Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)

Importance of the Topic

Fat-soluble vitamins A, D, E, and K are essential micronutrients with diverse roles in human health. Quality control of oil-based capsules and tablets requires reliable separation techniques to quantify active forms and isomers in complex matrices. Traditional normal-phase methods often use large volumes of hazardous solvents, are time-consuming, and suffer from limited isomeric resolution. UltraPerformance Convergence Chromatography (UPC²) offers a greener, faster, and high-resolution alternative that can streamline routine analysis.

Study Objectives and Overview

This application study evaluated the performance of the Waters ACQUITY UPC² system across six commercial fat-soluble vitamin formulations: vitamin A, vitamins A + D₃, vitamin D₃, vitamin E, vitamin K₁, and vitamin K₂. The primary goal was to demonstrate that a single UPC² platform with method adjustments can resolve active ingredients and stereoisomers from various excipients rapidly and reproducibly.

Methodology and Instrumentation

The system comprised an ACQUITY UPC² Binary Solvent Manager, Sample Manager, Convergence Manager, Column Manager, and a PDA detector controlled by Empower 3 software. Columns used included BEH 3.0 × 100 mm (1.7 µm) and HSS C18 SB 3.0 × 100 mm or 2.1 × 150 mm (1.8 µm). Sample preparation involved dissolving oil-filled capsule contents in iso-octane or sonicating powdered tablets, followed by filtration.

Results and Discussion

• Vitamin A: A gradient from 97:3 to 90:10 CO₂/methanol (0.2% formic acid) over 3 min resolved cis- and trans-retinyl palmitate isomers (1.325 and 1.394 min) and retinol, separated from excipient peaks at 2.0–2.5 min.
• Vitamins A + D₃: Using a 2.1 × 150 mm column with a 99:1 to 90:10 gradient over 10 min, cis- and trans-palmitate (2.626, 2.851 min) and cholecalciferol (6.862 min) were cleanly separated from fish-oil excipients.
• Vitamin D₃: Under the same conditions as A + D₃, cholecalciferol eluted at 6.867 min, enabling clear quantitation against a sunflower-oil background.
• Vitamin E: A rapid gradient (98:2 to 95:5 CO₂/methanol) over 1.5 min on a BEH column yielded baseline separation of α-, β-, γ-, and δ-tocopherols within 90 s.
• Vitamin K₁: An isocratic 99% CO₂/1% methanol:acetonitrile (1:1) method separated two phylloquinone stereoisomers (2.062, 2.226 min) with Rs > 2.0; UV spectra confirmed similar chromophores.
• Vitamin K₂: An isocratic 95:5 CO₂/methanol run on HSS C18 SB identified menaquinone-7 as the predominant form (1.388 min), matching the label claim.

Benefits and Practical Applications

UPC² enables rapid analysis (run times from 90 s to 10 min) with minimal organic solvent consumption, high sensitivity, and effective isomer separation. Laboratories can adopt a single platform for diverse fat-soluble formulations, improving throughput and reducing costs associated with solvent procurement and disposal.

Future Trends and Opportunities

• Integration with mass spectrometry for structural confirmation and trace-level quantitation.
• Automation of sample preparation workflows to increase throughput.
• Application expansion to other lipophilic bioactives, such as carotenoids and sterols.
• Development of standardized UPC² methods for regulatory and pharmacopeial compliance.

Conclusion

The ACQUITY UPC² system delivers a robust, fast, and environmentally friendly approach to analyzing fat-soluble vitamin formulations. Its ability to resolve key isomers and active compounds in a single platform positions UPC² as a compelling alternative to traditional chromatographic methods.

Reference

Aubin A. Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC²). Waters Corporation; 2012.