Food Research International 43 (2010) 886–892

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Food Research International

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Fourier transform infrared (FTIR) spectroscopy for analysis of extra virgin olive
oil adulterated with palm oil
A. Rohman a,c, Y.B. Che Man a,b,*
a
Halal Products Research Institute, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia
b
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor D.E., Malaysia
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University, 55281 Yogyakarta, Indonesia

a r t i c l e i n f o a b s t r a c t

Article history: Fourier transform infrared (FTIR) spectroscopy has been developed for analysis of extra virgin olive oil
Received 29 July 2009 (EVOO) adulterated with palm oil (PO). Measurements were made on pure EVOO and that adulterated with
Accepted 9 December 2009 varying concentrations of PO (1.0–50.0% wt./wt. in EVOO). Two multivariate calibrations, namely partial
least square (PLS) and principle component regression (PCR) were optimized for constructing the calibra-
tion models, either for normal spectra or its first and second derivatives. The discriminant analysis (DA)
Keywords: was used for classification analysis between EVOO and that adulterated with PO and the other vegetable oils
Fourier transform infrared (FTIR)
(palm oil, corn oil, canola oil, and sunflower oil). Frequencies at fingerprint region, especially at 1500–
spectroscopy
Extra virgin olive oil
1000 cm 1, were exploited for both quantification and classification. Either PLS or PCR at first derivative
Palm oil spectra revealed the best calibration models for predicting the concentration of adulterated EVOO samples,
Multivariate calibration with coefficient of determination (R2) of 0.999 and root mean standard error of cross validation (RMSECV) of
Discriminant analysis 0.285 and 0.373, respectively. DA was able to classify pure and adulterated samples on the basis of their FTIR
spectra with no misclassified group obtained. In addition, DA was also effective enough to classify EVOO
samples as the distinct group from the evaluated other vegetable oils.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The adulteration of food products is of primary importance for

In the recent years, olive oil has received great attention
because of its biological activities and sensory qualities, and has
become a very important agricultural product for the Mediterra-
nean countries (Gurdeniz, Tokatli, & Ozen, 2007). The epidemiolog-
ical studies showed that olive oil-rich diet can prevent
cardiovascular diseases, reduce plasma triacylglycerol (TAG),
increase high density lipoprotein (HDL)-cholesterol levels, improve
the postprandial lipoprotein metabolism, reduce blood pressure
and the risk of hypertension, have anti-cancerogenic effects in ani-
mal models and in human cell lines. It does not promote obesity
and increases the lipolytic activity in adipose tissue, and prevents
age-related cognitive decline and dementia, as reviewed by
García-González, Aparicio-Ruiz, and Aparicio (2008). These effects
are largely attributed to the high content of the monounsaturated
fatty acid, oleic acid (C18:1) (Fomuso & Akoh, 2002). In addition,
the high levels of natural antioxidants found in olive oil provide
the health benefits (Jansen & Birch, 2009).
* Corresponding author. Address: Halal Products Research Institute, Universiti
Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia. Tel.: +60 3 89430405;
fax: +60 3 89439745.
E-mail address: yaakobcm@gmail.com (Y.B. Che Man).
consumers, food processors and industries. From the legislative
point of view, the quality standards were established through the
requirement of quality labels that specify the chemical composition
of each product (Gallardo-Velázquez, Osorio-Revilla, Zuñiga-de Loa,
& Rivera-Espinoza, 2009). The adulteration frequently involved the
replacement of high-cost ingredients with cheaper substitutes. Ol-
ive oil is frequently subjected to be adulterated with other edible
oils of lower commercial value (Flores, Ruiz Del Castillo, Blanch, &
Herraiz, 2007). Although the adulteration is done by economic rea-
sons, the action can affect the quality of food, where olive oil is a
component (Christy, Kasemsumran, Du, & Ozaki, 2004). Therefore,
the development of rapid and non-expensive analytical techniques
capable of detecting such adulterations in olive oil is currently
highly demanded.
Several analytical techniques have been developed for detection
and quantification of adulteration and authentication of olive oil,
such as mass spectrometry using new type of ion source, direct
analysis in real time (Vaclavik, Cajka, Hrbek, & Hajslova, 2009),
nuclear magnetic resonance (NMR) spectroscopy (Jafari, Kadivar,
& Keramat, 2009), infrared spectroscopy (Gurdeniz & Ozen, 2009;
Lerma-Garcia, Ramis-Ramos, Herrero-Martinez, & Simo-Alfonso,
2010), Raman spectroscopy (Heise, Damm, Lampen, Davies, &
Mcintyre, 2005), fluorescence (Poulli, Mousdis, & Georgiou,

0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2009.12.006
 A. Rohman, Y.B. Che Man / Food Research International 43 (2010) 886–892
2007), gas chromatography (Jafari et al., 2009), high performance
liquid chromatography (Flores, Ruiz Del Castillo, Herraiz, & Blanch,
2006) and differential scanning calorimetry (Chiavaro et al., 2009).
Some of these methods are time consuming, expensive, generally
destructive of the sample material, and require a high degree of
technical knowledge when interpreting the data.
Today, the application of Fourier transform infrared (FTIR) spec-
troscopy has increased in food studies, and particularly has become
a powerful analytical tool in the study of edible oils and fats (Guil-
len & Cabo, 2000). The power of FTIR as a quantitative tool lies in
its ability to readily carry out the multi-component analyses (van
de Voort, 1992). There have been several studies concerning with
the characterization, classification, and authentication of edible
fats and oils using infrared spectroscopy (Dobson, 2001). Combined
with chemometric methods, infrared (IR) spectroscopy is an
emerging analytical technique to verify the authenticity of edible
oils and fats, due to its simplicity, rapidity, and ease of sample
preparation (Yang, Irudayaraj, & Paradkar, 2005).
Infrared spectroscopy, combined with chemometric, has been
used for detection of olive oil adulterated with hazelnut oil (Baeten
et al., 2005; Groselj, Marjan Vracko, Fernández Pierna, Baeten, &
Novic, 2008), with sunflower and corn oils (Özdemir & Öztürk,
2007), sunflower oil (Tay, Singh, Krishnan, & Gore, 2002), corn
oil, hazelnut oil, soya oil, and sunflower oil (Kasemsumran, Kang,
Christy, & Ozaki, 2005). FTIR spectroscopy was also used for
authentication of EVOO from sunflower, corn, soybean and hazel-
nut oils (Lerma-Garcia et al., 2010), EVOO from sunflower and corn
oils (Gurdeniz & Ozen, 2009), and EVOO with sunflower, soybean,
sesame, and corn oils (Vlachos et al., 2006).
Multivariate calibration is a useful chemometric and often used
for analysis of complex mixture, as it enables the rapid and simul-
taneous determination of each component in complex mixture
without time consuming and with minimal sample preparation
(Maggio et al., 2009). The most commonly used multivariate cali-
bration methods are partial least square (PLS) and principle com-
ponent regression (PCR). Both of these methods are based on
data reduction and inverse calibration, where there is a possibility
to calibrate for the desired component while implicitly modeling
the other source of variation (Paradkar, Sivakesava, & Irudayaraj,
2002). Another chemometrics technique commonly used is dis-
criminant analysis, one of the classification methods, which finds
the relationships between a set of descriptive variables (FTIR spec-
tra) and a qualitative variable (class of sample) (Ballabio & Todes-
chini, 2009).
Fig. 1. FTIR spectra of extra virgin olive oil and palm oil at frequency of 4000–650 cm
887
However, there is no information available related to the use of
FTIR spectroscopy combined with chemometrics for analysis of ex-
tra virgin olive oil (EVOO) adulterated with palm oil (PO). There-
fore, in this research, we developed FTIR spectroscopy combined
with multivariate calibrations of PLS and PCR for quantitative anal-
yses of EVOO adulterated with PO. Furthermore, discriminant anal-
ysis was used for classification of EVOO adulterated with PO and
other vegetable oils.
2. Materials and methods
2.1. Sample preparation
Extra virgin olive oil (EVOO), palm oil (PO), sunflower oil, corn
oil, and canola oil were purchased from the local market in Serd-
ang, Selangor, Malaysia. For quantitative analysis using PLS and
PCR calibrations, a set of 27 samples containing EVOO and PO
was mixed together in accurately weighted proportions of 1–50%
wt/wt in chloroform and shaken vigorously to ensure the total
homogenization. For validation, 32 independent samples were
built. In discriminant analysis, firstly, EVOO and PO were mixed
to obtain a series of standard or trained sets of 15 pure and 27
adulterated samples containing 1–50% of PO. The samples contain-
ing PO were assigned as adulterated, while a series of pure EVOO
was marked with EVOO and classified using FTIR spectra. Secondly,
EVOO and that adulterated with some vegetable oil samples were
compared and classified using discriminant analysis.
2.2. Instrumentation
FTIR spectrometer (A Nicolet 6700 from Thermo Nicolet Corp.,
Madison, WI) equipped with a deuterated triglycine sulphate
(DTGS) as a detector and a KBr/germanium as beam splitter, inter-
faced to Computer operating under Windows-based, and con-
nected to software of the OMNIC operating system (Version 7.0
Thermo Nicolet), was used during FTIR spectra acquisition. The
instrument was maintained with the automatic dehumidifier to
diminish water vapor interference. A few drops of each sample
were positioned in contact with attenuated total reflectance
(ATR) on a multi-bounce plate of crystal at controlled ambient
temperature (25 °C).
All FTIR spectra were recorded from 4000 to 650 cm 1, co-add-
ing 32 interferograms at a resolution of 4 cm 1 with strong
apodization. These spectra were subtracted against background
1
.
888 A. Rohman, Y.B. Che Man / Food Research International 43 (2010) 886–892

Table 1 it possible to dry the ATR plate. The plate cleanliness was verified
Functional groups and modes of vibration in the spectrum of extra virgin olive oil by collecting a background spectrum and compared to the previous
(EVOO) and palm oil (PO).a
one. These spectra were recorded as absorbance values at each
Frequency Functional group assignment data point. The sample measurements were replicated three times.
(cm 1)
3005 cis double-bond stretching
2.3. Chemometrics
2924 and 2852 Asymmetrical and symmetrical stretching vibration of
methylene (ACH2) group
1743 Ester carbonyl functional group of the triglycerides Chemometrics analysis, including quantification using PLS and
1465 Bending vibrations of the CH2 and CH3 aliphatic groups PCR calibrations and discriminant analyses, was performed using
1417 Rocking vibrations of CH bonds of cis-disubstituted olefins the software TQ AnalystTM Version 6 (Thermo electron Corpora-
1402 @CAH bending vibration
1377 Bending vibrations of CH2 groups
tion, Madison, WI). The spectral regions where the variations were
1236 and 1160 CAO stretching observed were chosen for developing PLS and PCR calibrations as
1117 and 1098 Stretching vibration of the CAO ester group well as for discriminant analysis, especially in the fingerprint re-
1030 CAO stretching gion. The optimum number of PLS and PCR factors was determined
962 Bending vibration of CH functional groups of isolated trans-
by cross validation, employing cancellation one standard at a time
olefin
850 @CH2 wagging by plotting the number of factors against the root mean standard
721 Overlapping of the methylene (ACH2) rocking vibration error of cross validation (RMSECV) and determining the minimum
and to the out of plane vibration of cis-disubstituted olefins factor. The predictability of the models was tested by computing
a
Source: Guillen and Cabo (1997) and Lerma-Garcia et al. (2010). root mean square error of prediction (RMSEP) as used by Gurdeniz
and Ozen (2009).

air spectrum. After every scan, a new reference air background 2.4. Determination of fatty acid compositions
spectrum was taken. The ATR plate was carefully cleaned in situ
by scrubbing with hexane twice followed by acetone and dried Fatty acid compositions of EVOO, palm oil, and other vegetable
with soft tissue before filling in with the next sample, and made oils were determined as fatty acid methyl esters (FAME’s) accord-

Table 2
The main component of fatty acids in extra virgin olive oil (EVOO), palm oil, and other vegetable oils used in this study.

Fatty acid EVOO Palm oil Sunflower oil Canola oil Corn oil
Capric (C10:0) 0.01 ± 0.001 0.35 ± 0.003 0.01 ± 0.001 0.01 ± 0.001 0.02 ± 0.001
Lauric (C12:0) 0.01 ± 0.001 0.12 ± 0.001 0.02 ± 0.001 0.01 ± 0.001 0.04 ± 0.002
Myristic (C14:0) 0.01 ± 0.001 1.36 ± 0.09 0.06 ± 0.002 0.15 ± 0.001 0.12 ± 0.001
Palmitic (C16:0) 10.78 ± 0.25 42.75 ± 1.12 6.70 ± 0.42 3.84 ± 0.22 10.15 ± 0.62
Stearic (C18:0) 3.30 ± 0.13 4.64 ± 0.18 3.03 ± 0.17 1.65 ± 0.08 2.42 ± 0.08
Oleic (C18:1) 74.98 ± 1.72 39.33 ± 0.96 18.42 ± 0.80 61.43 ± 1.37 26.24 ± 0.72
Linoleic (C18:2) 7.77 ± 0.35 10.37 ± 0.56 67.95 ± 1.27 21.85 ± 0.64 58.60 ± 1.05
Linolenic (C18:3) 0.63 ± 0.04 0.41 ± 0.003 0.87 ± 0.005 9.70 ± 0.40 1.15 ± 0.09

1
Fig. 2. Overlay of FTIR spectra of EVOO in the mixture with PO in the region of 1500–1000 cm (A), together with its 1st derivative (B) and 2nd derivative (C).
 A. Rohman, Y.B. Che Man / Food Research International 43 (2010) 886–892 889

Table 3
1
Multivariate calibration for determining of EVOO content adulterated with palm oil at frequencies of 1500–1000 cm using PLS and PCR techniques.

Multivariate calibration Spectra Equation R2 RMSECV RMSEP

Calibration Validation Calibration Validation
PLS Normal y = 1.000x 0.089 y = 0.995x + 0.165 0.999 0.998 0.453 0.693
1st derivative y = 1.001x 0.146 y = 0.998x + 0.001 0.999 0.998 0.285 0.616
2nd derivative y = 0.998x 0.268 y = 0.971x + 0.635 0.991 0.980 1.44 2.27
PCR Normal y = 1.000x 0.115 y = 0.993x + 0.211 0.999 0.998 0.446 0.706
1st derivative y = 1.001x 0.166 y = 0.999x 0.057 0.999 0.998 0.373 0.680
2nd derivative y = 1.004x 0.518 y = 0.982x + 0.004 0.994 0.987 0.942 1.96

The entire range of spectra looks very similar for the two oils to
the naked eyes. This is due to the similar chemical composition
of EVOO and PO, in term of fatty acid compositions (Table 2). How-
ever, if one examines the spectra closely, there is a minor differ-
ence between two spectra which can be observed at frequency of
1402 cm 1 (a) caused by @CAH bending vibration and at
1030 cm 1 (b) which is attributed by CAO stretching.

3.2. Quantification

Quantification of EVOO contents in the adulterated EVOO sam-

Fig. 3. The calibration models of PLS (A) and PCR (B) for the relationship between
actual and FTIR predicted values of EVOO adulterated with PO using 1st derivative
spectra at 1500–1000 cm 1.
ing to method described by Nor Hayati, Che Man, Tan, and Nor Aini
(2009). Standard FAME’s (Sigma Chemicals, St. Louis, MO) were
used to calculate the percentage of fatty acids based on its peak
area.
3. Results and discussion
3.1. Spectra analysis
The importance of IR spectroscopy in the identification of
molecular structures originates from the much information con-
tent obtained and the possibility to assign certain absorption bands
related to its functional groups. In fats and oils, most of the peaks
and shoulders of the spectrum are attributable to the specific func-
tional groups (Bendini et al., 2007). The triglycerides were the prin-
cipal components in fats and oils and, consequently, it dominate
the spectra of fats and oils. Fig. 1 exhibited the FTIR spectra of extra
virgin olive oil (EVOO) and palm oil (PO). These spectra showed the
typical characteristic of absorption bands for common triglycerides
(Safar, Bertrand, Rober, Devaux, & Genot, 1994). The analytical
evaluation of the palm oil and EVOO spectra is given in Table 1.
ples was performed using partial least square (PLS) and principle
component regression (PCR) algorithms. For PLS and PCR, the sam-
ples of EVOO adulterated with PO were divided into the calibration
and the validation sets. The calibration set consisted of 27 samples,
whereas the validation set consisted of 32 samples. The division
into sets was done in order to obtain the similar mean values
and standard deviations so that both sets spanned the full range
of EVOO contents (Wang, Lee, Wang, & He, 2006).
PLS has an ability to use information of sample spectra from
wide spectral frequencies and then correlates between spectral
absorption change and its concentration of analytes whilst concur-
rently computing other spectra which may disturb analyte spectra
(Che Man, Syahariza, Mirghani, Jinap, & Bakar, 2005). Meanwhile,
PCR is a type of factor analysis where the spectral and concentra-
tion data are incorporated into the model in one step (Smith,
2002).
Fig. 2 shows FTIR spectra of EVOO mixed with PO in the range of
1500–1000 cm 1. The spectral regions where the variations were
observed were chosen for developing PLS and PCR regressions,
especially in the fingerprint regions at 1500–1000 cm 1. These fre-
quencies were also used for performing the discriminant analysis.
In the PLS and PCR calibration models, the evaluation of the
method linearity was carried out in order to show a proportional
relationship between responses (absorbances) versus analyte con-
centrations. The results obtained from the PLS and PCR calibrations
in terms of R2, RMSECV, and RMSEP either for normal spectra or its
derivatives are presented in Table 3.
Both PCR and PLS calibrations in the first derivative spectra re-
vealed the highest of R2 and the lowest of RMSECV compared with
other spectra treatments (Table 3). Using first derivative spectra at
1500–1000 cm 1, the relationship between actual value of EVOO
content and FTIR predicted value showed a good correlation with
R2 of 0.999 (Fig. 3).
In order to validate the developed model, cross validation using
‘leave one out’ technique was used. Cross-validation evaluates the
data by excluding selected samples in the regression model and
then constructing a model for the remaining samples. The model
is evaluated using the samples excluded from the model and the
error values for the predicted observations are computed. The
new samples are then excluded from the model set and a new
model is constructed. This procedure is repeated until all samples
in the PLS and PCR models have been excluded once (Gurdeniz
890 A. Rohman, Y.B. Che Man / Food Research International 43 (2010) 886–892
Fig. 4. The ‘leave one out’ validation models of PLS (A) and PCR (B) for the
relationship between actual and FTIR predicted values of EVOO adulterated with PO
using 1st derivative spectra at 1500–1000 cm 1.
Fig. 5. Root mean standard error of cross validation (RMSECV) versus principal
component (PC) factors.
Fig. 6. The Coomans plot of extra virgin olive oil (EVOO) and palm oil: (h) EVOO; (D) adulterated samples with palm oil.
et al., 2007). After predicting all the observations once by the cross-
validation technique, the R2 and RMSECV values were computed
for the relationship between actual EVOO value and FTIR predicted
value at frequency regions of 1500–1000 cm 1. The R2 final values
of 0.998 and RMSECV of 0.285 (using PLS) and R2 of 0.998, RMSECV
of 0.373 (using PCR) were achieved, respectively (Fig. 4).
In PLS calibration model, verification and justification of spectra
regions in the first derivative spectra used for constructing the
models were performed by calculating the predicted residual error
sum of squares (PRESS) value for different principal component
(PC) value (Manaf, Che Man, Hamid, Ismail, & Syahariza, 2007).
The PRESS value is a direct measure on how well a calibration pre-
dicts the concentrations left out during a cross validation (Smith,
2002). It is computed from the errors in the predictions obtained
from standards by cross validation and plotted as a function of
the number of factors employed in the calibration (Fig. 5). The opti-
mum number of factors corresponds to the point at which the
PRESS plot reaches a minimum or begins to level off (Sedman,
Van de Voort, & Ismail, 1997). From PRESS values, it can be in-
formed that the optimal factor number is four, as revealed in
Fig. 5, that express that RMSECV obtains a stable value, minimally
after four-factor.
3.3. Discriminant analysis
Discriminant analysis can be used to determine the class of
EVOO which is similar to PO by computing the distance from each
class center in the Mahalanobis distance units. Once a classification
model has been obtained, the membership of unknown objects to
one of the defined classes can be predicted (Ballabio & Todeschini,
2009).
The frequency regions used for PLS quantification was em-
ployed for DA, namely at frequencies of 1500–1000 cm 1. Firstly,
pure EVOO and that in mixture with adulterant of PO were classi-
fied into two groups, known as pure EVOO and adulterated oils.
Both classes were classified using DA and the Coomans plot was
created. The Coomans plot is a useful method to visualize the clas-
sification results. It is formed by calculating two independent prin-
cipal component (PC) models and plotting the residual distances of
samples from each two models (Gurdeniz et al., 2007). Fig. 6 dem-
onstrates the Coomans plot for the classification of EVOO adulter-
ated with addition of 1–50% of PO using seven PCs. The x-axis
shows the Mahalanobis distance to the EVOO, while the y-axis
shows the distance to the adulterated oil class. The Mahalanobis
distance is useful manner to determine the similarity or dissimilar-
ity of set values from unknown samples to set of values measured
from a collection of known samples (Tay et al., 2002). The Coomans
plot in Fig. 6 clearly shows the separated group of EVOO and that
 A. Rohman, Y.B. Che Man / Food Research International 43 (2010) 886–892
Fig. 7. The Coomans plot of EVOO and other vegetable oils: (h) EVOO; (D) adulterated with other vegetable oils.
adulterated with PO. Within the adulterated class, it is noted that
the samples containing a higher percentage of adulterants are
more inclined toward the right side of the plot, indicating longer
distance from the EVOO axis. In this study, the DA model classified
100% of all samples accurately according to its group, meaning that
there is no samples were wrongly classified into the mistake group,
which could happen sometimes because of the close similarities in
the chemical composition among groups (Manaf et al., 2007).
In the second study, pure EVOO samples were compared to dif-
ferent vegetable oils (palm oil, sunflower oil, corn oil, and canola
oil) and subjected to DA in order to classify the test samples into
EVOO and vegetable oils. Fig. 7 exhibits the Coomans plot of EVOO
and other vegetable oils using six PCs. It can be seen from the plot
that the two groups of pure EVOO and other vegetable oils are well
separated, with each groups located closer to their respective axis.
There is no misclassification which is reported for the DA in this
model.
4. Conclusions
FTIR spectroscopy combined with multivariate calibrations and
discriminant analysis can be used to monitor the adulteration of
extra virgin olive oil with much cheaper oil-like palm oil. PLS
and PCR calibrations can be successfully used to quantify the level
of EVOO contents at frequencies of 1500–1000 cm 1. Discriminant
analysis allows one to make a classification of EVOO and potential
adulterant of vegetable oils.
Acknowledgments
The first author thanks to The Ministry of National Education,
Republic of Indonesia for its scholarship and to Faculty of Phar-
macy, Gadjah Mada University for its approval to pursue Ph.D. pro-
gram in Halal Products Research Institute, Universiti Putra
Malaysia (UPM), Malaysia.
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