Matrix-Tolerant Liquid Chromatography–Tandem Mass Spectrometry Quantification of Vitamin B12 in Protein–Carbohydrate Systems

Abstract Quantification of vitamin B 12 in fortified and encapsulated protein–carbohydrate systems remains analytically challenging due to matrix-induced ionisation effects and the frequent reliance on cyanide-assisted conversion and solid-phase clean-up procedures. The aim of this study was to develop and validate a simplified, matrix-tolerant analytical method for reliable vitamin B 12 determination in encapsulation carrier systems without derivatization, enzymatic digestion, or solid-phase extraction. An optimised methanol–water extraction protocol, including evaluation of sequential extraction efficiency, was combined with ultra-high-performance liquid chromatography coupled to tandem mass spectrometry and applied to model whey protein isolate–maltodextrin matrices as well as additional carrier systems, including hydroxypropyl methylcellulose, gum Arabic, and zinc oxide. The extraction procedure achieved recoveries of 96–113% across different formulations, with negligible matrix effects (− 1.01 to + 0.80%). Validation demonstrated linear response ( R 2 > 0.99), limits of detection and quantification of 0.01 and 0.03 µg mL⁻ 1 , respectively, and intra-day and inter-day precision below 10%. The proposed workflow provides a streamlined alternative for vitamin B 12 analysis in encapsulated carrier systems, reducing sample preparation complexity while maintaining analytical performance.