Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system

Abstract

It is estimated that up to 50% of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O(2). The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t(90) = 1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.

Measurement of antioxidant activity

“…In the recently developed CHROMAC (chromium reducing antioxidant capacity) assay, formation of Cr 3+ complex from reduction of Cr 6+ complex by antioxidants gave rise to absorbance increase at 540 nm (Isik, Sahin, & Demir, 2013). Quaranta et al (2013) proposed an enzyme-based method for evaluating reducing power of antioxidants. This method does not involve metal ions, but rather indirectly measures the reduction of azobismethylene quinone to syringaldazine by antioxidants.…”

Identification and Anti‐oxidant Capacity Determination of Phenolics and their Glycosides in Elderflower by On‐line HPLC–CUPRAC Method

“…As identification and quantification of anti-oxidant compounds in plant matrices may give rise to isolation and purification problems, certain assays have been modified recently for on-line HPLC determinations with post-column detection (Dapkevicius et al, 1999(Dapkevicius et al, , 2001Ogawa et al, 1999;Koleva et al, 2000Koleva et al, , 2001Raudonis et al, 2012;Quaranta et al, 2013). An on-line method combining separation of components in the complex matrix and evaluation of anti-oxidant capacity can provide important benefits for detecting/quantifying active compounds in complex mixtures.…”

“…The primary aim of this study was to evaluate the anti‐oxidant properties of the methanolic extracts of S. nigra (flower part) by on‐line HPLC–CUPRAC (cupric reducing anti‐oxidant capacity) assay (Çelik et al ., ) with post‐column derivatisation. Certain assays have been developed for on‐line monitoring and measurement of anti‐oxidant capacity/activity by the use of HPLC (Dapkevicius et al ., ; Ogawa et al ., ; Koleva et al ., , ; Dapkevicius et al ., ; Raudonis et al ., ; Quaranta et al ., ). The most widely used assays in post‐column applications are free radical decolorisation methods, based on the scavenging of chromogenic free radicals DPPH (2,2’‐diphenyl‐1‐picryl hydrazyl) (Koleva et al ., ) or ABTS (2,2’‐azinobis‐3‐ethyl‐benzothiazoline‐6‐sulphonic acid) (Koleva et al ., ).…”

Antioxidant components of Viburnum opulus L. determined by on-line HPLC–UV–ABTS radical scavenging and LC–UV–ESI-MS methods

“…On-line HPLC-UV-ABTS method has been shown to be superior in sensitivity, selectivity, and relative simplicity for antioxidant capacity analysis, and applied for quick identification of antioxidant components of various plant extracts, foods, and beverages (He et al, 2010;Pellegrini, Rio, Colombi, Bianchi, & Brighenti, 2003). Among the increasing number of methods available to determine the antioxidant capacity of food extracts are online HPLC-FRAP assay (Burnaz, 2013;Raudonis, Raudone, Lakstas, & Janulis, 2012), online HPLC-CUPRAC assay (Celik, Ozyürek, Güclü, & Apak, 2010), online HPLC-DTNB assay (Ozyürek et al, 2012), and enzyme-based online assay (Quaranta et al, 2013).…”