SIMULTANEOUS ANALYSIS OF VITAMIN A AND D3 IN VITAMIN PREMIXES AND CONCENTRATES BY ULTRA-PERFORMANCE CONVERGENCE CHROMATOGRAPHY/PDA

Importance of the Topic

Simultaneous quantification of fat-soluble vitamins A and D3 is critical for ensuring nutritional quality and regulatory compliance in food and dietary supplements. Traditional HPLC methods require separate assays, extensive cleanup, long runtimes, and high solvent consumption. A streamlined analytical approach can reduce analysis time, solvent usage, waste, and improve laboratory throughput.

Objectives and Study Overview

The study aimed to develop and validate a robust, high-throughput assay using UltraPerformance Convergence Chromatography (UPC2) with photodiode array detection for simultaneous determination of vitamin A (retinyl acetate) and vitamin D3 (cholecalciferol) in vitamin premixes and concentrates. Method metrology, performance criteria, and comparison with conventional HPLC were evaluated.

Methodology and Instrumentation

• Sample preparation: 1.5 g of sample spiked with vitamin D2 internal standard, saponified with ethanol, KOH, and sodium ascorbate at 80–85 °C for 1 h, extracted with n-hexane/diethyl ether, washed, and filtered into autosampler vials.
• Chromatographic conditions: ACQUITY UPC2 BEH 3.0 mm × 100 mm, 1.7 μm column; mobile phase A: CO2; B: isopropanol; gradient from 0.5% to 20% B over 9.9 min; flow 1.7 mL/min; column temperature 55 °C; backpressure 2,000 psi; injection volume 7 μL; detection at 260 nm.
• Instrumentation: ACQUITY UPC2 system, photodiode array detector, Empower 3 software.

Main Results and Discussion

Calibration curves exhibited excellent linearity with R² values of 0.9998 for vitamin A and 1.0000 for vitamin D3 across seven concentrations. Intermediate precision over three months yielded relative standard deviations of 5.1% for vitamin A and 5.7% for vitamin D3, within the AOAC requirement of ≤8%. Method ruggedness tests altering flow rate, pressure, and temperature by ±2% showed no significant impact on quantification or chromatographic resolution, maintaining a D2/D3 resolution >1.6. Direct injection of hexane extracts eliminated laborious solvent exchange.

Benefits and Practical Applications

• Simultaneous analysis reduces total run time to approximately 12 minutes per sample.
• Significant reduction in solvent consumption: only CO2 and 0.12 mL of isopropanol per test.
• Elimination of sample purification steps streamlines workflow and decreases exposure to harmful solvents.
• Enhanced resolution and reproducibility support reliable quality control in production environments.

Future Trends and Potential Applications

Integration of supercritical fluid chromatography methods into routine analytical protocols can extend to other fat-soluble vitamins and lipid-soluble micronutrients. Coupling UPC2 with mass spectrometry may further improve sensitivity and selectivity. Miniaturized and automated on-line extraction systems could enhance throughput. Emphasis on green analytical chemistry will drive adoption of low-solvent, high-efficiency techniques.

Conclusion

The validated UPC2-PDA method offers a robust, efficient, and environmentally friendly solution for the concurrent quantification of vitamins A and D3 in complex matrices, outperforming traditional HPLC in speed, solvent usage, and operational simplicity.

References

• Aubin A. Analysis of fat soluble vitamin capsules using UltraPerformance Convergence Chromatography UPC2. Waters Application Note No. 720004394EN (2012).
• AOAC SMPR 2012.003. Standard Method Performance Requirements for vitamin A in pre-blends, pre-mixes, and pure materials.
• AOAC SMPR 2012.004. Standard Method Performance Requirements for vitamin D3 in pre-blends, pre-mixes, and pure materials.