Effect of Ascorbic Acid and Reducing Agents on the Photolysis of Cyanocobalamin

A study of the photolysis of cyanocobalamin (vitamin B12) (B12), aquocobalamin (B12a) and hydroxocobalamin (vitamin B12b) (B12b) in the presence of ascorbic acid (vitamin C) (AH2) has been carried out in the pH range 2.0-12.0 using a visible radiation source. It involved the development of a multicomponent spectrophotometric method for the simultaneous determination of B12, its photoproduct aquocobalamin (B12a)/hydroxocobalamin (B12b), and AH2 at 550, 525 and 265 nm (pH 4.0). These wavelengths correspond to the absorption maxima of the three compounds and thus provide high specificity and sensitivity to the method. The method has been validated with respect to various analytical parameters. The recovery of the method for the three compounds ranges from 97.1-103.0% with a RSD value of ± 3%. The accuracy of the analytical method has been shown by the linearity of the kinetic plots in the concentration range studied. The method is simple, rapid and convenient for the purpose of assay and can also be used for the determination of B12 and B12b only at 550 and 525 nm in photolysed solutions. B12b only in the presence or absence of AH2 in photolysed solution can be determined at 525 nm. The photolysis of B12 and B12a/B12b in the presence of AH2 at pH 2.0-12.0, under aerobic conditions, follows first-order kinetics and the values of apparent first–order rate constants (kobs) at pH 2.0 to 12.0 range from 0.37–2.63×10–4 s–1 and 0.21–6.35×10–4 s–1, for B12 and B12a/B12b, respectively. The second–order rate constants (k2) for the photochemical interaction of AH2 and B12 and AH2 and B12a/B12b range from 0.20 (pH 2.0)–1.09 ×10–2 M–1s–1 (pH 5.0) and 5.88 (pH 2.0)–91.08 ×10–2 M–1s–1 (pH 5.0), respectively. The values of the k2 for AH2– B12a/B12b interaction are 30–80 times greater than those of AH2–B12 suggesting a greater susceptibility of B12a/B12b to photodegradation compared to that of B12 in this pH range. The k2–pH profiles for both B12 and B12a/B12b are bell–shaped curves indicating the effect of AH2 ionization on the rates of their interaction. The complete discoloration of B12 and B12a/B12b solutions on prolonged photolysis indicates the formation of corrin ring cleavage (oxidation) products in acid and alkaline solutions. These oxidation products do not absorb in the visible region and, therefore, do not interfere with the assay of B12 and B12a/B12b in the visible region. Reaction schemes for the mode of photodegradation of B12 and B12b in the presence of AH2 have been presented. The involvement of a short lived ligand-to-metal charge transfer (LMCT) intermediate state in the photolysis of B12 and the formation of different singlet states, on the photoexcitation of B12a/B12b, leading to the photodissociation of Co-OH bond by different mechanisms have been suggested. The photolysis of B12-cysteine and B12a/B12b-cysteine complexes has also been studied in the pH range 2.0-12.0 using a two-component spectrometric method by absorpbance measurements at 550 and 531 nm, the absorption maxima of B12-CY and B12b-CY complexes, respectively. The kinetics of these systems has been evaluated and the association constants for these complexes have been determined. The photolysis of B12 and B12b in the presence of ferrous ions has also been studied and the second-order rate constants for their photochemical interaction have been determined. The rates of interaction are affected by the pH of the medium as a result of the conversion of Fe2+ ions to Fe (OH)2 above pH 5.0.