Analysis of Choline and its Metabolites

Importance of the Topic

Choline and its metabolites, including trimethylamine (TMA) and trimethylamine N-oxide (TMAO), serve as key biomarkers in physiological and disease-related processes. Reliable quantification of these small polar compounds in biological samples supports research in cardiovascular health, gut microbiome interactions and metabolic disorders.

Study Objectives and Overview

This application note demonstrates a rapid hydrophilic interaction liquid chromatography–mass spectrometry (HILIC-MS) method for simultaneous analysis of choline, TMA and TMAO. The goal is to achieve high throughput and sensitivity with clear chromatographic separation within a short run time.

Methodology and Used Instrumentation

A HILIC approach was implemented on a Shim-pack Velox HILIC column (50 × 2.1 mm, 2.7 µm) mounted on a Nexera X2 UHPLC system. The gradient elution employed mobile phase A (50 mM ammonium formate with 0.1% formic acid in water) and mobile phase B (acetonitrile), programmed from 70% B to 5% B over 3 minutes, with a total cycle of 4 minutes per injection. Key chromatographic parameters:

• Flow rate: 0.4 mL/min
• Column temperature: 40 °C
• Injection volume: 3 µL

Mass spectrometric detection used an LCMS-8060 triple quadrupole with electrospray positive ionization. Gas flows and temperatures were optimized:

• Nebulizing gas: 3 L/min
• Drying gas: 10 L/min
• Heating gas: 10 L/min
• DL temperature: 250 °C
• Interface temperature: off
• Block heater: 400 °C

Main Results and Discussion

The method achieved baseline separation of choline, TMA and TMAO within a 4-minute cycle. Chromatograms demonstrated sharp, symmetrical peaks with retention times at approximately 1.2 min (TMA), 1.5 min (TMAO) and 1.8 min (choline). Linearity was confirmed over relevant concentration ranges, and limits of detection were in the low nanomolar range. Precision tests showed relative standard deviations below 5% for all analytes.

Benefits and Practical Applications

The fast HILIC-MS workflow offers:

• High sample throughput suitable for large cohorts
• Minimal sample preparation for polar analytes
• Robust quantification of biomarkers in clinical and nutritional studies

Potential applications include monitoring gut microbial metabolism, assessing cardiovascular risk markers and investigating dietary interventions.

Future Trends and Potential Applications

Advances may include coupling with high-resolution MS for broader metabolome coverage, integration into automated sample-to-data platforms and expansion to related quaternary amines. Emerging microflow HILIC systems could further reduce solvent consumption and enhance sensitivity.

Conclusion

This application demonstrates a reliable, high-throughput HILIC-MS method for choline and its key metabolites. The combination of Shim-pack Velox HILIC chromatography and the LCMS-8060 system delivers rapid separation, high sensitivity and reproducible quantitative performance, meeting the needs of biomarker analysis in research and quality control.

Reference

Application News an_01-00238 (Shimadzu Corporation, First Edition Aug. 2023).