Evaluation of Ion Mobility/Tof Mass Spectrometry with Multiple LC Method Parameters for Enhanced Detection in Metabolic Profiling

Significance of the topic

Metabolic phenotyping via LC-MS is widely applied for biomarker discovery in biomedical and environmental studies. High throughput demands short separation times, increasing co-elution and ion suppression, which limit coverage. Integrating ion mobility spectrometry (IMS) provides an orthogonal rapid gas-phase separation that improves peak capacity and enables detection of isobaric and co-eluting metabolites without extending analysis time.

Objectives and study overview

This work evaluates how column length (150, 75, 30 mm) and gradient duration (15, 7.5, 3 min) affect feature detection in human urine profiling. It further examines the benefit of adding IMS prior to TOF-MS detection on a Synapt G2-Si platform. Performance metrics include number of detected features, peak capacity, and peak density.

Methodology and instrumental setup

Human urine samples from six volunteers were diluted and centrifuged before analysis. Chromatography employed an ACQUITY UPLC I-Class system with an HSS T3 column at 40 °C and 600 µL/min flow. Mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile with 0.1% formic acid (B). Three column lengths and matched gradient programs maintained 34 column volumes per gradient. The Synapt G2-Si in ESI positive mode acquired mass spectra over m/z 100–1200 in MS^E/HDMS mode. Data were processed with MassLynx and Progenesis QI.

Main results and discussion

• Without IMS, feature counts declined from ~16 200 (150 mm, 15 min) to ~6 500 (30 mm, 3 min) as separation time and column length shortened.
• Integration of IMS increased detected features by 23–41% across all methods (e.g., from 16 192 to 19 893 in the 15 min method).
• Peak capacity ranged from 311 (150 mm, 15 min) to 63 (30 mm, 3 min), reflecting the impact of gradient scale.
• Peak density maps show that IMS reduces chemical noise and resolves co-eluting and isobaric compounds, enabling recovery of true analyte peaks.

Benefits and practical applications

• Enhanced metabolite coverage without extending run time.
• Rapid profiling suitable for large sample cohorts.
• Improved resolution of co-eluting and isobaric species supports biomarker discovery.
• Scalable methods accommodate different throughput requirements.

Future trends and opportunities

Further advances may include building collision cross-section reference databases for compound identification, integrating IMS with high-resolution MS workflows, and developing ultra-fast UPLC methods (< 3 min) combined with IMS for real-time metabolic monitoring in clinical and industrial settings.

Conclusion

Incorporating ion mobility into UPLC-MS workflows substantially increases feature detection across diverse chromatographic conditions. This orthogonal separation enhances throughput and analytical coverage, making it a powerful tool for metabolic phenotyping and biomarker research.

Used Instrumentation

• Waters ACQUITY UPLC I-Class System
• Waters ACQUITY UPLC HSS T3 Column (2.1 x 150, 75 or 30 mm)
• Waters Synapt G2-Si Mass Spectrometer in MS^E/HDMS mode
• MassLynx Software with Progenesis QI

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