Analysis of Choline Metabolites by Hydrophilic Interaction Chromatography (HILIC) with LC/MS/MS

Significance of the Topic

Choline and its related quaternary amine metabolites play essential roles in cellular function, neurotransmission, and membrane integrity. Their high polarity necessitates specialized chromatographic techniques, making hydrophilic interaction chromatography (HILIC) a critical tool for reliable retention and quantitative analysis in metabolomics.

Objectives and Study Overview

This application note investigates the performance of two superficially porous HILIC phases—Agilent Poroshell 120 HILIC-Z and HILIC-OH5—for the separation and detection of choline, betaine, acetylcholine, and glycerophosphocholine by LC/MS/MS. The study focuses on mobile phase additive optimization and direct comparison of chromatographic behavior.

Methodology and Instrumentation

The analytical method consisted of:

• Columns: Poroshell 120 HILIC-Z and Poroshell 120 HILIC-OH5 (2.1 × 100 mm, 2.7 μm).
• Mobile phases: A: water with 10 mM ammonium acetate (adjusted to pH 4.5); B: acetonitrile. Gradient: 10–50% A (0–5 min), 50–60% A (5–6 min), hold 60% A until 8 min.
• Flow rate: 0.30 mL/min; column temperature: 30 °C; injection volume: 1 μL.
• Detector: Agilent 6460 triple quadrupole LC/MS with Jet Stream ESI in positive ion mode. Parameters: drying and sheath gas temperatures at 250 °C, drying gas flow 5 L/min, sheath gas flow 11 L/min, nebulizer pressure 45 psi, capillary voltage +3500 V, MRM acquisition.

Results and Discussion

Screening of mobile phase additives demonstrated that a mid-pH buffer containing 10 mM ammonium acetate yielded the best compromise of retention, peak shape, and sensitivity. On the HILIC-Z column, acetylcholine and choline exhibited sharp, symmetric peaks with high signal intensity. The HILIC-OH5 phase provided stronger retention and enhanced response for betaine but at the expense of peak symmetry for acetylcholine and choline. Under optimized conditions, all four metabolites were baseline-separated within 8 minutes.

Benefits and Practical Applications

This HILIC-MS/MS method offers rapid, sensitive, and reproducible quantification of choline metabolites in complex biological or environmental samples. Its robustness and throughput make it ideal for metabolomic studies, clinical diagnostics, pharmaceutical research, and QC laboratories.

Future Trends and Potential Applications

Future developments may include coupling HILIC to high-resolution mass spectrometry to expand analyte coverage, advancing superficially porous particle chemistries for tailored selectivity, and embedding the workflow in automated platforms for high-throughput screening. Extending this approach to other polar metabolite classes could further broaden its utility in systems biology and clinical research.

Conclusion

The optimized method using the Agilent Poroshell 120 HILIC-Z column with 10 mM ammonium acetate at pH 4.5 delivers efficient, high-resolution separation and sensitive detection of choline and its metabolites. This approach underpins robust metabolomic analyses and routine laboratory workflows.