| Summary: | <p>To confirm or disprove previous hypotheses, cyclic voltammetry of 0.5 mM cholecalciferol (vitamin D<sub>3</sub>) at glassy carbon electrode (GCE) and platinum disk electrode (PtE) in pure acetonitrile and water‑ethanol mixture at 50 mV·s<sup>‑1</sup> has been used to investigate the oxidation mechanism. The oxidation occurs in two one-electrone steps. According to calculation of the highest electron density in cholecalciferol molecule which is evidently delocalized over carbon atoms of the three conjugated double bonds (C19, C10, C5-C8) points to part of the molecule involved in oxidation processes. An oxidation peak (at +0.925 V vs. Ag/AgCl) was used to develop direct voltammetric method based on differential pulse voltammetry for the vitamin D<sub>3</sub> determination at GCE performed in 40% ethanol containing 0.1 M LiClO<sub>4</sub>. Under optimization of analytical procedure, it was found that a composition of the supporting electrolyte used significantly affects a current response of oxidation peak obtained. Satisfactory sensitivity was achieved in the 1:1 water‑ethanol mixture containing 0.05 M lithium perchlorate as as supporting electrolyte. The linear range for vitamin D<sub>3</sub> determination was <br /> 2.4 × 10<sup>-6</sup> - 3.5 × 10<sup>-4</sup> M with the detection limit of 8.0 × 10<sup>-7</sup> M. This work demonstrates a fact that the GCE is suitable electroanalytical device for analysis of various food supplements and medicaments.</p> <br />
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