Assessing the activity of natural food antioxidants - Oxidation in Foods and Beverages and Antioxidant Applications - page 352

commonly in the form of extracts, can potentially contain a large number of
structurally related antioxidants which are difficult to isolate and identify
without using sophisticated procedures or instrumentation. Recently reported
methodologies that combine on-line separation and radical scavenging activity
of antioxidants are emphasized in this section. Analytical parameters such as
sample preparation procedures (drying, milling), extraction methods (agitation,
ultrasonic or microwave assisted), extraction temperature, solvent systems
(aqueous or organic), and sample clean-up or fractionation all influence the
composition and, consequently, the activity of the tested extracts. The
formation of artifacts cannot be excluded (characteristic is the formation of
carnosol or other diterpenes from carnosic acid upon storage and extraction of
rosemary) in certain cases, and therefore have to be considered together with
interferences due to the presence of other components (e.g., pigments, ascorbic
acid). It has been argued that kinetic parameters (e.g., EC 50 , T EC50 , AE) offer a
more comprehensive assessment of antioxidant capacity versus percent
inhibition. 152 The selection of suitable reference compounds, where necessary,
is not often straightforward. However, in general, a reference compound should
be structurally related to the antioxidants of interest. The widespread use of
Trolox is understandable because it is inexpensive and stable, but is not always
justified.
14.10 On-line chemical characterization and assessment of
antioxidants present in complex mixtures
A number of the so-called `high resolution screening assays' (HRS) were
developed during the last decade. 153,154 Such assays combine HPLC with a fast
post-column reaction, often with a solution of a chromogen free radical. 154 The
radical scavengers present in an extract are then identified by means of a UV
detector as negative peaks. The instrumentation employed for the reaction with
DPPH · is depicted in Fig. 14.3.
Integration of the respective negative peaks is used to calculate the % RSA of
each of the individual components that could scavenge the free radical. The
DPPH · and ABTS · assays 155,156 have been successfully adapted to HRS
systems, and have been optimized in order to increase sensitivity. 153
Recent advances in this field include in-line coupling of diode-array (DAD) or
ultra violet detectors (UV) with mass spectrometers (MS), or sample preparation
with solid phase extraction (SPE) followed by characterization with nuclear
magnetic resonance (NMR) spectrometers. LC-ESI/MS has been successfully
applied on line with DPPH · assay to simultaneously separate, characterize and
assess scavenging activity of complex natural phenolic mixtures. 157 Such
procedures offer information about the reactivity of individual compounds with
the radical; however, the kinetics of these reactions are overlooked, which means
that AE values cannot be obtained.