Quantitative Determination of Fat-Soluble Vitamins in Infant Formula by LC-MS/MS Method with Supported Liquid Extraction (SLE)

Significance of the Topic

Fat-soluble vitamins A, D3, E and K1 are essential micronutrients in infant formula, contributing to vision, calcium absorption, antioxidative protection and blood coagulation. Their accurate measurement is critical for ensuring nutritional compliance and infant health. However, low concentrations, lipophilicity and instability during extraction present significant analytical challenges.

Objectives and Overview of the Study

This study aimed to establish and validate a rapid, sensitive LC–MS/MS method for the simultaneous quantitative determination of fat-soluble vitamins in infant formula. Supported liquid extraction (SLE) was applied for sample cleanup, and method performance was benchmarked against FAPAS quality control materials.

Methodology and Instrumentation

Sample preparation involved:

• Saponification of 0.1 g milk powder with water, pyrogallol, KOH and overnight mixing;
• pH adjustment to 6–7 using formic acid to optimize vitamin retention on the SLE column;
• Extraction on an SLE cartridge using butylated hydroxytoluene in hexane under gravity and vacuum;
• Evaporation under nitrogen and reconstitution in methanol followed by 0.22 µm filtration.

Chromatographic separation used a Shim-pack GIST C18 column with gradient elution of 0.1% formic acid in water and methanol. Detection was performed in APCI positive MRM mode on the Shimadzu LCMS-8045. Optimized transitions and collision energies ensured selective monitoring of each vitamin.

Main Results and Discussion

Calibration curves from 10 to 400 ng/mL exhibited excellent linearity (R2 > 0.999 for all vitamins). Limits of detection ranged from 1.6 to 2.8 ng/mL and limits of quantitation from 4.8 to 8.5 ng/mL. Intra-day precision showed %RSD ≤ 3.4 for concentration and ≤ 0.7 for retention time. Analysis of FAPAS QC material yielded recoveries between 87.4% and 101.8% with overall %RSD below 10%, confirming accuracy and precision. Inter-day repeatability remained within 10% for vitamins A and E, and under 15% for D3 and K1.

Benefits and Practical Applications

This method delivers rapid, reliable quantitation of four key fat-soluble vitamins with minimal sample handling and solvent consumption. The use of SLE simplifies cleanup and eliminates extensive liquid–liquid extraction. The protocol is well suited for routine quality control and nutritional analysis in dairy and infant formula industries.

Future Trends and Potential Applications

Advances may include integration of isotope-labeled internal standards for enhanced accuracy, automation of SLE workflows, expansion to high-throughput multi-vitamin panels and coupling with high-resolution mass spectrometry. Miniaturized extraction techniques and on-line sample cleanup are expected to further streamline analysis.

Conclusion

A robust LC–MS/MS method using supported liquid extraction was developed for quantifying vitamins A1, D3, E and K1 in infant formula. The approach demonstrates high sensitivity, excellent linearity and reproducibility, and meets stringent QA/QC requirements without the need for elaborate extraction procedures.

References

1. Karrar E, Mohamed Ahmed IA, Manzoor MF, Sarpong F, Wang X. Food Chemistry. 2022;373:131436.
2. Chew YL. Shimadzu Application News AD-0208.
3. Food Analysis Performance Assessment Scheme (FAPAS). QC Material Data Sheet T21125QC.