A Single-Injection UPC2 Method for Fast and Simultaneous Separation of Nine Fat-Soluble Vitamins

Importance of the Topic

Fat-soluble vitamins and carotenoids play critical roles in human health, serving functions from vision and bone health to antioxidant protection and blood coagulation. Accurate, simultaneous quantification of these analytes in dietary supplements and fortified foods is essential to ensure regulatory compliance, prevent overdosing risks, and maintain consumer safety.

Objectives and Study Overview

This study introduces a single-injection UltraPerformance Convergence Chromatography (UPC 2) method for the rapid separation of nine fat-soluble vitamins and two carotenoids in under four minutes. The goal was to replace multiple conventional liquid chromatography assays with a single, high-throughput approach that maintains high resolution and reproducibility.

Methodology and Instrumentation

Sample Preparation:

• Standards of seven vitamins (A acetate, A palmitate, D2, E, E acetate, K1, K2) and two carotenes (β-carotene, lycopene) dissolved in MTBE at 0.1 mg/mL.

UPC 2 Conditions:

• System: Waters ACQUITY UPC 2 with PDA detection
• Column: ACQUITY UPLC HSS C18 (3.0 × 100 mm, 1.8 μm)
• Mobile phase A: CO2; B: acetonitrile
• Gradient: 2 % B (0–2 min), ramp to 20 % B (2.5–3.5 min), return to 2 % B by 4 min
• Flow rate: 1 mL/min; temperature: 30 °C; back pressure: 2500 psi; injection volume: 1 μL

Main Results and Discussion

The optimized method achieved baseline separation of all nine species within four minutes. Critical resolution of vitamin K1 and K2 was attained by using C18 stationary phase with acetonitrile modifier. Elution order correlated with analyte LogP values: more polar vitamins eluted at low organic content, whereas nonpolar carotenes required higher acetonitrile. Reproducibility was excellent: retention time RSDs <0.25 % and peak area RSDs <1 % (except 2.7 % for vitamin A acetate) across six injections.

Benefits and Practical Applications

Compared with conventional RP and NP LC methods, the UPC 2 approach:

• Reduces analysis time by 4–10×
• Eliminates multiple assays for different analyte classes
• Enhances laboratory throughput and lowers solvent consumption
• Meets stringent regulatory quality control requirements for dietary supplements and fortified foods

Future Trends and Potential Applications

Integration of UPC 2 with mass spectrometry could expand detection of additional micronutrients. Method adaptation for complex matrices such as emulsions or biological fluids will further extend its applicability. Continued development of greener modifiers and automated sample handling will drive sustainable, high-throughput vitamin analysis.

Conclusion

A robust UPC 2 method for nine fat-soluble vitamins and two carotenoids was established, offering rapid, reproducible, and high-resolution separation in a single injection. This approach streamlines analytical workflows, ensuring reliable quality control in nutraceutical and food industries.

Reference

• Santos J et al. J. Chromatogr. A. 2012;1261:179–188.
• Aubin A. Waters Application Note 720004394en, 2012.
• Blake CJ. J. AOAC Int. 2007;90(4):897–910.
• Gomis DB et al. J. Chromatogr. A. 2000;891:109–114.
• Salo-Vaananen P et al. Food Chem. 2000;71:535–543.