Elsevier

Journal of Chromatography A

Volume 1210, Issue 2, 14 November 2008, Pages 121-134
Journal of Chromatography A

Review
Antioxidant activity assays on-line with liquid chromatography

https://doi.org/10.1016/j.chroma.2008.09.061Get rights and content

Abstract

Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper both electrochemical and chemistry-based assays are reviewed and discussed. The focus is on the mechanisms involved and differences between the assays, rather than on the matrix or analytes. With 45 applications high resolution antioxidant screening has now become an almost routine tool for the rapid identification of antioxidants in plant extracts, foods and beverages. The methods based on true reactive oxygen species (ROS) provide the most realistic measure of antioxidant activity. Unfortunately these methods are difficult to set up and control and have not been applied since they were reported. The methods based on electrochemical detection are more practical, but have still received only limited attention for practical screening purposes. The methods based on a single relatively stable reagent such as DPPHradical dot and ABTSradical dot+ have become most popular, because of their simple set-up and ease of control. The methods have been combined with on-line DAD, MS and NMR detection for rapid identification of active constituents.

Introduction

Oxygen is of vital importance to aerobic organisms. Under controlled circumstances, e.g. inside mitochondria, endoplasmatic reticulum, or peroxisomes, oxygen generally serves a metabolic purpose [1], [2], [3], [4]. Even under normal circumstances though a small percentage of the electrons passing through the mitochondrial electron transport chain leaks out and combines with molecular oxygen to form reactive oxygen species (ROS) [1], [2], [5], [6]. Biologically important ROS include superoxide anion (O2radical dot), singlet oxygen (O2 1Δg), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), hydroxyl radical radical dotOH, peroxynitrite ONOO and, peroxy and alkoxy radicals [1], [7]. These ROS can cause damage at various sites in the cell (membranes, cytoplasm and nucleus). To avoid damage, the cell contains various enzymes and antioxidants to provide protection in these cell compartments [1], [2], [3], [4], [8], [9]. However, numerous factors (e.g. disease, drugs, pollution and malnutrition) can adversely affect production of ROS and/or protection mechanisms, leading to oxidative stress.

Oxidative stress can be defined as “an imbalance between oxidant formation and antioxidant-repair capacity” [2]. It has been established that oxidative stress is involved in numerous human diseases, including cancer, atherosclerosis, Alzheimer’s disease, inflammation, asthma, rheumatoid arthritis [1], [2], [3]. Furthermore, cellular damage as a result of ROS is believed to be involved in the process of ageing [1], [2], [3]. There is abundant scientific consensus to acknowledge the general importance of antioxidants in counteracting the effects of oxidative stress [1], [2], [3], [4], [10]. Providing protection against oxidative damage with antioxidants is also an important issue in preservation of food (products), cosmetics and many other consumer products [11], [12], [13], [14], [15]. Consequently, antioxidants and antioxidant (bio)chemistry are currently the subject of intensive research interest [1], [2], [3], [4]. Besides research under real circumstances, including in vivo studies [16], assessing the potency of (potential) antioxidants requires in vitro model systems to investigate antioxidant activity under relatively simple and controlled circumstances [7]. Various batch type assays have been developed for this purpose [17]. These assays are well suited for the determination of antioxidant activity of individual pure antioxidants, or total antioxidant activity of mixtures of antioxidants or antioxidants in complex matrixes. In the latter case(s) however, determining the contribution of individual antioxidants in the mixture is a difficult task.

To avoid the time-consuming and skilful work associated with activity guided or bioassay guided fractionation, so-called high resolution screening (HRS) was developed. This technique combines an efficient separation technique like GC or HPLC with a fast post-column (bio)chemical detection step. HPLC can be combined with many different assays, for GC this is mostly restricted to pheromone research with insect antennas [18], [19]. A schematic lay-out of the hardware of high resolution antioxidant screening by HPLC is presented in Fig. 1. A major constraint in high resolution screening is finding a (bio)assay that is compatible in terms of time-scale and (bio)chemical conditions with HPLC separations. In the past 10 years several HRS assays have been published [20], [21], [22], [23], [24].

As the interest in antioxidants is on the rise, high resolution assays for the rapid screening of antioxidants in multi-component extracts have been independently developed by an American [25], a Japanese [26], [27], a Dutch [28], [29], [30], a Spanish [31], and an Italian group [32]. In the most popular approach, a coloured, relatively stable radical such as DPPH or ABTS, is added post-column to the HPLC flow, and antioxidants are detected by a decrease in absorbance at visible wavelengths due to the conversion of radicals to their non-coloured reduced form. Since the first papers in 1996, about 45 application papers on on-line antioxidant analysis have been published, most of them in the last 3 years. Although some of them have been briefly discussed in some more general reviews [33], [34], [35], [36], [37], no comprehensive review that specifically focuses on on-line antioxidant activity screening has yet appeared. In this paper we review and discuss all on-line antioxidant activity assay papers and some relevant flow injection assays (FIA). Special emphasis is put on the mechanism underlying these assays and the surplus value offered by these assays as well as their limitations. Both on-line antioxidant activity assays using electrochemical detection and chemistry-based methods are discussed and compared.

At this point it is good to recall the difference between an antioxidant and a radical scavenger as often these terms are both used in antioxidant literature. In principle, any radical scavenger can be an antioxidant if it terminates radical chain propagation. Radical scavengers are the largest class of antioxidants. In addition antioxidant classes such as metal chelators, oxygen scavengers, UV light absorbers and enzymatic antioxidants (dismutases, catalases and peroxidases) are recognised (note that, e.g. superoxide dismutase is in essence also a scavenger). Moreover, antioxidants will react with ROS in a mode that is largely determined by the ROS. The same antioxidant may react as a scavenger with superoxide anion, but be nitrated (as a substitute substrate) by peroxynitrite.

In the sections discussing applications of on-line assays for antioxidant screening, a more practical approach is taken and all papers making use of on-line assays for the rapid pinpointing of antioxidant peaks in chromatograms are reported. The more interesting ones are briefly discussed. The focus is on the evolution and differences between the assays, rather than on the matrix or analytes.

Section snippets

Categories of antioxidant activity assays

In this review, three major categories of antioxidant activity assays will be distinguished:

  • 1.

    Assays involving actual ROS–oxidizable substrate interactions,

  • 2.

    Assays involving a relatively stable single oxidizing reagent.

  • 3.

    Assays relating antioxidant activity to electrochemical behaviour.

With the first category of assays, the oxidizing agents are those also active in biological systems and playing a role in consumer product deterioration. These are applied to oxidise a substrate of which the

Conclusion

The first on-line HPLC high resolution screening for antioxidants in plant extracts took place by means of a multi-channel coulometric array electrochemical detector in 1997 [96]. With this approach Guo et al. detected antioxidants in many herbs, fruits and vegetables (see Table 3) [96]. One year later Mannino et al. used on-line amperometric detection for the same purpose [32]. Both coulometric and amperometric assays were successfully applied in subsequent years (see Table 3) but not on a

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