SPECTROPHOTOMETRIC DETERMINATION OF ASCORBIC ACID BY HORSERADISH PEROXIDASE

Vladimir Đurić, N. Deletic
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Abstract

L-ascorbic acid is one of the essential nutrients and most common food supplements, fortificants, and preservatives. It is commercially available as solutions, drops, tablets, capsules, crystal powder, beverage mixtures, multivitamin formulations, and multi antioxidant formulations. The usual daily dose is from 25 mg to 1.5 g. Ascorbic acid is a distinctly reducing agent with low redox potential (0.18 and 0.08 V at pH 4.5 and 6.4, respectively). Based on ascorbate property, numerous methods for its quantitative determination are developed, from titrimetric, electrochemical, and chromatographic methods, to fluorometric and kinetic ones. Enzyme peroxidase is interfered with by ascorbic acid, which decreases the oxidation speed of its co-substrates during hydrogen peroxide decomposition by peroxidase. Absorbance changes at the wavelength of corresponding reagents are in correlation with ascorbate concentration. During this study, benzidine and o-tolidine have been used as chromogenic reagents. Reaction conditions were optimized for various buffer systems, calibration curves were constructed, and limits of detection (0.04 μmol/L) and quantification (0.12 μmol/L) were calculated. Using calibration charts, it was possible to detect ascorbic acid within limits from 0.4 to 10 μmol/L. The optimized method was applied for the determination of ascorbic acid in pharmaceutical products. The method was characterized by exceptional sensitivity and accuracy, but only for preparations not containing substances that affect enzyme peroxidase.
辣根过氧化物酶分光光度法测定抗坏血酸
l -抗坏血酸是一种必需营养素,也是最常见的食品补充剂、强化剂和防腐剂。它是商业上可用的溶液、滴剂、片剂、胶囊、结晶粉末、饮料混合物、多种维生素配方和多种抗氧化剂配方。通常每日剂量为25毫克至1.5克。抗坏血酸是一种明显的还原剂,具有较低的氧化还原电位(在pH为4.5和6.4时分别为0.18和0.08 V)。基于抗坏血酸的性质,从滴定法、电化学和色谱法到荧光法和动力学法,开发了许多定量测定方法。过氧化物酶受到抗坏血酸的干扰,在过氧化物酶分解过氧化氢的过程中,抗坏血酸降低了其共底物的氧化速度。相应试剂波长处吸光度变化与抗坏血酸浓度相关。本研究以联苯胺和邻甲苯胺为显色试剂。优化了不同缓冲体系的反应条件,建立了标定曲线,计算了检测限(0.04 μmol/L)和定量限(0.12 μmol/L)。通过校正图,可以在0.4 ~ 10 μmol/L范围内检测到抗坏血酸。该方法可用于药品中抗坏血酸的测定。该方法具有特殊的灵敏度和准确性,但仅适用于不含影响酶过氧化物酶的物质的制剂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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