Rapid Separation of Vitamin K1 Isomers and Vitamin K2 in Dietary Supplements Using UltraPerformance Convergence Chromatography with a C18 Column

Importance of the Topic

Vitamin K1 plays a critical role in blood coagulation and bone metabolism, and its bioactivity depends on the geometric configuration of its isomers. Accurate quantification of trans-phylloquinone and separation from the inactive cis isomer is essential for assessing nutritional supplements’ true potency. Conventional HPLC methods often require long run times and specialized columns, making rapid quality control challenging.

Objectives and Overview of the Study

This application note presents a study aimed at developing a fast, reliable, and solvent-efficient method for separating vitamin K1 trans- and cis-isomers alongside vitamin K2 (MK-4) in dietary supplements. Using UltraPerformance Convergence Chromatography (UPC 2) on a standard C18 column, the method seeks to reduce analysis time and organic solvent consumption compared to existing C30-based HPLC approaches.

Methodology and Instrumentation

The separation employed an ACQUITY UPC 2 system with a PDA detector and Empower 3 software. Key parameters included:

• Column: ACQUITY UPC 2 HSS C18 SB, 3.0 × 100 mm, 1.8 μm
• Mobile phase A: compressed CO₂; B: acetonitrile/methanol (50:50, v/v)
• Gradient: isocratic 0.5% B for 2 min, ramp to 20% B over 1.5 min, hold 0.5 min
• Flow rate: 3.00 mL/min; column temperature: 50 °C; injection: 20 μL

Sample preparation involved dissolving standards in iso-octane and extracting tablet powders, followed by filtration.

Main Results and Discussion

The UPC 2 method achieved baseline separation of cis-K1, trans-K1, and MK-4 within three minutes, with a total run time of four minutes including column reequilibration. The cis/trans critical pair resolution was 1.7. Ten-replicate analyses showed retention time RSDs below 0.1% and peak area RSDs under 1.1%. Limits of quantitation ranged from 0.04 to 0.06 µg/mL, and calibration curves exhibited excellent linearity (R²>0.998). Analysis of a commercial supplement revealed 11.2% cis-K1 relative to total K1 content with high repeatability.

Benefits and Practical Applications

The UPC 2 method offers:

• Five-fold faster analysis compared to HPLC on C30 columns
• Reduced organic solvent use (<1 mL per injection)
• Use of a standard C18 column—no need for special C30 phases
• High resolution and sensitivity suitable for routine QA/QC in food and supplement laboratories

Future Trends and Possibilities of Use

Emerging applications may include extension to longer-chain menaquinones (e.g., MK-7 and higher), integration into automated high-throughput workflows, and coupling with mass spectrometry for structural confirmation. Further optimization could explore alternative modifier compositions and pressure/temperature tuning to enhance selectivity.

Conclusion

UltraPerformance Convergence Chromatography on a C18 column provides a rapid, robust, and environmentally friendly approach for the separation and quantitation of vitamin K1 isomers and MK-4. This method enhances throughput while maintaining high resolution and repeatability, meeting the needs of modern supplement analysis.

References

• AOAC Official Method 999.15. Vitamin K in milk and infant formulas by liquid chromatography. AOAC International; 2005.
• Woollard DC, Indyk HE, Fong BY, Cook KK. Determination of vitamin K1 isomers in foods by liquid chromatography with C30 bonded-phase column. J AOAC Int. 2002;85(3):682–691.
• Huang B, Zheng F, Fu S, Yao J, Tao B, Ren Y. UPLC-ESI-MS/MS for trans- and cis-vitamin K1 in infant formulas: method and applications. Eur Food Res Technol. 2012;235(5):873–879.
• Aubin A. Analysis of fat-soluble vitamin capsules using UltraPerformance Convergence Chromatography. Waters Application Note No. 720004394en; 2012.