Development of a spectrofluorometric method for selective and sensitive determination of zinc in human Plasma: Implications for olfactory function

Abstract

The relationship between zinc levels and olfactory function is an area of active research due to the role of zinc-dependent metalloenzyme, carbonic anhydrase, in maintaining olfactory function. In this study a spectrofluorometric method was developed for determining zinc levels in the human plasma of healthy participants and patients with olfactory dysfunction. The method relies on the fluorescence property changes of 4-Methylesculetin, which interacts with zinc. In this interaction, the oxygen atom of hydroxyl groups serves as a ligand, coordinating with zinc as a Lewis acid. As a result, the formed complex exhibits significant fluorescence enhancement attributed to the rigid chelate structure, thereby enhancing quantum yield. The resulting complex displays emission peak at 520 nm, which is distinct from the emission peak of 4-Methylesculetin at 460 nm. This bathochromic shift indicates heightened excited-state energy, attributed to zinc coordination and complex formation. The reaction conditions were carefully optimized to ensure selective zinc determination in plasma samples. The developed method demonstrates linearity over a concentration range of 200–4000 ng/mL, with a lower limit of quantification of 115 ng/mL. When applied to quantify plasma zinc levels, the method revealed significantly decreased zinc levels in patients with olfactory dysfunction compared to healthy individuals. These results suggest a potential link between olfactory dysfunction and zinc levels (normal vs. patient; 1150 ± 19 ng/mL vs. 940 ± 14 ng/mL, p < 0.05). The developed method was validated following the ICH M10 guidelines and provided a feasible investigative link between zinc and olfactory function.