Analysis of Water-Soluble Vitamins and their Metabolites - Performance Gains in Hydrophilic Interaction Chromatography (HILIC) with LC/MS/MS

Importance of the Topic

Accurate quantification of water-soluble vitamins and their metabolites is critical in fields ranging from nutritional science and clinical diagnostics to pharmaceutical development and quality control. These analytes are small, highly polar molecules that challenge conventional reversed-phase separations. Hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC–LC/MS/MS) offers a powerful solution, enabling sensitive, high-resolution analysis of these compounds.

Study Objectives and Overview

This study aimed to develop a robust HILIC–LC/MS/MS method for simultaneous separation and detection of 24 water-soluble vitamins and their metabolites. Major goals included:

• Evaluating the effect of mobile phase additives at pH 3.0, 7.0 and 9.0 on retention and resolution.
• Optimizing detection sensitivity and peak shape for phosphorylated metabolites.
• Comparing conventional stainless steel hardware with PEEK-lined components and assessing the benefits of a deactivator additive.

Methodology and Instrumentation

Sample Preparation:
A mixed standard solution was prepared by diluting individual vitamin stock solutions in acetonitrile or water mixtures (Table 1). Concentrations ranged from 0.02 to 10.8 µg/mL in the final mixture.

Chromatographic Conditions and Instrumentation:

• LC System: Agilent 1290 Infinity II pump, multisampler and column thermostat.
• Column: Agilent InfinityLab Poroshell 120 HILIC-Z, 2.1 × 100 mm, 2.7 µm, with optional PEEK-lined hardware.
• Mobile Phases: Ammonium formate or acetate solutions adjusted to target pH values; organic eluent was acetonitrile.
• Gradient: 0–1 min at 100% organic; 1–8 min linear gradient to 50% organic; total run time 10 min.
• Flow Rate: 0.30 mL/min; Column Temperature: 40 °C; Injection Volume: 0.5 µL.

Mass Spectrometry:

• Detector: Agilent 6460 triple quadrupole MS with Jet Stream ESI in both positive and negative modes.
• Drying Gas: 250 °C, 6 L/min; Sheath Gas: 325 °C, 12 L/min; Nebulizer Pressure: 35 psi.
• Voltages: Capillary 3,500 V (positive)/2,500 V (negative); Nozzle 500 V/1,000 V.
• Multiple Reaction Monitoring transitions optimized for each analyte.

Main Results and Discussion

Mobile phase at mid pH (pH 7.0 with 10 mM ammonium acetate) achieved the best overall retention and resolution. Moving from pH 3.0 to pH 7.0 improved peak shapes and retention for most analytes, while further increasing to pH 9.0 produced minimal additional benefit.

Steel–analyte interactions caused substantial peak tailing and signal loss for phosphorylated compounds. A simple phosphoric acid wash (0.5% in 90:10 acetonitrile:water) partially restored peak shape. Incorporating a deactivator additive into mobile phases effectively chelated residual metal sites and significantly sharpened peaks.

Employing PEEK-lined column hardware in combination with the activator additive yielded the highest plate counts and the lowest tailing factors for sticky metabolites such as thiamine diphosphate, pyridoxal 5′-phosphate and riboflavin phosphate.

Benefits and Practical Application

This optimized HILIC–LC/MS/MS approach delivers:

• Rapid, high-resolution separation of a broad panel of vitamins and metabolites in a single 10 min run.
• Enhanced sensitivity and reproducibility for phosphorylated analytes using deactivation strategies.
• Robust performance compatible with routine workflows in metabolomic profiling, nutritional studies and pharmaceutical QC.

Future Trends and Possibilities

Emerging directions include:

• Development of novel stationary phases with tailored surface chemistries to further minimize undesired interactions.
• Integration of ultrahigh-pressure HILIC systems for sub-2 µm particle columns and faster separations.
• Coupling with high-resolution mass spectrometers for untargeted metabolomics and structural elucidation.
• Automated in-line deactivation and column conditioning protocols to simplify method maintenance.

Conclusion

A mid-pH ammonium acetate mobile phase paired with Agilent InfinityLab Poroshell 120 HILIC-Z and strategic deactivation of metal surfaces provides a sensitive, high-throughput solution for water-soluble vitamin analysis. PEEK-lined hardware further enhances peak quality for challenging phosphorylated metabolites.

References

1. Hydrophilic Interaction Chromatography Method Development and Troubleshooting, Agilent Technologies Technical Overview, publication number 5991-9271EN, 2018.
2. InfinityLab Deactivator Additive User Guide, Agilent Technologies, publication number 5991-9516EN, 2018.