The Analysis of Vitamin B12 in Infant Formula

Importance of the Topic

Vitamin B12 is a crucial micronutrient required for infant development, supporting DNA synthesis, cellular metabolism, and neurological function. Its content in infant formula must be accurately measured at trace levels to ensure nutritional adequacy and regulatory compliance. Analytical methods that combine high sensitivity, selectivity, and rapid turnaround are essential for quality control in the food and beverage sector.

Study Objectives and Overview

This application note details the development and validation of a streamlined liquid chromatography method for quantifying free cyanocobalamin in infant formula. The target was to achieve a sub-ppb detection limit within a 3.5-minute runtime, referencing AOAC Official Method 2014.02 but omitting cyanide reagents in sample preparation.

Methodology

Sample preparation involved aqueous extraction, heat-assisted release of protein-bound cobalamins, and immunoaffinity cleanup to remove matrix interferences. The extract was evaporated and reconstituted prior to analysis.

Used Instrumentation

• Agilent 1260 Infinity II Prime LC system with flexible pump, multisampler, and multicolumn thermostat
• Agilent 1290 Infinity II diode array detector equipped with a 60 mm InfinityLab Max-Light cartridge flow cell
• Poroshell 120 SB-Aq column (3.0 × 100 mm, 2.7 µm) capable of 800 bar operation
• Agilent OpenLab CDS software version 2.7 for data acquisition and processing

Main Results and Discussion

• Limit of quantification achieved was 1.2 ng/mL, based on a 10:1 signal-to-noise ratio.
• Calibration across 1–250 ng/mL demonstrated excellent linearity (R2 ≈ 1.000).
• Six consecutive injections at 3 ng/mL yielded retention time RSD < 0.1% and peak area RSD 1.16%, confirming high repeatability.
• Recovery testing with NIST-certified reference material (NIST1869) gave an average of 88.8%, consistent with immunoaffinity column supplier claims (70–110%).
• Analysis of a commercial infant formula sample returned 7.1 ng/g of vitamin B12, with a clear chromatographic peak and minimal matrix interference.

Benefits and Practical Applications

Compared to traditional microbiological assays, this LC-DAD approach offers improved selectivity and precision, faster throughput, and elimination of cyanide reagents. The method is well suited for routine QC of fortified infant nutrition products and can be adapted for related food matrices.

Future Trends and Applications

Advancements may include coupling immunoaffinity cleanup with mass spectrometry for structural confirmation, miniaturized flow cells for enhanced sensitivity, and automation of sample preparation. Such developments will further streamline vitamin analysis in complex food systems.

Conclusion

A robust, fast, and sensitive LC-DAD method was established for vitamin B12 in infant formula, meeting regulatory needs and offering superior performance in routine laboratory workflows.

References

1. Zempleni J.; Gregory J. F.; Stover P. J.; Suttie J. W.; Green R. Vitamin B12. In Handbook of Vitamins, 5th Edition, Taylor & Francis, 2014, pp 447–489.
2. Heudi O.; Kilinç T.; Fontannaz P.; Marley E. Determination of Vitamin B12 in Food Products and Premixes by RP-HPLC and Immunoaffinity Extraction. J. Chromatogr. A 2006, 1101, 63–68.
3. Martin F.; Giménez E. C.; Konings E. New Methods for the Analysis of Water-Soluble Vitamins in Infant Formula and Adult/Pediatric Nutritionals. J. AOAC Int. 2016, 99, 19–25.
4. Gimenez E. C. Vitamin B12 Analysis in Food: AOAC Official Method 2014.02. J. AOAC Int. 2014, 97, 1397–1402.
5. R-Biopharm. EASI-EXTRACT VITAMIN B12 (LGE) Instruction Manual.