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7
Analysis and Authentication
Franca Angerosa*, Christine Campestre**, Lucia Giansante*
* CRA-Istituto Sperimentale per la Elaiotecnica, Viale Petruzzi, 65013 Città Sant’Angelo
(PE) – ITALY, ** Dipartimento di Scienze del Farmaco, Università degli Studi G.
D’Annunzio, Via dei Vestini, 31, 66100 Chieti - ITALY
Introduction
Olive oil, differently from most vegetable oils, is obtained by means of some
technological operations which have the purpose to liberate the oil droplets from the
cells of olive flesh. Due to its mechanical extraction, it is a natural juice and preserves
its unique composition and its delicate aroma, and therefore can be consumed with-
out further treatments. However, a refining process is necessary for making edible
lampante virgin olive oils. Lampante oils cannot be directly consumed because of the
presence of organoleptic defects or because chemical-physical constants exceeding the
limits established by International Organizations.
Consumers are becoming continuously more aware of potential health and thera-
peutic benefits of virgin olive oils and their choice is oils of high quality which pre-
serve unchanged the aromatic compounds and the natural elements that give the
typical taste and flavor.
Because of the steady increasing demand and its high cost of production virgin
olive oil demands a higher price than other vegetable oils. erefore, there is a great
temptation to mix it with less expensive vegetable oils and olive residue oils. On the
other hand even refined olive oils, due to high mono-unsaturated fatty acids content
and other properties, often have prices higher than those of olive residue oil or seed
oils. us, there are attempts to partially or totally substitute both virgin and refined
olive oils with pomace oil, seed oils, or synthetic products prepared from olive oil fatty
acids recovered as by-products in the refining process. e substitution or adultera-
tion of food products with a cheap ingredient is not only an economic fraud, but may
also have severe health implications to consumers. Such is the case of the Spanish
toxic oil syndrome (TOS), resulting from the consumption of aniline-denaturated
rapeseed oil that involved more than 20,000 people (World Health Organization,
1992; Wood et al., 1994; Gelpi et al., 2002).
erefore, there is always a need to protect consumers through effective and clear
regulations that assure uniformity of definitions, labelling rules, instrumental tech-
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114
F. Angerosa et al.
niques and methodologies, limits, and identity characteristics in all countries.
At the moment Codex Alimentarius, European Commission (EC), and Inter-
national Olive Oil Council (IOOC) generally give the same limits for the olive oil
identity characteristics. However, there are some differences between EC regulations
and IOOC Trade Standards due to the fact that this last organization must take into
account characteristics of all olive oils and pomace oils produced by all IOOC mem-
bers. ese characteristics can be different from those of European Union countries
because of different cultivars and climate conditions.
In the last 20 years a great analytical effort was made from food chemists and
many gas chromatographic, high pressure liquid chromatographic, and spectromet-
ric methodologies were developed to evidence possible frauds. Several analytical ap-
proaches are currently included in regulations of the European Community, the Draft
of Codex Alimentarius Standards, and the International Olive Oil Trade Standards.
e application of new reliable analytical approaches had, as a consequence, a
reduction of adulteration, but there are still problems with sophisticated practices.
ese are the addition of: i) hazelnut oil; ii) olive oils subjected to forbidden deodor-
ization in mild conditions; iii) olive oil obtained by second centrifugation of olive
pastes (remolido).
e evaluation of quality and the checking genuineness of olive oils is made on
the basis of analytical data of a number of parameters which must be within limit
values established by the European Commission (EC Reg No 2568/1991 and its lat-
est amendment EC Reg No 1989/2003), the Codex Alimentarius Norm (Codex Ali-
mentarius Commission Draft, 2003) and the IOOC Trade Standards (International
Olive Oil Council Trade Standards, 2003). e methods generally applied can be
divided into two groups: i) methods adopted by national and international organiza-
tions such as IOOC, Codex Alimentarius, and the European Commission; ii) meth-
ods not evaluated by standardizing bodies, but proposed by researchers, which are
either used to support nonconclusive results of official analyses, when sophisticated
adulterations have to be evidenced, or to obtain a rapid and a more complete evalua-
tion of olive oil quality.
Definitions
Olive oils can be distinguished in virgin olive oils mechanically or physically extracted
from olive fruits, olive oils coming from further refining treatments and olive pomace
oils, obtained by refining of the oil extracted from the olive pomace with a suitable
solvent. All categories of olive oils are summarized in Table 7.1. e European Regu-
lations do not permit the trade of refined olive oil or refined pomace olive, but allow
trading their blends with virgin olive oils. EC (EC Reg. No 2568/91) fixes the fol-
lowing categories: extra virgin, virgin and lampante, whereas IOOC and Codex also
include, among edible olive oils, the ordinary grade. Codex Alimentarius does not
consider oils not fit for human consumption.
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115
Analysis and Authentication
Quality Parameters
Olive oils are classified by different International Organisms according to their qual-
ity which is established on the basis of certain parameters. ese parameters verify
hydrolytic and oxidative processes that take place in the fruits and during the tech-
nological procedures for extracting and refining, and also during the oil preservation.
Parameters used by the different international organizations to check olive oil quality
are reported in Table 7.2.
Common to all international organizations are the determination of free fatty
acids, peroxide value, spectrophotometric absorbances in the UV region, organoleptic
characteristics, and halogenated solvents. In addition, the Codex Alimentarius and
IOOC Standards include insoluble impurities, some metals and unsaponifiable mat-
TABLE 7.1
Denition of all categories of olive oils according to the different
International Organizations
Category Denition according to EC, IOOC and Codex Alimentarius
Extra virgin olive oil Virgin olive oil having free acidity, as % of oleic acid, up to 0.8
and the other characteristics according to regulations in force
Virgin olive oil Virgin olive oil having free acidity, as % of oleic acid, up to 2.0
and the other characteristics according to regulations in force
Ordinary virgin olive oil Virgin olive oil having free acidity, as % of oleic acid, up to 3.3
and the other characteristics according to regulations in force.
EC does not include this category
Virgin lampante olive oil Virgin olive oil having free acidity, as % of oleic acid, greater than
3.3 and the other characteristics according to regulations in
force
Rened olive oil Olive oil obtained from virgin olive oil rening that preserves its
natural glyceridic composition, having free acidity, as % of oleic
acid, up to 0.3 and the other characteristics according to regula-
tions in force
Olive oil Oil obtained by blending rened olive oil and virgin olive oil
having free acidity, as % of oleic acid, up to 1.0 and the other
characteristics according to regulations in force
Crude pomace olive oil Oil extracted from olive pomace by means of a solvent having the
characteristics according to regulations in force
Rened olive residue oil Olive oil obtained from crude olive oil rening that preserves its
natural glyceridic composition, having free acidity, as % of oleic
acid, up to 0.3 and the other characteristics according to regula-
tions in force
Olive residue oil Oil obtained by blending rened olive residue oil and virgin olive
oil having free acidity, as % of oleic acid, up to 1.0 and the other
characteristics according to regulations in force
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116 F. Angerosa et al.
ter determinations. ese standards have common rules for sampling.
Methods for Olive Oil Quality Evaluation
Included in International Standards
Olive Oil Sampling and Laboratory Sample Preparation
[IOOC, Codex Alimentarius, EC according to EN ISO 61 and EN ISO 5555]
e different international organizations adopted the same rules for olive oil and
pomace olive oil sampling. An exception to the norms is made for many of the olive
oils and pomace oils formed by packages containing up to 100 liters. Detailed proce-
dures are described in the Annex I bis of EC Regulation No 2568/91.
Free Fatty Acids (Free Acidity)
[IOOC: COI/T.15/NC n.3 (2003); Codex Alimentarius according to ISO 660 or
AOCS Cd 3d-63(99), EC Reg. No 2568/91 Annex II]
Free acidity is the oldest parameter used for evaluating the olive oil quality since
it represents the extent of hydrolytic activities. e determination is carried out by
titration of free fatty acids of oils, diluted in a suitable mixture of solvents, with an
aqueous or ethanolic potassium hydroxide solution. Maximum levels (Table 7.3) have
been fixed by Regulations to establish the category, since it is tightly related to the
quality of raw material. Oils obtained from healthy fruits, regardless of the cultivar,
processed just after harvesting, show very low values of free acidity. But, if fruits are
damaged by fly (Bactrocera oleae) attacks or are submitted to a prolonged preservation
before processing, hydrolytic enzymes become active and the free acidity of the oil
slightly increases. e possible invasion of olives from molds causes a notable increase
of free acidity because of the presence of lipolytic enzymes in the mold.
TABLE 7.2
Quality parameters xed by the different International Organizations.
Parameter IOOC Codex Alimentarius EC
Sampling method x x x
Free acidity x x x
Peroxide value x x x
Absorbance in UV region x x x
Organoleptic assessment x x x
Volatile halogenated solvents x x x
α-tocopherol x x —
Cu, Fe, Pb, As determination x x —
Oil content in pomace residue — — x
Insoluble impurities x x —
Unsaponiable matter content x x —
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117
Analysis and Authentication
Peroxide Value (PV)
[Codex Alimentarius and IOOC: according to ISO 3960 or AOCS Cd 8b-90; EC
Reg. No 2568/91 Annex III]
e evaluation of the degree of olive oil oxidation is based on determinations of
both the primary and the secondary products of oxidation. e primary stage of oxi-
dation is the formation of hydroperoxides from polyunsaturated fatty acids through a
radicalic mechanism.
e analysis is carried out by an iodometric procedure, which involves the dis-
solution of oil in a mixture of acetic acid-chloroform, and the addition of an excess of
potassium iodide solution. Iodine formed is titrated with a standardized solution of
sodium thiosulfate. e level of hydroperoxides (PV) is expressed as milliequivalents
of active oxygen per kilogram of oil (meqO2/kg). A limit value of 20 meqO2/kg has
been established for virgin olive oils, 5 for refined ones and 15 for blends of virgin
olive oils with refined olive oils or refined olive pomace oils.
Peroxide value is a parameter which increases, and depends on the storage condi-
tions (oxygen admittance, light, preservation temperature and time). After reaching
a maximum, PV decreases because of the formation of secondary products, typical of
rancidity.
Absorbances in Ultraviolet Region
[IOOC and Codex Alimentarius: according to COI/T.20/Doc. No. 19 or ISO 3656
or AOCS Ch 5-91(01), EC Reg. No 2568/91 Annex IX]
e evaluation of the degree of olive oil oxidation can be made also by means of
the measurements of extinctions on oil sample diluted in an adequate solvent. Spe-
cific absorbances, conventionally indicated as K, are measured in the UV region at
the wavelengths corresponding to the maximum absorption of the conjugated dienes
and trienes, respectively at about 232 and 270 nm. e conjugated dienes and trienes
TABLE 7.3
Limits of free fatty acidity, as oleic acid percent, xed by the International Organiza-
tions for each olive oil category. nl = no limit
Category IOOC Codex Alimentarius EC
Extra virgin olive oil ≤ 0.8 ≤ 0.8 ≤ 0.8
Virgin olive oil ≤ 2.0 ≤ 2.0 ≤ 2.0
Ordinary virgin olive oil ≤ 3.3 ≤ 3.3 -
Lampante oil > 3.3 - > 2.0
Rened olive oil ≤ 0.3 ≤ 0.3 ≤ 0.3
Olive oil ≤ 1.0 ≤ 1.0 ≤ 1.0
Crude olive residue oil nl - nl
Rened olive residue oil ≤ 0.3 ≤ 0.3 ≤ 0.3
Olive residue oil ≤ 1.0 ≤ 1.0 ≤ 1.0
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118 F. Angerosa et al.
are formed in the autoxidation process from the hydroperoxides of unsaturated fatty
acids and their fragmentation products. e absorption around 270 nm could also
be caused by substances formed during earth treatment in the refining process. K232
evaluation is considered optional by IOOC Trade Standards. In addition to K232 and
K270, often, especially in trade negotiations, DK value is considered useful, calculated
according to the following equation:
DK = Kmax – [1/2(Kmax+4 + Kmax-4)] [1]
where Kmax is the maximum absorbance near 270 nm.
Table 7.4 summarizes specific absorbances at 232 and 270 nm and DK value for
each olive oil category.
Organoleptic Assessment of Virgin Olive Oil
[Codex Alimentarius: according to COI/T.20/Doc. No. 15; IOOC: COI/T.15/NC
n.3 (2003); EC Reg. No 2568/91 Annex XII]
Current regulations also compel determination of organoleptic characteristics of
virgin olive oils because they are considered as a very important criterion of quality
evaluation. Although values of free acidity, peroxide index, and absorbances in the
UV region are within limits fixed by regulations in force, virgin olive oils may have
some organoleptic defects which obviously lower their quality. e methodology for
evaluating organoleptic characteristics of virgin olive oils, known as Panel Test meth-
od, was developed in 1980’s by IOOC, and later included into EC legislation.
e method involves as a measurement instrument, a group of 8 to 12 persons,
suitably selected and trained to identify and evaluate the intensities of positive and
negative sensory perceptions. e group uses a vocabulary specifically developed for
TABLE 7.4
Limits of the absorbances at 232 and 270 nm and DK value for each olive oil category
xed by the different International Organizations.
IOOC Codex Alimentarius EC
Category K232 K270 ∆K K270 ∆K K232 K270 ∆K
Extra virgin olive oil ≤ 2.50 ≤ 0.22 ≤ 0.01 ≤ 0.22 ≤ 0.01 ≤ 2.50 ≤ 0.22 ≤ 0.01
Virgin olive oil ≤ 2.60 ≤ 0.25 ≤ 0.01 ≤ 0.25 ≤ 0.01 ≤ 2.60 ≤ 0.25 ≤ 0.01
Ordinary virgin olive oil nl ≤ 0.30 ≤ 0.01 ≤ 0.30 ≤ 0.01 — — —
Lampante oil nl nl nl — — nl nl nl
Rened olive oil nl ≤ 1.10 ≤ 0.16 ≤ 1.10 ≤ 0.16 nl ≤ 1.10 ≤ 0.16
Olive oil nl ≤ 0.90 ≤ 0.15 ≤ 0.90 ≤ 0.15 nl ≤ 0.90 ≤ 0.15
Crude olive residue oil nl nl nl — — nl nl nl
Rened olive residue oil nl ≤ 2.00 ≤ 0.20 ≤ 2.00 ≤ 0.20 nl ≤ 2.00 ≤ 0.20
Olive residue oil nl ≤ 1.70 ≤ 0.18 ≤ 1.70 ≤ 0.18 nl ≤ 1.70 ≤ 0.18
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119
Analysis and Authentication
the virgin olive oil assessment and taste virgin olive oils in pre-established conditions.
Official methodology fixes a number of facilities concerning volume and temperature
of oil sample, tasting room temperature and moisture, shape and size, and color of
tasting glass. Samples are randomly presented and tasters are requested to mark the
sensations they experienced during the tasting on a profile sheet and to evaluate their
intensity on an unstructured scale 10 cm long, ranked from 0 to 10. Data provided
by tasters are statistically processed to verify the reliability of the test.
e median value of the defect perceived with the higher intensity identifies the
oil category. For extra virgin olive oil and virgin olive oil categories, the median of
defects must be zero and the fruity value has to be greater than zero (Table 7.5).
Volatile Halogenated Solvents in Olive Oil
[IOOC and Codex Alimentarius: according to IOOC T20/DOC. No 8/Corr.1
(1990); EC Reg. No 2568/91 Annex XI]
Halogenated solvents such as chloroform, and tetrachloroethylene are contami-
nants that can be detected in trace amounts in virgin olive oils. eir determination is
carried out in the volatile fraction (isolated by a headspace technique) by GC coupled
to an Electron Capture Detector (ECD) or by direct injection of the oil into the gas
chromatograph by using suitable precolumns. In the latter case after a few injections
it is necessary to clean or to replace the precolumn with the disadvantage of discon-
tinuous work. e limit for each halogenated compound is fixed at 0.1 ppm, whereas
the sum of all of them must not exceed 0.2 ppm.
Metals
[Copper and Iron: IOOC and Codex Alimentarius according to ISO 8294 or AOCS
Cd 3-25 (02)]
[Lead: IOOC according to ISO 12193 or AOCS Ca 18c-91(97) or AOAC 994.02
(02)]
TABLE 7.5
Median limits of defects (Md) and fruity attribute (Mf) of virgin olive oil categories xed
by the International Organizations.
IOOC Codex Alimentarius EC
Category Md Mf Md Mf Md Mf
Extra virgin olive oil 0 > 0 0 > 0 0 > 0
Virgin olive oil ≤ 2.5 > 0 ≤ 2.5 > 0 ≤ 2.5 > 0
Ordinary virgin olive oil > 2.5 ≤ 6.0 0 > 2.5 ≤ 6.0 ≥ 0 - -
Lampante oil ≤ 2.5 0 ≤ 2.5 0 ≤ 2.5 0
Lampante oil > 6.0 >0 > 6.0 >0 > 6.0 >0
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120 F. Angerosa et al.
[Arsenic: IOOC according to AOAC 952.13 or 942.17 or 985.16]
Trace metals in vegetable oils may originate from endogenous factors connected
with plant metabolism, or hexogenous factors such as the soil, fertilizers, and process-
ing equipment.
ere are few metals reported to be present in olive oils: copper (few tens of
ng/g), iron, nickel (2 to 50 ng/g), manganese, cobalt, chromium (2 to 500 ng/g), tin
(3 to 15 ng/g), lead (<40 ng/g), mercury (2 ng/g), and cadmium (<10 ng/g). Iron is
certainly the element present at the highest concentration (70 to 3600 ng/g) (Gar-
rido et al., 1994). In refined olive oils, the metal content is lower than in virgin oils,
due to the refining process. Some transition metals (copper, iron) are related to the
oxidative stability of olive oils because of their catalytic effect on the decomposition
of hydroperoxides.
Both IOOC and Codex Alimentarius have fixed the same legal limits only for
some metal concentration in olive oils. Iron content must not exceed 3 ppm, while a
limit of 0.1 ppm was established for copper, lead, and arsenic. Most of the procedures
reported in the literature for the determination of trace elements involve the use of
atomic absorption spectrometry (AAS) equipped with a graphite furnace, with an
ashing pretreatment of the sample before the analysis, or just a dilution of the olive oil
sample in methyl isobutyl ketone (Garrido et al., 1994; Martin-Povlillo et al., 1994).
Karadjova et al. (1998) made the electrothermal atomic absorption spectrometric
determination of several metals in olive oil using universal modifiers for their thermal
stabilization during the pretreatment step.
Other techniques such as Inductively Coupled Plasma Atomic Emission Spec-
trometry (ICP AES) (Murillo et al., 1999) and voltammetry (Galeano Diaz et al.,
2003) are also used.
Very recently, derivative potentiometric stripping analysis (dPSA) was utilized to
evaluate trace metals in olive oil (La Pera et al., 2002; Dugo et al., 2004). e dPSA
provides a sensitive and convenient procedure for trace metal determination, repre-
senting an attractive alternative to spectroscopic and voltammetric techniques. e
method has a slow dry ashing step with respect to sample pre-treatment but it requires
a short time of analysis.
α-Tocopherol
[IOOC and Codex Alimentarius: according to ISO 9936]
e tocopherol content varies widely in relation to olive varieties and ripeness,
to processing, and also to storage conditions of oils with values ranging from 100
to 300 ppm, for oils of good quality (Beltran et al., 2005; Psomiadou et al., 2000).
During processing (e.g. deodorization), tocopherol concentration decreases drasti-
cally (Hernandez Rabascall and Riera Boatella, 1987). Because of its activity against
oxidation (Blekas et al., 1995), α-tocopherol is added to refined olive oils to improve
their stability.
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121
Analysis and Authentication
Tocopherols can be determined after saponification by RP-HPLC with ampero-
metric detector (Dionisi et al., 1995), or by the direct injection of the oil, dissolved
in a suitable solvent, into a normal or a reversed phase HPLC apparatus with UV
or spectrofluorimetric detector. eir individual content is calculated using calibra-
tion factors determined from standard solutions (International Standard ISO 9936).
Besides the HPLC methods, tocopherols and tocotrienols can be determined also by
gas chromatographic technique, after trimethylsilyl-derivatization (Rovellini et al.,
1997).
Moisture and Volatile Matter Content
[IOOC and Codex Alimentarius: according to ISO 662]
e oil sample is heated at 105°C on a sand-bath, until moisture and volatile
substances are completely removed. Table 7.6 reports limit values fixed by IOOC and
Codex Alimentarius for each olive oil category.
Insoluble Impurities in Petroleum Ether
[IOOC and Codex Alimentarius: according to ISO 662]
Oil sample is treated with an excess of solvent, the solution is filtered, and the
filtered dried and weighted at 105°C. Limits are shown in Table 7.7.
Not Included in International Standards
Other analyses, in addition to official ones, are useful to complete the assessment of
olive oil quality. ey are measurements related to the level of antioxidants, the state
of oxidation, the hydrolysis, the shelf life and the possible presence of contaminants
and volatile compounds, especially those related to organoleptic defects arising from
TABLE 7.6.
Limits of moisture and volatile matter percentages xed by IOOC and Codex Alimen-
tarius for each olive oil category.
Category IOOC Codex A.
Extra virgin olive oil ≤ 0.2 ≤ 0.2
Virgin olive oil ≤ 0.2 ≤ 0.2
Ordinary virgin olive oil ≤ 0.2 ≤ 0.2
Lampante virgin olive oil ≤ 0.3 -
Rened olive oil ≤ 0.1 ≤ 0.1
Olive oil < 0.1 ≤ 0.1
Crude olive oil ≤ 1.5 -
Rened olive residue oil ≤ 0.1 ≤ 0.1
Olive residue oil ≤ 0.1 ≤ 0.1
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122 F. Angerosa et al.
microbiological/fermentative or chemical oxidative processes.
Phenolic Compounds
Virgin olive oils contains phenolic substances responsible for their stability against
oxidation (Baldioli et al., 1996; Servili and Montedoro, 2002; Del Carlo et al., 2004),
beneficial properties in relation to human health (Visioli et al., 2002; Boskou et al.,
2005) and for bitter, pungent, and astringent sensory notes (Gutierrez-Rosales et al.,
2003; Angerosa and Di Giacinto, 1995; Andrewes et al., 2003; Mateos et al., 2004).
Phenolic compounds are transferred into the oil during the olive processing, but their
concentration is dramatically reduced during refining (Servili and Montedoro, 2002;
Servili et al., 2004) and storage of oils (Cinquanta et al., 1997; Okogeri and Tasioula-
Margari, 2002).
Phenolic fraction includes simple phenols, tyrosol, and hydroxytyrosol, deriva-
tives of hydroxybenzoic and hydroxycinnamic acids, aglycons of some glucosides,
namely oleuropein, demethyloleuropein, ligstroside, and verbascoside (Montedoro et
al., 1993; Angerosa et al., 1995, 1996a; Cortesi et al., 1995; Bianchi, 2003). Free and
esterified cinnamic and elenolic acids, some flavones, hydroxy-isochromans (Bianco
et al., 2002), pinoresinol and 1-acetoxypinoresinol (Brenes et al., 2000; Owen et al.,
e analysis of phenolic substances involves their extraction from the oil, a clean-
up step, the separation into single compounds, and finally their quantification. Several
Authors (Morales and Tsimidou, 2000; Tsimidou, 1998; Pirisi et al., 2000; Hrncirik
and Fritsche, 2004) have recently reviewed the different analytical approaches for
the determination of phenolic compounds, in order to explain the controversial data
reported in the literature. Extraction is usually performed by liquid-liquid partition
with mixtures of methanol and water, in different ratios, or absolute methanol or tet-
rahydrofuran. Some researchers consider that the mixture methanol:water 80:20 v/v
TABLE 7.7
Limits of the percentage of insoluble impurities in petroleum ether for each olive oil
category xed by IOOC and Codex Alimentarius for each olive oil category.
Category IOOC Codex A.
Extra virgin olive oil ≤ 0.1 ≤ 0.1
Virgin olive oil ≤ 0.1 ≤ 0.1
Ordinary virgin olive oil ≤ 0.1 ≤ 0.1
Lampante virgin olive oil ≤ 0.2 -
Rened olive oil ≤ 0.05 ≤ 0.05
Olive oil < 0.05 ≤ 0.05
Crude olive oil - -
Rened olive residue oil ≤ 0.05 ≤ 0.05
Olive residue oil ≤ 0.05 ≤ 0.05
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2000) have also been identified (see also Chapter 5).
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123
Analysis and Authentication
allows the best recoveries (Montedoro et al., 1992), others obtained better or at least
similar recoveries by adopting absolute methanol (Angerosa et al., 1995). e use of
tetrahydrofuran seems to increase significantly recoveries in relation to those obtained
with methanol:water 60:40 v/v (Cortesi et al., 1995). Solid phase extraction (SPE)
with amino-modified C18 or polyvinylpyrrolidone packing material cartridges were
also applied, but a selective retention of phenols was observed (Mannino et al., 1993;
Favati et al., 1994; Cortesi et al., 1995). e use of C18 or end capped C18 cartridges
provided unsatisfactory recoveries, probably due to the different interactions between
the sorbing material and the analyte (Liberatore et al., 2001). e comparison of dif-
ferent methods (liquid-liquid extraction and C8, C18, and diol stationary phases) to
isolate phenolic compounds gave evidence that the better results were obtained with
liquid-liquid extraction in terms of recovery of the phenolic fraction (Bendini et al.,
2003).
e methanolic extract of oil samples can be used to determine the total phenolic
content colorimetrically. An aliquot of the phenolic extract is diluted with water, then
0.5 mL of Folin-Ciocalteau reagent, and a sodium carbonate solution are added, and
finally the solution is spectrophotometrically examined at 725 nm, using a calibration
curve obtained with caffeic acid standard solutions (Gutfinger, 1981). e response
of single phenols to the Folin–Ciocalteau reagent is quite different. However, in spite
of the drawbacks of the method, it remains a good practical, means to evaluate the
stability of the virgin olive oil as suggested by Blekas et al. (2002).
e most common technique to separate the phenolic fraction into single com-
pounds is RP-HPLC, by using acidic water-methanol or water-acetonitrile mixtures,
and detection at 280 nm. Detection can be made also by means of amperometric
technique or capillary zone electrophoresis (Mannino et al., 1993; Bendini et al.,
2003). Cortesi et al. (1995) applied a MS detector operating in chemical negative
ionization mode for characterizing phenolic compounds.
MS detection under EI at 70 eV or CI mode of single phenolic compounds, de-
rivatized as trimethylsilyl ethers and separated by HRGC, have proved to be a useful
tool to elucidate the structure of phenols present in virgin olive oils (Angerosa et al.,
1995, 1996a; Liberatore et al., 2001).
1H and 13C NMR were also used to identify phenolic compounds (Montedoro et
al., 1993; Sacchi et al., 1996) and products of degradation of oleuropein by β-gluco-
sidase (Limiroli et al., 1995). More recently, separation and identification of phenolic
compounds were achieved by means of the hyphenated LC-SPE-NMR technique
(Christophoridou et al., 2005), thus enabling the identification of several new phe-
nolic components, which had not been reported previously in the polar part of olive
oil.
Volatile Compounds
Virgin olive oils can be affected by several sensory defects related to some volatile
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124 F. Angerosa et al.
compounds that can arise from microbiological deterioration or fermentations of ol-
ive fruits or by chemical oxidative processes (Angerosa, 2002; Morales and Aparicio,
2005). Some volatiles have been related to positive attributes by means of statistical
techniques (Morales et al., 1995; Aparicio et al., 1996a; Angerosa et al., 2000a), oth-
ers to the defects that more usually can be perceived in virgin olive oils, so that volatile
determination can support results of sensory analysis. Fusty defect was related to the
presence of 2-methyl butan-1-ol + 3-methyl butan-1-ol (Angerosa et al., 1996b),
winey attributes to ethanol, ethyl acetate and acetic acid (Angerosa et al., 1996b;
Morales et al., 2000), whereas musty perceptions to compounds with eight carbon
number (Angerosa et al., 1999a). Some aldehydes were found to be related to rancid-
ity, especially trans-2-heptenal, hexanal and trans-2-pentenal (Solinas et al., 1987).
Also the ratio hexanal/nonanal represents an appropriate way to detect the beginning
and the evolution of the autoxidation process (Morales et al., 1997).
e analytical approach to volatile determination requires an isolation method.
Good results are obtained using techniques that involve an enrichment step such as
dynamic headspace, supercritical fluid extraction (SFE) and solid phase microextrac-
tion (SPME) (Morales et al., 1994; Angerosa et al., 1997a; Vichi et al., 2003; Cavalli
et al., 2003). Among the techniques with an enrichment step the most popular one is
the isolation by means of dynamic headspace. Volatile compounds of an oil sample,
submitted to a given temperature, are purged with an inert gas at a controlled flow,
obliged to pass through a trap where they are retained. ey are later desorbed ther-
mically (Morales et al., 1994) or by solvent (Angerosa et al., 1997a) and injected into
the gas chromatograph for their separation and quantification. e construction of
calibration curves is needed to perform their quantification, otherwise volatile com-
pounds are expressed as ppm of a suitable standard.
A special method which allows to quantify volatiles is the technique known as
Stable Isotope Dilution Assay (SIDA) that uses deuterated forms of the volatiles to
be quantified (Guth and Grosch, 1993). e method, although has a very good sen-
sitivity, is poorly applied since it requires the preparation of a number of deuterated
compounds not commercially available.
Partial Glycerides
Monoacylglycerols. Monoacylglycerols do not naturally occur in olive fruit, but are
formed during olive processing. eir production is affected by the conditions of both
the olive storage and the oil extraction.
Currently, the proposed methodology includes oil extraction with acetonitrile,
TLC separation on silica gel plate, and recovery of monoacylglycerols with diethyl
ether, GC analysis of silylated fraction (Leone et al., 1989). Paganuzzi (1987) sug-
gested an impregnation of the silicagel layer with boric acid and controlled silylation
conditions to avoid isomerization between 2- and 1-monoacylglycerols.
Diacylglycerols. Diacylglycerols may originate from both incomplete biosynthesis of
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125
Analysis and Authentication
triacylglycerols and partial hydrolysis enzymatically mediated. e measurement of
total diglycerides is helpful for evaluating the quality of olives used for the oil produc-
tion.
e ratio of 1,2- to 1,3-diacylglycerols can be considered a good parameter of
freshness of virgin olive oils, since only 1,2-diacylglycerols are practically present in
fresh oils whereas 1,3-ones are formed during the oil preservation (Figure 7.1). A high
value of this ratio is related to oils of very high quality level for which high prices can
be justified (Pérez-Camino et al., 2001).
Diacylglycerols can be determined by HRGC of the oil previously silylated (Se-
rani et al., 2001).
Pérez-Camino et al. (1996) have set up a simple analytical method for diacylg-
lycerol determination, using a solid-phase extraction (SPE) of these compounds from
the oil, followed by GC on a polar column. With the aid of this method, the isom-
erization of diacylglycerols during the isolation and GC analysis is negligible (Pérez-
Camino et al., 1996; Conte et al., 1997).
Also NMR spectroscopy has been applied in the determination of mono- and di-
Fig. 7.1. HRGC diacylglycerol prole of (A) a newly extracted virgin olive oil; (B) the same
sample after 6 months of preservation. 1: 1,2-C34-diglycerides; 2: 1,3-C34-diglycerides; 3:
1,2-C36-diglycerides; 4: 1,3-C36-diglycerides. Angerosa et al, unpublished data
OliveOil2.indb 125 3/31/2006 1:12:34 PM
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126 F. Angerosa et al.
glycerides. Sacchi et al. (1990, 1991) quantified the contents of partial glycerides us-
ing 1H and 13C NMR, whereas Spyros and Dais (2000) used 31P NMR spectroscopy,
after derivatization of free hydroxyls of partial glycerides with a specific phosphorous
reagent.
Accelerated Oxidation Tests
Accelerated oxidation tests are useful to evaluate the effects of natural or chemical
antioxidants on olive oil resistance toward oxidation, and to compare the storage
stability of different oils.
Oven Test. Oil is put into an oven at a constant temperature and the oxidation is fol-
lowed by chemical and sensory tests. e resistance of olive oil to oxidation is repre-
sented by the time expressed in days necessary for the beginning of oxidation.
Rancimat-OSI. It is based on the change of conductivity of the distillate collect-
ed from an oil subjected to an accelerated oxidation at a prefixed temperature. e
change of conductivity is due to the production of formic and other carboxylic acids
because of the oxidation of secondary products during the forced oxidation.
Pigments
e green-yellowish color is due to various pigments, i.e. chlorophylls, pheophytins
and carotenoids. e levels of these compounds has been traditionally determined
with spectrophotometrical methods measuring the total content in chlorophylls and
carotenoids with value ranging, as the chlorophylls are concerned, from 1 to 10 ppm,
and for the carotenoids, from a few up to 100 ppm.
According to the analytical procedure proposed by Minguez-Mosquera and Gan-
dul-Rojas (1992), both chlorophyllic and carotenoid pigments can be preliminary
separated with solid-phase extraction (SPE) on octadecyl columns and liquid-liquid
extraction. ey can be well detected and identified by HPLC coupled with a diode
array detector (DAD) (Minguez-Mosquera and Gandul-Rojas, 1992; Gandul-Rojas
et al., 1999; Mangos and Berger, 1997) or a fluorimetric detector (Endo et al., 1992.).
Simultaneous detection of oil pigments such as pheophytins, carotenoids and tocoph-
erols can be achieved by HPLC, using isopropanol-hexane mixtures as mobile phases,
and a programmable UV/Vis spectrophotometer (Psomiadou and Tsimidou, 1998;
Seppanen et al., 2003). Chlorophylls a and b, pheophytins a and b, and β-carotene
can be measured at their appropriate absorption maxima (430, 452, 409, 433, and
452 nm respectively).
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127
Analysis and Authentication
Contaminants
Pesticides. To protect olive groves from Bactrocera oleae, the key insect pest of olive
fruit, and from other parasites, several pesticides are used, such as synthetic pyre-
throids, organochlorine, and especially, organophosphorous insecticides (Lentza-
Rizos and Avramides, 1995). In general, since most pesticides are nonpolar, residues
tend to be distributed mostly in the olive oil. Due to an increasing awareness of the
possible risks involved with the widespread use of pesticides, strict regulation of maxi-
mum residue limits (MRLs) for these contaminants have been fixed by the Codex
Alimentarius Commission of the Food and Agriculture Organization (FAO), and also
by the European Community (Lacoste et al., 2004).
e analytical methodology, for determining pesticide residues in oils, uses dif-
ferent procedures, as reviewed by Lentza-Rizos and Avramides (1995). e main dif-
ferences between existing methods lie in the sample clean-up step; the final deter-
mination is usually carried out by GC, in combination with one or more element
specific detectors, such as ECD or NPD. e simplest and most rapid technique was
that used by Morchio et al. (1992) who injected oil samples, previously diluted 1:1
with acetone, directly into a gas chromatograph. is method leads to a rapid decrease
in column resolution, due to the lipidic material, so it can be used only for a few
samples.
No clean-up is necessary in the method described by Dugo and coworkers (2005)
because there were no interferences in the GC coupled with FPD detector.
Most of the clean-up methods for pesticide determination applied to olive oil are
based on liquid-liquid partitioning with solvent of different polarity, such as hexane
and acetonitrile (Cabras et al., 1997), followed by Florisil or size-exclusion chroma-
tography (Barrek et al., 2003) or solid phase extraction (SPE) (Ramesh and Balasub-
ramian, 1998). Lentza-Rizos et al. (2001) have also used a low-temperature lipid pre-
cipitation for the rapid analysis of pesticides, obtaining good recoveries. Alternative
procedures which include supercritical fluid extraction (SFE) with CO2 containing
3% acetonitrile have shown an equivalence with liquid extraction (Hopper, 1999).
To further improve the detectability of analytes, and reduce the working time,
very recently a fully automated method has been developed. It employs an on-line
combination of RP-HPLC and GC by using an oven transfer adsorption-desorption
interface and does not require any sample pre-treatment step other than filtration
(Sanchez et al., 2004).
Polycyclic Aromatic Hydrocarbons (PAHs). Polycyclic Aromatic Hydrocarbons (PAHs)
are a well-known group of chemical contaminants widely distributed in the environ-
ment and considered to be carcinogens, as recently evidenced by the Scientific Com-
mittee on Food of the European Union (http://europa.eu.int/comm/food/fs/sc/scf/
index_en.html). Due to their high lipophilic characteristics, PAHs can contaminate
oils and fats. Olive pomace could be significantly contaminated by PAHs, during its
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128 F. Angerosa et al.
drying because this process is done in direct contact with combustion fumes. PAHs,
however, can be removed from edible oils by treatment with activated charcoal. On
February 2005 EC (Reg No 208/2005) established the maximum concentration (2
µg/kg) for benzo(a)pyrene considered as a marker of the presence and the effects of
cancer producing PAHs in edible foods. EC did not adopt any method as official to
quantify the amount of benzo(a)pyrene, but the methodologies to be used must sat-
isfy some criteria such as a recovery range, limit values for precision, quantification,
etc.
Identification and assessment of PAHs in olive oils have been carried out by sev-
eral methods. PAHs have been extracted by solvent-solvent partition, with a previous
saponification step (Stijve and Hischenhuber, 1987), while clean-up of the extract
has been performed by column chromatography on silica gel, alumina, Sephadex, or
Florisil (International Standard ISO 15302; Moret and Conte, 2000). To avoid long
handling times and the use of large volumes of organic solvents, solid-phase extrac-
tion (SPE) has been successfully used instead of packed chromatography column,
due to a wide range of available sorbents and extraction conditions (Cortesi et al.,
2001; Barranco et al., 2003; Moret and Conte, 2002; Weisshaar, 2002; Moreda et
al., 2004). Very recently also supercritical fluid extraction (SFE) has also been used to
isolate PAHs from vegetable oils (Lage Yusty and Cortizo Davina, 2005).e separa-
tion and quantification of PAHs is usually performed on the extract by reverse phase
HPLC with fluorescence detection (International Standard ISO 15302; Moreda et
al., 2004), but also high-resolution gas chromatography coupled with a FID detector
or mass spectrometry has been applied (Menichini et al., 1991; Guíllen et al., 2004).
Cortesi et al. (2001) performed the PAH analysis by coupled HPLC multiwavelength
UV detection and programmed fluorimetry. Hyphenated techniques like liquid chro-
matography coupled with GC appear to be very promising (Bogusz et al., 2004). Us-
ing isotope dilution technique, Diletti et al. (2005) have quantified the PAH content
at level below 1 ppb, with a GC-MS method.
Methods for Checking Olive Oil Genuineness
Included in International Standards
e Official methodologies include measurements of some chemical and physical
constants, such as iodine value, refractive index determination, and specific color re-
actions, which may be useful in revealing adulteration with seed oils. Nowadays, these
methods have been completely replaced by modern chromatographic and spectro-
metric determinations that provide more information and may lead to more conclu-
sive results. e current methods to check olive oil genuineness are founded on two
essential principles: 1) the different composition of olive oil; 2) the changes occurring
in some constituents due to refining; 3) the differences in the composition between
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129
Analysis and Authentication
virgin olive oil and pomace olive oil.
Chemical classes that are affected by botanical origin are fatty acid composition,
triacylglycerols, and sterols whereas those influenced by the kind of extraction (me-
chanical or by solvent) are aliphatic alcohols, waxes, and the triterpene dialcohols.
Refining process modifies the natural olive oil composition and some new com-
pounds are produced. ese are some sterols, sterolic hydrocarbons, and trans isomers
of unsaturated fatty acids (Lanzón et al., 1994; 1999; León-Camacho et al., 1999,
2001, 2004; Mariani et al., 1992; Paganuzzi, 1984; Strocchi and Savino, 1989).
e limits adopted by International bodies are reported in Table 7.8.
Fatty Acid Composition
[IOOC: according to COI/T.20/Doc. No. 24 or AOCS Ch 2-91(02); Codex Alimen-
tarius: according to COI/T.20/Doc. No. 24 and ISO 5508 or AOCS Ch 2-91(02) or
AOCS Ce 1f-96(02); EC Reg. No 2568/91 Annex X A]
e determination is performed by gas chromatographic analysis of fatty acid
methyl esters, after transesterification of olive oil triglycerides on very polar capil-
lary columns. Preparation of fatty acid methyl esters (FAME) can be carried out in
a methanolic medium with alkaline, acid, or alkaline and acid catalysis. Methyla-
tion with diazomethane represents an alternative procedure for free acids. Cert et al.
(2000) have statistically assessed the precision and reproducibility in the preparation
of FAME using (1) cold methylation with methanolic potassium hydroxide and (2)
hot methylation with sodium methylate followed by acidification with sulfuric acid
in methanol and heating. In oils with low acidities, the results obtained for both
methylation methods were equivalent. However, the olive pomace oil sample (acidity
15.5%) showed significant differences between the fatty acid composition obtained
with the two methylation methods. e methylation with the alkakine and acid ca-
talysis did not yield an increase of the trans-isomers.
Wide ranges of percentages are seen for the more abundant fatty acids, whereas
limit values are fixed for the minor ones (myristic acid, linolenic acid, arachidic acid,
eicosenoic acid, behenic acid, and lignoceric acid). e oleic acid, the most represen-
tative fatty acid of olive oil, ranges from 55% to 83%. Hot climate modifies the fatty
acid composition of olive oils. North African oils have a lower percentage of oleic acid
and higher percentages of linoleic and palmitic acids than oils from the Mediterra-
nean basin. Minor fatty acids are prominent in seed oils. For instance, linolenic acid,
always ≤ 0.9% in olive oil, can reach 8% in seed oils. e limit for linolenic acid has
been fixed at 1% to include some genuine Moroccan olive oils, characterized by 1% of
linolenic acid. e level of other minor fatty acids is useful to reveal seed oil adultera-
tion. High amounts of eicosenoic and behenic acids are characteristic of soybean and
rapeseed oils, erucic acid of rapeseed oils, and lignoceric acid of peanut oil. e limits
fixed for fatty acids are not useful for detection of frauds if the addition of seed oil is
OliveOil2.indb 129 3/31/2006 1:12:35 PM
Fatty acids profiles are very different in edible oils (Table 7.9).
Copyright © 2006 by AOCS Press
130 F. Angerosa et al.
of about 5% (Christopoulou et al., 2004).
e detection becomes harder when the composition of oils in the mixture is very
similar, such as in the case of hazelnut and sunflower oils, or oils obtained from seed
plants biotechnologically modified. In these cases, other analyses are requested.
Trans Unsaturated Fatty Acids
[IOOC and Codex Alimentarius: according to COI/T.20/Doc. No. 17 and ISO
15304 or AOCS Ce 1f-96(02); EC Reg. No 2568/91 Annex X A]
Virgin olive oils contain only cis isomers of unsaturated fatty acids. In the refining
process there is a partial isomerization of unsaturated fatty acids the extent of which
is related to the conditions of the process. e official method of trans unsaturated
fatty acids determination involves the quantitative conversion of triacylglycerols into
methyl esters followed by High Resolution Gas Chromatography (HRGC) quan-
tification of trans fatty acid methyl esters, by using capillary columns covered by a
(1) Oils with a wax content between 300 and 350 mg/kg are considered to be:
lampante olive oil if the total aliphatic alcohol is less than or equal to 350 mg/kg or if the percentage
of erythrodiol and uvaol is less than or equal to 3.5
crude olive pomace oil if total aliphatic alcohols are greater than 350 mg/kg and the percentage of
erythrodiol and uvaol is greater than 3.5.
(2) b-sitosterol is the sum of D5,23-stigmastadienolo, chlerosterol, β-sitosterol, sitostanol, D5-avenas-
terol and D5,24-stigmastadienol
(3) Percentages of other fatty acids: C16= 7.5-20.0; C16:1= 0.3-3.5; C17:0= ≤ 0.3; C17:1= ≤ 0.3;
C18:0= 0.5-5.0; C18:1= 55.0-83.0; C18:2= 3.5-21.0
Notes:
a) The results of the tests must be expressed to the same number of signicant digits as that speci-
ed for each characteristic. The last signicant digit must be rounded up to the next digit if the non-
signicant digit that follows is greater than 4.
b) An oil has to be placed in a different category or declared not in conformity in terms of purity if any
one of the characteristics exceeds the limit.
c) The limits for the characteristics (1) and (3) do not have to be respected simultaneously for all olive
pomace oils.
Table 7.8
Identity characteristics of olive oil categories xed by IOOC. Limits adopted by EC and
Codex Alimentarius are the same for the olive oil categories.
Categories Waxes
mg/
kg(1)
Saturated
acids in 2-
position of
triacylglyc-
erol %
Stigma-
stadienes
mg/kg
DECN42 Trans
oleic
isomers
%
Trans
linoleic
+ trans
linolenic
isomers %
Chole-
sterol
%
Bras-
sica-
sterol
%
Campe
-sterol %
Extra virgin
olive oil
≤250 ≤1.5 ≤0.15 ≤0.2 ≤0.05 ≤0.05 ≤0.5 ≤0.1 ≤4.0
Virgin olive oil ≤250 ≤1.5 ≤0.15 ≤0.2 ≤0.05 ≤0.05 ≤0.5 ≤0.1 ≤4.0
Ordinary virgin
olive oil
≤250 ≤1.5 ≤0.15 ≤0.2 ≤0.05 ≤0.05 ≤0.5 ≤0.1 ≤4.0
Lampante virgin
olive oil
≤300 ≤1.5 ≤0.50 ≤0.3 ≤0.1 ≤0.1 ≤0.5 ≤0.1 ≤4.0
Rened olive oil ≤350 ≤1.8 –≤0.3 ≤0.2 ≤0.3 ≤0.5 ≤0.1 ≤4.0
Olive oil ≤350 ≤1.8 –≤0.3 ≤0.2 ≤0.3 ≤0.5 ≤0.1 ≤4.0
Crude pomace
olive oil
>350 ≤2.2 –≤0.6 ≤0.2 ≤0.1 ≤0.5 ≤0.2 ≤4.0
Rened olive
residue oil
>350 ≤2.2 –≤0.5 ≤0.4 ≤0.35 ≤0.5 ≤0.2 ≤4.0
Olive residue oil >350 ≤2.2 –≤0.5 ≤0.4 ≤0.35 ≤0.5 ≤0.2 ≤4.0
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131
Analysis and Authentication
cianopropylsilicone stationary phase. To avoid artificial increases of isomers, a cold
methylation with methanolic potassium hydroxide or diazomethane, an analysis tem-
perature no higher than 225°C and a cleanliness control of the injector are recom-
mended (León-Camacho, 2001). Peaks formed by ethyl or other esters, produced
when the column has an insufficient polarity, could overlap with the trans-linolenic
acid methyl ester one, and give wrong results.
e presence of trans isomers of olive oil unsaturated fatty acids is not a specific
kind of adulteration.
Fatty Acid in the 2-Position of Triacylglycerol
[IOOC and Codex Alimentarius: according to ISO 6800:199 or AOCS Ch 3-91(97),
EC Reg. No 2568/91 Annex VII]
It is well known that unsaturated fatty acids are oriented during the biosynthesis
of triacylglycerols to 2-position and only a very low amount of saturated ones esteri-
(1) Oils with a wax content between 300 and 350 mg/kg are considered to be:
lampante olive oil if the total aliphatic alcohol is less than or equal to 350 mg/kg or if the percentage
of erythrodiol and uvaol is less than or equal to 3.5
crude olive pomace oil if total aliphatic alcohols are greater than 350 mg/kg and the percentage of
erythrodiol and uvaol is greater than 3.5.
(2) b-sitosterol is the sum of D5,23-stigmastadienolo, chlerosterol, β-sitosterol, sitostanol, D5-avenas-
terol and D5,24-stigmastadienol
(3) Percentages of other fatty acids: C16= 7.5-20.0; C16:1= 0.3-3.5; C17:0= ≤ 0.3; C17:1= ≤ 0.3;
C18:0= 0.5-5.0; C18:1= 55.0-83.0; C18:2= 3.5-21.0
Notes:
a) The results of the tests must be expressed to the same number of signicant digits as that speci-
ed for each characteristic. The last signicant digit must be rounded up to the next digit if the non-
signicant digit that follows is greater than 4.
b) An oil has to be placed in a different category or declared not in conformity in terms of purity if any
one of the characteristics exceeds the limit.
c) The limits for the characteristics (1) and (3) do not have to be respected simultaneously for all olive
pomace oils.
Table 7.8 continued.
Stigma-
sterol %
β-sito
sterol
(2)
D7-
stigma
stenol %
Total
sterols
mg/kg
Erithro-
diol +
Uvaol
%(3)
C14:0% C18:3 % C20:0 % C20:1
%
C22:0 % C24:0 %
< camp ≥93.0 ≤0.5 ≥1000.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.2 ≤0.2
< camp ≥93.0 ≤0.5 ≥1000.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.2 ≤0.2
< camp ≥93.0 ≤0.5 ≥1000.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.2 ≤0.2
< camp ≥93.0 ≤0.5 ≥1000.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.2 ≤0.2
< camp ≥93.0 ≤0.5 ≥1000.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.2 ≤0.2
< camp ≥93.0 ≤0.5 ≥1000.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.2 ≤0.2
< camp ≥93.0 ≤0.5 ≥2500.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.3 ≤0.2
< camp ≥93.0 ≤0.5 ≥1800.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.3 ≤0.2
< camp ≥93.0 ≤0.5 ≥1600.0 ≤4.5 ≤0.05 ≤1.0 ≤0.6 ≤0.4 ≤0.3 ≤0.2
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132 F. Angerosa et al.
TABLE 7.9
Fatty acid composition of the main seed oils according to Codex Alimentarius.
Safower- Sunower-
Rapeseed Safower- seed Sunower- seed
Fatty Olive Rapeseed oil (low seed oil (high Soyabean -seed oil (high Peanut Maize Grapeseed
acid oil oil erucic acid) oil oleic acid) oil oil oleic acid) oil oil oil
C12:0 ND ND ND ND ND-0.2 ND-0.1 ND-0.1 ND ND-0.1 ND-0.3 ND
C14:0 0.0-0.05 ND-0.2 ND-0.2 ND-0.2 ND-0.2 ND-0.2 ND-0.2 ND-0.1 ND-0.1 ND-0.3 ND-0.3
C16:0 7.5-20.0 1.5-6.0 2.5-7.0 5.3-8.0 3.6-6.0 8.0-13.5 5.0-7.6 2.6-5.0 8.0-14.0 8.6-16.5 5.5-11.0
C16:1 0.3-3.5 ND-3.0 ND-0.6 ND-0.2 ND-0.2 ND-0.2 ND-0.3 ND-0.1 ND-0.2 ND-0.5 ND-1.2
C17:0 0.0-0.3 ND-0.1 ND-0.3 ND-0.1 ND-0.1 ND-0.1 ND-0.2 ND-0.1 ND-0.1 ND-0.1 ND-0.2
C17:1 0.0-0.3 ND-0.1 ND-0.3 ND-0.1 ND-0.1 ND-0.1 ND-0.1 ND-0.1 ND-0.1 ND-0.1 ND-0.1
C18:0 0.5-5.0 0.5-3.1 0.8-3.0 1.9-2.9 1.5-2.4 2.0-5.4 2.7-6.5 2.9-6.2 1.0-4.5 ND-3.3 3.0-6.5
C18:1 55.0-83.0 8.0-60.0 51.0-70.0 8.4-21.3 70.0-83.7 17.0-30.0 14.0-39.4 75.0-90.7 35.0-69.0 20.0-42.2 12.0-28.0
C18:2 3.5-21.0 11.0-23.0 15.0-30.0 67.8-83.2 9.0-19.9 48.0-59.0 48.3-74.0 2.1-17.0 12.0-43.0 34.0-65.6 8.0-78.0
C18:3 0.0-1.0 5.0-13.0 5.0-14.0 ND-0.1 ND-1.2 4.5-11.0 ND-0.3 ND-0.3 ND-0.3 ND-2.0 ND-1.0
C20:0 0.0-0.6 ND-3.0 0.2-1.2 0.2-0.4 0.3-0.6 0.1-0.6 0.1-0.5 0.2-0.5 1.0-2.0 0.3-1.0 ND-1.0
C20:1 0.0-0.4 3.0-15.0 0.1-4.3 0.1-0.3 0.1-0.5 ND-0.5 ND-0.3 0.1-0.5 0.7-1.7 0.2-0.6 ND-0.3
C20:2 ND ND-1.0 ND-0.1 ND ND ND-0.1 ND ND ND ND-0.1 ND
C22:0 0.0-0.2 ND-2.0 ND-0.6 ND-1.0 ND-0.4 ND-0.7 0.3-1.5 0.5-1.6 1.5-4.5 ND-0.5 ND-0.5
C22:1 ND >2.0-60.0 ND-2.0 ND-1.8 ND-0.3 ND-0.3 ND-0.3 ND-0.3 ND-0.3 ND-0.3 ND-0.3
C22:2 ND ND-2.0 ND-0.1 ND ND ND ND-0.3 ND ND ND ND
C24:0 0.0-0.2 ND-2.0 ND-0.3 ND-0.2 ND-0.3 ND-0.5 ND-0.5 ND-0.5 0.5-2.5 ND-0.5 ND-0.4
C24:1 ND ND-3.0 ND-0.4 ND-0.2 ND-0.3 ND ND ND ND-0.3 ND ND
ND = not detectable
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133
Analysis and Authentication
fies this position of glycerol. According to specific distribution rules there is a promi-
nent concentration of saturated fatty acids in 1- and 3- positions.
Oils with a fatty acid composition identical to that of genuine olive oils can be
chemically prepared by esterifying by-products of olive oil refining process. In these
products the 1,3 random, 2 random distribution cannot be reproduced. erefore the
amount of saturated fatty acids is notably higher in the 2-position than in genuine
oils.
e determination of fatty acids in the 2-position of glycerol includes 1) neutral-
ization, if the free acidity exceeds 3%, 2) chromatographic separation on a column of
alumina, 3) partial hydrolysis of triacylglycerols mediated by porcine pancreatic lipase
for a defined time, 4) isolation of monoacylglycerols in the 2-position by TLC, 5)
methanolic transesterification and 6) HRGC analysis of methyl esters.
EC regulation fixed limits for the sum of palmitic plus stearic acid percentages,
for the different olive oil categories (1.5% for virgin olive oils, 1.8% for refined olive
oils, and 2.2% for pomace oils); percentages higher than limits evidence the addition
of esterified oil.
DECN42 Values
[IOOC and Codex Alimentarius: according to COI/T.20/Doc. No. 20 or AOCS Ce
5b-89(97); EC Reg. No 2568/91 Annex XVIII]
e availability in the market of desterolized oils with fatty acid composition
very similar to that of olive oils lead to the quest of new methods to reveal possible
adulterations. From a practical point of view, it is very useful to cluster triglycerides
with the same chromatographic behavior by Equivalent Chain Number (ECN). ECN
is the actual carbon number minus twice the number of double bonds per molecule.
For an example glycerol trilinoleate has an ECN equal 42 (3 x 18 =54, 2 x (3 x 2) =12,
54-12 = 42).
Olive oil, differently from the most seed oils, has mainly triglycerides with ECNs
44, 46, 48, and 50; triglycerides with ECN40 and ECN42 are absent or found at
trace amounts, respectively. erefore, the evaluation of ECN42, which varies accord-
ing to content of glycerol trilinoleate, is an effective tool to detect more unsaturated
oils. More effective information can be drawn from DECN42, the difference between
theoretical ECN42 (calculated by a special computer programme based on the GC
determination of fatty acid composition and 1,3-random, 2-random distribution
theory) and the experimental ECN42 (determined by HPLC technique). e current
HPLC method for determining triacylglycerols is based on the resolution into single
glycerides, according to both molecular weight and total number of double bonds.
e separation is made in isocratic conditions, using a mixture of acetonitrile and ac-
etone as mobile phase. e detection is performed by means of an RI detector. RI de-
tector has the disadvantage that it is greatly affected by both temperature and compo-
sition of the mobile phase. erefore, any increase of temperature should be avoided
OliveOil2.indb 133 3/31/2006 1:12:36 PM
Copyright © 2006 by AOCS Press
134 F. Angerosa et al.
to reduce inevitable deflection of baseline. is is obtained with suitable thermostated
cells. Under these experimental conditions, the resolution into single glycerides is not
complete and it can be only partially improved with the use of a 4 mm i.d. (internal
diameter) RP-18 column with 4 µm particle diameter. Moreda and coworkers (2003)
successfully overcame the poor reproducibility of the mobile phase composition by
replacing the mixture of acetonitrile and acetone with proprionitrile.
DECN42 must not exceed 0.2 for extra and virgin olive oils, 0.3 for lampante
and refined olive oils, 0.5 for refined pomace oil and olive pomace oil, and can reach
0.6 for crude pomace oil. DECN42 is a very useful and effective tool in detecting the
presence of most of the vegetable oils (El-Hamdy and El-Fizga, 1995). However, the
fixed limits for DECN42 are not sufficient to detect percentages lower than or equal
to 5% of hazelnut, peanut, and mustard oils in mixtures with olive oils according to
Christopoulou et al. (2004).
Sterol Composition
[IOOC and Codex Alimentarius: according to COI/T.20/Doc. No. 10 or ISO 12228
or AOCS Ch 6-91(97); EC Reg. No 2568/91 Annex V]
Sterol content, and especially sterol profile, are quite characteristic of each botanical
erefore the determination of sterol composition is widely applied as an effec-
tive and reliable means to detect the adulteration with foreign oils. e content of
some sterols, such as campesterol, stigmasterol, and β-sitosterol, decreases during the
refining process since they suffer a dehydration. e resulting oils with a low sterol
content (desterolized) can be used to adulterate olive oil. In this case sterol composi-
tion does not give conclusive information but the suspicion can be supported by the
determination of both total amount of sterols, which must be ≥ 1,000 ppm, and
dehydration by-products of sterols. Also some stigmastadienols, such as D5,23- and
D5,24-stigmastadienols, not naturally occurring in virgin olive oils, but formed during
the refining process may be present (Amelotti et al., 1985).
Olive oil must contain not more than 0.5% of cholesterol. Higher percentages
of this sterol evidence the presence of animal fats, palm oil, or its fractions. A limit
of 0.1% of brassicasterol has been fixed. Higher values indicate the adulteration with
oils from the Brassicaceae family. Percentages higher than 0.5% of D7-stigmastenol
A maximum value of 4.0% has been fixed for campesterol, present at high levels
in soybean, rapeseed, and sunflower oils. In addition as campesterol percentage is
greater than that of stigmasterol, this relation is useful to evidence mixtures with soy-
bean oil. Some genuine virgin olive oils showing a campesterol content exceeding the
upper limit established by EU regulations (Rivera del Alamo et al., 2004). e appar-
ent β-sitosterol (the sum of contents of D5,23- and D5,24-stigmastadienols, chlerosterol,
β-sitosterol, sitostanol, and D5-avenasterol) must cover 93%.
OliveOil2.indb 134 3/31/2006 1:12:36 PM
species (Table 7.10).
indicate the adulteration with sunflower oil (Figure 7.2).
Copyright © 2006 by AOCS Press
135
Analysis and Authentication
TABLE 7.10
Sterolic composition of main seed oils according to Codex Alimentarius.
Rapeseed Safowerseed Sunowerseed
oil (low Safower- oil (high Soyabean Sunower- oil (high Peanut Maize Grapeseed
erucic acid) seed oil oleic acid) oil seed oil oleic acid) oil oil oil
Cholesterol ND-1.3 ND-0.7 ND-0.5 0.2-1.4 ND-0.7 ND-0.5 ND-3.8 0.2-0.6 ND-0.5
Brassicasterol 5.0-13.0 ND-0.4 ND-2.2 ND-0.3 ND-0.2 ND-0.3 ND-0.2 ND-0.2 ND-0.2
Campesterol 24.7-38.6 9.2-13.3 8.9-19.9 15.8-24.2 6.5-13.0 5.0-13.0 12.0-19.8 16.0-24.1 7.5-14.0
Stigmasterol 0.2-1.0 4.5-9.6 2.9-8.9 14.9-19.1 6.0-13.0 4.5-13.0 5.4-13.2 4.3-8.0 7.5-12.0
beta-sitosterol 45.1-57.9 40.2-50.6 40.1-66.9 47.0-60.0 50.0-70.0 42.0-70.0 47.4-69.0 54.8-66.6 64.0-70.0
delta-5-avenasterol 2.5-6.6 0.8-4.8 0.2-8.9 1.5-3.7 ND-6.9 1.5-6.9 5.0-18.8 1.5-8.2 1.0-3.5
delta-7-stigmastenol ND-1.3 13.7-24.6 3.4-16.4 1.4-5.2 6.5-24.0 6.5-24.0 ND-5.1 0.2-4.2 0.5-3.5
delta-7-avenasterol ND-0.8 2.2-6.3 ND-8.3 1.0-4.6 3.0-7.5 ND-9.0 ND-5.5 0.3-2.7 0.5-1.5
Others ND-4.2 0.5-6.4 4.4-11.9 ND-1.8 ND-5.3 3.5-9.5 ND-1.4 ND-2.4 ND-5.1
Total sterols (mg/kg) 4500- 2100- 2000- 1800- 2400- 1700- 900- 7000- 2000-
11300 4600 4100 4500 5000 5200 2900 22100 7000
ND not detectable
OliveOil2.indb 135 3/31/2006 1:12:36 PM
Copyright © 2006 by AOCS Press
136 F. Angerosa et al.
Sterol detemination involves: a. the saponification of the oil sample, after the ad-
dition of a suitable internal standard (e.g.α-cholestanol) with an ethanolic potassium
hydroxide solution, b. the extraction of unsaponifiable matter with diethyl ether, c. the
isolation of sterolic fraction by means of TLC on a plate impregnated with potassium
hydroxide and d. the quantification of single sterols, previously silylated, by HRGC.
e analysis may show some problems because of an ineffective separation of sterolic
fraction from the unsaponifiable matter thin layer chromatography. It is possible that
small amounts of cycloarthenol and 24-methylene-cycloarthanol are scraped off with
the sterol band, thus overlapping with D7-stigmastenol and β-sitosterol respectively,
in the usual condition of analysis (Morales and León-Camacho, 2000).
Fig. 7.2. HRGC of sterolic fraction of a mixture of olive oil with 20% of sunower seed oil. 1:
cholesterol; 2: 24-methylencholesterol; 3: campesterol; 4: campestanol; 5: stigmasterol; 6:
D7-campesterol; 7: D5,23-stigmastadienol; 8: clerosterol; 9: β-sitosterol; 10: sitostanol; 11:
D5-avenasterol; 12: D5,24-stigmastadienol. IS: internal standard (α-cholestanol). Angerosa
et al, unpublished data
OliveOil2.indb 136 3/31/2006 1:12:43 PM
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137
Analysis and Authentication
Some researchers performed capillary GC analysis of silyl-derivatives of sterols
previously separated by means of isocratic HPLC (Cert et al., 1997). When compared
with the official TLC sterol determination method, the HPLC technique shows no
difference except of a higher D7-sterol recovery (Cert et al., 1997).
e official method is time consuming, therefore several researchers tried to sim-
plify it by removing the TLC step. Bello (1992) achieved the separation of the sterolic
fraction from the unsaponifiable matter using a commercial (Sep-Pak) silica cartridge
and petroleum ether-diethyl ether elution. Results obtained with this method were
in good agreement with those deriving from the time-consuming TLC step. A simi-
lar approach was also followed by Lechner et al. (1999) who, prior to capillary gas
chromatography, successfully applied SPE to separate sterols from the triacylglycerol
matrix. Another interesting approach is the extraction with a semicontinuous coun-
tercurrent supercritical carbon dioxide extraction (Ibanez et al., 2002).
Erythrodiol and Uvaol
[IOOC and Codex Alimentarius: according to IUPAC Method 2.431; EC Reg. No
2568/91 Annex VI]
A very high content of erythrodiol, uvaol, waxes, and aliphatic alcohols is ac-
cumulated in the flesh and skin of olive fruits so that oils obtained by solvent from
solid residue after the mechanical extraction of olive pastes is particularly rich in
these compounds. Percentages of erythrodiol and uvaol in relation to that of sterols
can provide a good means of differentiation between mechanically obtained oils and
solvent extracted. Since triterpenic dialcohols essentially occur as free or mono- and
di-esters of fatty acids, the determination of these ester classes can be useful for a bet-
ter identification of different kinds of olive oil (Mariani et al., 1998).
Triterpenic dialcohols are separated and analyzed with sterols, using the same
methodology. e sum of erythrodiol and uvaol, in the total sterol fraction, does not
exceed 4.5% in virgin and olive oils. In pomace oils it can be as high as 30%. Percent-
Such results have to be confirmed by wax level, since genuine virgin oils pro-
duced in certain regions contain erythrodiol and uvaol in percentages higher than the
fixed limits (Albi et al., 1990).
Alternatively to the GC official methodology, the separation of unsaponifiables
can be performed, by preparative HPLC with refractive index detection, preparation
of silyl-derivatives, and analysis by capillary GC. e HPLC determination of sterols/
dialcohols gave higher D7-sterol amount. All the other sterols and erythrodiol and
uvaol recoveries were similar to those of the official TLC sterol determination method
(Cert et al., 1997).
OliveOil2.indb 137 3/31/2006 1:12:43 PM
ages higher than 4.5% indicate blending with olive pomace oil (Figure 7.3).
Copyright © 2006 by AOCS Press
138 F. Angerosa et al.
Wax Content
[IOOC and Codex Alimentarius: according to COI/T.20/Doc. No. 18/Rev. 2 or
AOCS Ch 8-02(02), EC Reg. No 2568/91 Annex IV]
Waxes, esters of fatty acids with fatty alcohols, found in olive oil are esters C36,
C38, C40, C42, C44, and C46. As they accumulate in the skin of olives, higher
amounts of them can be detected in olive pomace oils rather than in olive oils (Bi-
Fig. 7.3. HRGC of both sterolic fraction and triterpenic dialcohols of a mixture of olive oil
with 15% pomace olive oil. 1: cholesterol; 2: 24-methylencholesterol; 3: campesterol; 4:
campestanol; 5: stigmasterol; 6: D7-campesterol; 7: D5,23-stigmastadienol; 8: chlerosterol;
9: β-sitosterol; 10: sitostanol; 11: D5-avenasterol; 12: D5,24-stigmastadienol; 13: D7-stigma-
stenol; 14: D7-avenasterol; 15: erythrodiol; 16: uvaol. IS: internal standard (α-cholestanol).
Angerosa et al, unpublished data
OliveOil2.indb 138 3/31/2006 1:12:46 PM
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139
Analysis and Authentication
anchi et al., 1994). Since the waxy fraction C40-46 esters are the least affected by
the dewaxing process (Amelio et al., 1993), the determination of the sum of C40-46
aliphatic waxes can be considered a reliable parameter to detect olive-residue oil in
olive oil (Grob et al., 1990).
For the determination of waxes separation by silica gel chromatography, after the
addition of a suitable internal standard (e.g. lauryl arachidate) is necessary. e waxy
fraction eluted with hexane:diethyl ether 99:1 is analyzed by GC, using a capillary
column and on-column injection. Limits are fixed by EC to guarantee the purity and
to classify the various grades of olive oil; these are: 250 mg/kg for virgin olive oils, 300
mg/kg for lampante olive oils, 350 mg/kg for olive, and refined olive oils. Contents
higher than 350 mg/kg are present in solvent-extracted oils. However wax quanti-
fication should be supported by erythrodiol and uvaol determination, since several
studies proved that wax content increases during the oil preservation (Mariani and
Venturini, 1996; Paganuzzi et al., 1997), because of a natural esterification of fatty
alcohols and free fatty acids. In oils with high free acidity the extent of esterification
is relevant.
e esterification of several different fatty acids and fatty alcohols can lead to
waxes with the same carbon atom number and therefore the content of a given wax
will be given by the sum of more peaks. León-Camacho and Cert (1994) suggested to
shorten the gas chromatographic column or to increase the carrier gas flow, to avoid
the splitting of wax peaks.
An attempt to make automatic wax content determination was made by Amelio
et al. (1993) who replaced column chromatography by a separation in HPLC and
automatic collection of wax fraction that later is analyzed by HRGC.
Recently Pérez-Camino and coworkers (2003) proposed a simplification of the
official method. ey isolated wax fraction from the oil using solid-phase extraction
on silica-gel cartridges. e fraction was later analyzed by capillary GC using on-col-
umn injection. e determination of aliphatic waxes had the same precision as the EC
official method.
Aliphatic Alcohol Content
[IOOC: COI/T.20/Doc. No. 26; Codex Alimentarius: NGD C 76-1989; EC Reg.
No 2568/91 Annex XIX]
In olive oils saturated linear fatty alcohols form a homologue series, mainly with
an even chain of carbon atoms which range from 20 to 32. Some seed oils have linear
fatty acids with an odd chain.
Aliphatic alcohols accumulate in the flesh and skin of olive fruits and, as a con-
sequence, they are contained in solvent extracted oils in higher amounts than in me-
chanically extracted oils (Christopoulou et al., 1996; Tacchino and Borgoni, 1983).
An aliphatic alcohol content higher than values usually found in genuine olive
oils may be indicative of a fraudulent addition of olive pomace oil, but it cannot be
OliveOil2.indb 139 3/31/2006 1:12:46 PM
Copyright © 2006 by AOCS Press
140 F. Angerosa et al.
considered conclusive since some genuine oils also show levels exceeding the proposed
limits. Erroneous evaluations could be made, due to an increase of the free alkanols
level after solvent crystallization in the dewaxing process (Amelio et al., 1993). In
these cases, other supporting analyses are necessary to confirm adulteration.
e alcoholic fraction is isolated from the unsaponifiable matter by TLC after
the addition of a suitable internal standard (e.g. 1-eicosanol); quantification is carried
out on the silyl derivatives, using GC on a capillary column. e separation of linear
from triterpenic alcohols and methylsterols by TLC before GC analysis is advisable
(Morales and León-Camacho, 2000). Depending on the mobile phase a band with
a Rf slightly higher than that of linear alcohols can be observed; this band is due to
a tertiary polyisoprenoid alcohol which is readily decomposed during GC to form a
hydrocarbon artifact, with a lower molecular weight (Lanzón et al., 1992).
Stigmastadienes
[IOOC and Codex Alimentarius: according to COI/T.20/Doc. No. 11 or ISO 15778-
1 or AOCS Cd 26-96(02); EC Reg. No 2568/91 Annex XVII]
Several unsaturated hydrocarbons with a steroideal structure, known as sterenes,
are formed by dehydration of sterols, during olive oil refining. Among them, stig-
masta-3,5-diene originates from the dehydration of β-sitosterol (Cert et al., 1994),
and it is considered as an effective marker of oils subjected to a bleaching process or to
a thermal treatment (Lanzón et al., 1994). Limits set by International bodies are stig-
mastadienes not more than 0.15 ppm in virgin olive oil, and 0.5 ppm for lampante.
Stigmastadiene determination is especially useful to evidence the addition of des-
terolized oils since the high temperatures needed for the removal of sterols during
refining process promote the formation of sterenes. Dehydration products from the
other sterols are also good tracers of olive oil adulteration with seed oils (Grob et al.,
1994a, 1994b; Mariani et al., 1995).
e official method involves the extraction of unsaponifiable matter, the frac-
tionation of steroidal hydrocarbons with silica gel column chromatography and GC
determined by RP-HPLC coupled with an UV detector since they have characteristic
absorptions due to the presence of a conjugated double bond system (Schulte, 1994;
Amelio et al., 1998). More effective is the sterene determination with on-line coupled
LC-GC-MS techniques (Grob et al., 1994b).
Spectrophotometric Analysis in the Ultraviolet Region
[IOOC: COI/T.15/NC n.3 (2003); Codex Alimentarius: according to COI/T.20/
Doc. No. 19 or ISO 3656 or AOCS Ch 5-91 (01), EC R e g . No 2568/9 1 An-
nex IX].
e detection of adulteration of virgin olive oils with refined olive oil and olive
OliveOil2.indb 140 3/31/2006 1:12:46 PM
analysis. A typical chromatogram is showed in Figure 7.4. Sterenes could also be
Copyright © 2006 by AOCS Press
141
Analysis and Authentication
residue oils can be carried out by measuring specific absorbances in the UV region
(Chiricosta et al., 1996) at the wavelengths typical of conjugated polyenes. Measure-
ments are made on an oil sample diluted in an adequate solvent. A number of prod-
ucts due to autoxidation of the oil interfere, since they adsorb in the same region. A
passage of the sample through an alumina (Di Sipio andTrulli, 2001, 2002) or a silica
gel column (Morchio et al., 2000) is necessary before the spectrophotometric analysis.
Some years ago the use of a modern refining process resulted in oils with negligible
UV absorption values. An admixture of such oils with virgin olive oil cannot be re-
vealed by UV absorbance measurements. Other analyses, e.g. trans isomer fatty acid
determination, are suggested to evidence this adulteration (Morchio et al., 1989).
Not Included in International Standards
Other methodologies to check olive oil genuineness, although not included in official
methods, can usefully support attempts to reveal adulteration. ese methods are
based on the analysis of both triacylglycerols and non-triacylglycerols components.
Triacylglycerols
e current method for determining triacylglycerols is based on the resolution into
Figure 7.4. HRGC steradienes prole of (A) a virgin olive oil; (B) a rened olive oil. 1: campesta-
dienes; 2: stigmastadienes. Angerosa et al, unpublished data
OliveOil2.indb 141 3/31/2006 1:12:48 PM
individual compounds using HPLC with a refractive index (RI) detector (Figure 7.5).
Copyright © 2006 by AOCS Press
142 F. Angerosa et al.
However, in the usual HPLC determination of triacylglycerols (Cortesi et al., 1990)
RI detection does not allow adoption solvent gradients which are essential for the
better separation of triacylglycerols.
Some researchers used light scattering detection with solvent gradients, thus ob-
taining efficient separations of triglycerides (Palmer and Palmer, 1989; Caboni et al.,
1992). More recently, the detection of triacylglycerols from vegetable oils has been
made with evaporative light scattering detectors (ELSD) which allow a solvent gradi-
ent to be used as mobile phase, improving their separation. ELSD show a sensitivity
200-400 times greater than the refractive index (RI) (Mancini et al., 1997).
Some ratios of major triacylglycerols were used to differentiate genuine olive oils
from mixtures with reesterified oils or to evidence the presence of hazelnut oil (Casa-
dei, 1987). Triacylglycerol profiles were also processed by chemometrical techniques,
Figure 7.5. HPLC triacylglycerol prole of a mixture of olive oil with 20% of rapeseed oil. 1:
LLL; 2: OLLn+PoLL; 3: PLLn; 4: OLL; 5: OOLn+PoOL; 6: PLL+PoPoO; 7: POLn+PPoPo+PPoL; 8:
OOL+LnPP; 9: PoOO; 10: SLL+PLO; 11: PoOP+SpoL+SOLn+SpoPo; 12: PLP; 13: OOO+PoPP;
14: SOL; 15: POO; 16: POP; 17: SOO; 18: POS+SLS. Angerosa et al, unpublished data
OliveOil2.indb 142 3/31/2006 1:12:54 PM
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143
Analysis and Authentication
to reveal olive oil falsification (Tsimidou et al., 1987a).
Trilinolein (LLL) content (Christopoulou et al., 2004) can give useful informa-
tion about possible adulteration with rich linoleic seed oils; however, the low LLL
content in some seed oils or the addition of canola oil up to 7.5% w/w cannot allow
the detection of adulteration (Salivaras and McCurdy, 1992).
e gas chromatographic approach is not widespread because of poor volatility
of triacylglycerols and high temperatures (350°C) required for the analysis. Several
years ago, GC methods were not able to determine triacylglycerol composition since
stationary phases could not resist the high temperatures needed to volatilize the sam-
ple. Nowadays the use of phenyl-methyl-silicone phases, able to endure temperatures
greater than 350°C for a long time, allows the separation of triglycerides according to
carbon atom number and unsaturation (Antoniosi Filho et al., 1993; Geeraert and
Sandra, 1987). To avoid losses or thermal decomposition in the split injection, an on-
column injection is generally used due to different volatility of triglycerides,. e gas
chromatographic method, although able to resolve triglycerides, has the disadvantage
of column deterioration.
Alcoholic Fraction
Refining leads to isomerization of the triterpenic fraction because of the opening of a
3-carbon atom ring and the translocation of a double bond in the side chain from the
24-28 to the 24-25 position (Paganuzzi, 1984; Strocchi and Savino, 1989; Lanzón et
al., 1999). us the detection of triterpenic isomers can serve as a means of revealing
illegal additions of refined oils to virgin olive oil.
e isolation of triterpenic alcoholic fractions is achieved by TLC fractionation
of the unsaponifiable matter, while the quantification is carried out by HRGC. Frega
and coworkers (1993) achieved the separation of the different compounds through
HRGC analysis of the silylated unsaponifiable matter. Some researchers (Mariani et
al., 1993) suggested to avoid the time-consuming isolation of unsaponifiable; they
carried out the separation of the oil previously silylated by silica gel column followed
by HRGC analysis.
e determination of alcoholic index (I.A.) can be a useful means to detect the
addition of olive pomace oil to olive oil, since it is significantly higher in olive residue
oils than in virgin olive oils (Camera, 1978/1980). Alcoholic index is a numerical
factor calculated from a ratio of areas of some peaks of the alcoholic fraction of the
unsaponifiable matter. It is given by the following equation
C22 (C22 + C24 + C26 + C28)
(I.A.) = ––––– × ––––––––––––––––––––– [2]
Cx (CA + 24MeCA)
where C22-C28 are the correspondent aliphatic alcohol areas, Cx is the area of geranyl-
geraniol and CA and 24MeCA are the areas of cycloartenol and 24-methylen-cyclo-
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144 F. Angerosa et al.
artanol respectively.
Other Absorptions in the Visible and UV Spectra
An interesting region of UV spectra is between 310 and 320 nm, where conjugated
tetraenes absorb. e display of derivative indices of dienes, triene, and tetraene bands
by three-dimensional graphs allows a discrimination of virgin olive oils, refined olive
oils, and seed oils even in the presence of autoxidation products according to Chiri-
costa et al. (1994). e derivative spectrophotometry is very rapid, has a low cost,
and provides evidence for the presence of seed oils at low percentages (Calapaj et al.,
1993).
Spectrophotometry in the visible region is a good means to detect the presence
of virgin olive oil in olive oil category. Olive oils are obtained by blending virgin olive
oils with refined olive oils. Virgin olive oils, differently from refined olive oils, show
emission at 673 nm due to the presence of chlorophyll. us the presence of virgin
olive oil, even in a small quantity, can be evidenced measuring spectrofluorimetrically
the emission at 673 nm. However, due to the variability of the chlorophyll content
in virgin olive oil related to agronomic and technological factors, it is not possible to
quantify the amount of virgin olive oil.
Hydrocarbons
Refining causes, in addition to a loss of volatile compounds (especially sesquiterpenes),
the appearance of hydrocarbons not naturally occurring in virgin olive oils such as
alkadienes (mainly n-hexacosadiene), stigmasta-3,5-diene, isomerization products of
squalene, isoprenoidal olefins from hydroxy derivatives of squalene, and steroidal hy-
drocarbons deriving from 24-methylene cycloartanol (Lanzon et al., 1994).
e procedure to determine squalene isomers, according to Mariani and cowork-
ers (1993), involves a chromatographic separation on a silica gel column (2% H2O)
of oil sample previously silylated, the isolation of squalene isomer fraction and its
analysis by HRGC on SE-52 columns with flame-ionization detection.
e carbon number profile of n-alkanes could be a good means to determine
adulteration of extra virgin olive oil with very low percentages of both crude rapeseed
and sunflower seed oils (Webster et al., 2000). Analysis of the n-alkane pattern by
Principal Component Analysis has been suggested as a possible means to identify
these adulterants at levels of about 0.5% (Webster et al., 2000).
Authentication
Food authenticity is an important issue that includes adulterations, varietal and geo-
graphical characterization, and verification of some properties of olive oils with De-
nomination of Protected Origin (DOP) (EEC Reg No 2081/92), through analytical
OliveOil2.indb 144 3/31/2006 1:12:54 PM
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145
Analysis and Authentication
methodologies.
Adulteration
Extra virgin olive oil has a much higher price compared to olive oils of other catego-
ries or olive pomace oils and seed oils. Because of this, its adulteration with cheaper
products can be an attractive practice.
International bodies such as the European Commission and IOOC defined strict
modern instrumental techniques replacing classical purity tests.
Current methodologies adopted in official methods resulted in a significant im-
provement of the control of olive oils, but some questions have been raised by re-
searchers who indicated that such methods are not able to reveal all sophisticated
adulterations (Paganuzzi, 1997) and, in addition, can classify some genuine oils out-
side their natural category (Proto, 1992).
Fatty acid composition can only give some but not conclusive information about
the possible presence in a mixture of linoleic rich vegetable oils. e adulteration is
detected by the DECN42 determination, which has proved to be very effective, and
by the analysis of sterols, which is especially useful for detecting the botanical origin
of the added seed oil.
Monoacylglycerol content could differentiate between genuine virgin olive oils
and oils fraudulently deacidified (Leone et al., 1989), whereas total diglycerides con-
tent is helpful for detecting a possible fraudulent raising of the category of a given
product (Leone et al., 1988).
Problems related to the detection of the addition of one of different kinds of
desterolized oils to olive oil have been overcome since processes needed for removing
sterols involve the production of trans unsaturated C18 fatty acids, of stigmasta-3,5-
diene and n-alkadienes (e.g. n-hexacosadiene) and isomerization products of squa-
lene. Isomerizations that convert D7 sterols into D8(14) and D14 sterols (Biedermann
et al., 1995) are of practical interest in such cases because they reveal the addition of
small amounts of desterolized sunflower oils.
Addition of refined pomace olive oil to refined olive oil is generally detected by
the determination of waxes, aliphatic alcohols, and erythrodiol+uvaol.
e adulteration of virgin olive oil with low proportion of refined olive oil, in
addition to the official determinations has already been discussed. e spectrofluo-
rimetric detection at 673 nm (Marini et al., 1990), typical of chlorophyll pigment,
can evidence the presence of virgin olive oil in other grades, although it is not pos-
sible to measure levels because of the wide variability of chlorophyll content. Atomic
absorption is considered only a preliminary screening tool, to detect the presence of
synthetic chlorophyll. In the latter, the central magnesium ion of the chlorophyll
molecule is replaced by a transition element, such as a copper ion. Serani and Piacenti
(2001b) have developed an analytical approach to detect copper pheophytin (E141),
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Copyright © 2006 by AOCS Press
146 F. Angerosa et al.
a coloring agent illegal in olive oil. More recently Del Giovine and Fabietti (2005)
have proposed a capillary zone electrophoresis (CZE) method, with a laser induced
fluorescence (LIF) detector, to determine copper chlorophyll from natural pigments.
Results obtained confirm a good repeatability, reproducibility, and accuracy of this
method, compared to other methods such as HPLC.
In the last years authenticity and adulteration of olive oil have been extensively
monitored using spectroscopic techniques which show advantages in terms of speed
and expense per test.
Mid-infrared (MIR; 4000-400 cm-1) and near-infrared (NIR; 15000-4000 cm-1)
spectroscopy have been successfully used for the detection of oil adulterants, provid-
ing direct molecular specific information without extensive sample preparation (Sato,
1994; Guíllen and Cabo, 1999). e MIR region is where the fundamental groups
appear. For vegetable oils, MIR spectra are dominated by the vibrations of polymeth-
ylene chains of triglycerides. Two distinct regions are present in a MIR spectrum: the
first (3100-1700 cm-1) is formed by well-resolved peaks. In this part of the spectrum
there is absorption due to the C-H stretching vibration of cis fatty acid (–CH-CH=)
that appears near 3005 cm-1 in triolein, shifts towards higher frequencies as the degree
of unsaturation increases. e corresponding trans form absorbs near 3025 cm-1. e
second part of a MIR spectrum (1500-700 cm-1) is called the fingerprint region and
shows overlapping peaks. e fingerprint region is closely related to the degree and
type of unsaturation, and also to the content of cis and trans isomers. e intensity of
the band near 1400 cm-1 depends on the percentage of monounsaturated acyl groups;
that of the band near 1160 cm-1 on the content of saturated acyl groups. e presence
or absence of bands near 915 cm-1, very weak in olive oil, can be useful in detecting
the existence of blends with high linoleic oils (Guíllen and Cabo, 1999).
NIR spectra generally contain a number of broad and overlapping bands, arising
from the overtones (first and second) and combinations of functional groups present
in oil samples. e most intense bands in the oil spectra can be found at 4260 and
4370 cm-1, and are characteristic of the combinations of C-H stretching vibrations
of –CH3 and –CH2 with other vibrations. e two bands at 5700 and 5750 cm-1
correspond to the first overtone of the C-H stretching vibration of –CH3, –CH2 and
–HC=CH-. e absorption band near 6010 cm-1 is due to C-H vibration of cis-un-
saturation. Fatty acids having cis double bond exhibit strong absorption bands in the
region around 6010 cm-1, and the intensity of these bands increases with increasing
unsaturation. In the region between 7700 and 9100 cm-1, the second overtone of the
C-H stretching vibration of –CH3, –CH2 and –HC=CH- can be found.
Data handling of MIR and NIR spectra is very difficult, and useful information
can be drawn only in combination with chemometrics. Lai et al. (1995) demonstrat-
ed the potential of MIR spectroscopy for the quantitative determination of the level
of refined olive and walnut oils in extra virgin olive oil. Downey et al. (2002) applied
discriminant analysis and PLS to NIR data for the quantification of sunflower adul-
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147
Analysis and Authentication
teration in extra virgin olive oils. e presence of adulterants, such as corn oil, sun-
flower oil, soya oil, walnut oil, and hazelnut oil in pure olive oil, could be predicted
on the basis of NIR data with very low error limit ranging from ± 0.57 to ± 1.32 %
w/w, as reported by Christy and coworkers (2004).
NMR spectroscopy has also played an ever-increasing role in the study of prop-
erties of vegetable oils, as a tool for authentication and quality assessment of virgin
olive oil. Recently, Vlahov (1999) and Sacchi and coworkers (1997) have reviewed the
usefulness of NMR to the study of olive oils.
1H NMR spectrum of any edible oil shows about 10 signals, due to protons of
the main components, triglycerides. e proportion of various acyl groups in oils of
different botanical origin provides a great deal of information which permits good
discrimination between oils of different composition (Guíllen and Ruiz, 2003a,
2003b). Fauhl et al. (2000) have applied discriminant statistical analysis to some
concrete signals of the 1H NMR spectra, to show the effectiveness in discriminating
between olive, hazelnut and sunflower oils. e high resolving power of 13C allows
the characterization of triglyceride mixtures, the fatty acid compositions, without dis-
tinguishing, however, the homologous chains, i.e. C16:0 and C18:0, that appear as a
single resonance.
e analysis of 13C NMR spectra discriminates among virgin olive oils, oils with a
high content of oleic acid, and oils with a high content of linoleic acid by using step-
wise discriminant analysis. Zamora et al. (2001) obtained a 97.1% correct validated
classification for different oils, suggesting that 13C NMR may be used satisfactorily for
discriminating some specific groups of oil. To obtain 100% correct classifications for
the different oils and mixtures, more information is needed than that obtained from
the direct analysis of the oils. More recently 31P NMR spectroscopy has also been ap-
plied for the detection of extra virgin olive oil adulteration (Fragaki et al., 2005).
Other techniques, such as carbon stable isotope ratio (Angerosa et al., 1997b;
Spangenberg et al., 1998), Curie-point Pyrolysis mass spectrometry (Py-MS) (Goo-
dacre et al., 1993), FT-Raman spectroscopy (Baeten et al., 1996) and electrospay
ionization-mass spectrometry (ESI-MS) (Goodacre et al., 2002) have been applied
for assessing the adulterations.
Current Problems
A series of olive oil adulteration problems is related to the addition of high oleic acid
oils, such as hazelnut oil. Other problems are a: the illegal addition to virgin olive
oils of olive oil subjected to forbidden deodorization under mild conditions that do
not cause the formation of hydrocarbons not naturally occurring in virgin olive oils,
trans isomers of fatty acids, or isomerization products of squalene, which are all useful
tracers of refined oils; b: the addition of oils obtained by a second centrifugation of
olive pastes (remolido).
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148 F. Angerosa et al.
Addition of Hazelnut Oil
Adulteration of olive oil with hazelnut oil is one of the most difficult to detect, due
to similar triacylglycerol composition, total sterol content, and fatty acid profile
(Benitez-Sanchez et al., 2003).
Detection of hazelnut oil in mixtures with olive oil is especially difficult at adul-
teration levels below 20%. A method of detecting the adulteration with pressed ha-
zelnut oil is based on the determination of filbertone, (E)-5-methylhept-2-en-4-one,
a characteristic volatile compound of hazelnut oil with a great flavor impact (Blanch
et al., 1998, 2000). Blanch et al. (1998) have tested different techniques, such as
simultaneous distillation-extraction (SDE) and supercritical fluid extraction (SFE)
to determine their suitability for the detection of filbertone. RPLC-GC was the most
satisfactory for detecting compositional differences between olive and hazelnut oils.
e off-line coupling of HPLC and 1H-NMR for detecting filbertone shows good
sensitivity and selectivity (Ruiz del Castillo et al., 2001). Peña et al. (2005) developed
a new methodology to detect low percentages of hazelnut oil very recently, combining
direct analysis of oil samples by headspace-mass spectrometry and various multivari-
ate statistical techniques. Low levels of pressed hazelnut oil adulteration can be evi-
denced by RP-HPLC analysis of the polar component (Gordon et al., 2001; Zabaras
and Gordon, 2004), using a marker present in the polar fraction of hazelnut oils, but
not in olive oils (Gordon et al., 2001).
Ollivier et al. (1999) proposed to search for α-amyrin and lupeol that are pres-
ent in great proportion in hazelnut and almond oils and absent in virgin olive oils to
detect possible adulteration of olive oil at levels of addition >5%.
Several ketosteroids (e.g. sitostan-3-one), recently identified (Mariani et al., 2001)
may also be used as markers to identify the addition of hazelnut oil to olive oil.
Due to the natural variability of tocopherol pattern in different oils and their
degradation during refining, detection of adulterations presents serious limitation.
Recently (Mariani et al., 1999a; Morchio et al., 1999) investigated the content of
tocopherols to detect the adulteration of olive oils with hazelnut oils. Olive oils con-
tain a higher percentage of β-tocopherol than γ-tocopherol compared with hazelnut
oils. Conversely, olive oils have traces of δ-tocopherol, whereas hazelnut oils contain
higher amounts. e authors have suggested that genuine oils should have a ratio
γ/β-tocopherol <5. However, tocopherol determination is not actually of great inter-
est because of the large variability of the tocopherol composition and the tocopherol
degradation during refining.
Some researchers have considered the possibility of using more than one param-
eter to solve the problem. Mariani et al. (1999b) have recently set up a chromato-
graphic method for the determination of esterified sterols which allows the detection
of admixtures with hazelnut oil by calculating the ratio in the esterified sterol frac-
tion. is ratio is always ≤ 1 for non-adulterated olive oils. e method permits the
detection of levels 5-10% of hazelnut oil, but it has to be combined with DECN42,
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149
Analysis and Authentication
to avoid falsely positive results (Vichi et al., 2001). Cert and Moreda (2000) used
the comparison of several triglyceride algorithms with a reference database, to detect
low percentages of hazelnut oil. ey evaluated the ratio R = rECN42/rECN44 where
rECNx is the ratio between the value experimentally determined by means of HPLC
and theoretical content calculated from the HRGC composition of C16 and C18
fatty acids, assuming a 1,3-random,2-random distribution of fatty acids in the triacyl-
glycerol with restrictions for saturated fatty acids in the 2-position. R value in relation
to the ratio oleic acid/linoleic acid allows one to assess the olive oil genuineness. To
overcome problems related to the possibility of some falsely positive results, IOOC
experts (International Olive Oil Council T.20/Doc no. 25 February 2004) proposed
to adopt, in addion to R, a decision tree, founded on the agreement between results
from several mathematical algorithms (calculated from theroretical and experimental
triacylglycerol composition) and those from a database built from genuine oils. Algo-
rithms take into account the following parameters: LLLeor, DOOL (where DOOL
has the same meaning DECN42 that is the difference between theoretical and experi-
mental OOL), DLLL, DECN44, and percent of linolenic acid.
Other potential discriminant factors, such as (LLL/ECN42)x100, ECN46/LLL
and (ECN44+ECN46)/LLL, useful for blends with seed oils, could not reveal adul-
teration at low percentages of hazelnut oil (Christopoulou et al., 2004).
More recently, spectroscopic techniques have been widely applied to assess adul-
terations. FT-Raman spectroscopy, together with Partial Least Squares and Genetic
Programming, were employed to verify the level of hazelnut oil added to virgin olive
oil, Results seemed to successfully predict the addition of hazelnut oil in the range
0-20% (López-Díez et al., 2003). Classification of hazelnut oil, olive oil, and other
types of oils was successfully achieved with FT-IR spectroscopy (Christy et al., 2004;
Ozen and Mauer, 2002). Depending on the adulterant oil, detection limits for olive
oil adulteration were as low as 2%, adulteration of virgin olive oil with hazelnut oil
could be detected only at levels 25% and higher (Ozen and Mauer, 2002). 1H-NMR
(Mannina et al., 1999) and 13C-NMR (Zamora et al., 2001) have been investigated
as alternative approaches to detect hazelnut adulteration, also in combination with
artificial neural networks (Garcia-Gonzalez et al., 2004) giving a detection limit of the
model around 8% of hazelnut oil.
Olive Oils Subjected to Forbidden Deodorization in Mild Conditions
e “deodorized oils” are olive oils that have been subjected to a deodorization under
low temperature and in vacuum to remove undesirable volatile compounds or to the
bleaching and deodorization under conditions that avoid significant modifications of
their composition.
Serani and coworkers (Serani et al., 2001; Serani and Piacenti, 2001a, 2001b,)
studied both the effects of thermal treatments on the transformation of pheohytins
and the effects of chemical-physical treatments on diglyceride composition. ey ob-
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150 F. Angerosa et al.
tained a mathematical function that allows determination if a virgin olive oil has
been subjected to a thermical treatment, and named this function Cold index. e
function is always > 0.10 in deodorized oils, whereas it is near zero or negative in the
majority of extra virgin olive oils (Serani and Piacenti, 2001a, 2001b). In addition the
same authors studied oils subjected to chemical and physical treatments and related
the absolute content of diglycerides with the free acidity of the oil and the isomeriza-
tion time of 1,2-diglycerides. e latter is calculated by kinetics of isomerization and
mathematically expressed as a function of the ratio between 1,2- and 1,3-diglyceride
isomers and free fatty acids (Serani and Piacenti 2001a; Serani et al., 2001). Treated
oils show isomerization times notably longer than genuine virgin olive oils. Limit
values were suggested to discriminate genuine virgin olive oils.
Mixtures of Virgin Olive Oil With Olive Oil Obtained by Second
Centrifugation of Olive Pastes (Remolido)
e processing of olive pastes obtained from the first centrifugation can be performed
immediately or after storing. Oils from the second centrifugation of fresh pastes show
characteristics very similar to those from the first centrifugation, but they are closer
to those of olive pomace oils, if pastes are processed after several days, because of a
greater amount of erythrodiol, waxes and free aliphatic alcohols.
ere are not consolidated methodologies for detecting addition of oils from
the second centrifugation. IOOC experts (International Olive Oil Council, T.20/
Doc. N. 39-1 1998 and T.20/Doc. N. 38-4 1998) proposed to determine both total
aliphatic alcohol content and alcoholic index (I.A.) to reveal fraudulent admixtures
with oils from the second centrifugation. Alcoholic index is significantly higher in
oils from the second centrifugation than in oils from the first one, in extra and virgin
categories, and in lampante grade. Alcoholic index has already been described in the
Alcoholic fraction section.
Varietal Characterization.
ere is a huge number of Olea europaea cultivars and some of them were recently
planted in new areas different from regions where they were autochthonous.
A great research work has been made in an effort to understand the modifica-
tions of the qualitative and quantitative composition of most oil fractions, according
to variety. Several investigations indicated that some parameters can be used to dif-
ferentiate oils from various cultivars (Aparicio and Luna, 2002; Stefanoudaki et al.,
2000). Esti and coworkers (1996a) found that the total content of alcohols could be a
useful tool for varietal characterization. On the other hand, Gandul-Rojas and Min-
guez-Mosquera (1996) reported differences in the contents of chlorophylls and carot-
enoids useful for discriminating some Spanish varieties. Fatty acids and unsaturated
and aliphatic hydrocarbons were used to distinguish Croatian cultivars (Koprivnjak
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151
Analysis and Authentication
and Conte, 1996). Koprivnjak et al. (2005) obtained to differentiate three different
Croatian varieties by applying a linear discriminant analysis (LDA) to n-alkanes of
oils obtained during four consecutive years.
Relationships between cultivars and sensory quality were investigated by several
researchers using sensory evaluations and volatile composition analysis (Aparicio and
Luna, 2002; Stefanoudaki et al., 2000; Cavalli et al., 2004; Tura et al., 2004).
e characterization of monovarietal virgin olive oils is very difficult since their
composition is affected by a number of variables such as pedoclimatic conditions
(Aparicio et al., 1994a; Morello et al., 2003), ripening degree of fruits, and extraction
systems. Moreover, there is also the difficulty related to the variability of the contents
of single compounds over the years.
e most important changes have been observed during the ripening process,
which in turn is affected by climate, agronomic practices, and irrigation (Romero et
al., 2002). Nitrogen fertilization would slaken fruit ripeness, a greater availability of
water as in the case of irrigation (Goldhamer et al., 1994) promotes the maturation,
thus causing a reduction of phenols, weakening of bitterness and a modification of
volatile composition and sensory profile (Salas et al., 1997).
Climate, and in particular temperatures, modify the metabolic activities of fruit
and affect unsaturated fatty acid content (Esti et al., 1996a; Beltran et al., 2004) and
phenolic content (Beltran et al., 2005).
Statistically significant changes were observed in triterpenic and sterolic fractions
(Esti et al., 1996a; Christopoulou et al., 1996) and in the diacylglycerol ratio (Vlahov,
1996). e volatile composition shows a different evolution pattern in relation to
fruit maturity and the extension of fruit pigmentation (Morales et al., 1996; Angero-
sa and Basti, 2001) with notable changes in sensory odor note intensities. Phenolic
compounds show a dramatic reduction that can reach about 60% during the last 4
months of fruit ripening (Škevin et al., 2003; Mousa et al., 1996; Esti et al., 1996b).
is decrease of phenolic compounds is responsible for a weakening of the bitter sen-
sory note.
All the mentioned variations in composition are greater in “cold” areas. Oil from
mountainous regions generally shows a higher content of linoleic acid, lower oxidative
stability, and lower concentrations of sterols, tocopherols, phenols, and chlorophylls
than oil from areas at low altitude (Aparicio et al., 1994a; Mousa et al., 1996).
Technological conditions during the oil extraction also modify the composition.
e concentration of volatile compounds and polyphenols in olive oils depends on the
type of grinding machines, malaxation conditions, and extraction system. A greater
recovery of phenolic compounds is observed by using metallic crushers. Conversely,
the amount of volatile compounds is significantly higher in oils obtained with a mill
stone (Angerosa and Di Giacinto, 1995). Malaxation time and especially tempera-
ture negatively affect the composition of metabolites arising from the lipoxygenase
pathway, reduce volatile compounds displaying pleasant odors and increasing those
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152 F. Angerosa et al.
giving less attractive perceptions (Morales and Aparicio, 1999; Morales et al., 1999;
Angerosa et al., 2001). In addition, due to oxidative mechanisms mediated by en-
dogenous peroxidases and polyphenoloxidases and interactions with polysaccarides,
phenolic compounds are reduced significantly (Servili et al., 2003) and this causes a
significant loss of bitterness.
Oils extracted by pressure are significantly more stable and have more intense
grass notes and bitter taste in relation to oils extracted by the three phase decant-
ers. is is attributed to a higher concentration of phenols and volatile compounds
(Aparicio et al., 1994b; Di Giovacchino et al., 1994; Angerosa et al., 2000b). Oils
from two-phase systems are characterized by a reduced loss of o-diphenols, tocopher-
ols (Jiménez-Márquez et al., 1995; Angerosa and Di Giovacchino, 1996), and volatile
compounds (Ranalli and Angerosa, 1996) as well as low levels of aliphatic and triter-
penic alcohols and waxes (Ranalli and Angerosa, 1996).
Because of the different influences of the processing conditions, the characteriza-
tion of oils from different cultivars can only be achieved through the information
from various glyceridic and nonglyceridic fractions. To obtain a reliable differentia-
tion of monovarietal oils, it is necessary to have a large set of oil samples representative
of all pedoclimatic, technological and agronomic variables, a large number of chemi-
cal compounds and/or sensory attributes, and to apply to them statistical techniques
or artificial intelligence algorithms. Bucci et al. (2002) claimed that good results can
be obtained by applying supervised chemometric procedures to official quality pa-
rameters, such as linear discriminant analysis (LDA) and artificial neural networks
(ANNs).
Giansante and coworkers (2003) used fatty acids, fatty alcohols, polycyclic triter-
penes, and squalene to discriminate oils from four cultivars. Experimental data were
processed by unsupervised and supervised chemometrics. PCA and SIMCA statistical
procedures were applied to triglycerides and sterols to distinguish oils from different
cultivars (Galeano Diaz et al., 2005).
Excellent results were obtained by Aparicio and his group by applying multi-
variate statistical procedures to several oil fractions as well as to volatile and sensory
descriptors (Aparicio et al., 1997). Volatile compounds are strongly related to sensory
descriptors. Sensory notes deriving by the construction of a statistical sensory wheel
(Aparicio and Morales, 1995; Aparicio et al., 1996a) were successfully used for the
characterization of cultivars (Aparicio et al., 1996b) by means of fuzzy logic profiles
(Calvente and Aparicio, 1995).
A completely different approach to the monovarietal oil characterization is based
on the evaluation of the percent distribution of volatile metabolites arising from
oxidation of linolenic acid (LnA) mediated by lipoxygenase. Metabolite content is
strictly connected with the cultivar variable because of the enzyme differences geneti-
cally determined and not significantly influenced by the environmental conditions
of olive growing areas (Angerosa et al., 1999b). erefore, cultivars are grouped and
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Analysis and Authentication
differentiated according to activity of hydroperoxide lyases (% trans-2-hexenal), acyl-
hydrolases (% of both trans-2-hexen-1-ol and cis-3-hexen-1-ol) and alcohol acyltrans-
ferase (% cis-3-hexenyl acetate), and the amount of trans-2-hexenal. (Angerosa et al.,
2004). Moreover, the percent distribution is the same in oils from the beginning of
purple coloring of the fruit. is means that the main metabolites from LnA are inde-
pendent of the degree of fruit ripening (Angerosa and Basti, 2001). e independence
of volatile compositions from the growing area and ripening stage, and the consis-
tency over years, suggest that the cultivar is the dominant factor in the formation of
the aroma. erefore, the determination of metabolites from LnA, together with the
concentration of trans-2-hexenal, could be considered an effective tool to differentiate
monovarietal oils (Table 7.11 ) (Angerosa et al., 2004).
One of the most innovative approachs to identify variety is the characterization
of virgin olive oils by DNA (Cresti et al., 1997). is is especially important for olive
oils with a DOP (Denomination of Protect Origin) designation. eir certification
implies that the oil composition related to cultivars grown in a given growing area,
is in accordance with the registration of the denomination. Labels generally report
the country of origin, but do not provide any detail about cultivars. Assay of DNA,
present in olive oil and even in refined oil (Hellebrand et al., 1998), can provide reli-
able information about varieties used for its production (Angiolillo et al., 1999). It is
TABLE 7.11
trans-2-hexenal (ppm) and percent distribution of C6 metabolites from enzymatic oxida-
tion of linolenic acid. Source: Angerosa et al, 2004.
% cis-3-
trans-2- % trans-2- % trans-2- % cis-3- hexenyl
Cultivar hexenal ppm hexenal hexen-1-ol hexen-1-ol acetate
Mastoidis 17.1 99.4 0.1 0.5 0.0
Coratina 43.5 97.8 1.5 0.7 0.0
Frantoio 53.4 96.6 1.2 0.7 1.5
Taggiasca 17.2 94.9 1.6 1.6 1.9
Canino 30.3 94.8 2.8 2.2 0.2
Picual 23.2 92.6 1.2 5.0 1.2
Leccino 47.3 89.0 10.1 0.9 0.0
Dritta 11.4 84.5 10.9 1.5 3.1
Bosana 12.1 82.7 10.1 2.0 5.2
Carolea 7.4 83.4 2.2 14.4 0.0
Provenzale 5.7 79.4 1.4 9.6 9.6
Nocellara del Belice 6.8 78.4 1.1 15.8 5.0
Gentile di Chieti 6.5 75.1 2.3 18.1 4.5
Maurino 6.3 74.4 2.3 20.9 2.4
Koroneiki 4.6 58.7 3.8 16.3 21.3
Pisciottana 11.0 52.6 4.7 32.9 9.9
Moraiolo 1.8 45.6 5.0 42.4 7.0
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154 F. Angerosa et al.
possible to construct a DNA database of varieties used for oil production by analyzing
DNA of leaves, since the profile of purified DNA from a monovarietal oil correspond
to the profile of DNA isolated from the leaves of the same cultivar (Busconi et al.,
2003). e DNA assay requires an amplification with suitable techniques (Testolin
and Lain, 2005) because of the low level in olive oils. e technique is useful in the
verification of the cultivar used for the production of monovarietal oils. However,
often DOP oils are produced by processing olives from two or more cultivars. In these
cases at the moment, even if the composition of different cultivars is known, DNA
analysis can only lead to the identification of the main variety used if this has a pro-
portion above 80%. erefore, the application of DNA assays for the identification of
production cultivars does not currently give conclusive results (Breton et al., 2004).
Characterization of Virgin Olive Oils by Geographical Origin
Verifying the declared origin or determining the origin of unidentified olive oil is not
yet an easy task. Standard limits, introduced by International bodies, are able to reveal
most of the adulterations, but they are not useful in differentiating oils according to
olive growing areas. Oil composition is an expression of biosynthetic genetically con-
trolled pathways, modulated through the action of specific enzymes whose activity
is affected by climate, cultivar, soil kind, and the extraction process. is means that
the identification of geographical origin can be achieved only when very strict rela-
tionships between compositional and sensory data, and the agronomic and climatic
characteristics of a given growing area are understood. On the assumption that the
composition of virgin olive oils is related to the geographical area where they are pro-
duced, oils with the Denomination of Protected Origin (DOP) designation and In-
dication Geographical Protect (IGP) are marketed within countries of the European
Union. In fact the control of a DOP or IGP products is obtained by administrative
measures of oil production. Many efforts are made by researchers to control these
commodities by objective analytical methods.
Researchers trying to elucidate relationships between composition and geograph-
ical origin use HRGC and HPLC methods to determine major and minor compo-
nents of olive oils. Experimental data are generally processed by multivariate statistical
procedures or expert systems for the classification of the olive oils. Interesting results
were obtained by applying many multivariate procedures, but the more encouraging
differentiations were made by means of expert systems that use a very large database.
For the creation of a database, all the possible information about climate, cultivars,
growing area, altitude, longitude, latitude, etc, must be taken into account. In ad-
dition, sampling should include oils produced in many olive crops to obviate to the
variability induced by olive producing year.
e major components of olive oil give useful information which may be used to
differentiate the oils. Statistical procedures have been applied to fatty acids (Tsimidou
and Karakostas, 1993; Stefanoudaki et al., 1999). e effect of latitude, which dif-
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Analysis and Authentication
ferentiates oils of the North regions from those of the South areas, is clearly delineated
by fatty acids and triacylglycerols (Tsimidou et al., 1987b; Tsimidou and Karakostas,
1993; Alonso and Aparicio, 1993). However, more information can be drawn from
minor constituents. For instance, the longitude may be indicated by the triterpe-
nic alcohol content which decreases from coastal to inland regions (Aparicio et al.,
1994a).
Chemometric methods have been applied to sterolic composition and triglycer-
ides (Galeano Diaz et al., 2005), fatty acids, fatty alcohols, and triterpenes (Giansante
et al., 2003; Bianchi et al., 2001), triglycerides (Brescia et al., 2003), volatile com-
pounds (Vichi et al., 2003), whereas unsaturated and aliphatic hydrocarbons were
used to differentiate Croatian oils (Koprivnjak and Conte, 1996; Koprivnjak et al.,
2005). An expert system, labelled SEXIA, has been successfully applied to data of un-
saponifiable components, also sometimes including volatile compounds and sensory
descriptors (Aparicio and Alonso, 1994; Aparicio et al., 1994c; Morales et al., 1995;
Aparicio et al., 1996b).
Recently, emergent techniques were also investigated for their ability to differen-
tiate geographical origin of virgin olive oils. Angerosa and coworkers (1999c) applied
stable isotope ratio to gain information about the geographical origin of oil samples.
13C NMR spectroscopy was able to discriminate monovarietal oils from different Ital-
ian production areas (Shaw et al., 1997; Vlahov et al., 2001, 2003; Vlahov 2005).
is result was explained by the differences in fatty acid composition. Satisfactory
results were obtained by Sacchi (Sacchi et al., 1998) and Sacco et al., (2000), who
applied Principal Component Analysis or Hierarchical Clustering to high-field 1H
NMR spectroscopic data of minor components. ey obtained a very good classifi-
cation of oil from traditional cultivars with respect to the region of origin. However
samples from new cultivars were not correctly classified. is indicates a strong con-
tribution of olive variety on chemical composition of virgin olive oils.
FT-IR and NIR, in combination with different multivariate procedures were also
tested as a means to differentiate oils from different producing countries (Downey et
al., 2003; Tapp et al., 2003).
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