HPLC Analyses of Nucleotides in Powdered Infant Formula and Liquid Infant Formula

Importance of the Topic

Infant formulas are designed to mimic breast milk and support optimal growth. Nucleotides, while present at low levels, play critical roles in intestinal development, immune function, lipid metabolism and cognitive performance. Accurate quantification of nucleotides in powdered and liquid infant formulas is essential for quality control, regulatory compliance and ensuring nutritional adequacy.

Objectives and Study Overview

This study presents a validated high-performance liquid chromatography (HPLC) method for simultaneous determination of five nucleotide monophosphates (CMP, UMP, GMP, IMP, AMP) in powdered and liquid infant formulas. Method development was guided by ISO 20638:2015(E) and AOAC Official Method 2011.20 to optimize sample pretreatment and analytical parameters for precision and robustness.

Methodology

Sample pretreatment involves protein precipitation and cleanup with a strong‐anion exchange solid-phase extraction (SPE) cartridge to remove matrix interferences. Extracts are filtered and diluted prior to analysis. Chromatographic separation uses a Shim-pack GIST C18-AQ column (250 × 4.6 mm, 5 μm) on a Nexera XR HPLC. The mobile phase gradient employs 10 mmol/L KH2PO4 buffer (pH 5.6) and methanol, at 0.6 mL/min and 40 °C. Detection is by photodiode array at 250 nm for IMP, 260 nm for AMP, GMP, TMP (ISTD) and 270 nm for CMP, UMP.

Used Instrumentation

• Nexera XR HPLC system
• Shim-pack GIST C18-AQ column
• PDA detector
• SPE cartridges (strong-anion exchange)
• UV-visible spectrophotometer (UV-1900i) for standard purity assessment

Main Results and Discussion

Calibration curves for all five nucleotides exhibited excellent linearity (r2 ≥ 0.99997). Method repeatability was demonstrated with retention time RSDs < 0.1% and peak area RSDs < 0.6% in standard solutions, and < 1.1% in formula samples. Recoveries in spiked powdered and liquid formulas ranged from 87% to 105%, meeting ISO/AOAC criteria. Quantitative analysis of commercial formulas revealed total nucleotide contents at 90%–103% of labeled values.

Benefits and Practical Applications

• Simultaneous quantification of multiple nucleotides in one run improves laboratory throughput.
• Optimized SPE cleanup reduces matrix effects and enhances accuracy.
• High precision and recoveries support routine quality control in infant formula manufacturing and regulation.

Future Trends and Applications

Future developments may include coupling HPLC with mass spectrometry for enhanced sensitivity, expanding analyte panels to include di- and triphosphates, and applying the method to human milk studies to deepen understanding of early nutrition.

Conclusion

The described HPLC method on Nexera XR with Shim-pack GIST C18-AQ offers reliable, accurate and reproducible analysis of five key nucleotides in infant formulas, aligning with international standards for nutritional quality assessment.

References

1. VYH Yu. Journal of Pediatrics and Child Health. 2002;38(6):543–549.
2. José Maldonado et al. Early Human Development. 2001;65(Suppl 2):S69–S74.
3. ISO 20638:2015(E). Infant formula—Determination of nucleotides by liquid chromatography.
4. Brendon D. Gill et al. Journal of AOAC INTERNATIONAL. 2015;98(4):971–979.