Normal phase liquid chromatography assay for vitamin K1 isomers

Significance of the Topic

The analysis of vitamin K1 isomers via normal phase liquid chromatography (NP-LC) addresses critical needs in pharmaceutical quality control and nutritional research. Reliable separation of phylloquinone and phytomenadione ensures accurate potency assessment and compliance with regulatory standards, making method robustness and reproducibility essential.

Goals and Overview of the Study

This application brief demonstrates the capabilities of the Thermo Scientific Vanquish Core HPLC system, equipped with a normal phase kit, for routine NP-LC analysis of vitamin K1 isomers. The objective was to develop a fast, stable, and pharmacopeia-compliant assay with high precision and resolution.

Methodology and Instrumentation

A Hypersil Silica column (5 µm, 250 × 4.6 mm) was used with a nonpolar mobile phase of heptane/diisopropyl ether/octanol (1000:3.3:0.7 v/v/v). A special solvent equilibration procedure—stirring heptane with water and decanting—accelerated column conditioning, reducing equilibration from 24 h to 2 h. Chromatographic parameters included 0.8 mL/min flow rate, 22 °C column temperature, 20 °C autosampler temperature, 50 µL injection volume, and UV detection at 254 nm. Data acquisition and analysis employed Chromeleon 7.3 CDS.

Instrumentation Used

• Vanquish Core System Base
• Vanquish Quaternary Pump CN
• Vanquish Split Sampler CT
• Vanquish Thermostatted Column Compartment C
• Vanquish Diode Array Detector CG with 10 mm flow cell
• Normal Phase Kit (P/N 6036.3972)

Main Results and Discussion

Six consecutive injections yielded retention time RSDs of 0.08% (cis) and 0.11% (trans), and peak area RSDs of 0.12% and 0.09%, respectively, all within the European Pharmacopoeia requirement of <1% RSD. The cis/trans resolution of 5.1 exceeded the minimum of 4. Peak asymmetry factors were near ideal (0.99 and 1.01), with no tailing observed. Low system backpressure (<20 bar at 0.4 mL/min) indicated compatibility with NP solvents, while a flow rate increase and capillary addition offered flexibility for backpressure optimization.

Benefits and Practical Applications

• High precision and reproducibility support quality control in pharmaceutical and supplement manufacturing.
• Accelerated equilibration protocol saves laboratory time and solvent consumption.
• System robustness to organic solvents ensures minimal contamination and maintenance.
• Pharmacopoeial compliance guarantees regulatory acceptance.

Future Trends and Possibilities for Utilization

Advances may include miniaturized NP-LC formats, greener solvent systems, and AI-driven method optimization. Integration with automated workflows and real-time monitoring could further enhance throughput and data quality. Emerging stationary phases may improve selectivity for structurally similar isomers.

Conclusion

The Thermo Scientific Vanquish Core HPLC system, equipped with a normal phase kit and using a silica column, provides a rapid, reliable, and highly reproducible assay for vitamin K1 isomers. This method meets stringent pharmacopeial standards and offers robust performance for routine analytical workflows.

References

• European Pharmacopoeia Monograph “all-rac-Phytomenadione” (Ph. Eur. 10.6, 6202 (01/2022:3011))