Antioxidants and Bioactive Compounds in Fruits - Fruit Ripening: Physiology, Signalling and Genomics - page 107

(2005) suggested for the determination of
phenolic acids, hydroxycinnamic acid
derivatives, fl avonols and anthocyanins.
The quantifi cation of total carotenoids
using absorbance-based equations was
proposed by Nagata and Yamashita (1992).
In addition, there are protocols based on
derivatization of the studied compounds:
for example, the evaluation of fl avanol
content is performed by the reaction of
fl avanol with vanillin to produce a red
chromophore moiety (Tabart et al. , 2010).
Overall, these methods are rapid, low cost
and easy, and inexpensive instrument
set-up is required; however, other com-
pounds often interfere in these measure-
ments. The Folin-Ciocalteu reagent may
react with non-phenolic reducing sub-
stances like AsA, citric acid and sugars,
which are found at high concentrations in
fruit (Apak et al. , 2007), and produce
erroneous readings.
Traditionally, fruit extracts are subjected
to fractionation and isolation procedures in
order to obtain pure compounds for their
identifi cation. This laborious and time-
consuming step is also a prerequisite for
performing bioassays such as the evalu-
ation of antioxidant activity. Nowadays,
high-resolution screening techniques have
been developed that combine a separation
technique with fast post-column (bio)
chemical detection to allow the rapid
pinpointing of antioxidant compounds in
complex fruit extracts, without previous
isolation procedures (Niederlander et al. ,
2008). This on-line technique has been
applied successfully in large-scale routine
analysis of complex plant extracts.
Bandoniene and Murkovic (2002) demon-
strated the on-line 2,2-diphenyl-1-picryl-
hydrazyl (DPPH) method for evolution of
antioxidant activity of individual phenolic
compounds from different apple cultivars.
HPLC coupled with different detectors
has been thoroughly exploited for the
qualitative and quantitative determinations
of individual phytochemicals in fruits.
Reversed-phase chromatography is used for
the phytochemical study of polar com-
pounds such as phenolics, terpenes and
AsA, whilst less-polar compounds such as

carotenoids and tocopherols are analysed
by normal-phase chromatography. Mass
and UV-Vis detectors are the most com-
monly used in phytochemical analysis of
fruits. Techniques such as HPLC-MS with
diode array (HPLC-DAD-MS) and MS with
electrospray ionization (MS-ESI) have been
used widely for the identifi cation and
quantifi cation of phenols, carotenoids and
vitamin C in fruits (Sancho et al. , 2011).
Fluorescence detectors have also been used
for these purposes for the determination of
fl uorescent secoiridoid compounds of olive
fruits or the determination of triterpenic
acids with on-line derivatization (Li et al. ,
2011). Fluorescence detection is also
preferred for the quantifi cation of phyto-
chemicals at lower concentrations. The
combination of LC with electrochemical
detection is often used for the quan-
tifi cation of catechins (Trojanowicz, 2011).
On the other hand, the application of GC is
quite limited because: (i) an extra step of
derivatization is usually required; and (ii)
many phytochemicals are degraded at high
temperatures. However, GC is often used
for the analysis of terpene composition.
The derivatization of fl avonoids and
phenolic acids for GC-MS analysis has also
been described (Proestos et al. , 2006).
NMR spectroscopy is one of the most
powerful instrumental analysis techniques
for phytochemical characterization. One-
and two-dimensional methodologies pro-
vide an array of experiments for the
structure elucidation of phytochemicals.
NMR spectroscopy allows the simul-
taneous detection and quantifi cation of
individual phytochemicals of all groups in
a complex fruit extract, thus avoiding time-
consuming and tedious chromatographic
techniques for their separation. Recently,
Charisiadis et al. (2011) demonstrated the
utility of ultrahigh-resolution hydroxyl
group 1 H-NMR analysis, creating new
directions for phytochemical analysis.
Nowadays, NMR fi ngerprints are also used
to compare the phytochemical composition
of different fruits (Ali et al. , 2011; Kim et
al. , 2011).
The antioxidant activity/antioxidant
capacity per se is a well-established