Simultaneous determination of vitamin A, D and four vitamin E isomers in infant formula by offline solid phase extraction and online two-dimensional liquid chromatography

Significance of the Topic

Accurate determination of fat soluble vitamins in infant formula ensures compliance with nutritional standards and safeguards infant health. Vitamins A, D and E isomers play distinct physiological roles and their quantification at low levels in complex dairy matrices demands sensitive, reliable methods.

Study Objectives and Overview

This work presents a streamlined analytical approach for simultaneous quantification of vitamin A, vitamins D2 and D3, and four vitamin E isomers in infant formula. By combining offline solid phase extraction (SPE) with online two-dimensional liquid chromatography (2D-LC) on a Thermo Scientific Vanquish Core HPLC system, the method aims to simplify sample preparation, reduce toxic solvent use, and enhance sensitivity relative to conventional liquid–liquid extraction protocols defined in Chinese national standards.

Methodology

• Sample Preparation and Saponification
  5 g of infant formula powder were dissolved in warm water, spiked with ascorbic acid and BHT, then saponified with ethanol and KOH at 80 °C for 30 min.
• Pretreatment Comparison
  
  • Liquid–Liquid Extraction (LLE): Multiple petroleum ether/diethyl ether extractions, washes, rotary evaporation and nitrogen drying.
  • Solid Phase Extraction (SPE): HyperSep Retain PFP column conditioned with methanol and water, load saponified sample, wash with methanol/water, elute vitamins with acetone, dry and reconstitute in methanol.
• Chromatographic Conditions
  
  • 1st Dimension Column: FOODKIT 2 ADE 3.0 × 150 mm, 2.6 µm with DAD and FLD for vitamins A and E isomers.
  • 2nd Dimension Column: FOODKIT 1 & 2 ADE 4.6 × 100 mm, 2.6 µm with DAD for vitamin D2 and D3.
  • Gradient elution and valve-switching schedule optimized to achieve baseline separation in 24 min total cycle time.

Used Instrumentation

• Thermo Scientific Vanquish Core HPLC System: dual pump, split sampler, column compartment, diode array detector, fluorescence detector, column switching valves and vacuum manifold.
• Columns and Consumables: HyperSep Retain PFP SPE cartridge, FOODKIT 2 ADE and FOODKIT 1 & 2 ADE columns.
• Software: Thermo Scientific Chromeleon 7.3 for data acquisition and processing.

Main Results and Discussion

• Chromatographic Performance: Complete baseline separation of four vitamin E isomers and vitamin D2/D3 within a 24 min 2D-LC run.
• Pretreatment Efficiency: SPE reduced sample prep time from ~3 h (LLE) to ~1 h and eliminated large volumes of toxic solvents.
• Method Validation:
  
  • Linearity: Correlation coefficients (R²) above 0.9993 over relevant concentration ranges.
  • Detection Limits: 1.4–4.0 ng/mL across all analytes.
  • Precision: Intra-day repeatability RSD <1% for all vitamins.
  • Recovery: SPE recoveries between 100–112% and LLE recoveries between 106–119% for spiked samples.
• Sample Analysis: Two commercial infant formulas yielded consistent vitamin contents with both pretreatment methods, confirming method accuracy and robustness.

Benefits and Practical Applications

The SPE–2D-LC method offers higher throughput, reduced solvent consumption and improved sensitivity and reproducibility, making it ideal for routine quality control in dairy and infant nutrition laboratories and for compliance with food safety regulations.

Future Trends and Potential Applications

Potential developments include extension to additional fat‐soluble nutrients, integration with mass spectrometry detectors for enhanced selectivity, automation of SPE workflows, miniaturized chromatographic systems for green analytical chemistry, and adaptation to other complex food matrices.

Conclusion

The optimized offline SPE combined with online 2D‐LC on a Vanquish Core HPLC platform delivers a robust, efficient solution for simultaneous quantification of vitamins A, D2, D3 and E isomers in infant formula. This approach outperforms traditional LLE in speed, safety and analytical performance, supporting stringent quality and regulatory requirements.

References

1. GB 5009.82-2016, National Food Safety Standard – Determination of Vitamins A, D and E in Foods.
2. Kamal-Eldin A, Görgen S, Lampi AM. Normal phase high performance liquid chromatography of tocopherols and tocotrienols. J Chromatogr A. 2000;881:217–227.
3. Chen JB. Rapid detection of vitamins A, D and E in food by on-line purification two-dimensional HPLC. J Food Saf Qual. 2019;10(6):1726–1733.
4. Zhang YH, Zhu XY, Yang XL, Jin Y. Simultaneous qualification of vitamins A, D₃ and E in fortified infant formulae by automated online 2D column switching LC. Chin J Anal Chem. 2013;41(5):771–775.
5. Zhang YH, Jin Y. Simultaneous determination of vitamins A, D3, and E in infant formula by online 2D‐LC. Chin J Chromatogr. 2015;33(3):291–297.