LC-MS/MS Method for the Analysis of Choline and Acetylcholine using a Syncronis HILIC 1.7 μm Column

Significance of the Topic

Choline and its derivative acetylcholine play vital roles in human health and neurochemical signaling. Choline must be obtained through diet and serves as a precursor of the neurotransmitter acetylcholine, which is involved in muscle control and memory. A rapid, reliable analytical method for these polar compounds is essential for research in neuroscience, clinical diagnostics, and quality control in food and nutritional supplements.

Objectives and Study Overview

This application note assesses the use of a Syncronis HILIC 1.7 micrometer column for fast liquid chromatography tandem mass spectrometry analysis of choline and acetylcholine. The primary aim is to achieve baseline separation within three minutes with high reproducibility and excellent peak shape.

Methodology and Instrumentation

• Stationary phase: Syncronis HILIC column 1.7 µm, 50 × 2.1 mm
• LC system: Thermo Scientific Accela 600 UHPLC
• Mobile phase: 15 mM ammonium formate pH 3.5 and acetonitrile (10:90 v/v)
• Flow rate: 0.6 mL/min; column temperature: 30 °C; injection volume: 2 µL
• MS detection: Thermo Scientific TSQ Vantage with heated electrospray ionization in positive mode
• MRM transitions: acetylcholine m/z 146→87.2; choline m/z 104→60.3

Used Instrumentation

• Accela 600 UHPLC system
• Syncronis HILIC 1.7 micrometer column
• TSQ Vantage mass spectrometer

Main Results and Discussion

The method produced a retention factor near 5 for acetylcholine and 1.9 for choline. Both analytes were fully separated in under three minutes. Precision testing over six replicate injections showed retention time RSDs below 0.1 percent and symmetrical peak shapes, indicating robust column performance.

Benefits and Practical Applications

• High throughput capability with analysis times under three minutes
• Excellent chromatographic reproducibility for confident quantitation
• HILIC retention mechanism tailored to highly polar quaternary amines

Future Trends and Opportunities

The shift toward sub-2 micrometer HILIC phases enables even faster, more sensitive analyses. Potential applications include biomarker discovery in neurodegenerative diseases and routine screening of functional food products. Integration with automated sample preparation and high-resolution mass spectrometry can further expand analytical scope.

Conclusion

The described LC-MS/MS workflow using a Syncronis HILIC 1.7 micrometer column achieves rapid, reproducible quantitation of choline and acetylcholine. It is well suited for laboratories requiring high throughput and robust analysis of polar analytes.

References

No external references were provided in the source document.