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

Importance of the topic

Choline is an essential water-soluble nutrient that supports liver function, lipid metabolism and cell membrane integrity. As a precursor to acetylcholine, a key neurotransmitter governing memory and muscle control, accurate measurement of both compounds is vital in nutritional studies, neuroscience research and clinical diagnostics.

Objectives and study overview

This application note evaluates the performance of a Thermo Scientific Syncronis HILIC 1.7 µm column coupled with LC-MS/MS for the rapid separation and quantitation of choline and acetylcholine. The aim was to achieve robust retention, excellent peak shape and high reproducibility in under three minutes per analysis.

Methodology and instrumentation

Sample Preparation:

• Standards of choline and acetylcholine (100 ng/mL) were dissolved in acetonitrile to ensure compatibility with the HILIC mobile phase.

Chromatographic Conditions:

• UHPLC system: Thermo Scientific Accela 600
• Column: Syncronis HILIC 1.7 µm, 50 × 2.1 mm
• Mobile phase: 15 mM ammonium formate (pH 3.5) / acetonitrile (10:90, v/v)
• Flow rate: 0.6 mL/min, injection volume 2 µL, column temperature 30 °C
• Wash solvents: weak wash 20:80 acetonitrile/water; strong wash 45:45:10 acetonitrile/acetone/isopropanol

Mass Spectrometry Conditions:

• Instrument: Thermo Scientific TSQ Vantage triple quadrupole MS with HESI source
• Polarity: positive; spray voltage 3000 V; vaporizer and capillary at 300 °C
• Sheath/auxiliary gas: 50/30 mTorr; collision pressure 1.5 mTorr
• Transitions: m/z 146→87.2 for acetylcholine (CE 13 eV), m/z 104→60.3 for choline (CE 17 eV)
• Cycle time 0.02 s; Q1/Q3 peak widths 0.02/0.7

Used Instrumentation

• Thermo Scientific Accela 600 UHPLC system
• Syncronis HILIC 1.7 µm, 50 × 2.1 mm column (PN 97502-052130)
• Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer

Main results and discussion

Under the optimized HILIC conditions, acetylcholine was retained with k′~5 and eluted at 0.87 min, while choline eluted at 1.96 min. Both analytes displayed sharp, symmetrical peaks and baseline resolution within three minutes. Six replicate injections yielded retention time RSDs of 0.1% for each compound, demonstrating exceptional method precision.

Benefits and practical applications

• Rapid turnaround time (<3 min) supports high-throughput workflows in pharmaceutical, clinical and nutritional laboratories.
• Excellent retention and peak shape for highly polar quaternary amines ensures reliable quantitation at low nanogram levels.
• Robust reproducibility facilitates routine QA/QC and method transfer between laboratories.

Future trends and opportunities

Advancements in HILIC materials may further enhance retention of polar metabolites. Integration with automated sample preparation and multiplexed MS platforms could expand the panel of analytes to include related neurotransmitters and biomarkers. Emerging bioinformatics tools will streamline data processing and support real-time decision making in clinical and research environments.

Conclusion

The Thermo Scientific Syncronis HILIC 1.7 µm column coupled with LC-MS/MS provides a fast, reproducible and sensitive method for the analysis of choline and acetylcholine. Its high retention, excellent peak shape and robust performance make it an ideal choice for laboratories conducting nutritional, neuroscientific and clinical studies.

References

• MacRitchie E, Meadows K. Application Note 20498. Thermo Fisher Scientific; 2012.