Simultaneous Analysis of Vitamins B7, B9, and B12 in Infant Formula

Importance of the Topic

Accurate quantification of vitamins B7 (biotin), B9 (folic acid) and B12 (cyanocobalamin) in infant formula is vital to ensure nutritional adequacy and regulatory compliance. These vitamins are essential for metabolism, DNA synthesis and red blood cell production but occur at trace levels in complex matrices.

Objectives and Study Overview

This application note details the development and validation of a robust HPLC-DAD method using an Agilent 1260 Infinity II Prime LC system for simultaneous analysis of B7, B9 and B12 in infant formula. The method was evaluated against NIST standard reference material and a commercial sample.

Methodology and Instrumentation

The chromatographic separation employed an Agilent InfinityLab Poroshell 120 SB-Aq column (3.0×100 mm, 2.7 µm) at 40 °C. A gradient of 0.0125 % formic acid in water and acetonitrile was delivered at 0.5 mL/min. Detection was performed by diode array detector with a 60 mm Agilent InfinityLab Max-Light cartridge flow cell monitoring wavelengths at 205 nm (B7), 268 nm (B9) and 361 nm (B12).

Sample preparation included enzymatic digestion with pepsin and sodium ascorbate, heat treatment, centrifugation, immunoaffinity cleanup using EASI-EXTRACT MULTI-VIT B IAC, elution with ammonium hydroxide in methanol, evaporation and reconstitution in mobile phase A.

Main Results and Discussion

Limit of quantification was established at 36.1 ng/mL (B7), 0.3 ng/mL (B9) and 1.5 ng/mL (B12). Calibration curves were linear (R²=1.000) over 50–5000 ng/mL (B7, B9) and 5–500 ng/mL (B12). Repeatability showed retention time RSD <0.1 % and peak area RSD <2 %.
Recovery from NIST 1869 SRM was 70.9 % (B7), 92.4 % (B9) and 80.8 % (B12), meeting validation criteria. Analysis of a commercial sample yielded 0.14 µg/g (B7), 1.00 µg/g (B9) and 0.0068 µg/g (B12).

Benefits and Practical Application of the Method

This approach enables simultaneous, sensitive determination of three B vitamins in a single run, improving laboratory efficiency and reducing sample handling. Immunoaffinity cleanup and concentration steps enhance selectivity and detection sensitivity in complex food matrices.

Future Trends and Potential Applications

Integration with mass spectrometry could further lower detection limits and expand vitamin panels. Automated sample preparation and miniaturized detector flow cells may increase throughput. Application may extend to other fortified foods and quality control workflows.

Conclusion

The validated HPLC-DAD method on the Agilent 1260 Infinity II Prime LC system provides reliable, high-throughput analysis of vitamins B7, B9 and B12 in infant formula, with excellent sensitivity, precision and recovery.

References

• U.S. Food and Drug Administration. Daily Value on the Nutrition and Supplement Facts Labels. 2024.
• Cellar NA, McClure SC, Salvati LM, Reddy TM. A new sample preparation and separation combination for precise, accurate, rapid, and simultaneous determination of vitamins B1–B9 in infant formula and related nutritionals by LC-MS/MS. Analytica Chimica Acta 2016;934:180–185.
• R-Biopharm. Instruction Manual for EASI-EXTRACT MULTI-VIT B (LGE).
• R-Biopharm. Technical Data Sheet for EASI-EXTRACT MULTI-VIT B (LGE).
• R-Biopharm. Validation Report for EASI-EXTRACT MULTI-VIT B (LGE).
• Ting LJ, Lin YL. Analysis of Vitamin B12 in Infant Formula. Agilent Technologies Application Note 5994-6881EN; 2023.