Quantitative HPLC determination of the antioxidant activity of capsaicin on the formation of lipid hydroperoxides of linoleic acid: A comparative study against BHT and melatonin

Article abstract of DOI:10.1021/jf980949t

The antioxidant activity of capsaicin, as compared to BHT and melatonin, was determined by the direct measurement of lipid hydroperoxides formed upon linoleic acid autoxidation initiated by AIBN. The formation of four isomeric lipid hydroperoxides was detected after reverse-phase HPLC separation. Data from three detectors, UV absorption, glassy carbon electrode electrochemical detection, and postcolumn chemiluminescence using luminol, were compared. Capsaicin was more effective than melatonin in suppressing the formation of lipid hydroperoxides but not as effective as BHT. The formation of capsaicin and BHT dimers was observed during oxidation, and the dimers were characterized using APCI MS(n).

Full text of DOI:10.1021/jf980949t

J. Agric. Food Chem. 1999, 47, 2563−2570
2563
Qu a n tita tive HP LC Deter m in a tion of th e An tioxid a n t Activity of
Ca p sa icin on th e F or m a tion of Lip id Hyd r op er oxid es of Lin oleic
Acid : A Com p a r a tive Stu d y a ga in st BHT a n d Mela ton in
David E. Henderson* and Adam M. Slickman
Chemistry Department, Trinity College, Hartford, Connecticut 06106
Susan K. Henderson
Chemistry Department, Quinnipiac College, Hamden, Connecticut 06518
The antioxidant activity of capsaicin, as compared to BHT and melatonin, was determined by the
direct measurement of lipid hydroperoxides formed upon linoleic acid autoxidation initiated by AIBN.
The formation of four isomeric lipid hydroperoxides was detected after reverse-phase HPLC
separation. Data from three detectors, UV absorption, glassy carbon electrode electrochemical
detection, and postcolumn chemiluminescence using luminol, were compared. Capsaicin was more
effective than melatonin in suppressing the formation of lipid hydroperoxides but not as effective
as BHT. The formation of capsaicin and BHT dimers was observed during oxidation, and the dimers
were characterized using APCI MSn.
Keyw or d s: Lipid hydroperoxide; capsaicin; butylated hydroxytoluene; melatonin; electrochemical
detection; HPLC; APCI-MS; antioxidant; linoleic acid autoxidation
INTRODUCTION
The search for plant-derived antioxidants is a topic
currently receiving a great deal of attention, partly due
to the suspected connection between lipid oxidation and
various pathologies such as cancer, heart disease, and
aging (Dix and Aikens, 1993). This study reports the
development of a simple, quantitative LC-EC procedure
for the comparison of antioxidant effectiveness by the
direct measurement of the first formed stable products
of lipid oxidation, namely, lipid hydroperoxides. This
procedure was used to compare the antioxidant (AO)
activity of capsaicin (CAP) to butylated hydroxytoluene
(BHT), a commonly used synthetic AO, and melatonin,
a hormone believed to have AO activity.
Capsaicin is the principle ingredient responsible for
the irritating and pungent effects of hot chile peppers
and has become more widely used as the popularity of
cuisines featuring hot and spicy foods has increased. In
a previous study (Henderson and Henderson, 1992) it
was observed that the oxidation of oleic acid at cooking
temperatures was inhibited by the presence of CAP.
CAP, shown in Figure 1, is an intriguing molecule for
use as an AO because it incorporates the phenolic
structure found in the widely used synthetic antioxidant
BHT. In addition, CAP contains an amide group which
has been associated with AO activity and is the most
probable site for free-radical attack in molecular model-
ing studies using Personal CAChe (Oxford Molecular
Group, Oxford, U.K.). Further, the amide group of CAP
is involved in thermal oxidation reactions of CAP
(Henderson, 1992). MEL (Figure 1) has been shown to
F igu r e 1. Structures of antioxidants.
exhibit AO activity (Pieri, 1994) and was included in
the study to evaluate possible effects of the amide group.
* To whom correspondence should be addressed (e-mail
david.henderson@trincoll.edu).
10.1021/jf980949t CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/15/1999

2564 J. Agric. Food Chem., Vol. 47, No. 7, 1999
Henderson et al.
F igu r e 2. Partial mechanism of oxidation of linoleic acid (Adapted from Porter, 1984).
It is noted that recent reports indicate that MEL’s AO
activity is not due to radical scavenging (Antunes, 1999).
It is widely accepted that the general pathway for
lipid autoxidation is initiated by the formation of a lipid
radical (L•) which reacts with molecular oxygen at a rate
limited only by the diffusion of the gas in the system.
The resulting lipid peroxyl radical (LOO•) is believed
to propagate a free-radical chain mechanism by ab-
stracting allylic hydrogen from a lipid molecule to
generate a new L• and the first stable molecular product
of this process, a lipid hydroperoxide (LOOH). This
process is shown schematically in Figure 2. Free-radical
oxidation can be interrupted by antioxidants which can
react with either alkyl or peroxyl radicals (Frankel,
1991).
The fatty acid chosen for this study, cis-9-cis-12-
octadecadienoic acid (linoleic acid), was selected for two
reasons. First, linoleic acid is rapidly oxidized in the
presence of the radical initiator 2,2′-azobisisobutyroni-
trile (AIBN) (Yamamoto et al., 1982). This is in contrast
to our initial studies which had indicated that the time
required to obtain a measurable level of oleic acid
oxidation at noncooking temperatures was too great to
be useful. Second, the LOOHs produced (Figure 2)
contain a conjugated diene group which is UV-active
with a molar absorptivity of 26 000 (Chan and Levett,
1977). This provided a detection method during the
development of the parameters for the other detectors
used in this study.
A number of recent studies have reported HPLC
methods that provide very sensitive and selective detec-
tion of LOOHs. The use of chemiluminescence detection
was first reported by Yamamoto et al. (1982), and the
use of reagents other than luminol has been reported
(Akaska et al., 1987; Ohshima et al., 1996). The mea-
surement of various LOOHs has been accomplished by
normal-phase (Hopia et al., 1996) and reverse-phase
HPLC separation (Korytowski et al., 1999) with various
modes of detection. The identification of the specific
LOOH isomers from linoleic acid in this report was
based upon the work of Chan et al. (1975) and the more
recent work of Schneider et al. (1997). Figure 2 shows
the formation of two isomers; however, the hydroper-
oxide can form at either the 9 or 13 position for both
cis-trans and trans-trans forms. The specific mecha-
nisms for the formation of each isomer have been
reported in detail by Porter et al. (1984, 1995).
Electrochemical detection has also been used to
measure LOOHs. Photochemically generated cholesterol
hydroperoxides were analyzed by reverse-phase HPLC
with electrochemical detection performed in the reduc-
tion mode under anaerobic conditions (Korytowski et al.,

Formation of Lipid Hydroperoxides of Linoleic Acid
J. Agric. Food Chem., Vol. 47, No. 7, 1999 2565
through a reaction coil consisting of 6 ft of 0.010 in. i.d.
stainless steel tubing and subsequent fluorescence detection
at 430 nm.
1991, 1999). Terao et al. (1988) reported the measure-
ment of arachidonic acid hydroperoxides and their
hydroxy derivatives by reverse-phase HPLC using
combined UV absorption and reductive electrochemical
detection.
One goal of this study was to select the optimal
detector for ongoing studies of antioxidant effectiveness
of natural products. The UV detector is limited to
polyunsaturated fats, and the wavelength used is
absorbed by many other substances, leading to potential
interferences. Postcolumn chemiluminescence, like all
postcolumn methods, suffers from degraded chromatog-
raphy and instrumental complexity. Electrochemical
detection should offer a true measure of the hydroper-
oxide functional group and allow efficient and simple
detection of a wide range of conjugated and nonconju-
gated lipid hydroperoxides with minimal interferences.
Electrochemical detection was performed using a BAS-200
HPLC (West Lafayette, IN) with dual-channel glassy carbon
amperometric detection. The system was operated by a PC
running BAS Control and BAS Report software in Windows
95. Solvents were preheated to 50 °C and degassed with
helium. The column oven was operated at 50 °C throughout
the study. Injection was by means of a Hitachi AS4000
autosampler for initial studies and kinetics studies. Quantita-
tive comparison of antioxidants were performed using a CMA
200 refrigerated autosampler kept at 4 °C (Acton, MA). The
outlet of the electrochemical detector was connected to a Gilson
(Middleton, WI) 115 UV detector for initial studies and to a
Hewlett-Packard1040 diode array detector (Palo Alto, CA) for
later studies. The HP detector was operated by a HP 300
Chemstation for data acquisition and signal processing. Detec-
tion was done at 233 and 280 nm.
This study reports the first use of direct reverse-phase
HPLC analysis of LOOHs for the evaluation of AO
effectiveness. Hopia, Huang, and Frankel have previ-
ously used normal-phase HPLC for studies of the
antioxidants R-tocopherol and Trolox (Hopia et al.,
1996). Since LOOHs are the first nonradical products
formed in the lipid oxidation process and hydroperoxides
are known to be responsible for tissue damage and
toxicity in both oxidized food products and in vivo, their
direct measurement offers the best single measure of
the effectiveness of an AO. The analysis of small-chain
volatile acid decomposition products in the Rancimat
method or malonaldehyde in the TBA method (Pokorny
et al., 1985) would not be able to differentiate between
antioxidants which prevented LOOH formation and
those which allowed peroxides to accumulate by block-
ing their subsequent degradation to secondary products.
Thus, direct hydroperoxide measurement should provide
more specific information on the mechanisms and true
effectiveness of antioxidants.
The mobile phase for electrochemical detection was identical
to that for chemiluminescence except for the addition of 0.030
M tetraethylammonium perchlorate as supporting electrolyte.
Oxidative mode detection was optimized at +1300 mV vs a
AgCl reference electrode. The second channel of the detector
was operated in reductive mode at +600 mV vs a AgCl
reference.
Mass spectra were obtained using a VG-Trio-2 quadrapole
MS (Micromass, Danvers, MA) with LabBase software and
using the atmospheric pressure chemical ionization (APCI)
LC-MS interface of the LCQ (Finnigan, Piscataway, NJ ). The
LCQ included a TSP HPLC system with a TSP 6000 diode
array detector (Thermo Separations Products, Piscataway,
NJ ), which was used to determine the wavelengths of hydro-
peroxide peaks for this report. The APCI source was operated
at 1.3 mL/min flow of 70:29.5:0.5 methanol:water:acetic acid.
Sheath gas flow 70, auxiliary gas flow 10, discharge current 5
µA, capillary temperature 150 °C, vaporizer temperature 350
°C for LOOH and 400 °C for dicapsaicin. Lipid hydroperoxides
were analyzed in negative-ion mode, while the capsaicin dimer
analysis was done in positive-ion mode.
Sa m p le P r ep a r a tion for Oxid a tion Stu d y. A 0.30 M
solution of linoleic acid (180.4 g/mol) was prepared by dissolv-
ing 0.54 g of linoleic acid in 10.00 mL of methanol. Linoleic
acid was stored at -70 °C under nitrogen prior to use. A 0.20
M solution of AIBN (164.2 g/mol) was prepared by dissolving
32.8 mg of AIBN in 1.00 mL of methanol. Antioxidant solutions
of 0.20 M were prepared by dissolving 61 mg of CAP in 1.00
mL of methanol, 44 mg of BHT in 1.00 mL of methanol, and
46 mg of MEL in 1.00 mL of methanol. A series of duplicate
samples were prepared by placing 1.00 mL of the linoleic acid
solution in each of 10 amber autosampler vials. Two vials were
used as controls to monitor the lipid without the addition of
AIBN or AO. To an additional two vials, 0.10 mL of AIBN
solution was added in order to measure the concentration of
LOOH in the presence of a free-radical initiator without the
addition of AO. To the remaining five vials, 0.10 mL of AIBN
solution was added and 0.10 mL of either CAP, BHT, or MEL
was added in duplicate. All of the vials were sealed with PTFE
caps. Each of the vials was immediately analyzed by HPLC
and stored at room temperature in the autosampler for the
time series analysis. This procedure produced a 1:1 molar ratio
of AIBN to AO and a 12:1 molar ratio of linoleic acid to AIBN.
Samples for quantitative comparison of antioxidants and for
concentration dependence studies were prepared as for the
time series analysis; however, four different volumes, 0.10,
0.050, 0.010, and 0.0050 mL, of each of the AO solutions were
added to separate vials. Appropriate volumes of methanol were
added to each vial in order to bring the final volume to 1.20
mL. Vials were filled in air and sealed. They were then
transferred to a constant-temperature bath operating at 30.0
°C for 1 week. They were then stored at -70 °C until they
were analyzed and then kept at 4 °C during analysis.
MATERIALS AND METHODS
Rea gen ts. CAP, linoleic acid, luminol (3-aminophthalhy-
drazide), hemin chloride (bovine), BHT, MEL, AIBN, and
sodium carbonate were purchased from Sigma Chemical Co.
(St. Louis, MO). HPLC solvents were Fisher HPLC grade.
Tetraethylammonium perchlorate was prepared by mixing
equimolar quantities of tetraethylammonium hydroxide
(Aldrich Chemical Co., Milwauke, WI) and concentrated per-
chloric acid (Fisher Scientific, Springfield, NJ ) in 0 °C water.
The resulting precipitate was vacuum filtered, rinsed with
water, and air-dried.
In str u m en ta tion . Chemiluminescence detection was per-
formed using a Hitachi (Tokyo, J apan) model 6300 dual-pump
solvent delivery system equipped with a Hitachi AS 4000
intelligent auto sampler, Hitachi model D6100 data system,
Hitachi model L3000 photo diode array detector at 233 nm,
and Hitachi F1050 fluorescence detector with the lamp turned
off and the emission wavelength set at 430 nm. A column oven,
Hitachi L 5020, was used to maintain the column temperature
at 50 °C. A luminol solution containing 15 g of sodium
carbonate, 0.0025 g of bovine hemin, and 0.1234 g of luminol
was prepared. This solution was found to have a pH of 11.
The HPLC column mobile phase used was 70:30 methanol-
water. The addition of 2% acetic acid to the water improved
the separation of the hydroperoxide peaks and was used
throughout the study. Flow rates used were 1.3 mL/min for
the column mobile phase and 3.0 mL/min for the postcolumn
luminol solution. The HPLC column used was a Microsorb
Short One, C-18, 3 µm, 20 cm in length, equipped with a 2 cm
guard column (Ranin, Emeryville, CA). Postcolumn detection
was accomplished by passing the sample-luminol mixture
The oxidation products of capsaicin were purified for further
analysis by solid-phase extraction using 1000 mg C18 SPE

2566 J. Agric. Food Chem., Vol. 47, No. 7, 1999
Henderson et al.
cartridges (Alltech Assoc., Deerfield, IL). The cartridge was
prepared for use according to instructions. A 1.0 mL amount
of oxidized CAP was absorbed. Elution of free CAP was done
with 7 mL of 70% methanol-water. Oxidized CAP was then
eluted with 3 mL of 100% methanol. The product was dried
with anhydrous Na2SO4 and evaporated to dryness under
nitrogen. Trimethylsilyl derivatives of capsaicin oxidation
products were prepared using Tri-Sil (Pierce, Rockford, IL).
Syn th esis of 13-Hyd r op er oxy Lin oleic Acid . A standard
solution of 13-hydroperoxy-cis-9-trans-11-octadecadienoic acid
was prepared using the procedure of Hamberg and Samuels-
son, using type I-B soybean lipoxidase (Sigma). Linoleic acid
was reacted for 30 min under oxygen at room temperature.
Samples were extracted with dichloromethane and stored in
amber vials at -70 °C. Before use, the dichloromethane was
evaporated using a helium stream at reduced pressure.
Samples were then redissolved in methanol for spectroscopic
and HPLC analysis. Calibration curves for the UV and EC
detectors were run using this solution to produce seven
solutions in successive 1:4 dilutions. UV absorption at 233 nm
was used with an absorptivity of 26 000 (Chan and Levett,
1997) to determine the concentration of LOOH. Calibration
curves covered the range from 5400 ng of LOOH injected (in
10 µL) to 5.4 ng of LOOH. Peak areas of duplicate injections
were averaged.
RESULTS AND DISCUSSION
Dir ect HP LC An a lysis of Lip id Hyd r op er oxid es.
Chromatograms for the separation of LOOHs using (a)
electrochemical detection, (b) UV absorption detection,
and (c) chemiluminescence detection are shown in
Figure 3a, b, and c, respectively. The identification of
the peaks, which represent four geometrical isomers,
was based upon the work of Schneider et al. (1997).
Early in this study, three major peaks were obtained
for the LOOHs. The first major peak, labeled peak 2
(λmax ) 237 nm) in Figure 3, was identified as an
unresolved mixture of the 9- and 13-hydroperoxy cis-
trans isomers. The retention time of the enzymatically
synthesized 13-hydroperoxy-cis-9-trans-11-octadecadi-
eneoic acid matched this peak, confirming this part of
the peak assignment. Peak 3 was identified as 13-
hydroperoxy-trans-9-trans-11-octadecadienoic acid (λmax
) 238 nm), and peak 4 was identified as 9-hydroperoxy-
trans-10-trans-12-octadecadienoic acid (λmax ) 238 nm).
All previous reverse-phase HPLC separations indicate
that the trans-trans isomers elute after the cis-trans
isomers (Schneider et al., 1997).
During the course of the study, two additional peaks
appeared. Peak 1, a small peak preceding the 13-
hydroperoxy-cis-9-trans-11-octadecadieneoic acid peak,
was observed on all three detectors (λmax ) 234 nm) and
is thus believed to be a LOOH. However, the expectation
that the 9- and 13-hydroperoxides should be formed in
approximately equal concentrations argues against as-
signing this peak to one of these isomers. Schneider et
al. (1997) reported the presence of other, unidentified
hydroperoxides in their mass spectral study, and this
peak may correspond to one of those LOOHs found in
their study. Negative-ion APCI spectra of all four peaks
show two major ions, m/z 311 (M-H-) and 371 (M +
acetate-). MS-MS spectra yielded a peak at m/z 293
(M - H2O-). MS3 spectra were below detection limits
and did not yield useful information on the identity of
the isomers. The values of λmax observed for the lipid
hydroperoxide peaks were measured at 1 nm resolution
using the TSP detector. All were shifted to longer
wavelengths than those reported by Hopia et al. (1996).
This shift is attributed to the higher polarity of the
F igu r e 3. Chromatograms of 0.20 M linoleic acid, 0.001 M
capsaicin, 0.02 M AIBN mixture after extensive lipid oxidation.
Flow rate 1.3 mL/min; column temperature 50 °C; mobile
phase 70% methanol, 30% water, with 2% acetic acid. For EC
and UV detection, mobile phase was also 0.030 M in tetra-
ethylammonium perchlorate. Chemiluminescence luminol so-
lution added postcolumn at 3.0 mL/min. Peaks between 2 and
4 min in EC chromatogram are capsaicin and dihydrocapsai-
cin. Hydroperoxide peaks identified in text. Asterisk (/)
indicates the position of peak 5.
aqueous methanol mobile phase when compared with
the nonpolar hexane-diethyl ether mobile phase used
for normal-phase HPLC.
A fifth small peak was observed between the cis-
trans peak and the two trans-trans peaks. This peak
has not been identified and is not included in the
quantization since it is below the detection limits on
most samples. It was not definitively detected in either
positive or negative APCI LC-MS chromatograms.
The retention time of each peak slowly increased as
the column aged, though the data were stable within
the time scale of several days. This was due to a buildup
of free linoleic acid on the column. Since linoleic acid is
not detected by EC, A233, or CL detectors, it had no
effects other than those on retention time. Periodic
flushing of the column with methanol restored the
retention times to their initial values. LC-MS studies
which did detect the linoleic acid showed that with a
short methanol flush after every three analyses, linoleic

Formation of Lipid Hydroperoxides of Linoleic Acid
J. Agric. Food Chem., Vol. 47, No. 7, 1999 2567
Ta ble 1. Lin ea r Regr ession for EC a n d UV Detector
Ca libr a tion Cu r ves for 13-c-t-LOOH
correlation
detector
slope
intercept
coeficient
UV
EC
1.035
1.033
-2.148
8.88
0.99999
0.99995
acid accumulation was completely eliminated and never
coeluted with the analytes.
Com p a r ison of Detector s for Lip id Hyd r op er -
oxid es. One objective of this study was the comparison
of the EC detector with UV and CL detectors noted
above. Hydroperoxides are amenable to both oxidation
and reduction in electrochemical systems. Both modes
were explored using glassy carbon electrodes. The
transition from oxidation to reduction for the LOOH
peaks occurs at approximately +600 mV vs AgCl in the
mobile phase used. The highest sensitivity was observed
with the highest positive potential which could be
achieved without excessive background, +1300 mV. At
this potential, the possibility of interferences by other
electrochemically active species is a concern. The second
cell of the detector was operated at +520 mV at which
the LOOH’s were reduced very selectively.
F igu r e 4. Time series plot of the log of total lipid hydroper-
oxide peak height (peaks 2-4) using chemiluminescence
detector. Solutions 0.20 M lipid, 0.020 M AIBN, and 0.020 M
antioxidant in air.
The calibration curves prepared using UV and EC
detection at 1300 mV showed excellent linearity over
the 3 orders of magnitude studied. Linear regression
results for both detectors for the enzymatically produced
13-cis-9-trans-11-LOOH are given in Table 1. The data
for the EC detector were divided by 266 to normalize
the differences in the numerical values of peak area
produced by the two data systems. The correlation
coefficients of 0.9999 for both detectors are indicative
of the excellent quantitative capability of both detectors.
At quantities of 100 ng and greater, the reproducibility
between injections of the same sample was excellent
with precision better than 2% relative standard devia-
tion. Below 95 ng, the precision of both detectors
decreased in a similar fashion. More significantly, while
the peaks at 5.9 ng were clearly distinguishable in
chromatograms for both the EC and UV detectors, the
peak areas were 50% smaller than those predicted by
the linear regression line. This is interpreted as evidence
of LOOH instability at these low concentrations. Similar
instability has been observed for other peroxides studied
in this laboratory and may be due to adsorption on
surfaces or decomposition on contact with metal com-
ponents in the injection system. The EC detector
calibration curves at 520 mV were comparable to those
at 1300 mV but an order of magnitude less sensitive.
No peak was detectable below 95 ng of injected LOOH.
Calibration curves for 13-cis-9-trans-11-LOOH on the
CL detector were not run. The data for the time series
studies was used to estimate the relative sensitivity and
reproducibility as compared with the UV detector. In
general, the CL detector suffered from greater base line
noise and lower overall sensitivity than the UV detector.
The CL detector also suffers from increased instrumen-
tal complication, and it was the most difficult to operate
in a reliable and reproducible manner. The postcolumn
volume required for mixing reagents and delay time for
reaction significantly degrades the chromatographic
separation. This is shown clearly in Figure 3. The only
advantage of the CL system is the ability to determine
unequivocally the presence of hydroperoxide in a specific
peak.
F igu r e 5. Comparison of the log of total cis-trans hydrop-
eroxide peak height and the log of total trans-trans hydrop-
eroxide peak height as a function of time. Lipid solutions as
in Figure 4.
the samples in an autosampler at room temperature and
sampling them repetitively over a period of more than
2 weeks. The study was conducted separately on both
the LC/EC-UV instrument and on the UV/chemilumi-
nescence system. The results of these studies are
summarized in Figure 4 and showed that the addition
of AIBN to linoleic acid caused rapid production of
LOOHs. The rate of increase in LOOH concentration
slows after 2000 min (about 33 h). This behavior may
be due to depletion of AIBN after 2000 min or to the
system approaching a steady state in which the LOOH
is decomposing. Throughout the entire time period
studied, the amount of LOOH produced in the presence
of AIBN was substantially higher than that observed
for linoleic acid alone. Both BHT and CAP consistently
showed a lower concentration of LOOH produced in the
presence of AIBN than that of the linoleic acid without
AIBN. This data is consistent with the expected behav-
ior of antioxidants.
Figure 5 compares the formation of the cis-trans
isomers to the formation of the trans-trans isomers as
a function of time. In the blank, the peak height of the
cis-trans isomer peak was approximately equal to the
sum of the height of the two trans-trans isomer peaks,
indicating that the air oxidation of linoleic acid produced
equal amounts of the four isomers. When linoleic acid
is oxidized in the presence of AIBN-generated free
radicals, the trans-trans isomers clearly predominate
Tim e Ser ies Stu d y of An tioxid a n ts. The formation
of LOOHs as a function of time was studied by placing

2568 J. Agric. Food Chem., Vol. 47, No. 7, 1999
Henderson et al.
F igu r e 7. Comparison of antioxidant effectiveness of four
antioxidants at 0.002 M concentration. Conditions as in Figure
6.
F igu r e 6. Comparison of antioxidant effectiveness of four
antioxidants at 0.001 M concentration. Data from electro-
chemical detector at 1300 mV. All peak heights normalized to
0.020 M AIBN solution and 0.20 M linoleic acid in air without
antioxidant. Oxidation at 30 °C for 8 days.
over the cis-trans isomers. In the presence of the
antioxidants CAP and BHT (not shown), the cis-trans
isomers appear first during oxidation and predominate
over the trans-trans isomers. Linoleic acid without the
radical initiator produces approximately equal quanti-
ties of the cis-trans and trans-trans isomers. The
formation of the cis-trans isomers are kinetically
favored. Their formation does not require bond rotation
or the removal of oxygen from the intermediate hydro-
peroxy radical (Figure 2) (Porter, 1984). The presence
of CAP and BHT favors cis-trans product formation by
a small extent while reducing the total LOOH formation
substantially.
Qu a n tita tive Stu d ies of An tioxid a n t Effica cy.
Quantitative determination of the production of each
of the isomers specified above was based on peak heights
using UV and electrochemical detection at 1300 mV.
Electrochemical detection was used for all quantization
because it was more selective than UV absorption
detection and proved to be more sensitive and much
more reproducible than chemiluminescence detection.
On the basis of the results of the time series study, it
was determined that a time of 10 000 min (7 days) was
adequate to achieve significant lipid oxidation, even in
the presence of the antioxidants, to allow reliable
quantization of the relative effects of the various anti-
oxidants with all three detectors.
Quantitative determination of the four hydroperoxide
isomers was performed on blank samples containing
only linoleic acid, samples containing linoleic acid and
AIBN, and samples containing linoleic acid, AIBN, and
each of the individual antioxidants. The concentration
of each of the four isomers obtained at varying molar
concentrations of each of the antioxidants used is shown
in Figures 6-9. Figure 6 shows the normalized results
for each isomer peak and the total LOOH formation at
0.001 M of each of the antioxidants. The results were
normalized against samples containing only linoleic acid
and AIBN. BHT was, by far, the most effective AO, in
that the total of the lipid peroxides formed decreased
by about 80% of those formed without AO. CAP was
found to decrease the production of LOOHs by about
30%. Melatonin, under these conditions, increased the
formation of LOOHs by about 10%, suggesting it acts
as a pro-oxidant. At this low concentration of AO, the
ratio of isomers formed in the presence of each of the
antioxidants is similar.
F igu r e 8. Comparison of antioxidant effectiveness of four
antioxidants at 0.010 M concentration. Conditions as in Figure
6.
Figure 7 demonstrates the effect of doubling the AO
concentration to 0.002 M. This increases the effective-
ness of all of the antioxidants. The production of LOOHs
was decreased by 90% and 60% for BHT and CAP,
respectively. The increased amount of MEL increased
the formation of LOOHs by 20%, again indicating the
pro-oxidant behavior under these conditions.
The concentration of each of the antioxidants was
increased to 0.01 M, and Figure 8 shows that the level
of LOOHs produced decreased substantially. A com-
parison of BHT, CAP, and MEL indicated that the
reduction in overall LOOH formation was about 97%,
78%, and 28%, respectively. It is noteworthy that at this
concentration, MEL switches from pro-oxidant to AO
behavior. Figure 9 shows the effect of increasing the
concentration of AO to 0.02 M. The formation of LOOHs
overall was reduced by about 98% using BHT, by about
88% using CAP, and by about 10% using MEL.
The relationship between the concentration of linoleic
hydroperoxides produced and the inverse of the concen-
tration of AO is shown in Figure 10. The data shown
for both BHT and CAP show a simple linear relationship
with correlation coefficients greater than 0.99. The
steepness of the slope of the line should be inversely
related to the effectiveness of the AO, again indicating
the effectiveness of CAP and the greater effectiveness
of BHT.
The data shown for MEL indicate that a more
complex relationship exists between the concentration
of AO and the concentration of hydroperoxides produced.

Formation of Lipid Hydroperoxides of Linoleic Acid
J. Agric. Food Chem., Vol. 47, No. 7, 1999 2569
F igu r e 9. Comparison of antioxidant effectiveness of four
antioxidants at 0.020 M concentration. Conditions as in Figure
6.
F igu r e 11. Structure of dicapsaicin from oxidation of cap-
saicin.
not been determined. The oxidation peak for BHT was
collected and subjected to GC-MS. It was identified as
the dimer of BHT.
The presence of both BHT and CAP dimers in this
reaction is consistent with the explanation that both of
these act as antioxidants by the same process, the
donation of a hydrogen from the aromatic ring to a more
reactive radical.
CONCLUSIONS
There are significant differences in the effectiveness
of BHT, CAP, and MEL as antioxidants. BHT proved
to be the most effective AO in this study. Both BHT and
CAP appear to operate by a simple mechanism which
does not change over the range of concentrations
studied, with effectiveness being linearly related to the
reciprocal of AO concentration.
F igu r e 10. Plot of total lipid hydroperoxide peak height as a
function of the reciprocal of antioxidant concentration. Least-
squares lines for BHT and capsaicin with correlation better
than 0.99. Line for melatonin shows nonlinear relation-
ship.
Under the conditions of this study, melatonin showed
very limited AO activity at higher concentrations and
pro-oxidant activity at low concentrations. This finding
is consistent with recent reports that melatonin is not
a free-radical scavenger antioxidant (Antunes, 1999).
This leads the authors to conclude that the amide group
present in the CAP molecule probably does not play a
major role in its AO activity under free-radical oxidation
conditions. Thus, the AO behavior observed for CAP
under these conditions is due primarily to the phenolic
moiety in the molecule. Previous work on the antioxi-
dant effects of CAP under high-temperature conditions
showed considerable involvement of the amide group in
formation of a range of amides and imides (Henderson,
1992). Further studies of the formation of lipid hydro-
peroxides under thermal conditions and the effects of
CAP and BHT on this process will be conducted to
clarify the mechanism of CAP during thermal oxidation.
This study demonstrates the ability to analyze LOOHs
directly and to use this analysis to evaluate the ef-
fectiveness and mechanism of action of antioxidants.
Ongoing studies in our laboratories are directed at
evaluating the effectiveness of other natural product
compounds as potential antioxidants in lipid systems.
In addition, studies are underway to evaluate the effect
of oxygen pressure on the effectiveness and mechanisms
of AO action. The HPLC methodology developed here
is also being employed to study thermal oxidation of
lipids.
The negative slope for melatonin at low concentrations
is consistent with its action as a pro-oxidant under these
conditions. The nonlinearity of these data is indicative
of changes in the mechanism of action as a function of
concentration.
Ma ss Sp ectr a l An a lysis of Ca p sa icin Oxid a tion
P r od u cts. During the course of oxidation reactions
involving both BHT and CAP with AIBN, a peak at
retention times much longer than that of the lipid
hydroperoxides was observed which increased regularly
as a function of time of oxidation. For the oxidation of
samples containing pure capsaicin, a single oxidation
peak was determined to be due to a coupling reaction
to produce a capsaicin dimer at m/z 608. When capsaicin
samples containing dihydrocapsaicin and other homo-
loges of capsaicin were oxidized, the multiple HPLC
peaks contained a mixture of dimers representing all
possible combinations the various homologues. The
MS-MS spectra of the CAP dimer showed major peaks
at 273, 420, and 440. The peak at 440 was identified as
the loss of one amide side chain from the dicapsaicin
and shifted to 442 for the dihydrocapsaicin dimer (m/z
612). The peak at 273 is the methoxyphenol core of the
dimer after loss of both side amide chains. The peak at
420 is the same for both capsaicin and dihydrocapsaicin
and has not yet been assigned. APCI spectra of the
silylated dicapsaicin shows the presence of two phenolic
groups indicative of the structure shown in Figure 11.
The exact position of the dimerization on the ring has

2570 J. Agric. Food Chem., Vol. 47, No. 7, 1999
Henderson et al.
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Received for review August 24, 1998. Revised manuscript
received April 12, 1999. Accepted May 10, 1999. We gratefully
acknowledge the support of the Trinity College Faculty
Research Committee and the Quinnipiac College Sabbatical
Leave Committee for their support of this work. The generous
donation of HPLC instrumentation by Bristol Myers Squibb
is also gratefully acknowledged.
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