HPLC analyses of nucleotides in powdered infant formula and liquid infant formula

Significance of the topic

Infant formulas are formulated to mimic the nutritional profile of breast milk, with nucleotides now recognized as key semi-essential nutrients that support intestinal development, immune function, lipid metabolism and cognitive development. Accurate quantification of nucleotide content is critical for quality control, nutritional labeling and regulatory compliance in the infant nutrition industry.

Objectives and overview

This study presents a validated high-performance liquid chromatography (HPLC) method for simultaneous determination of five nucleotides (CMP, UMP, GMP, IMP and AMP) in powdered and liquid infant formulas. The method follows ISO 20638:2015(E) and AOAC Official Method 2011.20, aiming to demonstrate precision, accuracy and compliance with declared product contents.

Methodology and sample preparation

Sample preparation involves:

• Weighing 1 g powdered formula or measuring 10 mL liquid formula into a 50 mL tube.
• Adding internal standard (TMP) and extracting with a methanol–water mixture.
• Purification using strong-anion exchange solid-phase extraction cartridges to remove matrix interferences.
• Elution, dilution, filtration (0.2 µm) and direct injection into the HPLC system.

Instrumentation used

The analysis employed:

• Shimadzu Nexera XR HPLC system
• Shim-pack GIST C18-AQ (250 mm × 4.6 mm, 5 µm) column
• PDA detector with wavelength channels at 250 nm (IMP), 260 nm (AMP, GMP, TMP) and 270 nm (CMP, UMP)
• Anion exchange SPE cartridges (Chromabond SB) and TORAST Disc 0.2 µm filters

Main results and discussion

Calibration curves prepared with mixed standards (STD1–STD4) showed excellent linearity (r2 ≥ 0.99997) over the working range. Repeatability tests yielded retention-time RSDs < 0.1% and peak-area RSDs < 0.6% for standards; similar precision was maintained for formula samples (area RSDs < 1.1%). Spike-and-recovery experiments (n = 6) produced recoveries between 87% and 105%, confirming method accuracy. Measured nucleotide contents corresponded to 90% (powdered A) and 103% (powdered B) of label claims, with comparable results in liquid formula.

Benefits and practical applications of the method

This robust HPLC-UV method supports routine quality control in infant formula production by offering:

• High precision and accuracy in complex matrices
• Compliance with international ISO and AOAC standards
• Efficient sample cleanup reducing matrix effects
• Flexibility for both powdered and liquid formulations

Future trends and potential applications

Emerging directions include:

• Automation of sample preparation workflows for higher throughput
• Integration with mass spectrometry for enhanced sensitivity and identification
• Expansion to other bioactive components in nutritional products
• Development of greener chromatographic solvents and consumables

Conclusion

The described HPLC-UV method on a Nexera XR platform with Shim-pack GIST C18-AQ column demonstrates excellent precision, accuracy and repeatability for nucleotide analysis in infant formulas. It meets ISO/AOAC requirements and provides a reliable tool for industry laboratories to ensure product quality and regulatory adherence.

Reference

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