Fast Analysis of Fat-Soluble Vitamins in Infant Milk Powder by Heart Cutting 2D-LC

Importance of the Topic

The accurate and rapid determination of fat-soluble vitamins A, D, and E in infant milk powder is critical to ensure compliance with nutritional regulations and to protect infant health. Conventional methods for vitamin D analysis often demand extensive sample cleanup and long runtimes. The heart-cutting two-dimensional liquid chromatography (2D-LC) approach provides a streamlined workflow, combining targeted purification and fast separation in a fully automated sequence.

Objectives and Study Overview

This study aimed to develop and validate a heart-cutting 2D-LC method for simultaneous quantification of vitamins A, E, and D3 in infant formula milk powder. Key goals included reducing total analysis time, minimizing sample preparation complexity, and achieving low detection limits and high reproducibility under routine testing conditions.

Methodology

The overall workflow comprised:

• Sample Preparation: Saponification and extraction following Chinese National Standard GB 5413.9—2010. Ten grams of milk powder were dissolved, treated with ascorbic acid and KOH, saponified at 53 °C for 45 min, extracted twice with petroleum ether, neutralized, dehydrated over sodium sulfate, and reconstituted in methanol.
• Heart-Cutting 2D-LC: A short C18 first-dimension column enabled rapid separation of vitamin A and E. Between 4.4 and 4.6 min, the valve switched to capture the vitamin D fraction on a C18 cartridge. The second-dimension column then achieved baseline separation of D2 and D3 under isocratic ACN/MeOH elution.

Instrumentation

• First dimension: Agilent 1260 Infinity Quaternary Pump (G1311B), Autosampler (G1329B), Thermostatted Column Compartment (G1316A) with 2-position/6-port valve, Diode Array Detector (G4212B) with 10 mm flow cell.
• Second dimension: Agilent 1260 Infinity Degasser (G1322A), Binary Pump (G1312B), Diode Array Detector (G4212B) with 60 mm ultrasensitive flow cell.
• Columns: ZORBAX RRHT Extend-C18 (4.6×50 mm, 1.8 µm), ZORBAX Eclipse PAH (2.1×100 mm, 3.5 µm), ZORBAX Extend-C18 capture column (4.6×12.5 mm, 5 µm).
• Software: Agilent OpenLab CDS ChemStation Edition C01.05[35].

Main Results and Discussion

The total analysis time was 14 min per injection. Vitamins A and E were quantified from the first-dimension chromatogram; vitamin D3 was isolated and analyzed in the second dimension. Calibration curves exhibited excellent linearity for vitamin A (2.5–12.5 µg/mL, R²=0.99996), vitamin E (50–250 µg/mL, R²=0.99984), and vitamin D3 (0.05–0.25 µg/mL, R²=0.99988). The limit of detection for D3 corresponded to 10 µg/kg (400 IU/kg) in milk powder. Reproducibility was high: retention time RSD<0.3% and peak area RSD<1% for all three vitamins.

Benefits and Practical Applications

• Single injection workflow reduces hands-on time and solvent consumption.
• Simultaneous quantification of multiple vitamins increases laboratory throughput.
• Minimal sample cleanup without the need for offline solid-phase extraction.
• High sensitivity and specificity for low‐level vitamin D detection.

Future Trends and Potential Applications

Emerging developments may include coupling heart-cutting 2D-LC with mass spectrometry for enhanced selectivity, expanding the method to additional fat-soluble nutrients, miniaturization through microflow columns, and integration of on-line sample preparation modules. Digital workflows and AI-driven data analysis are likely to further optimize method development and routine quality control.

Conclusion

The described heart-cutting 2D-LC method offers a rapid, reliable, and fully automated solution for routine determination of vitamins A, E, and trace D3 in infant formula. It meets regulatory requirements while delivering high sensitivity and reproducibility, ultimately improving laboratory efficiency and cost-effectiveness.

Reference

1. Heudi O. et al. J. Chrom. A 2004, 1022, pp 115–123.
2. Anon. GB 5413.9—2010 Determination of Vitamin A, D, E in Foods for Infants and Young Children. Chinese National Standards, Beijing.