H. Takamura et al., J. Near Infrared Spectrosc.

3, 219–225 (1995) 219

Determination of Lipid Oxidation in Edible Oils by NIR

H. Takamura et al., J. Near Infrared Spectrosc. 3, 219–225 (1995)

Determination of lipid oxidation in edible

oils by near infrared spectroscopy
Hitoshi Takamura, Noriko Hyakumoto, Naoko Endo and Teruyoshi Matoba
Department of Food Science and Nutrition, Nara Women’s University, Nara 630, Japan.

Tamako Nishiike
Division of Human Life and Environmental Sciences, Graduate School of Human Culture, Nara Women’s University,
Nara 630, Japan.

The relationship between near infrared (NIR) second derivative spectra and lipid oxidation was investigated to
develop a method for the determination of lipid oxidation in edible oils by NIR spectroscopy, using peroxide value
(POV) as the index of oxidation. Although several absorption peaks were found in the difference second derivative
spectra of oxidised edible oils, the intensity of the peak at 2084 nm only was highly correlated to POV. In the
spectra of purified hydroperoxides of methyl oleate and methyl linoleate, the intensity of the peak at 2084 nm was
also highly correlated with POV, which demonstrates that the absorption is due to hydroperoxide. In addition,
this peak shifted and weakened after reduction of hydroperoxide to hydroxide, which shows the absorption is
specific for the hydroperoxyl group. These results suggest that 2084 nm is the key wavelength for lipid peroxide
and can be used for the determination of lipid oxidation in edible oils.
Keywords: lipid oxidation, edible oil, peroxide value, near infrared.

Introduction peroxides by NIR spectroscopy.14 However, this

The near infrared (NIR) technique has been
widely used for quantitative analyses of food com-
ponents, such as moisture, protein, carbohydrate
and lipid.1–7 It is also used to determine the quality
of foods. In our laboratory, spectral analysis for
protein determination by NIR has been studied for
many years.8–12 Lipid is also an important nutrient
and easily deteriorates by oxidation and hydrolysis.
Lipid analysis by NIR spectroscopy was first re-
ported by Holman and Edmondson in 1956.13 Hol-
man et al. also reported the detection of lipid
Part of this study was presented at the 9th Symposium on NIR
Spectroscopy in November 1993, Osaka, Japan, and at the 7th
International Conference on Near Infrared Spectroscopy in
August 1995, Montréal, Canada.
method is not yet commonly adopted. In order to
determine the deterioration of edible oils, peroxide
value (POV), carbonyl value (COV) and acid value
(AV) are commonly used. POV is the amount of
hydroperoxides which are the primary products in
lipid oxidation. COV is the amount of carbonyl
compounds (aldehydes and ketones) which are the
secondary products in lipid oxidation. AV is the
amount of free fatty acids which are produced by
the hydrolysis of oil. These values are generally
determined by titrimetric or colorimetric methods,
which need a long time for the analysis, ample
samples, hazardous reagents and skill. Therefore,
the development of simple and easy methods is
required. In this study, we have found a specific
absorption peak of lipid peroxides and developed a
method for the determination of lipid oxidation in

© NIR Publications 1995, ISSN 0967-0335

220
edible oils by NIR spectroscopy using POV as the
index of oxidation.
Materials and methods
Materials
Canola, olive, safflower, soya bean and cotton
seed oils were purchased from Nacalai Tesque
(Kyoto, Japan). Methyl oleate (>99%) and methyl
linoleate (>99%) were from Wako Pure Chemicals
(Osaka, Japan) and Tokyo Chemical Industry (To-
kyo, Japan), respectively. t-Butyl hydroperoxide
was from Wako Pure Chemicals. Other reagents
were from Nacalai Tesque and Wako Pure Chemi-
cals.
Preparation of oxidised lipids
Canola, olive and safflower oils were purified by
florisil column chromatography to remove toco-
pherols.15 Soya bean and cotton seed oils were not
purified. These oils were then oxidised by autooxi-
dation at 50°C. Canola, olive and safflower oils
were oxidised until the POV reached approximately
600. Soya bean and cotton seed oils were oxidised
until the POV reached 30. Methyl linoleate was also
oxidised by autooxidation. Methyl oleate was oxi-
dised by UV-induced oxidation. Methyl oleate was
irradiated with a UV lamp (254 nm, 10 W) from a
distance of 14 cm for 3 h, and then kept in the dark
at 50°C for a couple of weeks. Methyl oleate and
methyl linoleate hydroperoxides were separated by
silica gel column chromatography from oxidised
methyl oleate and methyl linoleate, respectively.16
Methyl linoleate hydroxide was prepared by reduc-
tion of methyl linoleate hydroperoxide with sodium
borohydride according to Hamberg and Sa-
muelsson.17
Samples
Lipid samples were analysed by a NIR spec-
trometer and then by the chemical method for POV.
t-Butyl, methyl oleate and methyl linoleate hydro-
peroxides were mixed with unoxidised methyl li-
noleate before analysis. Methyl linoleate hydroxide
was also mixed with unoxidised methyl linoleate.
Determination of Lipid Oxidation in Edible Oils by NIR
NIR determination
NIR transmittance spectra (1100–2500 nm, 2 nm
intervals) of lipids were determined in a 1 mm cu-
vette cell using the NIRSystems (Pacific Scientific)
Model 6250 Research Composition Analyser at
30°C. Data processing and regression analysis were
carried out by using NSAS software (Ver. 3.27)
from NIRSystems.
POV determination
POV was determined according to the AOAC
standard method.18
Results and discussion
Change in NIR spectra of edible oils
during oxidation
Figure 1 shows the raw spectra (a), the second
derivative (segment = 10, gap = 0) spectra (b) and
the difference second derivative spectra (c) of oxi-
dised canola oil. The POV range was approximately
0–600. The raw spectra were too broad to show the
spectral changes due to oxidation. The second de-
rivative spectra showed the absorption peaks due to
oxidation. However, these peaks were small com-
pared to other peaks. The difference second deriva-
tive spectra, which were calculated by subtracting
the second derivative spectrum of unoxidised oil,
had clear peaks due to oxidation. Several peaks in
the negative direction, which are true absorption
peaks in second derivative spectra, were confirmed
at 1468, 1684, 1744, 1916, 2084 and 2244 nm. The
NIR spectra of oxidised olive and safflower oils
were similar to those of canola oil. These results
suggest that these absorption peaks are derived from
lipid oxidation. Previously, Holman et al. reported
that lipid peroxide absorbed at 1460 and 2070 nm
by using raw NIR spectra. 14 This difference may be
due to the treatment of the spectra.
Correlation between NIR spectra and
POV in oxidised oils
Correlation between the intensity of the absorp-
tion peaks of the NIR second derivative spectra and
POV in oxidised canola, olive and safflower oils was
analysed. Table 1 shows the regression coefficient
H. Takamura et al., J. Near Infrared Spectrosc. 3, 219–225 (1995)
Figure 1. NIR spectra of unoxidised and oxidised canola
oils. The raw spectra (a), the second derivative spectra (b)
and the difference second derivative spectra (c) of unox-
idised and oxidised canola (POV = 0–600) oils are shown.
The difference second derivative spectra were calculated
by subtracting the second derivative spectrum of unoxi-
dised oil.
(K 1), correlation coefficient (R) and standard error
(SE) values for the absorption peaks of oxidised
oils. The R values were large enough for the peak at
2084 nm to be seen in all three oxidised oils, which
suggests that this peak is due to lipid oxidation. In
221
Figure 2. The difference second derivative spectra of
t-butyl, methyl oleate and methyl linoleate. The differ-
ence second derivative spectra of t-butyl hydroperoxide
(a), methyl oleate hydroperoxide (b) and methyl linoleate
hydroperoxide (c) mixed with methyl linoleate were
shown. The difference second derivative spectra were
calculated by subtracting the second derivative spectrum
of unoxidised methyl linoleate. The POV of the spectra
with the largest peaks are approximately 1000.
addition, K 1 values were similar among the three
oils. Although the R values for the peak near 1744
nm were also large, K 1 values were different among
the three oils. Other peaks were not well correlated
to POV. These peaks seem not to be derived from
hydroperoxide but from the secondary oxidation
products. However, there is no evidence that the
222 Determination of Lipid Oxidation in Edible Oils by NIR

Table 1. Correlation between NIR second derivative spectra and POV of oxidised oils.

Canola Olive Safflower

nm K1 R SE nm K1 R SE nm K1 R SE

1450 –116076 –0.846 90.4 1462 –54496 –0.851 77.4 1466 –48714 –0.963 53.6
1684 –22830 –0.990 23.5 1686 –46221 –0.974 19.5 1682 –33608 –0.986 33.9

1744 –16865 –0.994 18.4 1746 –26323 –0.991 19.5 1742 –27565 –0.993 24.0
1916 –73748 –0.960 47.8 1916 –98441 –0.828 82.6 1912 –112560 –0.893 89.8

2084 –15997 –0.999 8.5 2084 –14995 –0.996 12.7 2084 –14931 –0.995 19.2

2244 –21512 –0.929 63.2 2242 –24133 –0.876 71.1 2240 –47348 –0.911 82.2
K 1, regression coefficient (slope).
R, correlation coefficient.
SE, standard error.
The POV range of the samples (n = 14–15) was 0–600.

absorption peak at 2084 nm is due to hydroperoxide absorption peak at 2084 nm were large enough for
since lipid oxidation forms conjugated double these lipid peroxides. In addition, the K 1 values for
bonds (CH=CH–CH=CH), hydroxyl groups (–OH), the absorption peak at 2084 nm were similar to
and carbonyl groups (C=O) other than hydroper- those for oxidised oils (Table 1), which demon-
oxyl groups (–OOH). strates that the absorption peak at 2084 nm is due to
hydroperoxide.
NIR spectra of t-butyl, methyl oleate and
methyl linoleate hydroperoxides and Change in NIR spectra by reduction of
correlation with POV methyl linoleate hydroperoxide
The difference second derivative spectra of t-bu- The difference second derivative spectra of
tyl, methyl oleate and methyl linoleate hydroperox- methyl linoleate hydroperoxide and hydroxide
ides are shown in Figure 2. These spectra were mixed with methyl linoleate are shown in Figures
calculated by subtracting the second derivative
spectra of unoxidised methyl linoleate from those
of hydroperoxides. Methyl oleate/linoleate hydro- Table 2. Correlation between peak intensity at 2084 nm of
peroxides showed the absorption peak at 2084 nm, NIR second derivative spectra and POV of methyl oleate
and methyl linoleate hydroperoxides.
while the absorption peak of t-butyl hydroperoxide
was at 2080 nm. This result suggests that the ab- Methyl oleate Methyl linoleate
sorption peak at 2084 nm is due to the hydroperoxyl
group. The difference between methyl oleate/ K1 R SE K1 R SE
linoleate hydroperoxides and t-butyl hydroperoxide
may be due to the conformation near the hydroper- –15415 –0.998 9.4 –15786 –0.997 20.0
oxyl group. K 1, regression coefficient (slope).
Regression coefficient (K 1 ), correlation coeffi- R, correlation coefficient.
cient (R) and standard error (SE) values for the SE, standard error.
absorption peak of methyl oleate/linoleate hydro- The POV range of the samples (n = 14–15) was 0–
peroxides are listed in Table 2. The R values for the 1000.
H. Takamura et al., J. Near Infrared Spectrosc. 3, 219–225 (1995)
Figure 3. The difference second derivative spectra of
methyl linoleate hydroperoxide and hydroxide mixed
with methyl linoleate. The difference second derivative
spectra of methyl linoleate hydroperoxide (a) and hydrox-
ide (b) mixed with methyl linoleate were shown. The POV
for lines a–e in Figure 3(a) are approxirnately 1000, 800,
600, 400 and 200, respectively. Lines a–e in Figure 3(b)
correspond to lines a–e in Figure 3(a) in the term of the
molar amount of hydroxide and hydroperoxide.
3(a) and 3(b), respectively. These spectra were cal-
culated by subtracting the second derivative spectra
of unoxidised methyl linoleate from those of hy-
droperoxide and hydroxide. The POV range in Fig-
ure 3(a) was approximately 0–1000. Lines a–e in
Figure 3(b) correspond to lines a–e in Figure 3(a) in
terms of the molar amount. The peak at 2084 nm is
hydroperoxide [Figure 3(a)] shifted and weakened
after reduction [Figure 3(b)]. This result indicates
that the NIR absorption peak at 2084 nm is due to
the hydroperoxyl (OOH) group of lipid hydroperox-
ide, not conjugate diene or hydroxide.
223
Table 3. Correlation between peak intensity at 2084 nm of
NIR second derivative spectra and POV of soya bean and
cotton seed oils with a low range of POV.
Soya bean oil Cotton seed oil
K1 R SE K1 R SE
–14567 –0.996 0.83 –16178 –0.994 1.37
K 1, regression coefficient (slope).
R, correlation coefficient.
SE, standard error.
The POV range of the samples (n = 6–7) was 0–30.
NIR analysis of oxidised oils with low
POV range
Unpurified soya bean and cotton seed oils were
oxidised to the POV range of 0–30 and then ana-
lysed by NIR and chemical methods. Table 3 lists
regression coefficient (K 1), correlation coefficient
(R) and standard error (SE) values for the absorption
peak at 2084 nm. The correlation between the
chemical values and NIR predicted values of POV
are shown in Figure 4. Although K 1 values were
somewhat different from other oils, SE values were
small when compared to the POV range, which
shows that POV in edible oil is measurable by using
2084 nm as the key wavelength.
Hong et al. reported the determination of POV
by NIR spectroscopy by using 1680, 1716, 2052,
2076 and 2464 nm in the second derivative spec-
tra.19 They described that the increase of absorption
at 2076 nm is due to the OH bond of hydroperoxide.
However, we found that the absorption peak at 2084
nm is due to the hydroperoxide. This difference may
be due to the difference in the calculation of second
derivative spectra or NIR spectrophotometer used.
In addition, they described that the decrease in ab-
sorption at 2052 nm is due to a decrease in the ester
bond. We also found a positive peak (which might
be a decrease in absorption) at 2052 nm (data not
shown). However, ester bonds are not cleaved by
oxidation, hence, this peak is thought to be an arti-
ficial peak formed by the calculation of the second
derivative spectra.
In this work we demonstrate that lipid oxidation
in edible oils can be determined by NIR spectros-
224 Determination of Lipid Oxidation in Edible Oils by NIR

Figure 4. The correlation between chemical values and NIR predicted values of POV in oxidised soya bean and cotton
seed oils. The correlation between chemical values and NIR predicted values of POV in (a) oxidised soya bean oil and
(b) cotton seed oil are shown. The oxidised oils were prepared as described in the Materials and methods section. The
NIR-predicted values were calculated from the absorbance at 2084 nm in the second derivative spectra.

copy using POV as the index of oxidation and that
2084 nm is the key wavelength for lipid peroxide.
As the NIR technique is a simple, easy and non-
destructive analysis, this method is expected to be
used as a new method for POV determination.
Acknowledgements
The authors thank Kazue Nagaki, Maki Nagao,
Mika Imatani and Hitomi Katsuki for their technical
assistance.
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Received: 30 September 1996
Accepted: 18 December 1996