Direct and Cobalt-Reduction Based Spectroscopic Determination of Vitamin B12

This study covers the development of a hybrid analysis method aimed at determining Vitamin B12 (cyanocobalamin) by utilizing both its chemical structure and redox properties. In the first stage of the study, a calibration curve was created over the characteristic absorbance peaks at a wavelength of 361 nm using ultraviolet-visible (UV-Vis) spectrophotometry. This curve was used as a reference for method accuracy and quantitative determination. In the second stage, an electrochemical reduction process was performed by utilizing the electroactive nature of Vitamin B12. In this process, the cobalt ion in the molecule was reduced from Co (III) to Co (II) by applying a certain potential to the solution. After the electrochemical process, the sample solutions were re-analyzed using a UV-Vis spectrophotometer, and the changes in the spectrum were examined. A significant improvement in analytical performance was observed when using the rB12 form. While the detection limit for B12 was determined as 0.004 mM, the linear range was 0.012–0.01 mM, and the correlation coefficient was 0.9975, the detection limit for rB12 decreased to 0.002 mM, the linear working range widened to 0.006–0.1 mM, and the correlation coefficient increased to 0.9993. Notably, the reduced form of B12 exhibited superior analytical performance compared to the non-reduced form, with improved sensitivity, wider linear range, and lower detection limit, highlighting the method’s enhanced applicability to redox-responsive analytes. This method also contributes to the development of new generation hybrid analysis methods for vitamins, pharmaceutical active ingredients, and biomolecules with redox properties.