ANALYSIS OF THIOL-TYPE COMPOUNDS IN PHARMACEUTICALS USING CUPRAC AND HPLC ASSAYS
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Multi-level defence systems (endogenous antioxidants, e.g., glutathione, cysteine, uric acid) counteract oxidative stress driven alteration of numerous biological molecules and their involvement in various diseases such as coronary and cardiovascular diseases, mutagenism, celi ageing, and diseases of the immune system. An antioxidant is any substance that, when present at low concentrations compared to those of an oxidizable substrate, signifîcantly delays or prevents oxidation of that substrate [1], and therefore, determination of total antioxidant capacity is important. Thiol-type (-SH) organic compounds are an important class of antioxidants. An important thiol-type antioxidant, N-acetylcysteine (NAC), exhibits direct and indirect antioxidant properties. its free thiol group is capable of interacting with the electrophilic groups of reactive oxygen species (ROS). The basic aim of this study is to apply the CUPRAC (cupric ion reducing antioxidant capacity) assay [2] originally developed in our laboratories to thiol-type antioxidants (e.g., N-acetylcysteine) and compare the results with other electron transfer (ET)-based assays (ABTS-persulfate and FRAP methods) and also with HPLC. Using the CUPRAC method, the total antioxidant capacity is measured by reduction of the chromogenic oxidant, Cu(II)-neocuproine reagent, with antioxidants to the Cu(I)-neocuproine chelate showing maximum absorbance at 450 nm. Thiol-type antioxidants, namely NAC, cysteine, cystine, homocysteine, homocystine, ete. were used in Standard solutions, and assayed using the normal (at room temperature), incubated (at 50 °C), hydrolyzed (at 80 °C) and hydrolyzed & incubated (at 80 °C) CUPRAC methods against trolox as the Standard reference compound. The same antioxidant solutions were erossassayed with ABTS/persulfate as the reference spectrophotometric method. Brunac eye drop (containing 5% N-acetylcysteine) and Trom effervescent tablet (containing 600 mg Nacetylcysteine) were simultaneously analyzed with CUPRAC and HPLC methods. The findings of the CUPRAC method for both pharmaceuticals (0.050 g mL' 1 NAC in Brunac; 619 mg NAC in Trom) were statistically alike with those of HPLC (0.054 g mL' 1 NAC in Brunac; 609 mg NAC in Trom).
[1] R. Apak,et al. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. , 2004, Journal of agricultural and food chemistry.
[2] B. Halliwell,et al. Free Radicals and Antioxidants in the Year 2000: A Historical Look to the Future , 2000, Annals of the New York Academy of Sciences.