Robust LC–MS/MS Method for Quantification of Ascorbic Acid in Infant Formula and Heat-Processed Donor Human Milk: A Stabilization and Matrix Effects Mitigation Approach

Authors

• Omar M. Al Kilani, University of Nebraska Medical CenterFollow
• Lee C. Winchester, University of Nebraska Medical CenterFollow
• Melissa Thoene, University of Nebraska Medical CenterFollow
• Colman Freel, University of Nebraska Medical CenterFollow
• Christopher Lee Shaffer, University of Nebraska Medical CenterFollow
• Anthony T. Podany, University of Nebraska Medical CenterFollow

Document Type

Article

Journal Title

Journal of Food Composition and Analysis

Publication Date

2026

Volume

158

Abstract

Ascorbic acid is an essential antioxidant for infant health; however, its quantification in infant formula and human milk is challenging due to matrix complexity, analyte instability, and lack of blank matrix equivalents. This study presents a simple and robust LC-MS/MS method that bypasses the need for standard addition using only 20 µL sample. The method showed excellent linearity (R² > 0.99), precision (CV < 15%), accuracy (±10%), and sensitivity (LOD < 0.0375 µg/mL). Stock and working solutions remained stable for months at −20 and −80 °C, respectively, avoiding daily preparation. Optimized extraction substantially reduced matrix effects. The method was externally validated using certified infant formula reference material and successfully applied to commercial samples, with incurred sample reanalysis showing <  20% variation. This validated method offers a practical tool for the routine quantification of ascorbic acid in heat-processed donor human milk and infant formula, with the potential for application to other matrices.

DOI Link

10.1016/j.jfca.2026.109452

ISSN

1096-0481

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

Recommended Citation

Al Kilani, Omar M.; Winchester, Lee C.; Thoene, Melissa; Freel, Colman; Shaffer, Christopher Lee; and Podany, Anthony T., "Robust LC–MS/MS Method for Quantification of Ascorbic Acid in Infant Formula and Heat-Processed Donor Human Milk: A Stabilization and Matrix Effects Mitigation Approach" (2026). Journal Articles: Pediatrics. 60.
https://digitalcommons.unmc.edu/com_peds_articles/60