Microflow Chromatography Provides Improved Sensitivity and Coverage of Polar Metabolites for Targeted Metabolomics

Importance of the Topic

Polar metabolite profiling is essential for biomarker discovery, drug toxicity screening and understanding metabolic pathway alterations in health and disease. These highly polar compounds present challenges in chromatographic retention and sensitivity when analyzed in complex matrices such as urine, plasma or cell extracts.

Objectives and Overview

This work describes a targeted microflow HILIC LC–MS/MS workflow using a M3 MicroLC system coupled to a QTRAP 6500+ mass spectrometer. The goal is to screen over 300 polar metabolites across multiple biological samples with enhanced sensitivity, selectivity and throughput compared to conventional analytical-flow methods.

Methodology

Biological samples (urine, plasma, MDCK cell extracts) underwent protein precipitation in a cold acetonitrile:acetone:methanol mixture, followed by centrifugation and evaporation. Dried extracts were reconstituted in high-organic buffer (95–100% mobile phase B) to enable injection volumes up to 5 µL on the microflow column without compromising chromatographic performance. A 30-minute HILIC gradient on a Luna NH2 column (150 × 0.3 mm, 3 µm) at 10 µL/min was employed. Positive and negative polarity switching (5 ms dwell, 50 ms settling) monitored 363 MRM transitions for 312 unique targets in a single run. Data processing used MultiQuant software with S/N thresholds for confident detection.

Used Instrumentation

• SCIEX M3 MicroLC system with integrated autosampler and column oven
• Luna 3 µm NH2 HILIC column (150 × 0.3 mm) with 1 µm inline filter
• SCIEX QTRAP 6500+ mass spectrometer with IonDrive Turbo V source and 25 µm electrode
• MultiQuant 3.0.2 for peak integration and quantitation

Main Results and Discussion

The microflow HILIC method achieved up to 60× improvement in signal-to-noise ratio (S/N) and on average 10× enhancement versus analytical-flow. It enabled confident detection (S/N ≥ 20) of 263 metabolites out of 312 targets, representing up to 50% greater coverage in MDCK cells, 35% in urine and 11% in plasma. High-organic reconstitution (100% mobile phase B) was critical for maximizing solubility and sensitivity of polar analytes. Consolidation into a single polarity-switching run reduced total analysis time and eliminated multiple injections per sample.

Benefits and Practical Applications

• Substantially increased sensitivity and metabolome coverage for polar analytes
• Reduced solvent consumption and operational costs through microflow chromatography
• Efficient single-method screening of hundreds of metabolites across diverse matrices
• Shortened cycle times enable higher laboratory throughput and rapid data turnover

Future Trends and Opportunities

Integration of microflow HILIC with high-resolution mass analyzers, ion mobility separations or data-independent acquisition could further expand metabolome coverage and facilitate untargeted biomarker discovery. Automation of sample preparation and online cleanup may improve reproducibility and throughput.

Conclusion

The described microflow HILIC LC–MS/MS workflow on the M3 MicroLC–QTRAP 6500+ platform provides dramatic gains in sensitivity, coverage and throughput for targeted polar metabolite analysis, offering a powerful solution for routine metabolomics in research and clinical laboratories.

Reference

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