Uleiul de Masline

Food Research International 54 (2013) 20252038
Contents lists available at ScienceDirect
Food Research International

journal homepage: www.elsevier.com/locate/foodres
Authenticity of olive oil: Mapping and comparing ofcial methods and

promising alternatives
Ramn Aparicio a,, Mara T. Morales b, Ramn Aparicio-Ruiz b, Noelia Tena a, Diego L. Garca-Gonzlez a
a
b
Instituto de la Grasa (CSIC), 4 Padre Garcia Tejero, 41012 Sevilla, Spain

Department of Analytical Chemistry, Universidad de Sevilla, 2 Prof. Garca-Gonzlez, 41012 Sevilla, Spain
a r t i c l e
i n f o
Article history:
Received 30 April 2013
Received in revised form 3 July 2013
Accepted 9 July 2013
Keywords:
Olive oil
Authenticity
Chromatography
Spectroscopy
Isotopic ratio
Trade standards
a b s t r a c t
Trade standards are continuously updated to give plausible solutions to situations created by fraudsters who
apply the most sophisticated procedures to their objectives of olive oil adulteration. Clustered inside targeted
and proling approaches, methods based on spectroscopic, isotopic and chromatographic techniques are
reviewed. Chromatographic methods, most of them being ofcial methods, compete with newer methods
based on spectroscopic, isotopic and trace element techniques for ensuring that the pace of research in the
detection of malpractices is rapid enough.
The speed of the analyses, the need of statistical interpretation of the results, the quality parameters of the
methods, limit of detection of the adulterants, and the applicability range among others are on the basis for
the absolute and comparative analyses of the most known methods, which results are unpacked in the paper.
The new frontiers of research in the eld of olive oil authenticity are also dissected together with the challenges
for the near future.
The extensive and deep analysis of the methods for quantifying the chemical compounds responsible for olive oil
authenticity will contribute to a better comprehension of the complex analytical world of olive oil for the analyst
working with this food product for the rst time, as well as for experienced professionals.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
The high price of olive oil and its reputation as a healthy and delectable oil makes it a preferred target for fraudsters. Thus, adulteration
may take place not only by accidental contamination during the stages
of oil processing but even more often by deliberate mislabeling of less
expensive olive oil categories or by the addition of less expensive edible
oils to virgin olive oil for the purpose of nancial gain.
Numerous adulterants have been found in virgin olive oil, and they
vary from rened olive oil, deodorized virgin olive oils, raw olivepomace oil and synthetic olive oilglycerol mixtures to almost all seed
oils (e.g. maize, cottonseed, hazelnut, rapeseed and sunower). In fact,
the admixtures of expensive olive oils with less expensive and lowergrade oils have been traditionally more than a potential problem in
countries that manufacture seed oils and import olive oils. This
procedure is harmful for new consumers who buy olive oil for its health
benets and strict purity control and are surprised receiving oil that
does not fulll their expectations (Garca-Gonzlez, Aparicio-Ruiz, &
Aparicio, 2009).
Several international institutions (e.g. International Olive Council
IOC and Antifraud Unit of the European Union OLAF among others)
are actively involved in anti-fraud regulations, which are focused on
Corresponding author. Tel.: +34 954 611550.
E-mail address: aparicio@cica.es (R. Aparicio).
0963-9969/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2013.07.039
tighter control of producing and importing countries, clear denitions

for olive oil products, uniform labeling regulations, and rapid, easy and
accurate instrumental techniques and analytical methodologies. The
nal objective is to avoid any image of a hypothetical uncontrolled distribution of adulterated olive oil into the market and to ensure fair trade
as well as the safety and consumer protection.
Advances in knowledge and technology, which have been needed in
the detection of malpractices by fraudsters, have required a considerable investment although no rapid and universal method has been ofcially recognized for all the authenticity issues yet; e.g. adulteration,
mislabeling, and misleading among others.
At this point, the most accepted denition for the genuineness of a
food product is: A product is authentic as long as it is rstly described
accurately by the label and secondly complies with the current legislation in force in the country where it is marketed or sold (Lees, 1998).
An authentic food is, in consequence, one which is truly derived from a
specied source where the term source must be clearly dened (e.g. a
particular category of olive oil). The ample number of olive oil categories,
which are clearly dened by current regulations (EC, 2013; IOC, 2011),
and the numerous edible oils that can be used in adulterations require
of a plethora of analytical techniques and methods for carrying out a
strict olive oil authenticity control.
This work is not a systematic revision of methods and instrumentation used in the authenticity of olive oil. There are several interesting
works already published for this purpose (Aparicio & Aparicio-Ruiz,
2026
R. Aparicio et al. / Food Research International 54 (2013) 20252038
2000; Aparicio, Aparicio-Ruiz, & Garca-Gonzlez, 2007; Aparicio, Conte,

& Fiebig, 2013; Aparicio et al., 1998; Ben-Ayed, Kamoun-Grati, & Rebai,
2013; Dais & Hatzakis, 2013; Frankel, 2010; Garca-Gonzlez, Baeten,
Fernndez-Piernas, & Tena, 2013). In contrast to these reviews, this
work presents an extensive and deep analysis of the most relevant compounds used as targeted analytes for virgin olive oil characterization
and authentication, the analytical problems derived from their determination by targeted and proling methods, and a critical approach of
their utility today, and future trends.
2. Discussion
2.1. Ofcial standards and promising alternatives: the state-of-the-art
Three are the different ordinances and legal sources that have traditionally ruled olive oil production and international trading although
there are also regulations in force in some producer states (e.g. Australia,
California). IOC trade standards have traditionally helped to design and
improve the other ordinances with which it shares much more than
90%. Particular regulations exist within the European Union (EU) to
control its olive oil market which represents 72% of the world production. As olive oils are subjected to worldwide trade, a further level of
regulation is needed, and this is provided by the Codex Alimentarius
Commission. EU regulations are in force for EU countries while IOC
trade standards and Codex standards are agreements that signatory
countries voluntarily have accepted to comply. They establish the limits
for each quality and purity criterion, including the precision values of
the applied methods, although IOC trade standards are more specic
than the Codex Alimentarius standards in, for instance, the labeling
aspects (IOC, 2011).
Today, most of the methods refer to ISO (International Standardization Organization) methods although some still refer to AOCS (American
Oils Chemists' Society) methods. In fact, IOC is an ofcial LiaisonMember of ISO/TC 34/SC 11 Animal and vegetable fats and oils,
which is a Subcommittee of ISO/TC-34 Food products. Based on this
relationship, IOC methods, which were developed for olive oil trade
standards, have been able to be compared with already checked ISO
methods that were developed for a wide number of fats and oils; sometimes, however, the peculiar characteristics of olive oils have allowed
some IOC particular methods to be accepted as ISO norms. The consequence is that the IOC methods are applied worldwide for the analytical
control of olive oils though alternative methods fostered by other associations should be taken into account as well; i.e. AOAC International,
Federation of Oil Seeds and Fats Association (FOSFA) and International
Union of Pure and Applied Chemistry (IUPAC).
In those days when the adulteration was very simple, the detection
of adulterants was relatively easier than nowadays. The fraudulent
practices, however, became more complex and ingenious as technology
advanced and it was widely accessible to everybody. In the objective of
determining chemical compounds that can be markers for olive oil
authenticity, many modern techniques have been proposed by
researchers and technologists. Thus, they have proposed methods
based on gas, liquid, gasliquid, quiral, silver-ion, mass, and supercritical
uid chromatographies, stable carbon isotope ratio analysis (SCIRA),
excitationemission uorescence (EEFS) and total synchronous uorescence (TSyF), pyrolysismass spectrometry (PyM), nuclear magnetic
resonance spectroscopy (NMR), and infrared and Raman spectroscopy
among others. However, any of those methods needs to be approved
or recommended by international associations to become an ofcial
standard. In fact, most of those numerous proposed methods can only
detect adulterations greater than 10%, which scarcely represent any
advantage over current tests and ofcial methods.
Conceptually the methods can be naturally divided into targeted
analyses based on denite information obtained from the fractionation of olive oil components and proling or non-targeted
analyses, which relies on the simultaneous contribution from
many known or unknown analytes belonging to a predened metabolic pathway (Baeten et al., 2005; Rezzi et al., 2005). The former
analyses, which quantify and identify series of chemical compounds,
analyte by analyte, search for compounds that do not appear, or only
at trace levels, in genuine olive oils but do appear in adulterated
ones. As these analyses reveal under what circumstances these
analytes appear in the adulterated oils, the information can also be
used to remove or diminish the amount of those selected markers
in an improved adulteration process; e.g. adding desterolized seed
oils that cannot be detected with methods based on the detection
of sterols. This approach requires not only considerable investment
in perfecting the classical methods, or in developing new methods,
but also in ensuring that the pace of research in the detection of malpractices is rapid enough.
The proling approach, which typically does not differentiate
between analytes and sometimes neither quantify them, aims to rapidly
determine the genuineness of olive oils based on information from
multi-target screening methods, which are gaining popularity as alternative to targeted approaches based on gas liquid chromatography
(GLC) or liquid chromatography (HPLC). In the case of proling techniques, the fraudsters have no information since there is not a particular
marker but the analysts may have problems interpreting the information because multivariate statistical procedures are needed to arrive at
correct conclusions, in addition obviously of plausible chemical or biochemical explanations, if analysts want to avoid that the authenticity
is not based on random parameters or noise.
The current limits for the physicochemical parameters involved in
each purity or quality criterion (Tables 1a1b) are results achieved,
however, from the chromatographic techniques. It is so because of
lower cost, rapid implementation and development, more versatility
for quantifying diverse analytes, and superior reproducibility of the
chromatography in comparison with other proposed techniques.
2.2. Targeted approaches
A standard method needs several years to be endorsed as ofcial
method from its submission to the regulatory institutions. The reasons
for that period of time can be found, among others, in that the proposals
are, in general, hyper-optimists due to a lax application of the statistical
procedures, an inadequate selection of the validation samples or a casual relationship between the adulteration and the selected chemical
markers.
The chemical compounds, whose contents allow determining the
difference between genuine and adulterated olive oils with regard to
their designations, are shown in Tables 1a1b. The chemical composition of olive oil has been traditionally clustered into major and minor
compounds; the former are, in large part, responsible for the olive oil
main characteristics while the latter are markers for their peculiarities.
This section, which has been structured around the series of chemical
compounds that are currently used in olive oil authentication, analyzes
the series from three viewpoints: a) the main reasons for analyzing
them; b) the current standards, with practical comments and suggestions if possible, for quantifying them; and c) potential alternatives to
ofcial methods for determining them.
2.2.1. Fatty acids
Fatty acids are, with a few exceptions, the major components of any
oil or fat. In small amounts they are present as free fatty acids but
usually form esters, most often with glycerol, to produce glycerides
(mono-, di- and tri-acylglycerols) and phospholipids but they can also
form esters with aliphatic alcohols of linear structure (waxes) or
terpenic structure (terpene and sterol esters).
2.2.1.1. Reasons for analyzing these compounds. The knowledge of the
fatty acid composition has widely been used for characterizing edible
oils since 1960s when seed oils with a modied fatty acid composition
2027
Table 1a
Olive oils purity and quality characteristics according to the International Olive Council (IOC, 2011).
Designations
(1)
(2)
(3)
(4)
(5)
(6a)
(7)
(8)
(9b)
Extra-virgin olive oil

Virgin olive oil
Ordinary virgin olive oil
Lampante virgin olive oil
Rened olive oil
Olive oil
Crude olivepomace oil
Rened olivepomace oil
Olivepomace oil
0.05
0.05
0.05
0.10
0.20
0.20
0.20
0.40
0.40
0.05
0.05
0.05
0.10
0.30
0.30
0.10
0.35
0.35
1000
1000
1000
1000
1000
1000
2500
1800
1600
4.5
4.5
4.5
4.5c
4.5
4.5
4.5d
4.5
4.5
250
250
250
300c
350
350
N350d
N350
N350
0.10
0.10
0.10
0.50
0.2
0.2
0.2
0.3
0.3
0.3
0.6
0.5
0.5
B
B
B
C
C
B
1.4%
1.4%
1.2%
2.50c
2.60c
Designations
(10b,e)
(11b)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)

Virgin olive oil
Rened olive oil
Olive oil
Crude olivepomace oil
Rened olivepomace oil
Olivepomace oil
0.22
0.25
0.30
1.10
0.90
2.00
1.70
0.01
0.01
0.01
0.16
0.15
0.20
0.18
0.8
2.0
3.3
N3.3
0.3
1.0
0.3
1.0
20
20
20
5
15
5
15
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.1
0.1
0.1
0.1
0.1
0.1
0.2f
0.2f
0.2f
4.0
4.0
04.0
4.0
4.0
4.0
4.0
4.0
4.0
bCamp
bCamp
bCamp
bCamp
bCamp
bCamp
bCamp
93.0
93.0
93.0
93.0
93.0
93.0
93.0
93.0
93.0
Note:
(1) Trans-oleic fatty acid (%).
(2) Sum of trans-linoleic & linolenic fatty acids (%).
(3) Total sterol content (mg/kg).
(4) Erythrodiol and uvaol content (% total sterols).
(5) Wax content: C40 + C42 + C44 + C46 (mg/kg).
(6) Stigmastadiene contents (mg/kg).
(7) Difference between the actual and theoretical ECN42 triacylglycerol content.
(8) Content of 2-glyceryl monopalmitate; B, 0.9 if total C16:0 14.0% or 1.0 if C16:0 N 14:0%; C, 0.9 if total C16:0 14.0% or 1.1 if C16:0 N 14:0%.
(9) Absorbency in ultra-violet at K232.
(10) Absorbency in ultra-violet at K270.
(11) Absorbency in ultra-violet (K).
(12) Free acidity (%m/m expressed in oleic acid).
(13) Peroxide value (in milleq. peroxide oxygen per kg/oil).
(14) 7-Stigmastenol (%).
(15) Cholesterol (%).
(16) Brassicasterol (%).
(17) Campesterol (%).
(18) Stigmasterol (%).
(19) The value of -Sitosterol is calculated as: 5,23-Stigmastadienol + Clerosterol + -Sitosterol + Sitostanol + 5-Avenasterol + 5,24-Stigmastadienol.
a
Total isomers which could (or could not) be separated by capillary column.
b
Quality characteristics.
c
When the oil has a wax content of between 300 mg/kg and 350 mg/kg it is considered a lampante olive oil if the total aliphatic alcohol content is 350 mg/kg or if the
erythrodiol + uvaol content is 3.5%.
d
When the oil has a wax content of between 300 mg/kg and 350 mg/kg it is considered a crude olive-pomace oil if the total aliphatic alcohol content is N350 mg/kg and if the
erythrodiol + uvaol content is N3.5%.
e
Maximum wavelengths of 268 if iso-octane is used and of 270 nm if cyclohexane is used.
f
Limit raised to 0.2 for olive-pomace oils.
similar to olive oil appeared; e.g. high oleic safower and sunower oils,
and high oleic rapeseed and soybean oils more recently. Furthermore, a
high percentage of myristic acid in labeled olive oils may indicate the
presence of seed oils, mainly fractionated palm oil, while high percentages of linolenic or eicosanoic or eicosenoic or behenic acids are associated to the presence of soybean or low erucic rapeseed oils, and a high
value of lignoceric to peanut oil.
On the other hand, the presence of trans isomers of oleic, linoleic and
linolenic acids in percentages above the approved levels (Table 1a)
is related to the presence of rened oils obtained from, for example, esteried olive oils, (partially-)hydrogenated seed oils or seed oils
desterolized at high temperatures, among others.
2.2.1.2. Ofcial methods: comments and suggestions. There is broad
agreement on the methodology for the quantication of fatty acids
(Tables 1a1b) although special attention should be paid to the methylation step, which can be carried out by acid or alkaline catalysis. The
analyst should bear in mind that methylation should be acid in case of
a medium or a higher concentration of free fatty acids since alkaline
catalysis is effective in trans methylation only.
2.2.1.3. Alternatives to ofcial methods. While GC is the technique used for

fatty acid quantication (Table 2a), spectroscopy is the alternative to the
ofcial method for quantifying trans fatty acids (Table 2a) in addition to
the determination of iodine value, saponication number and humidity
content. Reasons can been found in its rapid sample screening. Raman
bands near 1656 cm1 and 1670 cm1 are the consequence of cis and
trans isomer contents while Mid-IR bands around 860990 cm1 and
16501655 cm1 respectively correspond to the contents of trans and
cis isomers in olive oils (Ismail, Cocciardi, Alvarez, & Sedman, 2006;
Ismail, Nicodemo, Sedman, van de Voort, & Holzbaur, 1999).
In the eld of NMR (nuclear magnetic resonance), 1H NMR is the
best for the quantication of fatty acids although mathematical algorithms need to be used for the quantication of the sums of saturated,
monounsaturated, and polyunsaturated fatty acids from appropriate
signal intensities. As the signals overlap, the individual determination
of fatty acids cannot be carried out like GC with the exception of
linolenic whose methyl protons are detected at 0.96. The isomers cis
and trans have also been determined by using the allylic methylene
protons adjacent to cis and trans double bonds (Sedman, Gao,
Garca-Gonzlez, Ehsan, & van de Voort, 2010). 13C NMR, on the
2028
Table 1b
Olive oils purity and quality characteristics according to the International Olive Council (IOC, 2011).
Designations
(20a)
(21a)
(22a)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)

Virgin olive oil
Rened olive oil
Olive oil
Crude olive-pomace oil
Rened olive-pomace oil
Olive-pomace oil
Mf N 0
Mf N 0
Md = 0
0 b Md 3.5
3.5 b Md b 6.0d
Md N 6.0
(b)
15
15
15
15
15
15
30
30
30
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.2
0.2
0.2
0.2
0.2
0.2
0.3
0.3
0.3
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
(c)
(c)
(c)
(c)
(c)
(c)
(c)
(c)
(c)
Note:
(20) Organoleptic assessment: median of fruity attribute (Mf).
(21) Organoleptic assessment: median of defect (Md).
(22) Fatty acid methylesters (FAMEs) and fatty acid ethylesters (FAEEs).
(23) Unsaponiable matter (g/kg).
(24) Myristic acid (% m/m methylesters).
(25) Linolenic acid (% m/m methylesters).
(26) Arachidic acid (% m/m methylesters).
(27) Eicosenoic acid (% m/m methylesters).
(28) Behenic acid (% m/m methylesters).
(29) Lignoceric acid (% m/m methylesters).
(30) Other fatty acids (% m/m methylesters).
a
Quality characteristics;
b
(FAME + FAEE) 75 mg/kg or [75 mg/kg b (FAME + FAEE) 150 mg/kg and (FAEE/FAME) 1.5]
c
Palmitic: 7.520.0; palmitoleic: 0.33.5; heptadecanoic: 0.3; heptadecenoic: 0.3; stearic: 0.55.0; oleic: 55.083.0; linoleic: 3.521.0.
d
Or when the median of the defect is less than or equal to 3.5 and the median of the fruity attribute is equal to 0.
other hand, is used when information about trans fatty acids is required because the distance (5 ppm) between signals of the allylic
carbons of cis and trans double bonds (Gao et al., 2009). NMR instruments, however, require a large initial outlay and their methods
need more time for the analysis than GC methods.
2.2.2. Triacylglycerols
A large extent of olive oil main characteristics is the responsibility of
triacylglycerols (trihydric alcohols esteried with three fatty acids)
(TAG). The molecular structure of each individual TAG species has
three basic characteristics: (i) the total carbon number (CN), which is
the sum of the alkyl chain lengths of each one of the three fatty acids
(FAs); (ii) the degree of unsaturation in each FA; and (iii) the position
and conguration of the double bonds in each FA (Buchgraber,
Ulberth, Emons, & Anklam, 2004). Thus, the large number of possible
FA combinations on the glycerol backbone of TAGs makes the TAGs
very revealing although, for this same reason, their analysis is a very
challenging task.
2.2.2.1. Reasons for analyzing these compounds. At the beginning of 1980s,
Gegiou and Georgouli (1983) investigated the presence of re-esteried
oils in olive oils by the ratios of TAGs (i.e. 1-oleo-2,3-dipalmitin to 1,3dipalmito-2-olein, 1,3-dioleo-2-palmitin to 1-palmito-2,3-diolein, and
1,3-dioleo-2-stearin to 1-stearo-2,3-diolein). Later, Salivaras and
McCurdy (1992) explored the information of TAGs in the detection of
adulterations of virgin olive oil with canola oil from 7.5% while
El-Hamdy and El-Fizga (1995) detected adulterations with about 1% of
linoleic-rich vegetable oils (soybean, sunower and corn oils) though
the detection of olive oil admixed with non-rich linoleic acid seed oils
is much more difcult.
In this decade, the interest of researchers was focused on the differences between the experimental composition of TAGs and their
theoretical composition determined from fatty acids according to their
1,3-random 2-random distribution (Cortesi, Rovellini, & Fedeli, 1990).
From a theoretical viewpoint, the difference between both values should
be zero for an authentic olive oil sample but the analytical error for example, only some theoretical TAGs show good mathematical signicance
(Garca-Pulido & Aparicio, 1993) directed the attention to the difference between the real and theoretical ECN42 (equivalent carbon number
42) triacylglyceride content (ECN42). The IOC trade standard (IOC,

2011) and European regulation (EC, 2013) include a limit of |0.2| for
ECN42 determined in edible virgin olive oils (|0.3| for lampante virgin
olive oil), which makes possible detecting a fraudulent addition of seed
oils to olive oil, even at low percentages.
A different approach to TAG analysis was focused on the discrimination of natural TAGs from chemically synthesized ones that were
obtained from free fatty acids esteried with glycerol; synthesized
TAGs were sold as olive oil in the early 1970s. The biosynthesis of TAG
in vegetable oils, however, does not allow that high concentration of
saturated fatty acids as well as fatty acids with a carbon chain longer
than 18 carbon atoms to be present at 2-position of the molecule; 1%
of such acids esteried at the 2-position of glycerol is enough to conclude that the oil is not genuine. Thus, the analytical proposals focused
on the direct analysis of the reaction of lipolysis by capillary gas chromatography that Lercker, Moschetta, Caboni, and Frega (1985) improved
by removing the step of separation by TLC, which made the analytical
process easier and faster. The method was validated, and is included
in the Italian Regulation (NGD, 2002).
2.2.2.2. Ofcial methods: comments and suggestions. TAGs can be separated
by HPLC, according to the number of carbon atoms and their unsaturation,
using diverse sample preparations, stationary and mobile phases,
columns and detectors. The current discussion inside IOC is, however,
focused on the mobile phase: acetone/acetonitrile (1:1 v/v) adversus
propionitrile (Table 2a). A mobile phase of acetone/acetonitrile, in
isocratic elution, allows that critical pairs are effectively separated but
saturated long-chain TAGs are not sufciently dissolved with this
phase. The isocratic elution with propionitrile (Fiebig, 1985; Moreda,
Prez-Camino, & Cert, 2003) results in an improved separation of the
groups of peaks clustered as ECN42.
The selected particle size for reverse phase (RP)-18 column was
4 m because it was observed an increase of the olive oil TAG resolution
as the particle size diminished; in fact, the use of columns with a small
particle size minimizes the problems of integration and quantication
of HPLC peaks. The refractive index (RI) detector is also the most appropriate despite its drawbacks of low sensitivity, and intensity of the response depending on the saturation level of TAGs. Besides RI detectors
should not be placed near sun-lit windows and the analyst should be
2029
Table 2a
The standard methods for quantifying acyl lipids and fatty acids.
Compounds
Technique
Sample preparation
Chromatographic characteristics
Triacylglycerols
HPLC-RI
0.12 g oil in 0.5 mL hexane is charged into SPE-cartridge
Mobile phase ow-rate (0.6 to 1.0 mL/min)

Oven temperature: 20 C
Mobile phase: propionitrile
Column: RP-18 (4 m)
Detector: RI
(1 g of Si) and solution pulled through and, then, eluted

with 10 mL hexane-diethylether (87:13 v/v).
HPLC-RI
0.5 g oil in 10 mL acetone or acetone/chloroform (1:1 v/v).
Mobile phase ow-rate (0.6 to 1.0 mL/min)

Oven temperature: 25 C
Mobile phase: acetone/acetonitrile (1:1 v/v)
Column: RP-18 (4 m)
Detector: RI
2-Glyceryl monopalmitate (%)
GC-FID
Hydrolysis with pancreatic lipase.

Separation by LC or SPE.
Require silanization
Column: capillary 12 m 0.32 mm 0.100.30 m

Phase: methylpolysiloxane or 5% phenylmethylpolysiloxane.
Carrier gas: hydrogen
Operation conditions: temperature gradient
Injection mode: on-column
Fatty acids
GC-FID
Total fatty acids:

Methylation with cold methanolic solution of KOH or double
methylation in a methanolic medium with alkaline and acid catalysis.
Trans fatty acids:
Methylation with cold methanolic solution of KOH
Fatty acid in the 2-position:
Hydrolysis with pancreatic lipase previously and methylation in
a methanolic medium with alkaline and acid catalysis.
Column: capillary 25100 m 0.20.8 mm 0.10.2 m

Stationary phase: polyglycol, polyester or cyanopropylsilicone
Injection mode: split
Isolation on LC Si-column
Column: capillary 1215 m 0.250.32 mm 0.10.3 m

Stationary phase: 5% phenylmethylpolysiloxane
Injection mode: split or on-column
GC-FID
GC-FID
Waxes
GC-FID
Note: GC, gas chromatography; FID, ame ionization detector; HPLC, high performance liquid chromatography; RI, refractive index detector; SPE, solid phase extraction.
careful about possible bubbles of gas in the solvents, leaks in the system
and back pressure HPLC pump since they affect to chromatographic
baseline (Christie, 1992). The use of a thermostated system and isocratic
elution is recommended, and also to pass the pre-solved acetone sample
through a 0.2 m pore size lter, when determining raw olive-pomace
oils, to remove possible precipitates that can shorten the effective life
of the chromatographic column.
The initial method for the determination of the percentage of palmitic
acid at the 2-position of the triacylglycerols was substituted by the content of 2-glyceryl monopalmitate (IOC, 2006a). The method and the maximum percentages of the compound for each olive oil category were
included in the EU Regulation 702/2007 (EC, 2007) after analyzing the results of collaborative trials whose aim was to determine the content of 2glyceryl monopalmitate in genuine extra virgin olive oils with high content of palmitic acid. The result was to assign the value (%) for this compound according to the percentage of palmitic acid (Table 1a).
2.2.2.3. Alternatives to ofcial methods. GLC is also attractive for the
analysis of acylglycerols because of the linear response and selectivity
although GLC is not exempt of problems such as, for example, the
high temperature needed for a suitable separation of TAGs. Thus, the
capillary columns (30 m 0.25 mm 0.10.15 m) coated with 65%
phenylsiloxane + 35% methylsiloxane have been suggested because
they endure high temperatures for a long time. All the existing studies
of GLC analysis of olive oil TAGs suggest to use a cold on-column injection although the analysis takes longer. As the time that TAGs are inside
the column increases, the results are both resolution loss and peak widening; for example, trilinolein (LLL) results in a poor peak shape and a
less reliable measurement than using RP-HPLC.
Analysis of TAGs of oil samples by liquid chromatography coupled
with mass spectrometry (HPLCMS) is a widespread method because
MS supplies identication of non- or partially resolved HPLC peaks. In
fact, MS provides detailed information about the FAs composition of
TAG molecules though only a few ionization techniques are suitable
for coupling with HPLC. Thus, electrospray ionization (ESI) is an
excellent technique because it enables the identication of the individual acyls without the need for authentic reference standards while atmospheric pressure chemical ionization (APCI) coupled to HPLC is
very effective because it allows identifying the positional isomers
(Holapek, Jandera, Zderadika, & Hrub, 2003); other signals at lower
masses, related to diacylglycerols (DAG) fragments, have been studied
by Jakab, Hberger, and Forncs (2002).
Recently, matrix-assisted laser desorption ionization time-of-ight
mass spectrometry (MALDI-TOF-MS) is being used for a rapid differentiation of edible oils based on the detection of TAG molecules as their
sodium adducts and, sometimes, as their potassiated adducts, which
render low intensities. The method possesses a good resolution in TAG
mass range which contributes to distinguish vegetable oils whose TAG
molecules differ only by their degrees of unsaturation (Cozzolino & De
Giulio, 2011). An important advantage of this method is the rapid sample preparation because of the absence of analyte purication, chemical
modication or derivatization. Concerning applications in specic adulterations, an approach based on the laser desorption ionization (LDI)
coupled TOF-MS has been proposed for the detection of adulteration
of olive oil with sunower oil (Calvano, Palmisano, & Zambonin, 2005).
2.2.3. Waxes
The term wax has been applied to an ample variety of plant products
containing several kinds of fatty materials that are synthesized in the
epidermal cells of the olives. The pathways for the biosynthesis of wax
components comprise an acyl reduction, which yields primary alcohols
and wax esters, and a decarbonylation pathway that synthesizes
secondary alcohols, aldehydes, alkanes and ketones (Christie, 2012).
Wax esters are, however, the most common form of the waxes, and
they consist of fatty acids esteried to long-chain alcohols with similar
chain-lengths (Christie, 2011). The main waxes found in olive oil are
esters of even numbered of carbon atoms from C36 to C46.
2.2.3.1. Reasons to analyze these compounds. Because waxes are synthesized from very-long-chain saturated fatty acids, their presence in
2030
olive oils is of interest because their concentrations vary among the olive
oil categories, and the information about its presence can be used as indicator of purity (EC, 2013). In fact, the contents of waxes C36 and C38 are
usually higher than the waxes C40, C42, C44 and C46 in VOOs, on the contrary of the concentrations in rened and olive-pomace oils (Table 1a).
However, differences in the content of waxes cannot be used as a unique
criterion to detect the presence of olive-pomace oil in olive oil but together with the amount of the erythrodiol and uvaol (IOC, 2011).
2.2.3.2. Ofcial methods: comments and suggestions. All the methods for
the determination of waxes consist of a separation from the other
lipid constituents using silica gel chromatography and the subsequent
quantication using GC (Table 2a). Although there is a specic method
for the determination of waxes in olive oil (IOC, 2007), a new method
that allows the simultaneous determination of waxes, fatty acid methyl
esters and fatty acid ethyl esters by capillary gas-chromatography (IOC,
2010) is widely applied in laboratories because it saves time when the
objective is to determine the olive oil genuineness. The method is recommended for distinguishing between olive oil and olive-pomace oil, for
the detection of the presence of lower quality oils (ordinary, lampante)
in extra-virgin olive oils, and for the detection of the fraudulent addition
of some deodorized oils to extra virgin olive oils.
Although semipolar columns SE-54 or SE52 are used, better resolution is obtained with phenyl-methyl-silicone columns (Table 2a),
which can endure higher temperatures. Anyway, it is advisable to condition the column when used for the rst time. It can be done by
means of a gradual warm-up until it reaches 350 C, after approximately 4 h, and maintaining this temperature for at least 2 h. Once the
instrument has been regulated with the operating conditions (IOC,
2007), the signal has to be adjusted in sensitivity at least twice higher
than that required for the analysis. Furthermore, the baseline should
be linear, with no peaks of any kind, and must not have any drift.
2.2.3.3. Alternative to ofcial methods. Furthermore the replacing of silica
gel columns by solid phase extraction (SPE) cartridges (1 g of silica gel)
for the sample purication because they need smaller amount of sample
and a reduced volume of elution solvent, it has been proposed a new
method based on the isolation of alkyl esters and waxes by SPE and
their analysis by capillary column (DB5; 512 m long) gas chromatography with on-column injector and FID detector (Cert et al., 2011).
The method seems to be more rapid than current standard with lower
consume of organic solvents although there is no information about
its precision, limit of quantication and the other analytical parameters.
Another analytical alternative is based on the use of TOTAD (Through
Oven Transfer Adsorption Desorption) interface (Aragn, Toledano,
Cortes, Villn, & Vzquez, 2011). The oil, with an internal standard (C32
wax ester) diluted in n-heptane, is injected directly with no sample pretreatment step other than ltration. Normal-phase liquid chromatography (NPLC) separates the wax ester fraction from the triglycerides and
the TOTAD interface transfers it to the GC to be analyzed. Biedermann,
Haase-Aschoff, and Grob (2008) also proposed that samples were prepared by NPLC but, in this proposal, the pre-column is attached to the
inlet of the column of the GC GC instrument by means of a press-t
connector. The rst dimension is performed with a PS-255 column
(20 m 0.25 mm i.d) and the second-dimension with a SOP-50 (50%
phenyl polysiloxane) (1.5 m 0.15 mm i.d).
2.2.4. Sterols
Sterols make up an extensive series of compounds with analogous molecular structure, more than 200 have been reported in
plants, that are grouped into three classes (4,4-dimethylsterols,
4-monomethylsterols, and 4-desmethyl sterols) according to the number of methyl groups at the C-4 position. They are made up of a tetracyclic
cyclopenta[a] phenanthrene ring and a long exible side chain at the C-17
carbon atom (Piironen, Lindsay, Miettinen, Toivo, & Lampi, 2000). 4,4dimethylsterols and 4-monomethylsterols are metabolic intermediates
in the biosynthetic pathway leading to end-product, 4-desmethylsterols.

Thus, 4-desmethylsterols, the most abundant class in olive oil, are commonly called phytosterols while 4,4-dimethylsterols are called triterpenic
alcohols and 4-monomethylsterols are named methylsterols.
2.2.4.1. Reasons to analyze these compounds. Sterols, which comprise a
major portion of the unsaponiable matter, are the most important of
the olive oil minor compounds in authentication purposes. Because of
the ranges of concentration of 4-desmethylsterols are characteristic of
the genuineness of several edible oils, they are used widely and reliably
to detect fraudulent mixtures of olive oil with other oils. For example,
rapeseed oils contain signicant levels of brassicasterol while safower
and sunower seed oils contain high levels of 7-stigmastenol, and, in
the case of olive oil, sterols have a prole of high levels of -sitosterol
and 5-avenasterol, low levels of campesterol and stigmasterol.
The content of free brassicasterol would allow detection of the
addition of 2% rapeseed oil to olive oil. The addition of 5% sunower oil increases the stigmasterol, campesterol and 7-stigmasterol concentrations
signicantly above those found in non-adulterated olive oils. An addition
of 10% soybean oil to olive oil is easily detected by the concentration of
free campesterol and stigmasterol since their concentrations will double,
while an olive oil spiked with 10% rapeseed oil increases the concentration of free campesterol by ve, and there are evident increments in the
concentration of -tocopherol and campesteryl-C18-ester that are almost negligible in the olive oils. The addition of 10% grape seed oil
would also increase the concentrations of free campesterol and stigmasterol. The prole of sterols is known as the ngerprint of the
olive oil because also it varies among the cultivars (Garca-Gonzlez
et al., 2009).
Some of the sterols, however, are not detected in VOOs but in rened
olive oils (ROO) due to the rening process (e.g., 5,23-stigmastadienol)
what allows distinguishing ROOs from VOOs. Thus, 5,24-stigmastadienol
increases its concentration during the rening process while the concentration of 5-avenasterol decreases (Amelotti, 1985). More recently, the
quantication of free and esteried sterols was accepted as one of the
most successful proposals for the detection of the adulteration of rened
olive oil with rened hazelnut oil (Bowadt & Aparicio, 2003). A double
mathematical model based on three free and esteried sterols
(campesterol, 7-stigmastenol and 7-avenasterol) was able to detect
the presence of hazelnut oil in olive oil at percentages in the range
68% (Mariani, Bellan, Lestini, & Aparicio, 2006). Another model system,
although based on the sum of campesterol and stigmasterol, has been
recently proposed for determining the presence of other vegetable oils
(i.e. corn, soybean, sunower, and cotton seed) in olive oil (Al-Ismail,
Alsaed, & Ahmad, 2010).
When oils with low concentration of sterols (desterolized) are used
to adulterate olive oil, which makes difcult their quantication, the
conrmation can be obtained through the analysis of the dehydration
by-products of sterols, which increase spectacularly with that fraudulent practice.
2.2.4.2. Ofcial methods: comments and suggestions. Sterols are determined as the overall contribution of both the free and esteried forms
as their trimethylsilyl (TMS) ethers or sterol acetates (Table 2b), which
improves their peak shape, volatility and response factor (Cercaci,
Passalacqua, Poerio, Rodriguez-Estrada, & Lercker, 2007). For their determination, it is necessary to isolate previously the unsaponiable fraction, preferably by means of diethyl ether, since it guarantees the total
extraction of all the sterols (IOC, 2001a). On the other hand, the possible mobile phases for TLC, benzene/acetone (95:5, v/v) and hexane/
diethyl ether (65:35, v/v) yield excellent results, with no need for
multiple developments.
The individual separation of 4-desmethylsterols, however, is not
exempt from difculties, which can arise in determination of the
components present at low concentration. Such difculties, which are
assigned to the incomplete separation of the sterol fraction in thin-
2031
Table 2b
The standard methods for determining minor compounds.
Chemical series
Technique
Sample preparation
Chromatographic characteristics
Sterols
GC-FID
Unsaponiable-matter isolation TLC or HPLC.

Requires silylation
Column: capillary (2530 m 0.250.32 mm 0.150.30 m)

Operation conditions: isothermal
Erythrodiol + uvaol
GC-FID

Requires silylation

Operation conditions: isothermal
Aliphatic alcohols
GC-FID

Requires silylation

Aliphatic hydrocarbons and sterenes
GC-FID
Unsaponiable-matter isolation on LC Si-column

Note: GC, gas chromatography; FID, ame ionization detector; HPLC, high performance liquid chromatography; TLC, thin layer chromatography.
layer chromatography on silica gel, also produce high values of the coefcients of variation in repeatability and reproducibility. The problem
might be attributed to various causes resulting from an excess of the
unsaponiable deposited on the thin layer, the inadequate development
of the plate, and defective scraping of the sterol band. Empirical results
suggest that the optimum amount to deposit on the thin layer should be
around 200 L if the content of 4,4-dimethylsterols is high. The objective is to prevent that small amounts of cycloarthenol can cause errors
as it overlaps with 7-stigmastenol. The presence of 24-methylenecycloarthanol, which yields a peak at tr = 1.27 in relation to sitosterol, warns the analyst that the percentage of 7-stigmasterol can
be caused in part by the presence of cycloarthenol.
Although the sterols are thought to be the ngerprint of the edible
oils, Grob, Giuffr, Biedermann, and Bronz (1994) found that numerous
edible oils were adulterated with cheaper oils whose sterols had been
previously removed in order to make these admixtures undetectable.
This fraudulent practice should be detected by the amount of trans
unsaturated C18-fatty acids, which are produced from normal cis acids
during the deodorizing process, but some of the illegal processes of
removing sterols are carried out without forming fatty acid transisomers. To avoid the adulteration, IOC proposed that the total value
of sterols should be expressed in mg/kg instead of percentages (IOC,
2011). Thus, a genuine VOO cannot have less than 1000 mg/kg of sterols; below this value, it is reasonable to think that the olive oil might
have been spiked with desterolized seed oils. However, the investigation on this kind of adulteration also focused on the analysis of the
products resulting from the degradation of the olenic sterols, which
is discussed in the section of hydrocarbons.
The analytical determination of triterpene dialcohols is carried out
together with sterols, by scraping their thin layer chromatography
(TLC) band with the band of sterols (Table 2b). They are suitable
markers detecting the presence of olive-pomace oil in olive oil although
there are genuine varietal olive oils that do not t IOC trade standards
(e.g. Spanish var. Verdial de Huevar). Results are calculated, in percentages, as the ratio between the sum of the areas of erythrodiol + uvaol
and the sum of sterols + erythrodiol + uvaol. A VOO must not have a
value exceeding 4.5% (IOC, 2011). This information, however, has to
be assessed together with the wax or aliphatic alcohol content to ascertain the presence of olive-pomace oil.
2.2.4.3. Alternative to ofcial methods. Because of the potential problems in
using TLC to isolate the sterol fraction from the rest of the unsaponiable
material, as well as the high elution times needed, Grob, Lanfranchi, and
Mariani (1990) simplied the analysis of this and other fractions of the
unsaponiable fraction by the in-tandem technique LC-GLC. The technique, which removes almost all the manual work, is widely applied by
analysts at present because it allows the quantication of free and esteried sterols in short time. By adding pivalic acid anhydride, LC allows
collecting a fraction composed of free monoalcohols and the fatty acid esters of these alcohols, which are transferred to GC on-line. It also allows
collecting erythrodiol and uvaol. The whole analytical procedure is identical to traditional except for the substitution of TLC purication for
HPLC, which involves considerable saving in both time and solvent.
Later, Dionisi, Prodolliet, and Tagliaferri (1995) set up RP-HPLC with amperometric detection to detect the addition of palm and grape seed oils to
any tocotrienol-free vegetable oil (e.g., olive oil) at 12%.
In his permanent ght against the olive oil adulteration, Mariani
et al. (2006) developed a method for an independent and adequate
quantication of the nonpolar fraction (containing esteried sterols)
and the polar fraction (containing free sterols) of great success in the
detection of the adulteration with hazelnut oil. The proposed method
comprises three steps: (i) the preparation of the silica gel chromatography column, (ii) the separation of the free and esteried sterols, and
(iii) the independent quantication of free and esteried sterols by
GLC according to IOC standard method (IOC, 2001a).
2.2.5. Hydrocarbons
Sterol composition was an important obstacle to the availability of genetically modied seeds with fatty acid composition similar to olive oil
to be used in adulteration. However, Lanzn, Albi, Cert, and Gracin
(1994) was pioneer in discovering the presence of hydrocarbons as nalkanes, n-hexacosadiene, stigmasta-3,5-diene, isomerization products
of squalene, isoprenoidal polyolens produced from hydroxy derivatives of squalene and steroidal hydrocarbons from 24-methylene
cycloarthanol in rened oils as a consequence of the high temperature
in the rening process, compounds that were are not detected in any
virgin olive oil after analyzing thousands of samples (Aparicio, Alonso,
& Morales, 1994).
2.2.5.1. Reasons to analyze these compounds. The sterol dehydration
products, just above cited, are good markers for rened edible oils,
either deodorized or desterolized. The quantication of campestatriene
in virgin olive oils can indicate the presence of desterolized rapeseed
oil while the ratio between degradation products of sistosterol
2032
(stigmastadienes) and campesterol (campestadienes) is a good marker

for desterolized grapeseed or palm or soybean or sunower oils.
The quantication of 3,5-cholestadiene, resulting from cholesterol
dehydroxylation, indicates the presence of deodorized lards. Furthermore, the desterolized process involves isomerization so converting
7-sterol into 8(14)-and 14-sterols, which were not quantied until
Mariani, Venturini, and Grob (1995) set up a technique that avoided
their overlaps with 5-sterols and hence allowing the detection of
low percentages of a desterolized high oleic sunower oil in olive oil.
7-sterols are themselves useful for detecting desterolized sunower
oil in olive oil, and the presence of ergosterol has been associated to
the addition of low quality olive oils obtained from olives infested by
molds and yeasts (Biedermann, Grob, & Mariani, 1995; Biedermann,
Grob, Mariani, & Schmidt, 1996; Mariani, 1998).
2.2.5.2. Ofcial methods: comments and suggestions. The initial proposal
based on the quantication of stigmasta-3,5-diene (derived from
-sitosterol) (Lanzn & Albi, 1989) was almost immediately included
inside IOC trade standards (Table 1a). Although 3,5-stigmastadiene is
the major compound, the dehydration reaction is not selective for sitosterol. In fact, the yield of dehydration reaction is not the same for
every sterol, and because of this, the presence of sterenes with three double bonds depends on the presence of hydroxysterols originated by sterol
oxidation (Bortolomeazzi, De Zan, Pizzale, & Conte, 2000) (Table 2b).
The EU regulation (EC, 2013) and IOC trade standards (IOC, 2011) include a footnote (Table 1a) pointing out that the limits (i.e. 0.10 for
non-lampante virgin olive oils) is for the sum of all the isomers that
can (or cannot) be separated by CG capillary columns (Table 2b).
2.2.5.3. Alternative to ofcial methods. The ofcial procedure involves a
previous step of saponication followed by liquid chromatography on
silica gel column, in order to remove interfering compounds such as
squalene isomers and n-alkanes. However, despite the elegant initial
method and the high separative power of capillary columns, some overlaps occur, which may produce false results. In the attempt of perfecting
the ofcial method, approaches by using off-line or on-line preseparation by HPLC have been proposed, any of them being validated
with acceptable success (Gallina-Toschi, Bendini, & Lercker, 1996;
Verleyen et al., 2002).
2.3. Proling approaches
Because there is not a method qualied as ofcial, rapid and global
for all the authenticity issues of olive oil but particular solutions, numerous proposals have been designed to opt to that qualication or to compete with the current ofcial methods (Aparicio et al., 2013; Frankel,
2010). On the contrary to the target analysis, which is constrained to
quantitative analysis of a particular analyte or analytes, the proling
analysis represents a well established approach that can be considered
the precursor for metabolomics (Ryan & Robards, 2006). This approach
is not however easy if it has to accomplish the following four basic
requirements: (i) the proposed methodology has to improve the current
standard in any aspect (e.g. analysis time, reproducibility, limit of detection, analysis cost); (ii) the samples for training and testing the method
have to represent the whole universe (i.e. olive oil cultivars and producer countries); (iii) the new methodology validation step has to be
carried out with blind samples; and (iv) an inter-comparison study
has to be carried out as well. In addition, the proposed method should
attempt to select those variables that allow an extendable application.
Researchers have investigated on the application of other techniques
different from the chromatographic ones, which were analyzed in the
previous section, with the aim of implementing rapid methods or with
the objective of identifying and quantifying until today undetectable
analytes. Table 3 shows the proposed techniques, which are described
in depth in recent revision works (Aparicio et al., 2013; Frankel,
2010). The table includes targeted and proling approaches now
Table 3
Main characteristics of the techniques proposed for the authentication of olive oils.
Characteristics
Techniques
Separation
Structural & pattern recognition
Stable isotope analysis
Trace element analysis
In-tandem
GC, RPLCGC, HPLC.

NMR, MS, NIR, FTIR, FT-Raman, DSC, TG, SF.
IRMS.
ICP-AES, AAS, FAAS, ETA-AAS.
GCMS, HPLCMS, ICP-MS, CG GC, LC LC,
SFC, 2H-EAPyIRMS, 2H-GCPyIRMS.
Note: Gas-chromatography (GC); reversed phase liquid chromatography (RPLC); liquid

chromatography (HPLC); nuclear mganic resonance (NMR); near infrared spectroscopy
(NIR), Fourier transform infrared spectroscopy (FTIR) and Fourier transform Raman
spectroscopy (FT-Raman); isotope ratio mass spectrometry (IRMS); inductive coupled
plasma-atomic emission spectroscopy (ICP-AES); atomic absorption spectroscopy (AAS);
ame absorption spectroscopy (FAAS); electrothermal atomization-AAS; mass
spectrometry (MS), GCMS, LCMS and ICP-MS; elemental analyserpyrolysis
isotope ratio mass spectrometry (2H-EAPyIRMS) and 2H-GCPyIRMS; bidimesional
chromatography (GC GC, LC LC); supercritical uid chromatography (SFC);
synchronous uorescence (SF); differential scanning calorimetry (DSC) and
simultaneous thermogravimetry (TG).
clustered inside ve groups, dened by their most remarkable characteristics, which pushing back the frontiers of research in the eld of
olive oil authenticity. They vary from classical to very sophisticated
techniques, which are capable of discriminating minor differences associated with authenticity issues.
2.3.1. Vibrational spectroscopy
Infrared absorption and Raman scattering give information about
molecular vibrations, yielding a vibrational ngerprint of the molecules,
which have been used for the identication of oils with the help of
multivariate statistical algorithms. Successful discriminations between
authentic and adulterated olive oils are, however, mostly centered on
the identication of trans double bonds in samples labeled virgin olive
oils as their unsaturated fatty acids have to be in their cis form. The information from the ngerprint region, which has not been deciphered
with mathematical algorithms yet, have allowed reaching relatively
low percentages of detection but almost always circumscribed to a
kind of adulteration, the addition of rened edible oils to virgin olive
oils (Garca-Gonzlez, Baeten, Fernndez-Piernas, & Tena, 2013;
Garca-Gonzlez, Infante-Domnguez, & Aparicio, 2013). Its main disadvantage is that its lowest percentage of detection is higher than the
values reached with some ofcial methods (e.g. stigmastadienes)
although the technique is much more rapid. The ngerprint, however,
still hides information that could contribute to the characterization of
labeled monovarietal virgin olive oils in the future.
2.3.2. NMR spectroscopy
NMR instrumentation has been applied with undoubted success in
the authentication of many water-based foods. The detection by NMR
of possible anomalies in the chemical composition of adulterated olive
oils is, however, less interesting when results are compared with
those from ofcial standards; for instance, the resonance in the carbonyl
region ascribed to saturated fatty acids at the sn-2 position of glycerol.
13
C NMR is the preferred technique to obtain information about the
positional distribution of the saturated, oleyl, linoleyl, and linolenyl
chains on the glycerol moiety. 1H NMR technique is adequate for the
quantication of fatty acids, despite it is unable to determine individual
fatty acids (unlike gas chromatography), and suitable for the detection of
minor compounds, such as free fatty acids, diacylglycerol isomers, total
sterols, squalene, cycloarthenol, etc. 31P NMR is used for the detection
and quantication of phospholipids like phosphatidic acid, lysophosphatidic acid and phosphatidylinositol. The resonances of the
phosphitylated hydroxyl groups of phenolic compounds are used for
detecting simple phenols, lignans, and avonols. Finally, the application
of site-specic natural isotope fractionation-nuclear magnetic resonance
(SNIF-NMR) is focused mainly on investigating the intramolecular distribution of deuterium in fatty acids and triacylglycerols (Dais, 2013).
Empirical results show, however, that slight differences in NMR spectra are less reliable when this technique tackles a real adulteration problem. It is so because the information reported by fatty acids/TAGs is not,
in general relevant, in sophisticated adulterations (Table 4) and the information from identied minor compounds is related with VOO quality
rather than, for example, the detection of the presence of rened nonolive oils in olive oils or deodorized virgin olive oils in virgin olive oils.
The combination of multivariate statistical algorithms and databases
of NMR spectra has been used in determining the geographical provenance of virgin olive oils. Some of the publishing results are promising
in the steps of training and testing but, unfortunately, less valuable
when model is validated by external laboratories (the reproducibility
step). Reasons are in the enormous amount of external and internal
parameters affecting to virgin olive oil chemical composition in comparison with the small amount of useful information reported by NMR spectra
that is, besides, circumscribed to a few chemical series; SEXIA expert
system, for example, uses 64 different chemical compounds of 9 chemical
series, and hundreds of decision rules, only for determining the geographical provenance of Spanish virgin olive oils (Garca-Gonzlez, Tena, &
Aparicio, 2012). The use of supervised statistical procedures without a
strict procedure for selecting the variables of the mathematical models
is on the basis of good testing but poor validation results; the bibliography
is plenty of those hyper-optimistic proposals that besides are usually
based on banks of samples that do not represent the entire universe to
be studied (e.g., cultivar, geographical origin, ripeness) or even on chemical compounds that have not been identied but tentatively.
2.3.3. Stable isotope analysis
Pyrolysisisotope ratio mass spectrometry (PyIRMS) is a ngerprinting technique that, coupled with suitable multivariate data analysis
procedures, has been proposed for the detection of frauds in olive oil
(Guillou et al., 1999); its main advantage is to be rapid and no need of
sample cleanup.
Information collected by elemental analysispyrolysisIRMS (2H-EA
PyIRMS) has allowed detecting rened olive oil samples adulterated
with rened hazelnut oils at percentages higher than 10%. Its main
drawbacks, however, are the lack of D/H (deuterium/hydrogen) certied
reference material and the geographical origin dependence. Other studies, based on 18O isotopic abundance in olive oils, have been focused on
olive variety and geographical origin. Although compounds with low
oxygen content generate more problems than faced when chromatographic techniques are applied, authors have found correlation between
enrichment in heavy isotopes and latitude (Aramenda et al., 2007)
though the model has not been validated yet.
13C isotope ratio measurements have been scarcely used for olive
oil authentication (Angerosa, Camera, Cumitini, Gleixner, & Reniero,
1997) but for other foods (e.g., wine and juices) because the values of
this ratio by themselves did not allow fraud can be ascertained although
they could perhaps provide valuable complementary information
Table 4
Main authenticity issues and sub-issues, and their current examples.
Issue
Sub-issue
Paradigm
Adulteration
Detection of rened hazelnut

oils in ROOs
Detection of seed oils in VOOs
Geographical origin
Addition of cheaper oil

to olive oils
Addition of rened oils
to VOOs
Addition of low to high
oil categories
Inexact label
Agricultural system
Traceability
Organic vs. conventional
Cultivar
Varietal VOOs
Detection of deodorized VOOs

in VOOs
Detection of VOO from several
origins
Characterisation of PDOs
Addition of conventional to
organic VOOs
Authentication of monovarietal
VOOs
Note: PDO, protected denomination of origin; ROO, rened olive oil; VOO, virgin olive oil.
2033
(Kelly, Parker, Sharman, Dennis, & Goodall, 1997). Other attempts,

based on the ratios 13C16:0 adversus 13C18:1, have been proposed to
detect slight variations in the composition of fatty acids as result of
the presence of edible oils with similar proles to olive oil; i.e. high
oleic sunower, hazelnut, olive-pomace (Ogrinc, Koir, Spangenberg,
& Kidri, 2003). The variability in 13C values of fatty acids, however, is
affected by a combination of geographical provenance (mainly environmental conditions) and genetic factors (Woodbury, Evershed, & Rossell,
1998) which depreciates the proposal because of the percentages of
false positives. Bibliography does not describe that these methods had
been validated according to ISO 5725 (ISO, 1994) and hence they cannot
be described as alternatives to ofcial standards. Therefore, it seems
important to establish a database that provides isotopic information
for olive oils as a previous step to tackling olive oil authenticity with
these kinds of procedures. The protocol should be supplemented with
a calibration step referred to a primary standard method.
2.3.4. Calorimetry
Although differences in the solidliquid phase transitions of olive oil
and seed oils are known by researchers from many years ago, there is
little information in the literature because of the reproducibility of the
thermograms. The methods based on simultaneous thermogravimetry
(TG) and differential scanning calorimetry (DSC), which seem to be in
their preliminary stages of application for determining olive oil authenticity and quality, are currently focused on identifying deconvoluted
peaks related to olive oil chemical composition (i.e. triacylglycerols,
fatty acids and phenols) (Vecchio, Cerratini, Bendini, & Chiavaro,
2009). In terms of authenticity, the main problem is, once again, that
the information collected by the technique (i.e. TAGs and FAs) is not
enough as to determine if an olive oil is adulterated due to the large panoply of possible adulterants and, as the same time, to be more efcient
and precise than the current ofcial methods.
2.3.5. Multidimensional chromatography
Although chromatography offers enough resolution to yield quantitative information for chemical components, the probability of separation
of every component ranges, depending on sample complexity,
19%37%. The detection of current sophisticated adulterations, however,
requires techniques with increased resolution power that is not provided
by single dimension chromatography. Solution can be in the multidimensional chromatography, a separation technology that utilizes two
orthogonal separation mechanisms in order to increase the resolution
power and peak capacity of an experiment by increasing the selectivity
of the experiment (Cortes, Winniford, Luong, & Pursch, 2009). For example, GC GC has been especially helpful in identifying low level oddnumbered fatty acids and species where the results of mass spectra can
be ambiguous (Tranchida et al., 2009) while GC GC-FID has allowed
studying phytol esters, geranylgeraniol esters and the straight-chain esters of palmitic acids and the unsaturated C18 acids (Biedermann,
Bongartz, Mariani, & Grob, 2008). Bidimensional chromatography is not
thought for its application in olive oil authenticity, because of the instrument cost and need of analysts with particular skills among others, but
for helping in unsolved problems; for example, GC GCTOFMS has
been used for the characterization of fresh adversus stale olive oil at qualitative level (de Koning, Kaal, Janssen, van Platerink, & Brinkman, 2009)
whereas a GC GCTOF-MS was checked to determine polycyclic aromatic hydrocarbons (Purcaro, Morrison, Moret, Conte, & Marriott, 2007).
2.4. Rapid methods for olive oil authenticity
The efciency of the ofcial food control is dependent on the possibility to proceed to rapid tests for particular analytes. It is more remarkable in the current global market in which the international regulations,
which are stricter day by day, have shot up the number of samples and
the analytes to be analyzed. The immediate consequence has been an
increasing demand of rapid methods that allow a fast overall view of
2034
Table 5
Basic characteristics of the methods proposed for the current analytical challenges of the olive oil authenticity issues.
Issue
Addition of cheaper oils to olive oils
Objective
Analyte/signal
Technique
Level of applicability
Ofcial method?
Time of analysisa
Limit of detectionb
Advantages
Disadvantages
References
Detection of the presence of crude or rened hazelnut oil in virgin or rened olive oil.
Free and steried sterols.
Gas chromatography.
Particular due to the design of the mathematical algorithm.
No, but the method has been validated with blind trials by IOC.
Sample pre-treatment: 120 min; measurement: 45 min; data analysis: 15 min.
b8%
Easy to carry out. Low cost analysis. Fine repeatability.
Time-consuming. Poor reproducibility because of the need of extensively well-trained analysts.
Garca-Gonzlez, Viera, Tena, and Aparicio (2007); Mariani et al, (2006), Bowadt & Aparicio (2003).
Objective
Analyte/signal
Technique
Ofcial method?
Time of analysisa
Detection of the presence of any edible oil (crude or rened) in virgin or rened olive oil
Selected 13C & 1H NMR bands of the spectrum.
13
C NMR and 1H NMR spectroscopies.
Universal although has been checked with only a few adulterants.
No, but the adulteration with hazelnut oils have been validated with blind trials.
Pre-treatment: No; measurements: 4 h for 1H NMR and 1.45 h for 13C NMR; data analysis: 20 min applying procedures
of Articial Neural Networks (ANN).
N10% using bands from 13C NMR and 1H NMR for adulterations with hazelnut oils.
~15% using bands from 13C NMR or from 1H NMR for dulterations with hazelnut oils.
Good repeatability.
Time-consuming. Poor reproducibility. False positives. Hyper-optimist models.
Dais and Hatzakis (2013), Mannina, D'Imperio, Capitani, Rezzi, et al. (2009), Garca-Gonzlez, Mannina, D'Imperio, Segre, and Aparicio (2004).
Limit of detectionb
Advantages
Disadvantages
References
Objective
Analyte/signal
Technique
Ofcial method?
Time of analysisa
Limit of detectionb
Advantages
Disadvantages
References
Objective
Analyte/signal
Technique
Detection of the presence of any edible oil (crude or rened) in virgin or rened olive oil.
Infrared or Raman bands.
FTIR or FT-Raman.
Universal although has been checked with only a few adulterants.
No, some kinds of adulteration have been validated with blind samples.
FTIR: Pre-treatment: 5minc; measurement: 5 min; data analysis: 5 min applying ANN.
FT-Raman: Pre-treatment: nilc; measurement: 10 min; data analysis: 5 min applying ANN.
N10%
Rapid and easily implementable method.
Full checked with hazelnut oils only. A large set of spectra is required. Unstable mathematical equations.
Garca-Gonzlez, Baeten, Fernndez-Piernas, and Tena (2013), Garca-Gonzlez, Infante-Domnguez, and Aparicio (2013),
Baeten et al. (2005), Lpez-Dez, Bianchi, and Goodacre (2003), Tay, Singh, Krishnan, and Gore (2002).
Ofcial method?
Time of analysisa
Limit of detectionb
Advantages
Disadvantages
References
Detection of the presence of any edible oil (crude or rened) in virgin or rened olive oil.
Triglycerides and fatty acids.
HPLC for quantifying triacylglycerides and GC for quantifying fatty acids. A free program for determining differences between
empirical and theoretical TAGs
Universal. With almost all the edible oils.
No, but it is being validated with blind trials by IOC.
Pre-treatment: 60 min; measurement: 90 min; data analysis: 5 min.
~10% but it depends on the kind of edible oil.
Global and rapid method with individual information from FAMEs & TAGs.
Time-consuming. False positives. Unstable mathematical model.
Garca-Gonzlez, Viera-Macas, Aparicio-Ruiz, Morales, and Aparicio (2007), IOC (2006b)
Issue
Addition of rened oils to virgin olive oils
Objective
Analyte/signal
Technique
Ofcial method?
Time of analysisa
Limit of detectionb
Advantages
Disadvantages
References
Detection of the presence of any rened edible oil in virgin olive oils.
Hydrocarbons: stigmastadienes.
Gas chromatography.
Universal.
Yes (Table 1a)
Pre-treatment: 90 min; measurement: 20 min; data analysis: No.
2%
Global method. Excellent LOQ (0.1 ppm) & LOD (2%).
May fail detecting rened oils obtained under soft conditions (b175 C).
IOC (2001b), Lanzn, Cert, and Gracian (1994).
Objective
Analyte/signal
Technique
Ofcial method?
Time of analysisa
Limit of detectionb
Advantages
Disadvantages
References
Detection of the presence of any rened edible oil in virgin olive oils.
cis/trans FTIR or FT-Raman bands.
Spectroscopy by FTIR or FT-Raman.
Universal.
No, but the method has been validated with blind samples.
Pre-treatment: Nil; measurement: 10 min; data analysis: 10 min.
N8%
Rapid method.
Limit of detection. The method does not work properly with less unsaturated oils.
Baeten et al. (2005), Hourant, Baeten, Morales, Meurens, and Aparicio (2000), Baeten, Meurens, Morales, and Aparicio (1996).
Issue
Addition of low to high VOO categories
Objective
Analyte/signal
Detection of the presence of VOOs deodorized at low temperature in Extra-VOOs

Alkyl and ethyl esters
2035
Table 5 (continued)
Issue
cheaper
oilsVOO
to olive
oils
Addition of low
to high
categories
Technique
Ofcial method?
Time of analysisa
Correct classicationb (%)
Advantages
Disadvantages
References
Gas-chromatography
Universal, but perhaps it is circumscribed to VOOs coming from olives that have undergone any fermentative process.
Yes (Table 1a)
Pre-treatment: 60 min; measurement: 27 min; data analysis: 5 min
Not reported by IOC
Detect the presence of some deodorized VOOs in VOOs
Numerous false negatives. No detection of deodorized VOOs with rancid/wood/frozen and other non-fermentative defects.
IOC (2010), Prez-Camino, Cert, Romero-Segura, Cert-Trujillo, and Moreda (2008).
Issue
Geographical traceability of VOOs
Objective
Analyte/signal
Technique
Ofcial method?
Time of analysisa
Correct classicationb (%)
Determination of the geographical provenance (country, region, county, PDO, PGI) of VOOs
Several: fatty acids, alcohols, sterols, hydrocarbons etc.
Gas chromatography for chemical analysis and expert system (SEXIA) for data analysise.
Whole Spain and partially the other EU producer countries.
No, but SEXIA has been validated with hundreds of samples for years.
Pre-treatment: 180 min; measurement: 300 min; data analysis: 10 min using expert system
Average certainty factors (CF): 92% for Andalusian PDOs, 95% for Spanish regions, and 96% for the identication of major
EU producing countries/varieties among others
Results are associated to high CFs. It based on the largest VOO database.
Time-consuming. Several different chemical analyses. It constantly needs to be updated.
Garca-Gonzlez, Baeten, Fernndez-Piernas, and Tena (2013), Garca-Gonzlez, Infante-Domnguez, and Aparicio (2013);
Aparicio and Luna (2002); Aparicio (2000), Aparicio, Alonso, and Morales (1994).
Advantages
Disadvantages
References
Objective
Analyte/signal
Technique
Ofcial method?
Time of analysisa
Correct classicationb(%)
Advantages
Disadvantages
References
Determination of the geographical provenance of VOOs

2H, 13C or 18O
EA-IRMS or NMR
Universal
No.
Pre-treatment: nil; measurement: few minutes; data analysis: 5 min
Not reported by authors
Rapid method
Reproducibility. Need of a previous large database. Harmonization calibration procedure
Camin, Larcher, Perini, Bontempo, et al., 2010; Chiavaro et al., 2011; Alonso-Salces et al., 2010.
Objective
Analyte/signal
Technique
Ofcial method?
Time of analysisa
Correct classicationb(%)
Advantages
Disadvantages
Determination of the geographical provenance of VOOs

Multi-elements
ICP-MS or ICP-AES
Universal
No
Pre-treatment: 7590 min with digestions in microwave; measurement: 35 min; data analysis: 15 min using ANN.
Not reported by authors
Causal relationship between soil and oil. A large number of variables (elements). Repeatability.
Low concentration of elements in the oils. Need of information of soils for training the model. Interference of fertilizers
and fungicidesd.
Llorent-Martnez, Ortega-Barrales, Fernndez-de Crdova, Domnguez-Vidal, and Ruiz-Medina (2011), Benincasa, Lewis,
Perri, Sindona, and Antonio Tagarelli (2007), Zeiner, Steffan, and Cindric (2005).
References
a
b
c
d
Checked by the authors at their labs and in the course of collaborative analyses of European funded projects.
The best gure reached in the course of collaborative analyses with blind samples.
The measurement is carried with the entire oil but if the measurement is of the unsaponiable matter, 60 min has to be added to the total analytical procedure.
Foliar fertilizers can contain K, Fe, Mg, Mn, P and Zn in different proportions, together with other elements (i.e. B, Ca), which can be presented complexed with amino acids such in the
cases of Ca, Fe, Mg, Mn and Zn. Fungicides can contain Cu among other elements.
Other authors have proposed the study of particular geographical production zones by diverse series of compounds, and data are analyzed by an umpteen different number of statistical procedures, either unsupervised (e.g. PCA, MDS) or supervised (e.g. LDA, PLS).
the olive oil traceability and safety; a demand widely claimed by

producers, sellers and consumers. However, there is not a uniform
agreement on a valid denition for the term rapid method due to
the lack of conceptual clarity of the adjective rapid. The adjective is
not conditioned by a xed time limit even though rapid methods are
characterized obviously by time. In fact, the concept of being a rapid
method is not only dened in the time domain, but it also expresses
a general sense of convenience in lab work, with a automation compliance in all the analytical steps: sample pre-treatment or sample preparation, the chemical or physical analysis, the data analysis, and the
evaluation of the results.
As described previously, regulators and associations of consumers
have suggested numerous criteria for dening the authenticity of olive
oil. Adulteration, geographical origin, production system and variety
are the main authenticity issues associated with the olive oils although
their relevance depends on the current trends of the global market.
Table 4 shows the issues, their most relevant sub-issues and their current paradigms, which are circumscribed to olive oil adulterations that
have traditionally produced high benets to fraudsters. Solutions, when

possible, come from classical techniques to very sophisticated techniques
that are capable of picking up minor differences associated with the
authenticity issues. Some of them are responsible of irrefutable results
(i.e. stigmastadienes in the addition of rened to crude oils) but unfortunately only a few can be dened as rapid (Aparicio and Alonso 1994).
Table 5 describes the most common and successful methods proposed for the authenticity sub-issues of Table 4; they are compared
with standard methods when possible. All the methods have been
dissected according to the time needed for the whole analysis (sample
preparation, measurement and data analysis), the limits of detection
and quantication, when described by the authors, and their main
advantages and disadvantages.
2.5. Unsolved problems with the current methods
The large collection of analytical methods and techniques available
at present and the fact that the olive oil is the most regulated edible
2036
oil in the world have not avoided the existence of some olive oil adulterations that are still difcult to be detected. Reasons can be found in the
ample information about the chemical composition of any edible oil that
is at the disposal of everybody through internet. Thus, the fraudsters can
easily prepare fraudulent recipes mixing edible oils at percentages calculated by computer software. For example, an adulteration
composed of 65% rened olive oil, 15% rened hazelnut, 15%
desterolized sunower and 5% palm olein without free sterols is
undetectable although the economical benets of this fraudulent
practice might not really yield a prot. Furthermore, the industrial
technology allows the deodorization of virgin olive oils with sensory
defects at moderate temperature (Biedermann, Bongartz, Mariani, &
Grob, 2008), and the resulting oils might not be detected even with
the proposed methodologies based on chlorophylls and alkyl esters
(Garca-Gonzlez & Aparicio, 2010).
Short-sighted planning for the elaboration of regulations, which
mostly are not based on a large number of olive oil samples of almost
all the varieties and producing regions, is the main forthcoming problem that has abruptly increased with the numerous olive orchards located beyond the latitude of the Mediterranean basin (e.g. New Zealand,
Argentina). The consequence is an ample group of olive oil varieties
and olive tree orchards that produce genuine (virgin) olive oils with
chemical compositions that are, in one or more parameters, outside
the limits of the current trade standards. Those exceptions are not
only of olive tree orchards placed in latitudes that do not correspond
to those of the Mediterranean basin (e.g. some virgin olive oils produced
in Argentina and Australia) (Ceci & Carelli, 2007; Mailer, Ayton, &
Graham, 2010) as they also include Mediterranean varietal olive oils
(e.g. cv. Verdial de Huevar of Spain) (Garca-Gonzlez, InfanteDomnguez, & Aparicio, 2013). In this context, the purpose of olive oil
authenticity should not be exclusively focused on the labeling control
but also on protecting the genuineness of olive oils with regard to
their geographical origin and botanical variety.
The growing global market together with the increasingly stricter
international regulations has also increased the number of samples
that have to be analyzed by means of numerous analytical methods.
Therefore, there is a growing demand for a global procedure, which
could be able to reduce to one or two all the current ofcial methods,
or for rapid methods, since the current methods are going to collapse
recognized analytical laboratories (IOC, 2012). The former alternative
would be widely supported for all the olive oil actors although it
confronts with the wide variety of edible oils and their different chemical compositions. The current proposals are based on mathematical
algorithms designed to detect low concentrations of extraneous oils in
olive oil, which results unstable systems that oscillate between the
percentages of false positives and false negatives. Furthermore, each
new datum added implies rebuilding the mathematical decision rules
in an endless process that in most of cases makes things worse. It is
the so-called Jackknife Paradigm.
An alternative to global and rapid methods is based on the determination of DNA in the oils (Bracci, Busconi, Fogher, & Sebastiani, 2011),
which could achieve indubitable results in the authenticity of olive oil
although its current results are at the same order of magnitude of the
chromatographic techniques (Consolandi et al., 2008; Kumar, Kalon, &
Chaudhary, 2011), in addition to the difculties in extracting goodquality DNA without contamination from other sources (Woolfe &
Primrose, 2004). In the meantime, the solution may come from databases with chemical information, obtained by chromatographic techniques, which could be used to perfect certain limits of the ofcial
parameters and also to aid in the calibration of those spectroscopic techniques which are less time-consuming.
3. Challenges for the near future
The olive oil authenticity is continuously evolving to situations
explained by the consumer demands of a global market. IOC trade
standards, in addition to international and national regulations, are periodically upgraded in the light of new challenges created by fraudsters
and the advances in analytical instrumentation.
Sometimes, however, the problem lies in the semantic denition of
authenticity, which is not obvious. If the fatty acid composition determines the physical and chemical properties of the oil and its
potential applications (food industry is offering tailored oils for specic
purposes), mutants or genetically modied seed oils, for example,
represent a challenge in oil authenticity not only for detecting contamination or fraud, but also for the global meaning of authenticity (GarcaGonzlez & Aparicio, 2010). Furthermore, forthcoming problems might
not be only focused on the authenticity of the olive oil added to canned
sh or inside bottles labeled as spiced VOOs, or even in the detection of
deodorized oils in VOO, but in the authentication of VOO geographical
provenance and the current overlap between the concepts of quality
and authenticity.
The high number of protected designations of origins (PDO) has raised
even more the concern of producers and consumers about the particular
characteristics of their VOOs. The European Community regulation (EC,
2006), as result of the consumer demands, established a controlled labeling for PDOs with the objective of assuring the consumers' expectations
and a better protection of VOOs, which have proved to have particular
sensory and chemical characteristics, against falsication or mislabeling
because their higher market price. Regulations, however, do not suggest
any analytical procedure to verify the information provided on the label
but exclusively administrative controls. As a consequence, the geographical declaration of virgin olive oil, which is never controlled by physical
chemical parameters, is vulnerable to frauds.
The revolution in agricultural techniques, on the other hand, has
allowed to cultivate olive trees from varieties autochthonous from
Mediterranean countries, there where never were thought they could
be cultivated. When planted inside the Mediterranean Basin, the chemical composition of the varieties is within the limits of trade standards.
The latitude, altitude and climate of the new producing areas have
played a dirty trick on farmers because those physical parameters affect
biochemical pathways of some cultivars more than would be expected,
and the chemical composition of resulting oils does not t with current
trade standards. The solution can come from the development of decision trees that, using other chemical compounds, can act as safeguard
for the genuineness of those olive oils without modifying the current
limits although analysts should keep in mind that decision trees need
of strict statistical algorithms that avoid an unacceptable number of
false positives.
Therefore, the future work on geographical traceability should be focused on building an Olive Oil Map where the most productive cultivars
and all the approved PDOs are characterized by chromatographic
(Aparicio et al., 1994; Garca-Gonzlez et al., 2012) spectroscopic (Dais
& Hatzakis, 2013; Garca-Gonzlez, Baeten, Fernndez-Piernas, & Tena,
2013) and isotopic (Chiavaro et al., 2011) methods. The resulting databases, in conjunction with new procedures of classication and visualization techniques, would allow a better authenticity of the geographical
provenance of virgin olive oils.
The increasing importance of mathematical algorithms in the
successful control of the olive oil adulteration have encouraged some
researchers to believe that a strong mathematical relationship between
two factors can prevail over the lack of any scientic evidence that
explains that relationship is causal. Inappropriate application of statistical procedures, most of the time without the required validation tests,
allows reaching over-optimist conclusions that usually present umpteen exceptions in the model validation step; for example, models
based on the statistical procedures LDA (linear discriminant analysis)
without a selection of the variables by means of a stepwise algorithm.
Furthermore, that hypothetical success ghting against adulterations
has driven analysts to think that the same chemical compounds also
can be used in determining virgin olive oil quality, in a mixture of
concepts that is difcult to be understood.
Thus, new trade standards should be analyzed under a magnifying

glass to prevent that a casual relationship between chemical compounds and authenticity/quality can be seen as causal. One of paradigms
is the use of chemical compounds, which are consequence of the degradation of the initial virgin olive oil color (i.e. pyropheophytins PPP),
for explaining VOO freshness. The casual relationship between the increase of PPP with shelf-life and the causal relationship between shelflife and sensory quality has been used to relate PPP with the
overall grading of virgin olive oil quality (color is not among the quality
parameters) when there is not any scientic evidence that relates the
concentration of PPP with virgin olive oil avor (Aparicio, Morales, &
Garca-Gonzlez, 2012). Conclusions without scientic basis for example, NIR spectra explain intensities of sensory descriptors and concentrations of volatile compounds of virgin olive oils are unfortunately a
fact very common today.
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Food Research International 54 (2013) 20252038

Contents lists available at ScienceDirect

Food Research International

Authenticity of olive oil: Mapping and comparing ofcial methods and

Instituto de la Grasa (CSIC), 4 Padre Garcia Tejero, 41012 Sevilla, Spain

tighter control of producing and importing countries, clear denitions

R. Aparicio et al. / Food Research International 54 (2013) 20252038

2000; Aparicio, Aparicio-Ruiz, & Garca-Gonzlez, 2007; Aparicio, Conte,

R. Aparicio et al. / Food Research International 54 (2013) 20252038

Extra-virgin olive oil

Extra-virgin olive oil

2.2.1.3. Alternatives to ofcial methods. While GC is the technique used for

R. Aparicio et al. / Food Research International 54 (2013) 20252038

Extra-virgin olive oil

42) triacylglyceride content (ECN42). The IOC trade standard (IOC,

R. Aparicio et al. / Food Research International 54 (2013) 20252038

0.12 g oil in 0.5 mL hexane is charged into SPE-cartridge

Mobile phase ow-rate (0.6 to 1.0 mL/min)

(1 g of Si) and solution pulled through and, then, eluted

0.5 g oil in 10 mL acetone or acetone/chloroform (1:1 v/v).

Mobile phase ow-rate (0.6 to 1.0 mL/min)

2-Glyceryl monopalmitate (%)

Hydrolysis with pancreatic lipase.

Column: capillary 12 m 0.32 mm 0.100.30 m

Total fatty acids:

Column: capillary 25100 m 0.20.8 mm 0.10.2 m

Column: capillary 1215 m 0.250.32 mm 0.10.3 m

R. Aparicio et al. / Food Research International 54 (2013) 20252038

in the biosynthetic pathway leading to end-product, 4-desmethylsterols.

R. Aparicio et al. / Food Research International 54 (2013) 20252038

Unsaponiable-matter isolation TLC or HPLC.

Column: capillary (2530 m 0.250.32 mm 0.150.30 m)

Unsaponiable-matter isolation TLC or HPLC.

Column: capillary (2530 m 0.250.32 mm 0.150.30 m)

Unsaponiable-matter isolation TLC or HPLC.

Column: capillary (2530 m 0.250.32 mm 0.150.30 m)

Aliphatic hydrocarbons and sterenes

Unsaponiable-matter isolation on LC Si-column

Column: capillary (2530 m 0.250.32 mm 0.150.30 m)

R. Aparicio et al. / Food Research International 54 (2013) 20252038

(stigmastadienes) and campesterol (campestadienes) is a good marker

GC, RPLCGC, HPLC.

Note: Gas-chromatography (GC); reversed phase liquid chromatography (RPLC); liquid

R. Aparicio et al. / Food Research International 54 (2013) 20252038

Detection of rened hazelnut

Addition of cheaper oil

Detection of deodorized VOOs

(Kelly, Parker, Sharman, Dennis, & Goodall, 1997). Other attempts,

R. Aparicio et al. / Food Research International 54 (2013) 20252038

Addition of cheaper oils to olive oils

Addition of rened oils to virgin olive oils

Addition of low to high VOO categories

Detection of the presence of VOOs deodorized at low temperature in Extra-VOOs

R. Aparicio et al. / Food Research International 54 (2013) 20252038

Geographical traceability of VOOs

Determination of the geographical provenance of VOOs

Determination of the geographical provenance of VOOs

the olive oil traceability and safety; a demand widely claimed by

have traditionally produced high benets to fraudsters. Solutions, when

R. Aparicio et al. / Food Research International 54 (2013) 20252038

R. Aparicio et al. / Food Research International 54 (2013) 20252038

Thus, new trade standards should be analyzed under a magnifying

R. Aparicio et al. / Food Research International 54 (2013) 20252038

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