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Lipid Oxidation: Measurement Methods

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DOI: 10.1002/047167849X.bio050

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8
Lipid Oxidation:
Measurement Methods
Fereidoon Shahidi and Ying Zhong
Memorial University of Newfoundland,
St. Johns, Newfoundland, Canada

1. INTRODUCTION

Dietary lipids, naturally occurring in raw food materials or added during food
processing, play an important role in food nutrition and flavor. Meanwhile, lipid
oxidation is a major cause of food quality deterioration, and has been a challenge
for manufacturers and food scientists alike. Lipids are susceptible to oxidative
processes in the presence of catalytic systems such as light, heat, enzymes, metals,
metalloproteins, and micro-organisms, giving rise to the development of off-flavors
and loss of essential amino acids, fat-soluble vitamins, and other bioactives. Lipids
may undergo autoxidation, photo-oxidation, thermal oxidation, and enzymatic
oxidation under different conditions, most of which involve some type of free radi-
cal or oxygen species (1, 2). Among these, only autoxidation and thermal oxidation
are discussed here in detail.
Autoxidation is the most common process leading to oxidative deterioration and
is defined as the spontaneous reaction of atmospheric oxygen with lipids (3). The
process can be accelerated at higher temperatures, such as those experienced during
deep-fat frying, which is called thermal oxidation, with increases in free fatty acid
and polar matter contents, foaming, color, and viscosity (4). Unsaturated fatty acids

Baileys Industrial Oil and Fat Products, Sixth Edition, Six Volume Set.
Edited by Fereidoon Shahidi. Copyright # 2005 John Wiley & Sons, Inc.

357
358 LIPID OXIDATION: MEASUREMENT METHODS

are generally the reactants affected by such reactions, whether they are present as
free fatty acids, triacylglycerols (as well as diacyglycerols or monoacylglycerols),
or phospholipids (3). It has been accepted that both autoxidation and thermal
oxidation of unsaturated fatty acids occurs via a free radical chain reaction that
proceeds through three steps of initiation, propagation, and termination (5). A
simplified scheme explaining the mechanism of autoxidation is given below:

Initiation:
initiator
LH L + H

Propagation:
L + O2 LOO

LOO + LH LOOH + L

Termination:
2 LOO
LOO + L Nonradical products
L + L

As oxidation normally proceeds very slowly at the initial stage, the time to reach a
sudden increase in oxidation rate is referred to as the induction period (6). Lipid
hydroperoxides have been identified as primary products of autoxidation; decom-
position of hydroperoxides yields aldehydes, ketones, alcohols, hydrocarbons, vola-
tile organic acids, and epoxy compounds, known as secondary oxidation products.
These compounds, together with free radicals, constitute the bases for measurement
of oxidative deterioration of food lipids. This chapter aims to explore current
methods for measuring lipid oxidation in food lipids.

2. METHODS FOR MEASURING LIPID OXIDATION

Numerous analytical methods are routinely used for measuring lipid oxidation in
foods. However, there is no uniform and standard method for detecting all oxidative
changes in all food systems (7). Therefore, it is necessary to select a proper and
adequate method for a particular application. The available methods to monitor
lipid oxidation in foods can be classified into five groups based on what they mea-
sure: the absorption of oxygen, the loss of initial substrates, the formation of free
radicals, and the formation of primary and secondary oxidation products (8). A
number of physical and chemical tests, including instrumental analyses, have
been employed in laboratories and the industry for measurement of various lipid
oxidation parameters. These include the weight-gain and headspace oxygen uptake
method for oxygen absorption; chromatographic analysis for changes in reactants;
MEASUREMENT OF OXYGEN ABSORPTION 359

iodometric titration, ferric ion complexes, and Fourier transform infrared (FTIR)
method for peroxide value; spectrometry for conjugated dienes and trienes, 2-thio-
barbituric acid (TBA) value, p-anisidine value (p-AnV), and carbonyl value;
Rancimat and Oxidative Stability Instrument (OSI) method for oil stability index;
and electron spin resonance (ESR) spectrometric assay for free-radical type and
concentration. Other techniques based on different principles, such as differential
scanning calorimetry (DSC) and nuclear magnetic resonance (NMR), have also
been used for measuring lipid oxidation. In addition, sensory tests provide subjec-
tive or objective evaluation of oxidative deterioration, depending on certain details.

3. MEASUREMENT OF OXYGEN ABSORPTION

3.1. Weight Gain


Consumption of oxygen during the initial stage of autoxidation results in an
increase in the weight of fat or oil, which theoretically reflects its oxidation level.
Heating an oil and periodically testing for weight gain is one of the oldest methods
for evaluating oxidative stability (9). This method requires simple equipment and
directly indicates oxygen absorption through mass change. Oil samples are weighed
and stored in an oven at a set temperature with no air circulation. To avoid the influ-
ence of mass change by volatiles, samples can be preheated in an inert atmosphere.
Samples are then taken out of the oven at different time intervals, cooled to ambient
temperature, and reweighed; the weight gain is then recorded. The induction period
can be obtained by plotting weight gain against storage time. In some cases, the
time required to attain a 0.5% weight increase is taken as an index of oil stability
(7, 9, 10).
As a physical method for measuring lipid oxidation, the weight-gain method has
several drawbacks such as discontinuous heating of the sample, which may give rise
to non-reproducible results, and requiring long analysis time and intensive human
participation (7). Nevertheless, this method offers advantages such as low instru-
mentation cost as well as a high capacity and processing speed of samples without
limitation (7). Antolovich et al. (9) suggested that this technique may be extended
to more sophisticated continuous monitoring of mass and energy changes as in ther-
mogravimetry (TG)/differential scanning calorimetry (DSC). The weight-gain
method can also be used for measuring antioxidant activity by comparing the
results in the presence and absence of an antioxidant. Nevertheless, this method
is useful only when highly unsaturated oils, such as marine oils and vegetable
oils containing a high content of polyunsaturated fatty acids, are examined.

3.2. Headspace Oxygen Uptake


In addition to the weight-gain method, oxygen consumption can be measured
directly by monitoring the drop of oxygen pressure. Using headspace oxygen meth-
od, an oil sample is placed in a closed vessel also containing certain amount of oxy-
gen at elevated temperatures, commonly around 100 C. The pressure reduction in
360 LIPID OXIDATION: MEASUREMENT METHODS

the vessel, which is due to the oxygen consumption, is monitored continuously and
recorded automatically. The induction period as the point of maximum change in
rate of oxygen uptake can be calculated (11). A commercial instrument for this
method, known as Oxidograph, is available. In the Oxidograph, the pressure change
in the reaction vessel is measured electronically by means of pressure transducers
(7, 12).
Oxygen consumption can also be measured by electrochemical detection of
changes in oxygen concentration. However, the analysis of the graphical data
obtained has been the bottleneck for this technique. The use of a semiautomatic
polarographic method has been proposed as an improvement for evaluation of lipid
oxidation by determination of oxygen consumption (13). As described by Genot
et al. (13), this method is based on use of two oxygen meters with microcathode
oxygen electrodes, coupled to a computerized data collection and processing unit.
The headspace oxygen method is simple and reproducible and may be the best
analytical method to evaluate the oxidative stability of fats and oils (14). Its appli-
cation in measurement of lipid oxidation in food products other than fats and oils,
however, is limited because protein oxidation also absorbs oxygen (15).

4. MEASUREMENT OF REACTANT CHANGE

Lipid oxidation can also be assessed by quantitatively measuring the loss of initial
substrates. In foods containing fats or oils, unsaturated fatty acids are the main
reactants whose composition changes significantly during oxidation. Changes in
fatty acid composition provide an indirect measure of the extent of lipid oxidation
(15). In this method, lipids are extracted from food, if necessary, and subsequently
converted into derivatives suitable for chromatographic analysis (7). Fatty acid
methyl esters (FAME) are the derivatives frequently used for determination of fatty
acid composition, usually by gas chromatography (GC) (16). Similarly, iodine
value, which reflects the loss of unsaturation, can also be used as an index of lipid
oxidation (17).
Measurement of changes in fatty acid composition is useful for identification of
lipid class and fatty acids that are involved in oxidation reactions (7). However,
because the distribution of unsaturated fatty acids varies in different food systems,
for instance, the highly unsaturated fatty acids being located predominantly in
phospholipids of muscle foods, separation of lipids into neutral, glycolipid, phos-
pholipid, and other classes may be necessary (7, 15). Moreover, it is an insensitive
way of assessing oxidative deterioration. For comparison through calculation, oxi-
dation of 0.4% polyunsaturated fatty acids to monohydroperoxides would represent
a change of 16 meq oxygen/kg oil in peroxide value, whereas a change of less than
1.0 meq oxygen/kg oil could readily be detected by measuring peroxide value (12).
Additionally, the application of this method is limited because of its inability
to serve as an indicator of oxidation of more saturated lipids (7). Nevertheless,
its usefulness for measuring oxidation of highly unsaturated oils cannot be
underestimated.
MEASUREMENT OF PRIMARY PRODUCTS OF OXIDATION 361

5. MEASUREMENT OF PRIMARY PRODUCTS OF OXIDATION

5.1. Peroxide Value (PV)


Lipid oxidation involves the continuous formation of hydroperoxides as primary
oxidation products that may break down to a variety of nonvolatile and volatile
secondary products (8, 15). The formation rate of hydroperoxides outweighs their
rate of decomposition during the initial stage of oxidation, and this becomes
reversed at later stages. Therefore, the peroxide value (PV) is an indicator of the
initial stages of oxidative change (18). However, one can assess whether a lipid
is in the growth or decay portion of the hydroperoxide concentration by monitoring
the amount of hydroperoxides as a function of time (7).
Analytical methods for measuring hydroperoxides in fats and oils can be classi-
fied as those determining the total amount of hydroperoxides and those based on
chromatographic techniques giving detailed information on the structure and the
amount of specific hydroperoxides present in a certain oil sample (8). The PV repre-
sents the total hydroperoxide content and is one of the most common quality indi-
cators of fats and oils during production and storage (9, 18). A number of methods
have been developed for determination of PV, among which the iodometric titra-
tion, ferric ion complex measurement spectrophotometry, and infrared spectroscopy
are most frequently used (19).

5.1.1. Iodometric Titration Method Iodometric titration assay, which is based


on the oxidation of the iodide ion (I) by hydroperoxides (ROOH), is the basis of
current standard methods for determination of PV (9). In this method, a saturated
solution of potassium iodide is added to oil samples to react with hydroperoxides.
The liberated iodine (I2) is then titrated with a standardized solution of sodium thio-
sulfate and starch as an endpoint indicator (7, 9, 20). The PV is obtained by calcu-
lation and reported as milliequivalents of oxygen per kilogram of sample (meq/kg).
The official determination is described by IUPAC (21). Chemical reactions involved
are given below:

ROOH 2H 2KI ! I2 ROH H2 O 2K


I2 2NaS2 O3 ! Na2 S2 O6 2NaI

Although iodometric titration is the most common method for measurement of PV,
it suffers from several disadvantages. The procedure is time-consuming and labor-
intensive (18). As described by Ruiz et al. (18), the assay includes six steps: accu-
rate weighing of the sample, dissolution of lipids in chloroform, acidification with
acetic acid, addition of potassium iodide, incubation for exactly 5 minutes, and
titration with sodium thiosulfate. This technique requires a large amount of sample
and generates a significant amount of waste (18, 22, 23). Furthermore, possible
absorption of iodine across unsaturated bonds and oxidation of iodide by dissolved
oxygen are among potential drawbacks of this method (7, 9). Besides, lack of sen-
sitivity, possible interferences, and difficulties in determining the titration endpoint
362 LIPID OXIDATION: MEASUREMENT METHODS

are also the main limitations (8, 23). To overcome these drawbacks, novel methods
based on the same reaction have been developed, in which some other techniques
are adopted as modification of the classical iodometric assay. Techniques such as
colorimetric determination at 560 nm (24), potentiometric endpoint determination
(25), and spectrophotometric determination of the I 3 chromophore at 290 nm or
360 nm (26, 27) have been proposed. In addition, an electrochemical technique
has been used as an alternative to the titration step in order to increase the sensitiv-
ity for determination of low PV by reduction of the released iodine at a platinum
electrode maintained at a constant potential (7).

5.1.2. Ferric Ion Complexes Other chemical methods based on the oxidation of
ferrous ion (Fe2) to ferric ion (Fe3) in an acidic medium and the formation of
iron complexes have also been widely accepted. These methods spectrophotometri-
cally measure the ability of lipid hydroperoxides to oxidize ferrous ions to ferric
ions, which are complexed by either thiocyanate or xylenol orange (23, 28, 29).
Ferric thiocyanate is a red-violet complex that shows strong absorption at 500
510 nm (8). The method of determining PV by coloremetric detection of ferric thio-
cyanate is simple, reproducible, and more sensitive than the standard iodometric
assay, and has been used to measure lipid oxidation in milk products, fats, oils,
and liposomes (8, 23).
The ferrous oxidation of xylenol orange (FOX) assay uses dye xylenol orange to
form a blue-purple complex with a maximum absorption at 550600 nm (8). This
method is rapid, inexpensive, and not sensitive to ambient oxygen or light (30). It
can consistently quantify lower hydroperoxide levels; and good agreement exists
between the FOX assay and the iodometric method (30). The FOX method has
been successfully adapted to a variety of applications. However, because many fac-
tors, such as the amount of sample, solvent used, and source of xylenol orange, may
affect the absorption coefficient, knowledge of the nature of hydroperoxides present
in the sample, and careful control of the conditions used are required for accurate
measurements (8).

5.1.3. Fourier Transform Infrared Spectroscopy (FTIR) It has been recog-


nized that hydroperoxides can quantitatively be determined by IR spectroscopy
via measurement of their characteristic O-H stretching absorption band (31). An
absorption band at about 2.93 mm indicates the generation of hydroperoxides,
whereas the replacement of a hydrogen atom on a double bond or polymerization
accounts for the disappearance of a band at 3.20 mm. The formation of aldehydes,
ketones, or acids gives rise to an extra band at 5.72 mm. Furthermore, cis-, trans-
isomerization and formation of conjugated dienes can be detected through the
changes in the absorption band in the range of 10 mm to 11 mm (7).
A rapid Fourier transform infrared spectroscopy (FTIR) method based on the
stoichiometric reaction of triphenylphosphine (TPP) with hydroperoxides has
been developed and successfully applied to determination of PV of edible oils
(32). The hydroperoxides present in oil samples react stoichiometrically with
TPP to produce triphenylphosphine oxide (TPPO), which has an intense absorption
MEASUREMENT OF PRIMARY PRODUCTS OF OXIDATION 363

band at 542 cm1 in the mid-IR spectrum (8, 18). The band intensity is measured
and converted to peroxide value. The chemical reaction involved is given below:

ROOH TPP ! TPP O ROH

By using tert-butyl hydroperoxide spiked oil standards and evaluation of the band
formed at 542 cm1, a linear calibration graph covering the range of 1100 PV was
obtained (18). More recently, disposable polymer IR (PIR) cards have been used as
sample holders where unsaturated oil samples oxidize at a fairly rapid rate (33). In
the FTIR/PIR card method, warm air continuously flows over the sample allowing
oxidation to be monitored at moderate temperatures. At periodic intervals, indivi-
dual cards are removed and the FTIR spectra scanned (33). Another new FTIR
approach uses flow injection analysis (FIA), which offers exact and highly repro-
ducible timing of sample manipulation and reaction as well as a closed environment
with oxygen and light being easily excluded (18).
The FTIR spectroscopy is a simple, rapid, and highly precise method. It shows
excellent correlation with the iodometric method and avoids the solvent and reagent
disposal problems associated with the standard wet chemical method (18, 32). The
FTIR method provides an automated, efficient and low-cost means of evaluating
oxidation in oils undergoing thermal stress and has gained considerable interest
for quality control in the industry (8, 20, 34). However, there is a need to charac-
terize the spectral changes, assign wavelengths to more common molecular species
produced, and access potential spectral cross interferences (20). Recently, an
improved Fourier transform infrared attenuated total reflectance (FTR-ATR) meth-
od using the whole FTIR spectral data instead of particular wavenumbers has been
proposed (34).
In addition to the three major methods discussed above, other techniques have
also been employed in determination of PV, such as chemiluminescence and chro-
matography. Chemiluminescence method is based on detecting the chemilumines-
cent products generated during the reaction of hydroperoxides with substances such
as luminol and dichlorofluorescein (7, 35). This method was reviewd by Jimenez
et al. (36). High correlations have been found between chemiluminescence and
other standard methods, indicating that chemuliminescence could serve as an accu-
rate tool for determination of PV (37). However, this method has low sensitivity to
tert-butyl hydroperoxide, tert-butyl perbenzoate, diacyl peroxides, and dialkyl per-
oxides (35). Chromatographic techniques, mainly gas chromatography (GC) and
high-performance liquid chromatography (HPLC), have also been employed for
evaluation of lipid oxidation. These methods provide information about specific
hydroperoxides, whereas other assays measure their total amount. Chromatographic
methods require small amounts of sample, and interference from minor compounds
other than hydroperoxides can be easily excluded (8). HPLC shows advantages over
GC and has become a popular technique for hydroperoxide analysis. It operates at
room temperature, thus decreases the risk of artifact formation, and no prior deri-
vatization is required (8). A wide range of hydroperoxides can be analyzed using
either normal or reverse-phase HPLC. Thus, hydroperoxides, the primary products
364 LIPID OXIDATION: MEASUREMENT METHODS

and intermediates in lipid oxidation reaction, provide an important parameter for


evaluation of oxidation level. In addition, the inhibition of formation or action of
these unstable species by antioxidants can be used as a means of assessing antiox-
idant activity (9). Measurement of hydroperoxides is also carried out in accelerated
tests to establish the oxidative stability of a given oil. A case in point is the active
oxygen method (AOM), in which air is bubbled through fat or oil held at 98100 C
and PV is determined periodically (7, 38). The time required to reach a PV of 100
meq/kg is the AOM stability of the oil sample (7). This method is now considered
outdated and is replaced by other standard methods in the industry, although
product specifications still routinely give AOM values (38).

5.2. Conjugated Dienes and Trienes


It was discovered in 1933 that the formation of conjugated dienes in fats or oils
gives rise to an absorption peak at 230235 nm in the ultraviolet (UV) region. In
the 1960s, monitoring diene conjugation emerged as a useful technique for the
study of lipid oxidation (9). During the formation of hydroperoxides from unsatu-
rated fatty acids conjugated dienes are typically produced, due to the rearrangement
of the double bonds. The resulting conjugated dienes exhibit an intense absorption
at 234 nm; similarly conjugated trienes absorb at 268 nm (7). An increase in UV
absorption theoretically reflects the formation of primary oxidation products in fats
and oils. Good correlations between conjugated dienes and peroxide value have
been found (39, 40).
Ultraviolet detection of conjugated dienes is simple, fast, and requires no
chemical reagents and only small amounts of samples are needed. However, this

hydroperoxydiene oxodiene

O O
O
H
Reduction

hydroxydiene

O
H
conjugated triene

and
conjugated tetraene

Figure 1. Chemical reaction steps in conjugable oxidation products (COP) assay.


TABLE 1. Summary of Methods for Analysis of Primary Oxidation Products.

Method Principle Measurement Sensitivity Applications

Iodometric titration (PV) Reduction of ROOH with KI Titration with Na2S2O3 0.5-meq/kg fat Fats and oils
and measurement of I2
Ferric ion complexes (PV) Reduction of ROOH with Fe2 Absorption at 500510 nm 0.1-meq/kg fat Fats, oils and food lipids
and formation of Fe3 of the red complex with
complexes SCN
Absorption at 560 nm of the 0.5-meq/kg sample All samples
blue-purple complex with
xylenol orange
FTIR (PV) Reduction of ROOH with TPP Absorption at 542 cm1 of 0.2-meq/kg fat Fats and oils
TPPO
Chemiluminescence (PV) Reaction with luminol in the Chemiluminescence 1 pmol Fats and oils
presence of heme catalyst emission of oxidized
luminol
GC-MS (PV) Reduction of ROOH to ROH ROH derivatives From ng to fg depending on All samples
and quantitation of ROH technical details, amount
derivatives of sample and detection
system
UV spectrometry Estimation of conjugated Absorption at 230234 nm 0.2 meq/kg lipid All samples
(conjugated dienes and dienes and trienes and 268 nm
trienes)

NOTE: The oxygen absorption measurement and loss of double bonds for fatty acid analysis are not considered as primary changes in this table.
Adapted from (8).
366 LIPID OXIDATION: MEASUREMENT METHODS

method has less specificity and sensitivity than PV measurement (9, 12). Further-
more, the result may be affected by the presence of compounds absorbing in the
same region, such as carotenoids (7). To avoid these interferences, an alternative
spectroscopic method measuring conjugable oxidation products (COPs) has
been proposed. In this method, hydroperoxides and some decomposition products
are converted to more conjugated chromophores by reduction and subsequent
dehydration (Figure 1). The concentrations of the resultant conjugated trienes
and tetraenes are determined from their respective absorption at 268 nm and
301 nm and expressed as COP values (7, 12).
Table 1 summarizes different methods available for analysis of primary oxida-
tion products. Both chemical and instrumental methods are included in this
table.

6. MEASUREMENT OF SECONDARY PRODUCTS OF OXIDATION

The primary oxidation products (hydroperoxides) are unstable and susceptible to


decomposistion. A complex mixture of volatile, nonvolatile, and polymeric second-
ary oxidation products is formed through decomposition reactions, providing var-
ious indices of lipid oxidation (5). Secondary oxidation products include aldehydes,
ketones, alcohols, hydrocarbons, volatile organic acids, and epoxy compounds,
among others. Methods for assessing lipid oxidation based on their formation are
discussed in this section.

6.1. Thiobarbituric Acid (TBA) Test


The thiobarbituric acid (TBA) test was proposed over 40 years ago and is now
one of the most extensively used methods to detect oxidative deterioration of
fat-containing foods (41). During lipid oxidation, malonaldehyde (MA), a minor
component of fatty acids with 3 or more double bonds, is formed as a result of
the degradation of polyunsaturated fatty acids. It is usually used as an indicator
of the lipid oxidation process, both for the early appearance as oxidation occurs
and for the sensitivity of the analytical method (42). In this assay, the MA is reacted
with thiobarbituric acid (TBA) to form a pink MA-TBA complex that is measured
spectrophotometrically at its absorption maximum at 530535 nm (Figure 2) (9, 43, 44).
The extent of oxidation is reported as the TBA value and is expressed as milligrams

OH O OH
O O
N HN NH
+ H C CH2 C H
HS N OH S N OH O N S
H
TBA MA
TBA-MA adduct
Figure 2. Reaction of 2-thiobarbituric acid (TBA) and malonaldehyde (MA).
MEASUREMENT OF SECONDARY PRODUCTS OF OXIDATION 367

of MA equivalents per kilogram sample or as micromoles of MA equivalents per


gram of sample. It must, however, be noted that alkenals and alkadienals also react
with the TBA reagent and produce a pink color. Thus, the term thiobarbituric acid
reactive substances (TBARS) is now used instead of MA.
The TBA test can be performed by various procedures, among which four major
types have frequently been employed. These include test on the whole sample, test
on an aqueous or acid extract of sample, test on a steam distillate, and test on
extracted lipid from a sample (45). The test on a steam distillate (distillation meth-
od) is the most commonly used method for determining TBA value. Tarladgis et al.
(46) found that the distillation of an acidified sample was essential to liberate MA
from precursor or bound forms, to produce maximal color development and, espe-
cially, to separate TBARS from the food matrix (44). Although the distillation
method is the most popular TBA method, it is generally considered less accurate
and reproducible than the method using food extracts (15). However, trends
obtained in comparative studies always provide useful information that correspond
with other measurements. Comparison of different TBA test procedures has
been made by Hoyland et al. (46), Shahidi et al. (47), Pikul et al. (48), and
Wang et al. (49).
The TBA test is used frequently to assess the oxidative state of a variety of food
systems, despite its limitations, such as lack of specificity and sensitivity (44). As
already noted, many other substances may react with the TBA reagent and contri-
bute to absorption, causing an overestimation of the intensity of color complex (44).
Interferences may come from additional absorption of other alkanals, 2-alkenals,
2,4-alkdienals, ketones, ketosteroids, acids, esters, proteins, sucrose, urea, pyri-
dines, and pyrimidines, also referred to as TBARS (43, 50). For instance, the
reaction of TBA with various aldehydes leads to the development of a yellow
chromogen (aldehyde-TBA adduct) with an absorption maximum at 450 nm, which
overlaps with the pink peak at 532 nm resulting in erroneously high TBA values in
certain cases (43, 45, 51). Furthermore, the presence of barbituric acid impurities in
the TBA reagent may produce TBA-MA-barbituric acid and MA-barbituric acid
adducts that absorb at 513 nm and 490 nm, respectively, indicating that thiobarbi-
turic acid should be purified before use (43). In addition, nitrite can interfere in the
TBA test, whereas sulfanilamide could be added to samples to avoid the interfer-
ence when residual nitrite is present (52). In order to improve the specificity and
sensitivity of the TBA test, several modifications to the original TBA procedures
have been proposed, including reduction of the heating temperature to stabilize
the yellow color aldehyde-TBA complex (53), addition of antioxidants to sample
in an attempt to prevent oxidation during the test (54), extraction of the MA prior
to the formation of the chromogen (43), direct FTIR analysis of TBARS, and use of
HPLC to separate the complex before measurement or to characterize the individual
species of TBARS (9, 43).
Despite it limitations, the TBA test provides an excellent means for evaluating
lipid oxidation in foods, especially on a comparative basis. However, its use in bulk
oils is less common than the so-called para-anisidine value (p-AnV) detailed
below.
368 LIPID OXIDATION: MEASUREMENT METHODS

6.2. p -Anisidine Value (p -AnV)


The p-anisidine value (p-AnV) method measures the content of aldehydes (princi-
pally 2-alkenals and 2,4-alkadienals) generated during the decomposition of hydro-
peroxides. It is based on the color reaction of p-methoxyaniline (anisidine) and the
aldehydic compounds (55). The reaction of p-anisidine reagent with aldehydes
under acidic conditions affords yellowish products that absorb at 350 nm
(Figure 3) (7, 12). The color is quantified and converted to p-AnV. The p-AnV is
defined as the absorbance of a solution resulting from the reaction of 1 g of fat in
isooctane solution (100 ml) with p-anisidine (0.25% in glacial acetic acid) (12).
This test is more sensitive to unsaturated aldehydes than to saturated aldehydes
because the colored products from unsaturated aldehydes absorb more strongly at
this wavelength (12). However, it correlates well with the amount of total volatile
substances (55). The p-AnV is a reliable indicator of oxidative rancidity in fats and
oils and fatty foods (56). A highly significant correlation between p-AnV and flavor
scores and PV has been found (57). Nevertheless, some authors have indicated that
p-AnV is comparable only within the same oil type because initial AnV varies
among oil sources (58). For instance, oils with high levels of polyunsaturated fatty
acids might have higher AnV even when fresh (59).
This method is used less frequently in North America, but is widely employed in
Europe (38), particularly as a part of the Totox number, as explained below. Caution

O OH NH2
C C +
H C H CH3O
H
Malonaldehyde p-Methoxyaniline
(enolic form) (p-anisidine)

N OH

CH3O

NH2

CH3O

N NH

CH3O OCH3

Figure 3. Possible reactions between p-anisidine reagent and malonaldehyde.


MEASUREMENT OF SECONDARY PRODUCTS OF OXIDATION 369

must be exercised when performing this test because of toxicity of the anisidine
reagent (55).

6.3. Totox Value


The Totox value is a measure of the total oxidation, including primary and second-
ary oxidation products. It is a combination of PV and p-AnV:

Totox value 2 PV p-AnV

During lipid oxidation, it is often observed that PV first rises, then falls as hydro-
peroxides decompose (38). PV and p-AnV reflect the oxidation level at early and
later stages of oxidation reaction, respectively. Totox value measures both hydro-
peroxides and their beakdown products, and provides a better estimation of the
progressive oxidative deterioration of fats and oils (38). However, Totox value
has no scientific basis because it is a combination of two indicators with different
dimensions (7). Recently, Wanasundara and Shahidi used TBA values and defined
TotoxTBA as 2PV TBA using the TBA test in place of the p-AnV assay (60).

6.4. Carbonyls
The carbonyl compounds, including aldehydes and ketones, are the secondary oxi-
dation products generated from degradation of hydroperoxides, and are suggested
to be the major contributors to off-flavors associated with the rancidity of many
food products (9). The analysis of total carbonyl compounds, which is based on
the absorbance of the carbonyl derivatives, provides another approach to measure
the extent of lipid oxidation in fats and oils. In this method, the total carbonyl
content is measured by a colorimetric 2,4-dinitrophenylhydrazone procedure. The
carbonyl compounds formed during lipid oxidation are reacted with 2,4-dinitrophe-
nylhydrazine (DNPH) followed by the reaction of the resulting hydrazones with
alkali (Figure 4). The final colored products are then analyzed spectrophotometrically

R H R H
R C O + H2N N NO2 R C N N NO2

NO2 NO2

OH
H2O

R

R C N N NO2

NO2

Figure 4. Reactions between carbonyls and 2,4-dinitrophenylhydrazine.


370 LIPID OXIDATION: MEASUREMENT METHODS

at a given wavelength (7, 15). Many variations of this method using an alternative
solvent, reagent, wavelength, or workup have been reported. The determination of
total content of carbonyls has been used in different oxidative stability studies.
However, it has been criticized because the determination conditions cause degra-
dation of hydroperoxides into carbonyl derivatives, giving erroneous results (58).
Carbonyls produced from protein oxidation may also give rise to higher values
than those expected from lipid oxidation alone. The addition of triphenylphosphine
(TPP) prior to carbonyl determination has been proposed to avoid the interference
from hydroperoxides. Hydroperoxides are reduced by TPP, and neither TPP nor
TPPO, the oxidation products of TPP, interfere with the measurement of carbonyl
content (61). In quality assessment of used frying fats, where short-chain carbonyls
are already removed by distillation at the high temperature of the deep-frying,
selectivity can be improved by determination of higher carbonyl compounds instead
of the total carbonyls. HPLC is used to separate the DNPH derivatives of higher
carbonyls from those of short-chain carbonyl compounds (62).
Apart from detection of total carbonyl content, the analysis of individual carbo-
nyl compounds has gained popularity for following lipid oxidation. Hexanal, one of
the major secondary products formed during the oxidation of linoleic and other o6
fatty acids, serves as a reliable indicator of lipid oxidatin in foods rich in o6 fatty
acids (7). A strong linear relationship was reported between hexanal content, sen-
sory scores, and TBA values (63). Moreover, measurement of hexanal offers the
advantage of analyzing a single, well-defined end product for antioxidant efficiency
studies (9). Hexanal can be quantified by chromatography (64) or as the intensity of
the carbonyl band by NIR spectroscopy (65). Nevertheless, these methods may
require volatilization of hexanal, whereas hexanal volatilization may be hindered
due to covalent or other types of binding between hexanal and proteins in foods
and, thus, may affect accurate hexanal quantifications (66). More recently, an
indirect enzyme-linked immunosorbanct assay (ELISA) has been developed for
monitoring lipid oxidation through quantification of hexanal-protein adducts, which
are recognized by polyclonal or monoclonal antibodies (66).
Other carbonyl compounds, including propanal, pentanal, decadienal, etc., are
also used for evaluating lipid oxidation in foods. For instance, propanal is a recom-
mended indicator for lipid oxidation in foods that are high in o3 fatty acids, such as
marine oils (67, 68). In general, it is essential to use appropriate indicators when
assessing the oxidative deterioration of different food systems.

6.5. Oil Stability Index (OSI)


During lipid oxidation, volatile organic acids, mainly formic acid and acetic acid,
are produced as secondary volatile oxidation products at high temperatures, simul-
taneously with hydroperoxides (20, 69). In addition, other secondary products,
including alcohols and carbonyl compounds, can be further oxidized to carboxylic
acids (20). The oil stability index (OSI) method measures the formation of volatile
acids by monitoring the change in electrical conductivity when effluent from
oxidizing oils is passed through water (12). The OSI value is defined as the point
of maximal change of the rate of oxidation, attributed to the increase of conductivity
MEASUREMENT OF SECONDARY PRODUCTS OF OXIDATION 371

by the formation of volatile organic acids during lipid oxidation (70). However, this
method requires a somewhat higher level of oxidation (PV > 100) to obtain mea-
surable results than other methods in which hydroperoxides are the most important
products formed and detected (71). Therefore, to determine oil stability in the
laboratory, especially for some oils that are stable under normal conditions, the oxi-
dation process is accelerated by exposing oil samples to elevated temperatures in
the presence of an excess amount of air or oxygen (72, 73). The OSI method differs
from ambient storage conditions by using a flow of air and high temperatures
to accelerate oxidation (71). The OSI is an automated development of the active-
oxygen method (AOM), because both employ the principle of accelerated oxida-
tion. Nevertheless, the OSI test measures the changes in conductivity caused by
ionic volatile acids, whereas PV is determined in the AOM (7).
Two pieces of commercially available equipment, the Rancimat (Metrohm Ltd.)
and the Oxidative Stability Instrument (Omnion Inc.), are employed for determin-
ing the OSI value. Rancimat is a rapid automated method, which agrees well with
the AOM (71). In the Rancimat assay, a flow of air is bubbled through a heated oil,
usually at 100 C or above. For marine oils, temperatures as low as 80 C are often
used. Volatile compounds formed during accelerated oxidation are collected in dis-
tilled water, increasing the water conductivity. The change of conductivity is plotted
automatically and the induction period of the oil or the time taken to reach a fixed
level of conductivity is recorded (20, 74). The Rancimat assay enables continuous
monitoring of the oxidation process. As reported by Farooq et al. (75), analysis by
the Rancimat method is four to five times more rapid than that by the AOM. Excel-
lent correlation between Rancimat and conjugated dienes has been found (72).
However, the main shortcoming of this method is that only eight samples can be
included in each batch. Another appatatus, the Oxidative Stability Instrument, oper-
ates on the same principle as the Rancimat, and has the capacity of simultaneously
analyzing up to 24 samples (20). Various modifications have been proposed for
assessing lipid oxidation by the OSI method. These include the use of auxiliary
energies, such as microwaves to shorten the analysis time (72) and a combination
of the OSI method with chromatography to obtain specific information about vola-
tile products (76). The volatiles trapped during measurement by the Rancimat assay
can be analyzed by headspace-GC (HS-GC) with FID and GC-MS for quantifica-
tion of individual volatiles, thus improving the specificity of the assessment (76).
Although the OSI method is useful for quality control of oils, it is not recom-
mended for measurement of antioxidant activity for certain reasons. The high tem-
peratures used do not allow reliable predictions of antioxidant effectiveness at
lower temperatures. Volatile antioxidants may be swept out of the oil by the air
flow under test conditions, and also the oils are severely deteriorated when endpoint
is reached (12).

6.6. Hydrocarbons and Fluorescence Assay


Formation of saturated hydrocarbons, especially short-chain (C1-C5) hydrocarbons
such as ethane, propane, and pentane, can be measured for monitoring lipid oxida-
tion when aldehydes are either absent or undetectable (7, 15). Pentane content,
372 LIPID OXIDATION: MEASUREMENT METHODS

O RNH2
H C CH CHOH RN CH CH CHOH
Amine-Malonaldehyde Adduct
(Non-fluorescent)

RNH2

O O
H C CH2 C H RN CH CH CH NHR
Malonaldehyde Conjugated Schiff Base
(Fluorescent)

Figure 5. Reaction of lipid oxidation products such as malonaldehyde and amines.

determined by GC techniques, has been a useful parameter to assess rancidity of


fats and oils as well as freeze-dried muscle foods (7, 15). Significant correlations
existed between pentane levels and rancid odor scores (15).
It has been observed that the content of secondary oxidation products, such as
malonaldehyde (MA), decreases with increased lipid oxidation, which can be
explained by further reaction of MA with proteins. MA reacts with compounds con-
taining primary amino groups (proteins, amino acids, DNA, phospholipids) to form
fluorescent products (Figure 5) (37). A fluorescence assay has been successfully
used to assess lipid oxidation in muscle foods and biological tissues.
In addition to MA, hydroperoxides and other aldehydes also react with amino
compounds generating various fluorescent products with different excitation and
emission maxima (37). Significant correlations existed between this method and
the TBA value as well as oxygen absorption level, and appears to be a reliable

TABLE 2. Summary of Methods for Analysis of Secondary Oxidation Products.

Method Compounds Comments Applications

TBA TBARS, mainly Spectrometry technique All samples,


malonaldehyde It can be carried out especially fish
on whole sample oils
p-Anisidine Aldehydes, mainly Absorption at 350-nm Fats and oils
alkenals Standard method
Carbonyls Total carbonyls or Spectrometry technique Fats and oils
specific carbonyl and HPLC for total
compound formed or specific carbonyl
compounds
OSI method Volatile organic acids Monitoring changes in Fats and oils
(Rancimat & conductivity Rapid
Oxidative Stability and automated
Instrument)
Gas Chromatography Volatile carbonyls and Direct headspace Rapid All samples
hydrocarbons analysis

Adapted from (8).


MEASUREMENT OF FREE RADICALS 373

indicator of oxidative deterioration in muscle foods, especially in freeze-dried


products (37, 77).
Table 2 exhibits the summary of methods for analysis of secondary oxidation
products.

7. MEASUREMENT OF FREE RADICALS

The initial steps of lipid oxidation involve chain reactions of free radicals as impor-
tant short-lived intermediates. Oxidation level of fats and oils can be measured
directly by detecting the formation of radicals. Methods based on the detection
of radicals or on the tendency for the formation of radicals provide a good indica-
tion of initiation of lipid oxidation (78, 79).
Electron spin resonance (ESR), also referred to as electron paramagnetic reso-
nance (EPR) spectroscopy, relies on the paramagnetic properties of the unpaired
electrons in radicals and has been developed for assessing the formation of free
radicals originating in the early stages of oxidation and the onset of primary oxida-
tion (6, 78). The assay measures the absorption of microwave energy when a
sample is placed in a varied magnetic field (7). Quantification of radical concentra-
tions is complicated by comparison with stable paramagnetic compounds, such as
transition metals and nitroxyl radicals (78). However, the short lifetimes and low
steady-state concentration of the highly reactive lipid-derived radicals make it dif-
ficult to detect these radicals at concentrations lower than the minimum detectable
concentration of 109 M (78). To overcome this problem, various approaches have
been used, including pulse radiolysis and UV photolysis, continuous flow systems
and spin trapping, among which spin trapping has been the most widely employed
procedure (9). Spin trapping technique allows the accumulation of detectable con-
centrations of longer-lived radicals by addition to samples of a spin trapping agent,
which reacts with free radicals to form more stable spin adducts, but often at the
expense of the ability to identify the original radical (6, 9, 78). Nitroso compounds
and nitrones are the most common spin traps, both leading to nitroxyl type spin
adducts, such as a-phenyl-tert-butylnitrone (PBN) adducts (Figure 6) (78).

O
H H O
N+ + R Ph N
Ph CMe3 R CMe3

PBN

O
Me3C N O + R R N
CMe3
MNP
Figure 6. Formation of nitroxyl radical spin adducts.
374 LIPID OXIDATION: MEASUREMENT METHODS

ESR spectroscopy is of great value for the study of the early stages of lipid oxi-
dation and prediction of oxidative stability of fats and oils. It has high sensitivity
and allows mild conditions by applying significantly low temperatures and requires
little sample preparation (6, 78, 80). Strong linear correlations were found between
ESR and Rancimat and oxygen consumption analyses (6, 79). ESR has also been
used for evaluation of antioxidant activity (81). Nevertheless, spin traps used in the
ESR assay have been reported to exhibit widely differing trapping efficiencies for
different radicals and show both pro-oxidant and antioxidant effects (9, 82, 83).
Moreover, spin adducts can act as antioxidants, giving erroneous results of oxida-
tive stability of samples (9). However, even with these limitations, the ESR spectro-
scopy is a suitable method for measuring lipid oxidation in foods and in biological
tissues.

8. OTHER METHODS

8.1. Differential Scanning Calorimetry (DSC)


During lipid oxidation, fat or oil materials reveal a number of thermally induced
transitions, such as the transfer of oxygen molecules to unsaturated fatty acids
(exothermic process) (84). Therefore, thermal analysis can be applied in accelerated
oil stability tests. The differential scanning calorimetry (DSC) technique, which is
based on thermal release of oxidation reactions, has the potential as a nonchemical
method for assessing oxidative stability of fats and oils, indicating the onset of
advanced oxidation (termination) (6). It provides unique energy profile information,
which specifically measures the temperature and heat flows associated with lipid
oxidation as a function of time and temperature (85). The method uses isothermal
or nonisothermal conditions and a flow of oxygen as purge gas, with a calorimeter
measuring the heat flow into (endothermic) or out of (exothermic) an oil sample
undergoing oxidation changes (6, 84). The oxidation curves of the sample are
obtained with different heating time, and a dramatic increase for the evolved
heat can be observed with the appearance of a sharp exothermic curve during initia-
tion of oxidation. The endpoint is taken at the time where a rapid exothermic reac-
tion between oil and oxygen occurs and induction period (IP) determined
automatically by intersection of extrapolated baseline and tangent line (leading
edge) of the exotherm (Figure 7) (6, 84). The DSC also measures oxidation onset
temperature, the temperature at maximum reaction, and the ending temperature
(84). The isothermal and nonisothermal DSC show good agreement, suggesting
that both isothermal and nonisothermal DSC are suitable for oxidation studies of
oils (86). The DSC technique has recently been reviewed by Tan et al. (84, 87).
The DSC is a sensitive, effective, and consistent method for characterization of
the quality of oils at different stages of oxidation (20). It is simple and rapid, and it
requires no solvent or chemical reagent. As reported by Hassel et al. (89), oils sam-
ples, which required 14 days via AOM, could be evaluated in less than 4 hours
by DSC. Thus, DSC is a reliable alternative to current methods for monitoring lipid
OTHER METHODS 375

Exothermic

IP

50 100 150 200 250


Time (min)
Figure 7. Determination of induction period (IP) by DSC.

oxidation (85). The results from DSC show excellent correlations with other
accelerated methods and chemical analyses (6, 73, 85).

8.2. Nuclear Magnetic Resonance (NMR) Spectroscopy


High-resolution 1H NMR spectroscopy, in which hydrogen atoms (proton, 1H) with
various locations in the triacylglycerol (TAG) molecules are determined, has been
used to evaluate oxidative deterioration of fats and oils (7). The principle of NMR is
that hydrogen atoms in a strong magnetic field absorb energy, in the radiofrequency
range, depending on their molecular environment, in which changes occur during
the oxidation process (7). These changes may be monitored by NMR spectroscopy
as a reflection of oxidation level of food lipids. The oil sample is dissolved in
CDCl3 to avoid inference from solvent, and its NMR spectrum recorded, with tetra-
methylsilane (TMS) as an internal standard (7). The spectrum shows several groups
of signals, corresponding to the hydrogen atoms in different locations in the TAG
molecules (Figure 8). The total number of each of these differently located protons
can be calculated, from which ratios of aliphatic to olefinic protons (Rao) and ali-
phatic to diallylmethylene protons (Rad) may be obtained (7). Both ratios increase
steadily during lipid oxidation and may serve as an index of oxidative deterioration
of oil samples. This method was reviewed by Guillen et al. (90). NMR spectroscopy
has been used by many researchers, and the changes in Rao and Rad measured by
NMR correlated well with Totox values (91, 92), conjugated diene values, and TBA
values (93). In addition to 1H NMR, 13C NMR and 31P NMR are also powerful tools
to predict oxidative stability of oils (9496). 13C NMR enables direct observation
of carbon atoms. The selectivity and dispersion of 13C NMR spectra are very
high (96). 13C NMR assesses lipid oxidation by monitoring the changes of carbon
chains in TAG molecules, revealing the specific sites that oxidative degradation
376 LIPID OXIDATION: MEASUREMENT METHODS

b
H d g a a c a a e f g h
b H C O CO CH2 (CH2)n CH CH CH2 CH CH CH2 CH2 (CH2)n CH3
a H C O CO CH2 (CH2)n CH3
b H C O CO CH2 (CH2)n CH3
H
b

TMS
h

a d
e

b
f
c

8 7 6 5 4 3 2 1 0 PPM
1
Figure 8. H NMR spectrum of oxidized canola oil.

occurs (94). However, because the abundance of the NMR active 13C nucleus iso-
tope is only 1.12% of 12C, the sensitivity of 13C NMR is usually much lower than
that of 1H NMR (96).
NMR spectroscopy is a rapid, nondestructive, and reliable technique for asses-
sing lipid oxidation. It simultaneously measures both the primary and the secondary
oxidative changes in oils, and provides specific information on oxidative regions in
the TAG molecules. Thus, NMR spectroscopy is considered a more suitable means
for estimating lipid oxidation than chemical determinations.

8.3. Sensory Evaluation


For the food industry, the detection of oxidative off-flavors by taste or smell is the
main method of deciding when a lipid-containing food is no longer fit for consump-
tion (12). Terminologies and methodologies have been developed for sensory
evaluation of specific food products such as meats, peanuts, and vegetable oils
(97). In the edible oil industry, the AOCS (American Oil Chemists Society) Flavor
Quality Scale (revised) with separate grading and flavor intensity has been
employed for describing lipid oxidation (97), as summarized in Table 3. The
descriptive analysis, including the detection and the description of both the quali-
tative and quantitative sensory aspects of a product, is performed by a trained panel,
as the sensitivity to the off-flavors varies among different individuals (12, 97). The
sensory induction period of the product can be determined.
MEASUREMENT OF FRYING FAT DETERIORATION 377

TABLE 3. A Partial List of Terms Used to Describe


Oxidized Oil.

Flavor-Related Terms Process-Oriented Terms

Buttery Hydrogenated
Nutty Oxidized
Beany Reverted
Grassy Light-struck
Watermelon Rancid
Painty
Fishy

Adapted from (97).

Sensory evaluation of lipid oxidation has been conducted by many researchers


(98100). However, as a subjective method, the reproducibility of sensory analysis
is generally considered worse than that of chemical or instrumental methods. More
recently, use of an electronic nose to monitor the formation of volatile compounds
associated with off-flavors from lipid oxidation has been proposed to supplement
information from human sensory panels (101).

9. MEASUREMENT OF FRYING FAT DETERIORATION

Deep-fat frying is a popular method for food preparation, in which vegetable oils
not only are used as a heat-exchange medium, but also contribute to the quality of
fried products (7). However, lipid oxidation easily occurs at relatively high tem-
peratures, producing a complex series of compounds that exerts undesirable effects
on food flavor and quality (4). The measurement of lipid oxidation, therefore, is
essential to determine its effect on food and oil quality, as well as the useful life
of fats or oils subjected to frying. The oxidative changes in frying fats are charac-
terized by a decrease in the total unsaturation of the fat with increases in the free
fatty acid content, foaming, color, and viscosity as well as the content of polar com-
pounds and polymeric material (4). Quality evaluation of frying fats, may be carried
out in different ways. Physical methods estimate oxidative degradation by monitor-
ing changes in physical properties of frying fats, such as molecular weight, specific
gravity, smoke point, refractive index, chromatic parameter, viscosity, surface
tension, and dielectric constant (4). Generally, rejection point of frying fat is estab-
lished by sensory assessment. Chemical methods include the iodine value, saponi-
fication value, free fatty acid content, peroxide value, TBA value, or p-anisidine
value, among others. PV is less useful because hydroperoxides decompose at about
150 C, and no accumulation of peroxides can be detected.
The extent of oxidation can also be assessed by the analysis of oxidized fatty
acids by spectroscopic means such as IR and NMR techniques (102). Moreover,
GC-MS for volatile profile analysis (103) and HPLC for determination of DNPH
derivatives of nonvolatile higher carbonyl compounds (62) provide qualitative
378 LIPID OXIDATION: MEASUREMENT METHODS

9 7 5 3
COOH

CH3

Double bond may present on the positions of C4, C5, C7, C8, or C9.

(CH2)nH (CH2)nH (CH2)nH

R R R
R = (CH2)11nCOOH 1 < n < 11

(CH2)nH

(CH2)nH (CH2)12nCOOH
(CH2)8nCOOH

n=1&2 n=3&4
Figure 9. Chemical structures of cyclic fatty acids formed during deep frying.

and quantitative evaluation of oxidation in frying fats. Cyclic fatty acids (Figure 9),
which may contain hydroxy and keto groups, are formed during deep frying and can
be measured by chromatography after derivatization (4, 7). Furthermore, determi-
nation of polar material in frying fats is a reliable approach for oil quality evalua-
tion and is an official method in Europe. This method involves separation of fat into
a polar and nonpolar fraction via silica gel chromatography. Nonpolar fat can be
weighed and the total polar material calculated or determined directly by their elu-
tion from the silica gel column (4,7).
Routine analysis for frying fat deterioration has been reviewed by Gertz (104).
Usually, more than two methods are required when using chemical analysis because
no single group of compounds has been identified as a key indicator of oxidative
degradation of frying fats.

10. METHODS FOR MEASURING ANTIOXIDANT ACTIVITY

A variety of natural and synthetic antioxidants are used in fat-containing foods in


order to inhibit lipid oxidation with a wide range of efficiencies, depending on their
properties, concentrations, and processing conditions. The need to measure antiox-
idant activity is well documented. Although numerous methods have been proposed
for measurement of antioxidant activity, the essential features of any test are a sui-
table substrate, an oxidation initiator, and an appropriate measure of endpoint (9).
Therefore, certain aspects should be taken into consideration when selecting a test
for measuring antioxidant activity. These include the model food system used for
METHODS FOR MEASURING ANTIOXIDANT ACTIVITY 379

the test, and the means by which oxidation is accelerated and monitored (12). Nor-
mally, most assessments of antioxidant activity are performed in oil, or other model
systems, giving sensible prediction for the activity in oil or water-in-oil emulsions,
whereas the results may be misleading for oil-in-water emulsions (12). Further-
more, stripping of oils may be necessary in such evaluations because the endogen-
ous antioxidants in nonstripped oils are found to enhance the oxidative stability of
oils, thus giving rise to erroneous results in the efficiency of antioxidants under
investigation (105107). In addition to oils and fats, lipid substrates used for testing
antioxidant activity could be fatty acids, fatty acid ethyl esters or triacylglycerols
(9), and b-carotene (108110). In some cases, such as radical scavenging methods,
no substrate is used. Most test procedures involve initiators to accelerate oxidation.
The combination of increased temperature and oxygen supply, addition of metal
catalysts, and exposure of the reactants to light can reduce the oxidative stability
by a large amount (9, 12). Nevertheless, the elevated temperature may bring about
changes in the oxidation mechanism, thus causing difficulties in the prediction of

TABLE 4. Methods of Expressing Results of Antioxidant Activity Tests.

Method Dimensions

Induction period h, d
Time to reach a set level of oxidation (pre- h, d
induction period)
Rate of oxidation (pre-induction period) mol kg1 hr1, gL1 d1
Concentration to produce equivalent effect to mol kg1, gL1
reference antioxidant (pre-induction period)
Concentration of ROOH functional group after mequiv. kg1
set time period
Concentration of oxidation product after set mg kg1 (ppm w/w)
time period
Scale reading after set time period Absorbance, conductivity, etc.
Free stable radical quenching (DPPH) Percentage inhibition
EC50, concentration to decrease concentration
of test free radical by 50%
TEC50, time to decrease concentration of test
free radical by 50%
Total radical-trapping antioxidant parameter mmol peroxy radical deactivated L1
(TRAP)
ABTS assay, phycoerythrin assay TEAC (mM Trolox equivalent to 1-mM test
substance)
Phycoerythrin assay ORAC, oxygen radical absorbance capacity;
mmol of Trolox equivalents
FRAP assay Absorbance of Fe2 complex at 593 nm
produced by antioxidant reduction of
corresponding tripyridyltriazine Fe3
complex
Metal chelating assay Percentage of inhibition of ferrozine-Fe2
complex formation

NOTE: Also see Tables 1 and 2 for other tests applicable to antioxidant activity determination.
Adapted from (9).
380 LIPID OXIDATION: MEASUREMENT METHODS

antioxidant activity at low temperatures as compared with those at high tempera-


tures (9, 12). After the substrate is oxidized under standard conditions, the oxidation
is monitored by chemical, instrumental, or sensory methods. An appropriate mea-
sure of endpoint is essential for assessing antioxidant activity. Analytical strategies
for endpoint determination include measurement at a fixed time point, measurement
of reaction rate, lag phase measurement, and integrated rate measurement (9). The
resulting antioxidant activity is expressed using a wide range of parameters (Table 4).
Approaches proposed for testing antioxidant activity include measuring of the
current state of oil samples, as discussed above, and radical scavenging assays, which
are gaining popularity in the evaluation of antioxidant activity. Radical scavenging
methods measure the relative abilities of antioxidants to scavenge synthetic radicals
or natural in comparison with the antioxidant potency of a standard antioxidant
compound (111). Trolox (6-hydroxy-2,5,7,8-tetramethylchroma-2-carboxylic acid),
ascorbic acid, and quercetin are among the standard antioxidants frequently
used. The most commonly used synthetic radicals are DPPH (2,2-diphenyl-1-
picrylhydrazyl) and ABTS (3-ethylbenzthiazoline-sulfonic acid) radicals. DPPH test
(112116) and ABTS assay (117122) are simple, rapid, and involve no substrate.
However, it has been suggested that these artificial substrate-free methods do not
always adequately mimic the processes in food systems, which sometimes makes
them less valuable for predicting the effectiveness of the antioxidant in foods (9).
Other measurements of antioxidant activity include FRAP (ferric reducing-
antioxidant power) (123126), TRAP (total radical-trapping antioxidant parameter)
(123, 127), phycoerythrin assay (128, 129), and test of metal chelating capacity
(130, 131), among others. Reviews on methods for testing antioxidant activity
have been published (9, 12).

11. CONCLUSIONS AND RECOMMENDATIONS

Lipid oxidation may be assessed in many ways, among which changes in the initial
reactants and formation of oxidation products are most commonly assessed. Mean-
while, sensory analysis assesses both the subjective and, in some cases, objective
measurements of oxidative changes in foods. Each method shows both advantages
and disadvantages, thus it is important to select the most adequate method, depend-
ing on the system under investigation and the state of oxidation itself. The use of
two or more methods assessing both primary and secondary oxidation products is
highly recommended.

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