Separation of Isomers of Vitamin K1 Using Normal Phase HPLC

Significance of the Topic

Vitamin K1 plays a critical role in blood coagulation and overall nutritional status. Monitoring its cis and trans isomers and related epoxy derivatives is essential for food quality control, dietary supplement verification, and clinical studies.

Study Objectives and Overview

This application note describes a normal-phase HPLC approach to resolve cis-phylloquinone, trans-phylloquinone, and trans-epoxy phylloquinone. Key aims include assessing different silica stationary phases and evaluating mobile phase equilibration for reproducible retention and resolution.

Methodology and Instrumentation

Samples of vitamin K1 isomers and the trans-epoxy form were prepared at defined concentrations in a mobile phase of heptane, diisopropyl ether, and octanol (1000:3.3:0.67 v/v). Chromatographic separation was performed at ambient temperature with a flow rate of 0.4 mL/min over 30 minutes.

• Instrumentation: Dionex UltiMate 3000 RSLC system with UV detection at 254 nm.
• Columns: Thermo Hypersil Silica (5 μm, 250 × 4.6 mm) and Syncronis Silica (5 μm, 250 × 4.6 mm) for comparison.
• Sample handling: Finnpippettes and amber vials to ensure sample integrity.

Main Results and Discussion

The Hypersil Silica column provided baseline resolution of all three compounds with retention reproducibility (RSD < 1%). Resolution between trans-epoxy and trans-phylloquinone exceeded 3, while cis and trans peaks were baseline separated. Inadequate column equilibration (five column volumes) led to peak order reversal and reduced reproducibility. Use of a high surface area Syncronis Silica column increased retention but diminished resolution between the two trans forms, highlighting the impact of silica surface chemistry on selectivity.

Method Benefits and Practical Applications

This normal-phase HPLC method allows reliable quantification of vitamin K1 isomers in foods, supplements, and biological matrices. It offers:

• Robust separation of geometrical isomers and epoxy derivatives.
• Reproducible retention when proper equilibration protocols are followed.
• Flexibility to evaluate different stationary phases for optimal selectivity.

Future Trends and Potential Applications

Advancements may include sub-2 μm silica or core-shell particles for faster separations, green solvent systems to reduce environmental impact, and coupling with mass spectrometry for enhanced sensitivity. Method transfer to UHPLC platforms and automated equilibration routines will further improve throughput.

Conclusion

A normal-phase HPLC approach on Hypersil Silica successfully separates vitamin K1 isomers and the trans-epoxy derivative with high resolution and reproducibility. Proper mobile phase equilibration and careful selection of silica chemistry are critical for method performance. Variations in column packing can significantly affect selectivity, underscoring the need for specific column evaluation in method development.

References

• British Pharmacopoeia Commission. Phylloquinone monograph. British Pharmacopoeia.
• European Pharmacopoeia Commission. Phylloquinone monograph. European Pharmacopoeia.
• Aspey S. Separation of Isomers of Vitamin K1 Using Normal Phase HPLC. Thermo Fisher Scientific Application Note 20824; 2013.