Development of a MULTI-2D LC×LC-ESI/TPI-DUAL SOURCE-QTOF-MS for the analysis of complex samples

Significance of the Topic

Non-targeted analysis of complex samples often suffers from insufficient separation and limited ionization coverage. Multi-dimensional LC coupled with dual-source ionization and high-resolution MS greatly enhances selectivity and detection of isomeric and trace analytes.

Objectives and Study Overview

This study presents the development of a Multi-2D LC×LC platform integrated with dual ESI/TPI sources and qTOF-MS. Key goals include:

• Implementing Bayesian optimization for 1D and 2D gradient and column selection
• Integrating seamless switching between electrospray and plasma ionization
• Demonstrating applicability on natural extracts and complex environmental matrices

Methodology and Instrumentation

The workflow combines comprehensive two-dimensional LC with advanced ionization and mobility techniques:

• Column screening: C18, PFP, HILIC and others in 150×2.1 mm (1.7–3 µm) for 1D and 50×4.6 mm (2.6–5 µm) for 2D
• Bayesian optimization: Automated tuning of elution gradients to maximize peak capacity and separation orthogonality
• Modulation: 0.75 min loop injections at 80 µL/min with valve switching every 30 min
• Dual ion sources: Agilent Jet Stream ESI and tube plasma ionization (TPI) sharing a single housing
• Ion mobility: Drift tube IMS and SLIM TWIMS for collision cross section (CCS) determination
• Detection: Quadrupole time-of-flight mass spectrometer and diode array detector

Main Results and Discussion

Bayesian optimization improved peak capacity from ~104 to over 231 in test separations. Application to vermouth and Agrimonia eupatoria extracts showed enhanced profiling of phenolics and flavonoids. Dual-source MS provided complementary ionization efficiencies, lowering limits of detection for diverse analytes. DTIM-qTOF measurements resolved coeluting isomers by CCS differences, exemplified by 4-hydroxybenzoic and salicylic acids. Custom Python software (PSeaC) enabled clustering and recombination of modulated features for accurate quantitation.

Benefits and Practical Applications

This platform is suited for environmental analysis, food quality control, and natural product research. It offers:

• High selectivity for isomer resolution
• Expanded chemical coverage via dual ionization
• Enhanced sensitivity and lower LODs
• Comprehensive non-target screening capability

Future Trends and Opportunities

Advancements in SLIM-based high-resolution IM will further boost separation power. Machine learning integration can streamline feature annotation in 4D datasets. The modular source design allows future addition of APCI and APPI. Broader applications in metabolomics and lipidomics are anticipated.

Conclusion

The Multi-2D LC×LC-ESI/TPI dual-source qTOF-MS platform represents a significant advance for complex mixture analysis. Bayesian method development, dual ionization, and IM-MS integration deliver unparalleled separation, detection, and identification performance.

Reference

E. L. Schymanski et al. Anal Bioanal Chem 2015 407:6237–6255
L. Mondello et al. Nat Rev Methods Primers 2023 3:86
J. F. Ayala-Cabrera et al. Anal Chem 2022 94:9595–9602
A. Pape et al. Trends Anal Chem 2024 170:117420
J. C. May et al. J Am Soc Mass Spectrom 2021 32(4):1126–1137