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2 Analytical Techniques in Food Biochemistry 35neither ion is able to cause direct oxidative damage to easily ox-
idized biological molecules such as lipids, proteins, and DNA.
Additionally, the assay is unable to measure the effects of antiox-
idants that act strictly on HAT (mechanisms such as thiols and
proteins), leading to underestimation of serum AOC but may be
useful in combination with other assays to differentiate between
antioxidant mechanisms.
Both the TEAC and other ABTS (2,2′-azinobis[3-
ethylbenzothiazoline-6-sulfonic acid] di-ammonium salt) assay
as well as DPPH assay (1,1-diphenyl-2-picryl-hydrazyl) use in-
dicator radicals that may be neutralized by either HAT or ET
mechanisms. In the TEAC (and its related assays), the long
life anion ABTS molecule is oxidized to its cation radical form
(ABTS·+) by peroxyl radicals, forming an intensely colored liq-
uid. The AOC is determined by the ability of the antioxidant to
decrease the color by reacting with the cation, which is measured
with a spectrophotometer at 415, 645, 734, and 815 nm (Prior
et al. 2005). As with ORAC, the results are expressed in TE. This
assay offers advantages over some of the other assays described
previously. Its major advantage is that antioxidant mechanisms
as influenced by pH can be studied over a wide pH range. Addi-
tionally, the generation cation radical is soluble in both polar and
nonpolar solvents and is not affected by ionic strength. While
it measures both hydrophilic and lipophilic antioxidants, it does
tend to favor the hydrophilic species, but this can be overcome
by using buffered media or partitioning the antioxidants between
hydrophilic and lipophilic solvents. In terms of effort, the assay
is short (30 minutes) as the antioxidants react rapidly with the
generated cation radical The major objection to this assay is that
the ABTS radical is not found in mammalian tissues and thus
represents a synthetic radical source, but any molecule, such
as phenolic compounds, that have low redox potentials can re-
act with ABTS+(Prior et al. 2005). As with some of the other
assays, TEAC may have long initial lag phases, and the assay
may not go to completion before stopping itself, resulting in
OAC underestimation.
The theory of the DPPH assay is based on the ability of the
antioxidants to reduce it to its radical form, DPPH·,astable
nitrogen radical producing a deep purple color with the decrease
in color monitored by absorbance at 515 nm or by electron spin
resonance. The results are reported as EC 50 , which measures a
50% decrease in the DPPH·concentration, which is proportional
to the antioxidant concentration (Prior et al. 2005). This assay
is also quick and only requires the use of a spectrophotometer,
but results are difficult to interpret as compounds such as the
carotenoids also absorb maximally at 515 nm. The major disad-
vantage is that DPPH acts both as the radical probe and as the
oxidant. As a radical, DPPH is not involved in lipid peroxidation.
Many antioxidants react with peroxyl radicals, but DPPH may
be inaccessible to the antioxidants or may interact with them
very slowly.
The F-C assay, aka total phenolics assay, is a measure of the
AOC of the total phenolic content of a (food) sample. Since
its development in 1927 for tyrosine analysis, it has been de-
veloped and improved to measure the AOC of all the phenolic
molecules of a sample (Singleton and Rossi 1965). These phe-
nolic compounds are oxidized by a molybdotungsto-phosphoricheteropolyanion reagent with the reduced phenols, producing a
colored product that is measured maximally at 765 nm. While
the method is simple, it has a number of drawbacks. The method
must be followed exactly and explicitly to minimize variability
and erratic results. The assay suffers from a large number of
interfering but naturally occurring substances such as fructose,
ascorbic acid, aromatic amino acids, and other compounds that
are listed by Prior et al. (2005). However, this limitation was
overcome in a study on the phenolic content of urine by using
solid-phase extraction to remove the water-soluble compounds
from the sample (Roura et al. 2006). It should also be taken into
consideration that the phenolic composition in different samples
would react differently as is demonstrated by the F-C assay of
different foods (cf. Figure 7, Prior et al. 2005).
For analytical chemists, it becomes increasingly important to
understand the composition of the food matrix as the differ-
ent antioxidant assays may not be identical in their underlying
mechanisms. Additionally, the results must also take into ac-
count the effect of geography, soils, climatic conditions, variety,
seasonality, transportation, processing, etc. of a particular food,
as these can influence resulting antioxidant values (Dewanto et
al. 2002, Tsao et al. 2006, Xu and Chang 2008).
In light of the different underlying reaction mechanisms, the
different antioxidants, their target free radicals, and their en-
vironmental conditions, the food matrix, etc., the dilemma be-
comes apparent as to which assay to choose as much has to be
taken into consideration. Extraction methodologies, detection,
quantification, measuring the effectiveness of the vast array of
antioxidants against their target free radicals, as well as interpret-
ing the results (Prior et al. 2005) are just some considerations.
As can also be deduced, the different assays have their advan-
tages/disadvantages, and the result of one cannot be compared
to the results of another. None of the assays can be considered
as measurements of total antioxidant activity as antioxidants are
indeed varied.
As a result of all these challenges and concerns, 144 scientists
met at the First International Congress on Antioxidant Meth-
ods in 2004 to standardize these issues across all laboratories
worldwide, with all resultant works are to be published inThe
Journal of Agricultural Food Chemistry(Prior et al. 2005). We
look forward to the fruit of this enormous and invaluable effort.GAS CHROMATOGRAPHY—MASS
SPECTROSCOPYGC–MS is a common and powerful analytical technique used in
both agricultural and food industries, which combines the fea-
tures of both GC and MS. Developed in the 1950s by Roland
Gohike and Fred McLaffert, GC–MS first separates the prepared
sample (containing a mixture of compounds) into separate and
distinct compounds (by GC) and then positively identifies and
quantifies these compounds based on their ion fragment pat-
tern (by MS). This approach is advantageous as it identifies a
molecule by both its unique retention time as well as its unique
fragmentation pattern. The results are graphically illustrated in a
mass spectrum graph that yields both qualitative and quantitative
information about the substance(s) of interest.