LC-MS/MS Method for Sensitive Detection and Quantitation of 8 Water-Soluble vitamins in Infant Milk Powder

Significance of the Topic

Accurate determination of water-soluble B vitamins in infant milk powder is critical to ensure compliance with nutritional standards and support healthy growth. Conventional LC and HPLC methods often require extensive clean-up or are limited to a subset of vitamins. The adoption of a sensitive LC-MS/MS approach streamlines analysis and enhances detection in complex matrices.

Objectives and Study Overview

This study aims to establish and validate a rapid, accurate LC-MS/MS method for simultaneous quantitation of eight water-soluble B vitamins (B1, B2, B3 as nicotinic acid and nicotinamide, B5, B6, B7 and B9) in infant formula. The method employs stable isotope-labeled internal standards to correct for matrix effects and improve quantitation reliability.

Methodology

Sample Preparation:

• Weigh 1 g of powdered formula into a polypropylene tube.
• Add 10 mL water and vortex, followed by 30 mL of 1% acetic acid.
• Vortex, centrifuge and filter the supernatant through a 0.22 μm nylon filter.
• Dilute filtrate fivefold with water prior to injection.

Quantitation is based on calibration curves prepared from individual vitamin stocks and five isotope-labeled internal standards across 10–750 ng/mL.

Used Instrumentation

Shimadzu LCMS-8060 equipped with electrospray ionization in positive mode and a Shim-pack GIST C18-AQ column (2.1×50 mm, 1.9 μm). Mobile phase consists of 5 mM ammonium acetate with 0.1% formic acid in water (A) and methanol (B) under a gradient elution. MRM transitions were optimized for each analyte.

Main Results and Discussion

The method demonstrated excellent linearity (r2>0.999) over the specified range, limits of detection between 0.08 and 1.36 ng/mL, and quantitation limits from 0.23 to 4.13 ng/mL. Repeatability (n=6) yielded relative standard deviations below 4% for all vitamins. Recovery experiments in infant formula matrix ranged from 90.1% to 113.6%. Quantitation of a NIST 1849a reference material produced results within certified ranges.

Benefits and Practical Applications of the Method

This approach combines high sensitivity, simple sample preparation and robust quantitation for routine quality control of infant formula and related matrices. Isotope dilution compensates for matrix effects and improves accuracy.

Future Trends and Potential Applications

Further integration of high-throughput sample automation, expansion to additional micronutrients and coupling with data analytics will enhance screening workflows. Adaptation of this method to other food matrices and fortified products can support broader nutritional monitoring.

Conclusion

A validated LC-MS/MS protocol for eight water-soluble B vitamins in infant milk powder has been developed. It delivers reliable sensitivity, precision and accuracy suitable for regulatory and research laboratories.

References

1. Cellar NA, McClure SC, Salvati LM, Reddy TM. Anal Chem Acta. 2016;934:180–185.
2. Goldschmidt RJ, Wolf WR. Anal Bioanal Chem. 2010;397(2):471–481.