Rapid quantitative analysis of vitamins K1 and K2 with chromatographic resolution from matrix interferences for clinical research

Importance of the Topic

Vitamin K1 (phylloquinone) and vitamin K2 (menaquinones, including MK-4 and MK-7) play essential roles in coagulation and bone metabolism. Accurate quantification of these lipophilic, fat-soluble vitamins in biological samples is critical for clinical research but is complicated by matrix interferences, particularly phospholipids, and by typical multi-step sample preparations that are time-consuming.

Objectives and Study Overview

The study aimed to develop a streamlined sample preparation workflow based on protein precipitation, combined with rapid UHPLC separation, to achieve robust, sensitive, and selective quantification of vitamin K1, MK-4, and MK-7 in plasma. Key goals included:

• Minimize sample preparation complexity and processing time.
• Resolve target analytes from phospholipid and other matrix interferences by chromatographic means.
• Establish a high-throughput LC-MS/MS assay suitable for clinical research applications.

Methodology and Instrumentation

Sample Preparation:

• Charcoal-stripped human plasma as surrogate matrix.
• Spiking with vitamin K1, MK-4, MK-7 and deuterated internal standard (d7-K1).
• Protein precipitation using acetonitrile (1:3 volume ratio), followed by evaporation and reconstitution in initial mobile phase.
• High-throughput processing enabled by glass-coated WebSeal 96-well plates.

Chromatography and MS Conditions:

• UHPLC System: Thermo Scientific Vanquish Horizon with active pre-heater, binary pump, split sampler, and column compartment.
• Analytical Column: Thermo Scientific Accucore Biphenyl, 2.6 µm, 50 × 2.1 mm.
• Mobile Phases: A – water with 5 mM ammonium acetate and 0.1% formic acid; B – methanol with 0.1% formic acid.
• Gradient elution at 0.6 mL/min, 40 °C, 3.5 min run time, 10 µL injection.
• MS Detection: Thermo Scientific TSQ Endura triple-stage quadrupole with HESI-II probe in positive mode; key transitions: K1 (451.4 → 187.1), MK-4 (445.3 → 187.1), MK-7 (649.5 → 187.1).
• Monitoring of phospholipid neutral loss (m/z 184) to assess and avoid co-elution.

Main Results and Discussion

The assay achieved a lower limit of quantitation (LLOQ) of 0.1 ng/mL and linearity to 10 ng/mL for all analytes. Calibration curves exhibited R² ≥ 0.9917, with average bias within ±5.7% and precision (CV) below 3.2% across QC levels. Chromatographic separation effectively resolved vitamin K species from phospholipid interferences, as confirmed by neutral loss scanning of m/z 184, enhancing assay robustness against ion suppression. The simplified protein precipitation protocol, in combination with the biphenyl column, delivered rapid analysis with retention time stability and minimal matrix effects. Additional discussion highlighted that cis/trans isomer separation of vitamin K1 can be further optimized using an Accucore C30 column for enhanced shape selectivity.

Benefits and Practical Applications

• Rapid, high-throughput workflow suitable for clinical and pharmacokinetic studies.
• Reduced sample preparation complexity lowers labor and consumable costs.
• Strong selectivity and sensitivity support reliable monitoring of vitamin K status in patient samples.
• Adaptable to automated 96-well formats for large-scale studies.

Future Trends and Opportunities

Potential developments include:

• Expansion to simultaneous analysis of additional fat-soluble vitamins and metabolites.
• Integration of high-resolution mass spectrometry for structural elucidation and isomer-specific quantitation.
• Advanced column chemistries (e.g., C30 or mixed-mode) for improved isomeric separations.
• Automation and miniaturization of sample preparation to further increase throughput.

Conclusion

This work demonstrates that a simple protein precipitation protocol, when paired with biphenyl UHPLC and targeted MS detection, yields a rapid, sensitive, and robust assay for vitamins K1, MK-4, and MK-7 in plasma. The method’s minimal matrix effects and high throughput make it well suited for clinical research applications.

References

1. Guo X., Lankmayr E. Phospholipid-based matrix effects in LC–MS bioanalysis. Future Sci. 2011;3(4):349–352.
2. Ismaiel O. et al. Monitoring phospholipids for assessment of ion enhancement and suppression in ESI and APCI LC/MS/MS. J. Chromatogr. B. 2008;875(2):333–343.
3. Hillbeck D. Separation of a Mixture of Vitamin K Isomers Using a Solid Core HPLC Column at Sub-ambient Temperature. Thermo Fisher Scientific Application Note 20590.