 750 m/s wave velocity, and 27 V amplitude. For CO₂, flow was 65 mL/min, velocity 1000 m/s, and amplitude 40 V. Drift times and collision cross sections (CCS) were measured, followed by tandem MS (15 V collision energy) on mobility-resolved ions.

Main Results and Discussion

• Two benzocaine protomers exhibited distinct drift times: ~3.45 ms (CCS 131 Ų) and ~4.36 ms (CCS 147 Ų).
• Phenylalanine separated at ~3.81 ms (CCS 137 Ų), enabling clear resolution from both protomers.
• CO₂ drift gas provided superior separation compared to N₂ due to stronger ion–gas interactions.
• MS/MS of drift-separated species revealed unique fragments: both protomers yielded an m/z 138 ion; the faster protomer produced additional ions at m/z 120 and m/z 94, consistent with carbonyl oxygen protonation.

Benefits and Practical Applications

• Increased specificity in newborn metabolic screening, reducing false positives from benzocaine contamination.
• Ability to distinguish positional isomers and protomers in pharmaceutical and biochemical analyses.
• Adaptable to bioanalytical workflows for reaction monitoring, metabolite profiling, QA/QC, and research laboratories.

Future Trends and Opportunities

• Exploring alternative or mixed drift gases to further improve isomer separation.
• Integration with liquid chromatography and automation for high-throughput analyses.
• Extension to other structural isomer classes, including drug metabolites and peptides.
• Combining IM-MS datasets with machine learning for rapid pattern recognition and compound identification.

Conclusion

Traveling-wave ion mobility-MS on the SYNAPT G2-Si HDMS effectively separates benzocaine protomers from phenylalanine, with CO₂ drift gas offering enhanced resolution. Diagnostic MS/MS fragmentation confirms structural assignments, improving the accuracy of metabolic screening protocols and providing a versatile analytical tool.

Reference

1. Hunt P. Newborn Screening by Tandem Mass Spectrometry in Texas. 2012.
2. Snelling JR. Characterisation of Pharmaceutical and Polymer Formulations by Novel Mass Spectrometry Approaches. PhD thesis, University of Warwick. 2012.
3. Warnke S et al. Protomers of Benzocaine: Solvent and Permittivity Dependence. J Am Chem Soc. 2015 Mar 17.