Simultaneous Determination of Vitamins A and E in Infant Formula by ACQUITY UPC2 with PDA Detection

Significance of the topic

Fat-soluble vitamins A and E play critical roles in infant nutrition, supporting vision, immune function, and antioxidant protection. Reliable and efficient analysis of these vitamins in infant formula ensures product safety, accurate labeling, and compliance with regulatory standards.

Objectives and Study Overview

This study aimed to develop a rapid, simplified, and environmentally friendly method for simultaneous determination of multiple forms of vitamins A (retinyl acetate and palmitate isomers) and E (α-tocopheryl acetate and α-tocopherol) in commercial infant formula using UltraPerformance Convergence Chromatography (UPC2) with photodiode array (PDA) detection.

Methodology and Instrumentation

Sample Preparation:

• Liquid–liquid extraction without saponification.
• Direct injection for vitamin A; ten-fold evaporation concentration for vitamin E.

Chromatographic Conditions:

• System: Waters ACQUITY UPC2 with PDA detector.
• Column: HSS C18 SB, 3.0×100 mm, 1.8 µm.
• Mobile phase: Supercritical CO₂ with methanol gradient (3 % to 10 %).
• Flow conditions optimized for an eight-minute cycle, including equilibration.
• Detection wavelengths: 320 nm (retinyl esters), 283 nm (α-tocopheryl acetate), 293 nm (α-tocopherol).

Main Results and Discussion

The method achieved baseline separation of cis- and trans-retinyl esters and vitamin E forms in a single eight-minute injection. Key performance metrics included:

• Linearity: R² ≥ 0.9953 across relevant concentration ranges.
• Limits of quantitation (LOQ): 0.3 to 13.0 µg/mL.
• Repeatability: RSD ≤ 3.8 % (n=6).
• Recovery: 91 % to 94 % (n=3).
• Cycle time: Three times faster than conventional HPLC (eight vs. 25 min).
• Solvent use: 0.9 mL per injection, >90 % reduction compared to normal-phase methods.

Benefits and Practical Applications

This UPC2-PDA approach offers:

• High throughput with minimal sample preparation.
• Reduced solvent consumption and environmental impact.
• Accurate quantification of isomeric forms of vitamins A and E.
• Potential for routine quality control in infant formula manufacturing.

Future Trends and Applications

Advances may include:

• Extension to other fat-soluble vitamins and complex food matrices.
• Automation of sample preparation workflows.
• Coupling UPC2 with mass spectrometry for enhanced selectivity and sensitivity.
• Integration into online monitoring systems for continuous quality assurance.

Conclusion

The proposed UPC2-PDA method delivers rapid, precise, and eco-friendly analysis of vitamins A and E in infant formula, combining high resolution, low solvent usage, and streamlined workflows. It represents a powerful tool for modern analytical laboratories and the food industry.

Reference

1. DeVries JW, Silvera KR. Determination of vitamins A (retinol) and E (alpha-tocopherol) in foods by liquid chromatography: collaborative study. J. AOAC Int. 2002;85:424.
2. Chavez-Servin JL, Castellote AI, Lopez-Sabater MC. Simultaneous analysis of Vitamins A and E in infant milk-based formulae by normal-phase high-performance liquid chromatography-diode array detection using a short narrow-bore column. J. Chromatogr. A. 2006;1122:138.
3. Aubin A. Analysis of Fat-Soluble Vitamin Capsules Using UltraPerformance Convergence Chromatography UPC.2. Waters Application Note 720004394en. 2012 June.
4. ACQUITY UPC2 System brochure. Waters Corporation. 2012; p/n 720004225en. 2012 March.