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 physiology. Choline contributes to cell membrane structure and methylation pathways, while acetylcholine functions as a neurotransmitter central to muscle activation and memory processes. Accurate quantification of these polar quaternary amines supports research in nutrition, neuroscience, and clinical diagnostics.

Objectives and Study Overview

This study aims to develop and validate an efficient LC-MS/MS method for simultaneous measurement of choline and acetylcholine. By employing a high-performance hydrophilic interaction chromatography (HILIC) column with 1.7 µm particles, the work demonstrates rapid separation and robust quantification suitable for routine laboratory analysis.

Methodology and Instrumentation

The analytical approach integrates HILIC-based chromatographic separation with tandem mass spectrometry detection:

• Chromatography system: Thermo Scientific Accela 600 UHPLC
• Column: Syncronis HILIC 1.7 µm, 50 × 2.1 mm
• Mobile phase: 15 mM ammonium formate buffer (pH 3.5) and acetonitrile (10:90, v/v)
• Flow rate and temperature: 0.6 mL/min at 30 °C
• Injection volume: 2 µL with designed wash protocols for carryover control
• Mass spectrometer: Thermo Scientific TSQ Vantage with HESI ionization in positive mode
• MRM transitions: m/z 146 → 87 (acetylcholine), m/z 104 → 60 (choline)
• Optimized parameters: spray voltage 3000 V, vaporizer and capillary at 300 °C, collision energy 13 eV (acetylcholine) and 17 eV (choline)

Results and Discussion

The method achieved baseline separation of acetylcholine and choline within three minutes, with acetylcholine exhibiting a retention factor around 5. Peak shapes were sharp and symmetric under the selected HILIC conditions. Precision was demonstrated by replicate injections (n = 6), yielding retention time relative standard deviations of 0.1% for both analytes. The rapid cycle time and low backpressure (135 bar) highlight the method's suitability for high-throughput workflows.

Benefits and Practical Applications

• Fast analysis suitable for routine measurements in nutritional and clinical research
• High reproducibility ensures consistent data quality across batches
• HILIC selective retention enhances sensitivity for polar analytes without derivatization
• Compact run time and robust performance reduce solvent use and increase laboratory throughput

Future Trends and Potential Applications

Advancements may include coupling with microflow LC to further reduce solvent consumption, integration with isotopic internal standards for enhanced quantitation, and expansion to multiplexed assays covering related metabolites. Deploying such methods in clinical laboratories could support diagnostic panels for neurodegenerative diseases and nutritional status assessments.

Conclusion

The optimized LC-MS/MS protocol leveraging a Syncronis HILIC 1.7 µm column offers a reliable, fast, and reproducible platform for co-analysis of choline and acetylcholine. Its performance metrics make it attractive for diverse analytical settings, from research studies to quality control.

References

No external references were provided in the source document.