Automated electrochemical determination of beer total antioxidant capacity employing microdialysis online-coupled with amperometry

Highlights

• Fully automated microdialysis system with multiple samples handling capability, directly coupled to amperometric detector.

• Applied to analysis of beers, the automated microdialysis coupled amperometry procedure provides the results that correlate well with conventional DPPH assay.

• Programmable detection system, able to perform electrochemical activation and cleaning of sensing element prior to measurement step.

• Low capital cost and very low running expenses.

• Green procedure - minimal chemicals loading, no harmful chemicals needed.

Abstract

Electrochemical techniques are rarely applied for the evaluation of total antioxidant capacity of beers due to severe electrode inactivation (fouling) by adsorbed oxidation products of the species present in the samples. In this work, we show that microdialysis online-coupled with amperometry is suitable for beer analysis, permitting also an assay automation. The working electrode of the amperometric detector employs a carbon fiber microelectrode that is regenerated between the analyses by short (1 sec) rapid (50 Hz) sinusoidal potential cycling between 0 and 2.9 V vs Ag/AgCl. This step, along with the sample cleanup by microdialysis effectively removes electrode fouling. The suitability of this approach for beer analysis was studied using eight different beers produced by international manufacturers. The results were compared with standard Kaneda diphenylpicrylhydrazil (DPPH) assay. Steady state currents, obtained at 600 mV vs Ag/AgCl correlate well with the results obtained with Kaneda method and thus can be used for quantification of beers' antioxidant capacities. HPLC-ED of microdialysates with and without DPPH treatment were employed to get an insight into DPPH reactivity with beer components.

Introduction

Easily oxidisable species in beers, mostly belonging to the group of polyphenolic compounds, are responsible for beer flavour, colour and taste stability as well as for microbial resistance of beers [1]. Beneficial effects of these compounds, acting as antioxidants is well known [2]. Moderate drinking of beer has beneficial effect on human health, namely suppression of rise of blood plasma lipoproteins following cholesterol containing diet, which effect is thought to be directly linked to beer antioxidants [3]. Nevertheless, quantitative analysis of individual antioxidants in food in general and in beer samples in particular represents a demanding analytical problem, therefore the contents of antioxidants in complex matrices are often determined indirectly and expressed as a collective parameter denoted as total antioxidant capacity [4].

For beer analysis, Kaneda assay of free radical-scavenging activity determination [5] based on DPPH decolorization with UV–VIS read-out is widely popular due to its simplicity and reliability. It is based on the reaction of free radical reactive antioxidants contained in the beer sample with the violet-colored stable DPPH radical. The reaction causes gradual decolorization of the reaction mixture due to the reduction of DPPH into colorless product, which process can be monitored spectrophotometrically (at 525 nm) or by EPR spectroscopy. The sample reduction activity (RA) is given as a difference between the signal at the beginning of the measurement and after a given amount of time (typically 10–30 min) of the reaction

In beers, a considerable part of antioxidant capacity is linked to the content of phenolic compounds [6], therefore a considerable interest is devoted in the literature to this topic. Phenolic compounds identified in beer include phenolic acids, flavonoids, proanthocyanidins, tannins, and amino phenolic compounds [7], [8], [9], [10]. In a detailed paper by Zhao et al. [11], the phenolic profiles of 34 Chinese and worldwide produced beers were determined and compared with corresponding antioxidant activities' data obtained using different methods. The contributions of individual polyphenols (nine different polyphenols, predominantly phenolic acids were analysed by HPLC) and their sum were correlated by the authors with antioxidant activities' data obtained using different methods. The values of Pearson's correlation coefficients were rather poor, in the case of DPPH assay ranged from R = -0.022 for p-coumaric acid to 0.701 for caffeic acid while for the sum of all studied phenolic compounds the figure was only 0.407. The authors arrived at a conclusion that total phenolic content might not be a good predictor for beer antioxidant activity and due to significant contribution of unidentified non-phenolic substances reacting with DPPH or to synergistic interactions between the involved antioxidants.

A range of other methods than DPPH assay is also available to determine beer antioxidant capacity, including electrochemical ones, of which cyclic voltammetry (CV) [12], [13], differential pulse voltammetry [14], (flow) coulometry [15], [16] or amperometry [17], [18] are mainly used. Electrochemical methods are based on the common property of radical scavenging antioxidants - their oxidisability, as antioxidant species are characterised by relatively low redox potentials [19]. Generally, the analytical signal for all above mentioned electrochemical methods is a current flowing through the working electrode at particular applied potential. The current is directly proportional to the concentration of the oxidisable species in the sample.

While the use of electrochemical methods to determine easily oxidisable species in wines [20], juices [21], body fluids [22] and similar real matrices is widespread, they were rarely used for beers. Automated evaluation of antioxidant capacity of beers, involving the combination of CV records and indirect amperometric determination using 2,6-dichlorophenol-indophenol (DCI) utilized Pt electrode, electrochemically regenerated by applying 0.2 Hz rectangular wave between 5 and −3 V vs NHE [23]. In this arrangement, CVs were featureless, almost identical for all studied beers and served rather as an intra-assay control which was based on amperometry in stirred solution at 0.75 V vs NHE. After the addition of oxidized form of DCI into a tested beer sample the rise in amperometric current was observed, as a result of the DCI reduction by the beer antioxidants.

The similarity and non-existence of clearly resolved peaks in beer voltammograms can be overcome by arrays of working electrodes made of different materials and processing of the obtained signals by advanced mathematical procedures. Electrochemical responses due to oxidizable species significantly contributes to performances of these “electronic tongues”, allowing to discriminate between different beer brands [24] or to assess beer aging [25]. Recently, direct electrochemical evaluation of beers by CV using screen printed electrodes was suggested by Roselló et al. [26], offering an alternative to the strategy based on chromatographic fingerprinting or voltammetric electronic tongues. CVs of beers were measured in a narrow potential range (-0.5 to 0.5 V vs Ag/AgCl). Rather than clearly discernible peaks, the rise in current was observed near the positive potential limit. Advanced data treatment techniques (support vector machine discriminant analysis and artificial neural networks were applied on CV current–potential pairs) allowed for the discrimination among beer types.

In summary, the difficulty in using electrochemical approaches towards beer analysis is caused by a non-existence of well developed peak(s) on cyclic voltammograms and by severe electrode fouling complicating repeated or continuous use of the working electrode. In this work we show that coupling microdialysis sampling and amperometric detection employing electrochemically regenerated cylindrical carbon fiber microelectrode (CFME) as sensing element allows for efficient determination of the total content of oxidisable compounds in beers, providing the results well correlated to DPPH assay according to Kaneda [5]. The developed coupled microdialysis-amperometry method can be fully automated using e.g. 96 well plate format. Taking advantage of easy application of the CFM detector in HPLC manifold, HPLC-ED of microdialysates with and without DPPH treatment was employed to get an insight into DPPH reactivity with beer components.