Determination of Fat-Soluble Vitamins in Infant Formula Using Agilent Bond Elut Plexa Polymeric SPE with HPLC and LC/MS/MS

Significance of the Topic

The accurate quantification of fat-soluble vitamins A, D, and E in infant formula is essential for infant health, supporting vision, growth, immune function, and antioxidant defense. Regulatory bodies require strict compliance with fortified levels to avoid deficiency or toxicity. However, the high lipid content and diverse chemical properties of these vitamins create significant analytical challenges, necessitating robust, sensitive, and cost-effective methods for routine quality control and research purposes.

Study Objectives and Overview

This study aimed to develop and validate a streamlined workflow for determining vitamins A, D2/D3, and alpha-, beta-, gamma-, and delta-vitamin E in powdered infant formula. The approach integrates sample saponification, polymeric solid phase extraction (SPE) using Agilent Bond Elut Plexa cartridges, and dual detection strategies: HPLC-DAD for vitamins A and E, and LC/MS/MS for vitamins D2 and D3. Key performance metrics included reproducibility, recovery, simplicity, and cost-effectiveness.

Methodology

Sample Preparation and Cleanup:

• Weigh 1.0 g of infant formula powder and add 5 mL water to form a slurry.
• Spike internal standards (VD2-d3, VD3-d3) and add antioxidants (0.5 g ascorbic acid, 0.1 g BHT) to prevent degradation.
• Add 10 mL ethanol and 5 mL 50% KOH, then incubate at 80 °C for 45 min under gentle nitrogen flow for saponification.
• Perform SPE cleanup on Bond Elut Plexa cartridges: condition with MeOH and water, load sample, wash with water and 80/20 MeOH/water, elute vitamins with acetone, evaporate under nitrogen, reconstitute in MeOH, and filter.

Analytical Conditions:

• HPLC-DAD (vitamins A and E): Agilent InfinityLab Poroshell 120 PFP column (2.1×100 mm, 2.7 µm), 40 °C, mobile phases water/MeOH with 0.1% formic acid, gradient 80–100% MeOH over 9 min; detection at 325 nm (A) and 294 nm (E).
• LC/MS/MS (vitamins D2/D3): Agilent InfinityLab Poroshell 120 EC-C18 column (2.1×100 mm, 2.7 µm), 40 °C, mobile phases water/MeOH with 0.1% formic acid, 4.5 mM ammonium formate, 0.5 mM ammonium fluoride; gradient 88–100% MeOH over 17 min; positive electrospray MRM transitions for quantification.

Used Instrumentation

• Agilent 1290 Infinity II LC with flexible pump, multisampler, variable wavelength detector.
• Agilent 1290 Infinity II LC and 6470A triple quadrupole LC/MS with Agilent Jet Stream electrospray source.
• Agilent Bond Elut Plexa polymeric SPE cartridges (200 mg, 6 mL).
• Agilent Vac Elut 20 manifold.
• Organomation nitrogen evaporator with heating system.

Results and Discussion

Chromatographic separation on HPLC-DAD achieved baseline resolution of retinol and four vitamin E isoforms with sharp peak shapes. LC/MS/MS provided sensitive detection of vitamins D2 and D3, avoiding saturation and ion suppression by optimizing mobile phase additives. The inclusion of 4.5 mM ammonium formate and 0.5 mM ammonium fluoride doubled the signal intensity compared to formic acid alone. Calibration curves were linear over 0.05–200 µg/mL for vitamins A/E (R² ≥ 0.999) and 1–1 000 ng/mL for vitamins D2/D3 (R² ≥ 0.998). Method validation using spiked infant formula yielded recoveries between 81.2% and 97.3% with RSDs below 10%.

Benefits and Practical Applications

This method offers a simple, low-cost workflow suitable for routine analysis in research and industry. The two-platform detection ensures accurate quantification across a wide concentration range, addressing the distinct levels of vitamins A/E and D in infant formula. The polymeric SPE cleanup effectively removes lipids and proteins, improving robustness and reducing matrix effects.

Future Trends and Applications

Future developments may include automation of sample preparation, miniaturized extraction formats, and coupling with high-resolution mass spectrometry for non-targeted vitamin profiling. Green chemistry approaches, such as solvent reduction and reusable SPE materials, will further enhance sustainability. Expanded applications could target other fortified food matrices and stability studies.

Conclusion

The integrated saponification–SPE–dual detection method provides reliable, precise, and efficient quantification of fat-soluble vitamins in infant formula. Its high recoveries, low variability, and adaptability make it a valuable tool for quality control, regulatory compliance, and nutritional research.

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