Simplified Analysis Workflow – Determination of Vitamins B1, B2, B3, B5, and B6 in Infant Formula

Importance of the Topic

Accurate measurement of B vitamins in infant formula is critical to ensure nutritional adequacy and regulatory compliance. These water-soluble vitamins support energy metabolism and neurological development in infants.

Study Objectives and Overview

This study aimed to develop and validate a streamlined analytical workflow for simultaneous quantification of vitamins B1, B2, B3, B5, and B6 in powdered infant formula. It focused on simplifying sample preparation and enhancing detection sensitivity using the Agilent 1260 Infinity II Prime LC system with diode array detection.

Methodology and Instrumentation

A unified sample protocol employed trichloroacetic acid for protein precipitation, followed by centrifugation and filtration. Two HPLC methods were used: one for B1, B2, B3, B6 with a reversed-phase C18 column at 35 °C, a gradient of SDS/formic acid and acetonitrile, and DAD detection at 268 nm and 296 nm; another for B5 using phosphate buffer/methanol at 40 °C and 205 nm. The LC system comprised a flexible pump, multisampler, multicolumn thermostat, and a 60 mm InfinityLab Max-Light cartridge cell to boost sensitivity.

Main Results and Discussion

The methods demonstrated excellent linearity (R2 = 1.000) over relevant concentration ranges, with limits of quantification between 0.006–1.040 µg/mL. Repeatability showed retention time RSDs below 0.3% and area RSDs below 1%, except B5 (3.94%). Recovery testing with NIST SRM 1869 yielded 91–118% recoveries. Analysis of a commercial infant formula provided accurate vitamin contents consistent with label claims.

Benefits and Practical Applications

The workflow reduces complexity by eliminating enzymatic hydrolysis, shortens analysis time, and ensures robust performance for quality control laboratories. It supports precise nutritional labeling and compliance with food safety regulations.

Future Trends and Potential Uses

Advancements may include coupling to mass spectrometry for enhanced specificity, high-throughput automation, and miniaturized sample preparation. Extending the approach to additional micronutrients could further streamline formula testing.

Conclusion

The presented method offers a reliable, rapid, and sensitive solution for routine determination of key B vitamins in infant formula, meeting stringent analytical and regulatory requirements.

References

• Hanna et al. The Permanente Journal 2022, 26(2), 89–97.
• Ting and Lin, Agilent Technologies Application Note 2023.
• Ting and Lin, Agilent Technologies Application Note 2024.
• Fanali et al. Liquid Chromatography: Applications; Elsevier Science, 2013.
• Woollard and Indyk, Journal of AOAC International 2002, 85(4), 945–951.
• Romera et al. Journal of Dairy Science 1996, 79(4), 523–526.