Determination of Total Antioxidant Capacity Using Thiamine as a Natural Fluorescent Probe

This work proposes a spectrofluorometric method for the determination of total antioxidant capacity (CAO) in beverage samples, based on inhibition of thiochrome formation (λex = 370 nm, λem = 440 nm); a product of thiamine (vitamin B1) oxidation from K3Fe(CN)6. In the development of the method, gallic acid (GA) was used as a reference, and inhibition of thiochrome formation (in percentage) was used as the analytical response. The selectivity of the method was evaluated using seven different compounds (gallic acid, ascorbic acid, quercetin, butylhydroxytoluene (BHT), 6-hydroxy-2,5,7,8-tetramethylchroman-2 (Trolox), cysteine, and glucose). As proof of concept, the proposed method was applied in the determination of CAO in different samples, such as teas and infusions, red wines, and white wines. The results were compared with the Folin-Ciocalteu (FC), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+), and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) methods using the linear correlation between the methods (at 95% confidence). We observed excellent agreement between the proposed method and FC (correlation coefficient (r) = 0.9768) and ABTS•+ (r = 0.9842), compared with DPPH• method (r = 0.8502). For the determination of total CAO in beverages, the proposed method developed proved to be fast, sensitive, simple, and the results were in agreement with established assays.本研究提出一种荧光分光光度法（spectrofluorometric method），用于测定饮料样品中的总抗氧化能力（total antioxidant capacity，CAO），该方法基于抑制硫色素（thiochrome）的生成——硫色素为硫胺素（thiamine，维生素B1）经铁氰化钾（K3Fe(CN)6）氧化后的产物，其激发波长λex=370 nm，发射波长λem=440 nm。 在方法开发过程中，本研究以没食子酸（gallic acid，GA）作为参比物质，以硫色素生成的抑制率（百分比形式）作为分析响应信号。 本研究选取7种不同化合物，即没食子酸、抗坏血酸（ascorbic acid）、槲皮素（quercetin）、丁羟甲苯（butylhydroxytoluene，BHT）、6-羟基-2,5,7,8-四甲基色满-2（6-hydroxy-2,5,7,8-tetramethylchroman-2，Trolox）、半胱氨酸（cysteine）及葡萄糖（glucose），对该方法的选择性进行了评价。 作为概念验证，本研究将所提出的方法应用于多种样品的总抗氧化能力测定，涵盖茶及浸提液、红葡萄酒与白葡萄酒。 以95%置信度下的方法间线性相关性为基准，将本方法的测定结果与福林-酚法（Folin-Ciocalteu，FC）、2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)自由基（ABTS•+）法以及2,2-二苯基-1-苦肼基自由基（DPPH•）法进行了对比。 结果显示，本方法与福林-酚法（相关系数r=0.9768）、ABTS•+法（r=0.9842）均表现出极佳的一致性，而与DPPH•法的相关性仅为r=0.8502。 针对饮料样品总抗氧化能力的测定，本研究所开发的方法展现出快速、灵敏、简便的优势，其测定结果与现有成熟检测方法具有良好的一致性。