Quality control of choline as a dietary supplement by high performance liquid chromatography coupled to a charged aerosol detector

Importance of the Topic

The analysis of choline and its salts in dietary supplements is critical for ensuring product quality and regulatory compliance. Choline plays essential roles in cell membrane structure, lipid metabolism, and neurotransmission. Deficiencies can lead to serious health issues such as liver damage and muscle disorders. Reliable analytical methods support accurate labeling and safety assurance for supplements containing choline salts like bitartrate, chloride, and citrate.

Objectives and Study Overview

This study aimed to develop a single chromatographic method capable of directly detecting choline salts and their impurities without derivatization. The approach sought to quantify choline content, identify accompanying counterions, and detect low-level impurities in commercial supplement samples. A secondary goal was to demonstrate compatibility with both charged aerosol detection (CAD) and mass spectrometry (MS).

Methodology and Instrumentation

The method employs hydrophilic interaction liquid chromatography (HILIC) on a zwitterionic stationary phase with a gradient of ammonium acetate buffer and acetonitrile. Key components:

• Column: SeQuant ZIC-pHILIC (4.6×150 mm, 5 µm)
• Mobile phases:
    
  • A: 80.4 mM ammonium acetate pH 4.7/ acetonitrile (20/80, v/v)
  • B: 80.4 mM ammonium acetate pH 4.7/ water/ acetonitrile (20/30/50, v/v/v)
• Gradient: initial 90% A to 0% A over 20 min, then re-equilibration to 90% A by 28 min
• Flow rate & injection: 0.5 mL/min, 10 µL injection
• Detector: Charged aerosol detector (evaporation 144 °C; corona 35 °C; pressure filter 1.0)
• Optional MS: single quadrupole operated in full scan and SIM at m/z 148.2 for impurity identification

Main Results and Discussion

The method achieved baseline separation of choline and its counterions (acetate, citrate, bitartrate, chloride) in under 30 minutes. Calibration from 0.08 to 0.12 mg/mL exhibited linearity (r2>0.997), precision ≤3% RSD, and recoveries between 96.7% and 100.8%. CAD sensitivity allowed detection of impurities down to ng levels. Use of parallel MS confirmed the structure of a choline-related by-product, O-(2-hydroxyethyl)choline, eluting at approximately 6 min. Sodium and chloride contaminants were traced to glassware and minimized by using polypropylene vessels.

Benefits and Practical Applications

This single-run HILIC-CAD approach eliminates the need for precolumn derivatization, thus simplifying sample preparation and reducing interference. It provides:

• Direct detection of choline salts and counterions
• Low detection limits for trace impurities
• Compatibility with MS for structural confirmation
• Robust performance for raw material and finished product testing

Future Trends and Opportunities

Advancements may include coupling CAD with high-resolution mass spectrometry to expand impurity profiling and structural elucidation. Automation of sample handling and data analysis could enhance throughput for routine quality control. Emerging stationary phases may further improve separation of structurally similar analytes in complex matrices.

Conclusion

The described HILIC-CAD method delivers comprehensive analysis of choline dietary supplements, offering high sensitivity, precision, and the ability to identify counterions and impurities without derivatization. It meets pharmacopeial requirements and streamlines workflows for both routine QC and research applications.

References

1. Zeisel, S. H. Dietary choline: biochemistry, physiology, and pharmacology. Annu. Rev. Nutr. 1981, 1, 95–121.
2. Zeisel, S. H.; da Costa, K.-A. Choline: an essential nutrient for public health. Nutr. Rev. 2009, 67, 615–623.
3. Linus Pauling Institute. Micronutrient Information Center: choline.
4. United States Pharmacopeia. Choline monograph. Pharmacopeial Forum, PF stage.