628 Eur. J. Lipid Sci. Technol.

104 (2002) 628638

Apostolos Kiritsakisa, Chemical analysis, quality control and packaging

Antonis Kanavourasb,
Konstantinos Kiritsakisc
a and mainly to describe new trends in the analysis of triacylglycerols, phenolic and fla-
Department of Food Tech-
nology, School of Food
Technology and Nutrition,
Highest Technological
Education Institute of
Thessaloniki,
Thessaloniki, Greece
b School of Packaging,
Michigan State University,
East Lansing Michigan,
USA
c Ministry of Agriculture,
Department of Agriculture
Development, Polygyros,
Chalkidiki, Greece
issues of olive oil
The aim of the present review is to present some methods for the quality evaluation
vor compounds of olive oil. Thus, methods used to determine the olive oil quality and
its resistance to oxidative deterioration, mainly the conductivity ones, are included.
Concerning the new trends in olive oil analysis, methods such as nuclear magnetic
resonance spectroscopy, Fourier-transform infrared techniques, differential scanning
calorimetry, atmospheric pressure chemical ionization-mass spectroscopy, solid-
phase micro extraction using nonpolar and bipolar fibers and some others, are de-
scribed. Since proper packaging of olive oil provides conditions to assure adequate
product life destination and marketing, a presentation of the packaging material, pack-
aging to oil interactions and some other packaging issues are also pointed out.
Keywords: Olive oil analysis, packaging, quality evaluation.

1 Introduction lipase hydrolysis [2]. RP-HPLC utilizing various mobile

Olive oil is one of the oldest known vegetable oils mainly
produced in the countries surrounding the Mediterranean
Sea. It is a natural fruit juice, obtained from the fruit of the
tree Olea europea, with a unique composition and quality
[1]. Olive oil is one of the very few oils that can be con-
sumed in its natural form, thus preserving all its natural
constituents. Several methods of analysis have been pro-
posed and are used to evaluate the unique olive oil com-
position and quality, which are affected by several factors,
including packaging [1].
This review is focused on the chemical analysis and qual-
ity evaluation of olive oil. Official or unofficial analytical
Review
phases, temperature programs, solvent gradients, and
detectors have been tested. The major difficulty in the
HPLC triacylglycerol analysis is the selection of the de-
tector. Refractive index and UV-absorbance (190-237
nm) detectors are characterized by their low sensitivity.
Mass and laser-light-scattering detectors allow the record
of excellent chromatograms in gradient elution [3].
Triacylglycerols from olive oil show an asymmetry in the
distribution of fatty acids among positions sn-1, sn-2 and
sn-3 [2]. A combination of SI-HPLC and the chromato-
graphic resolution of diacyl-sn-glycerol derivatives can be
used, as part of a stereospecific analysis procedure, to
obtain the distribution of fatty acids in positions sn-1, sn-2

techniques for the isolation and identification of some of
the natural components of the oil and some of those de-
riving during oxidative and hydrolytic deterioration are
presented. Advanced quality analysis methods are de-
scribed and packaging olive oil issues are discussed.
2 Triacylglycerol analysis of olive oil
and sn-3 of the main triacyl-sn-glycerol species of extra
virgin olive oil.
The most common triacylglycerols in olive oil are OOO,
POO, OOL, POL and SOO. An example of the triacylglyc-
erol composition of extra virgin olive oil is shown in Tab. 1
[4].

The triacylglycerol structure of olive oil was determined by
several procedures involving e.g. separations into molec-
ular species by reversed-phase high-performance liquid
chromatography (RP-HPLC) and silver ion thin-layer
chromatography (SI-TLC) in combination with pancreatic
Correspondence: Apostolos Kiritsakis, Department of Food
Technology, School of Food Technology and Nutrition,
Highest Technological Education Institute, 54101 Thessaloniki,
Greece. Phone: +30-31-0791357, Fax: +30-31-0791360; e-mail:
kiritsak@food.teithe.gr
Olive oil triacylglycerols present in the olive fruit meso-
carp differ from those present in the seed (kernel). The
trilinolein content was found to be less than 0.2% in
mesocarp oil and eight times as much in seed oil [5].
Some olive oils contain up to 0.8% trilinolein. The triacyl-
glycerol analysis was used to describe and identify the
origin of oils as well as to detect certain adulterations.
Stefanoudaki et al. [6] reported that oils obtained from
high elevation trees in the island of Crete, Greece, had a
higher triacylglycerol composition in unsaturated fatty

2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0931-5985/2002/0910-0628 $17.50+.50/0
Eur. J. Lipid Sci. Technol. 104 (2002) 628638 Chemical analysis of olive oil 629

Tab. 1. Triacylglycerols identified in olive oil by HPLC with separation of the phenolic compounds of olive oil by RP-
a refraction index detector. HPLC requires the utilization of a gradient consisting of at
least two solvents: a diluted acid and an organic solvent.
Triacylglycerols in extra virgin [%]
olive oil by HPLC An RP-HPLC technique with isocratic elution was applied
to separate and quantify the major phenolic compounds
LLL 0.267 of the hydroalcoholic extracts of olive oils. Hydroxyty-
OLLn + PoLL 0.292 rosol, tyrosol, caffeic acid, p-hydroxyphenylacetic acid
PLLn 0.082
and homovanillic acid were analyzed. This method can be
OLL 3.855
applied for the analysis of small quantities and of the most
OOLn + PoOL 1.481
PLL + PoPoO 1.456 important phenolic compounds in olive oil. In order to min-
PLL + PoPoL + PoPo 0.921 imize the error analysis, the separation of the phenolic
OOL 15.690 compounds of virgin olive oil using, tyrosol, vanillic acid,
PoOO 1.982 syringic acid and o-coumaric acid as external standards,
PLO 8.810 was applied [7].
PoPP + SPoL + Oln 1.197
PLP + PoPP 1.310
OOO 31.720 Several compounds such as: tyrosol, hydroxytyrosol (Fig.
SOL 1.732 1), phenolic acids, (ferulic, syringic, caffeic and p-coumar-
POO 19.660 ic), oleuropein aglycone, deacetoxyoleuropein aglycone,
POP 3.720 elenolic acid and derivatives, other secoiridoid com-
GOO 0.265 pounds and flavone aglycones (luteolin and apigenin),
SOO 3.728 have been identified by HPLC-mass spectrometry (MS) in
POS + SLS 1.156
O oleic, Ln linolenic, P palmitic, L linoleic, S stearic, Po

palmitoleic, G eicosenoic (gadoleic) [4].

acids compared to those obtained from lower elevation

trees. Oils from trees cultivated in low elevation, showed
a higher content of saturated fatty acids than those culti-
vated in high elevation. The concentrations of palmitic
and palmitoleic acids increased with increasing altitude.

The triacylglycerol composition can be altered if olive

fruits are collected in different ripeness stages. According
to Kiritsakis [1], the ratio of monounsaturated to polyun-
saturated fatty acids decreases as the ripeness index in-
creases. The increase in polyunsaturated fatty acids was
shown to raise the susceptibility of the oil to oxidative de-
teriorations.

3 Phenolic compounds analysis

A colorimetric determination of the amount of total phe-
nols present in olive oil can be achieved measuring the
absorbance at 725 nm using the Folin-Ciocalteu reagent
against a suitable blank [1]. Results are usually ex-
pressed as mg of caffeic acid equivalents per kg of olive
oil.

Individual phenolic compounds are mainly analyzed by

HPLC techniques after extracting them from the oil by a
solid-liquid or solid phase extraction using proper organic
solvents. A chromatographic analysis of phenols can be
achieved in olive oils after the solid-phase extraction of
these compounds. According to the literature, the proper
Fig. 1. Structures of various phenols and their degrada-
tion into tyrosol and hydroxytyrosol in extra virgin olive oil
[8].
630 Kiritsakis et al. Eur. J. Lipid Sci. Technol. 104 (2002) 628638

olive oils obtained from different olive cultivars [8]. Figs. 2

and 3 show some individual phenols identified in olive oil
by different techniques.

The chromatographic features and the method used for

the expression of the concentrations might influence the
analysis results.

Caruso et al. [9] determined the phenolic components of

olive oil, particularly oleuropein aglycone, by atmospheric
pressure chemical ionization-MS (APCI-MS) (Fig. 3). Ty-
rosol, hydroxytyrosol, elenolic acid, deacetoxyligstroside
and deacetoxyoleuropein aglycones, ligstroside and oleu-
ropein aglycones, and 10-hydroxy-oleuropein were identi-
fied from olive oil methanolic extracts. The oleuropein and
its isomers present in the oil were quantified by APCI- Fig. 2. HPLC chromatogram corresponding to a phenolic
MS/MS analysis of the extracts without preliminary sepa- extract containing some standards [1].
ration from other phenolic compounds.

A simple analytical method for the quantitative determina-

tion of phenols, flavones and lignins in virgin olive oils was
developed by Mateos et al. [10]. The polar fraction was
isolated from small amounts of oil sample (2.5 g) by solid-
phase extraction (SPE) using diol-phase cartridges, and
the extract was analyzed by RP-HPLC coupled with diode
array UV detection. Chromatographic separation of
pinoresinol, cinnamic acid, and 1-acetoxypinoresinol was
achieved. For the first time, 2-(4-hydroxyphenyl) ethyl
acetate was detected in olive oils. The aldehydic structure
of the ligstroside aglycone was confirmed by nuclear
magnetic resonance (NMR) spectroscopy.

4 Analysis of flavor compounds of olive

oil
Efforts have been made to establish an instrumental
analysis of the flavor and aroma components of olive oil in
order to eliminate the disadvantages of subjective senso-
ry evaluation (panel test).

The flavor analysis of the oils was initially accomplished

by gas chromatography (GC) for separation of head space
compounds and by a mass spectrometry equipment for
the identification of the isolated compounds. Later the
dynamic headspace sampling technique combined to a
thermo-desorption of the trapped compounds into a GC
Fig. 3. Rapid phenolic analysis of virgin olive oil by at-
port, was introduced. Proper trapping materials and con-
mospheric pressure chemical ionization-MS (APCI-MS):
ditions have been tested for an accurate, quick and
(a) chromatogram of the total ion current (TIC); (b) ion
reproducible extraction and analysis of the flavor and off- chromatogram of the ion at m/z 377; (c) APCI-MS spec-
flavor compounds from olive oil samples [1, 11]. trum obtained by loop injection of the VOO extract; ion at
m/z 137 = tyrosol; m/z 153 = hydroxytyrosol; m/z 241 =
Guth and Grosch [12] applied aroma extract dilution elenolic acid; m/z 303 = deacetoxyligstroside aglycon;
analysis (AEDA) to evaluate the flavor components of m/z 319 = deacetoxyoleuropein aglycon; m/z 361 =
olive oil and determined fifteen compounds with different ligstroside aglycon; m/z 377 = oleuropein aglycon; m/z
flavor profiles. The following compounds are mainly con- 393 = 10-hydroxy-oleuropein [9].
Eur. J. Lipid Sci. Technol. 104 (2002) 628638
tributed to the flavor, given in parentheses: (Z)-3-hexenal
(green), ethyl 2-methylbutyrate, ethyl isobutyrate, ethyl
cyclohexanoate (fruity), (Z)-2-non-enal (fatty) and 4-
methoxy-2-methyl-2-butanethiol (blackcurrant-like).
Sacchi et al. [13] applied high-field (600 MHz) NMR spec-
troscopy for the direct analysis of olive oil. NMR spectra of
virgin olive oils show a large number of structural and
compositional data related to different olive oil qualities.
The analysis of aldehydes seems to be interesting, giving
a picture of the flavor profile of the oil. The possibility of
determining additional minor components such as phe-
nols or alcohols present in the oil opens new application
possibilities for this specific instrumental flavor quality as-
sessment of virgin olive oil. Caja et al. [14] applied a si-
multaneous distillation-solvent extraction followed by GC-
MS for the identification of the volatile compounds of olive
oil. They have also proposed an alternative approach,
which allows the direct injection of the oil sample and in-
volves the use of on-line coupled reversed-phase liquid
chromatography-gas chromatography (RPLC-GC).
The methods used for the analysis of the natural flavor
compounds of olive oil can be applied for determining the
volatiles formed during oil oxidation [11]. The ratio of
hexanal/nonanal can be considered as an indicator of the
level of oxidation of olive oil.
Electronic nose and advanced statistical techniques have
been recently implemented as a way to differentiate ran-
cid and defected olive oil [15]. The technique is based on
the reaction of a series of semi-conductive organic poly-
mer sensors with a stream of volatile compounds isolated
from the headspace. The proper selection of the conduct-
ing polymers is a very important step [16]. Guadarrama et
al. [17] reported that they designed and used an electron-
ic nose for olive oil organoleptic characteristics identifica-
tion, using sixteen polymeric sensors with different elec-
trode position conditions and polymers and claimed that it
was able to distinguish between oils from different quali-
ties as well as between different oils prepared from vari-
ous cultivars and geographic regions.
Ricci et al. [18] carried out studies to demonstrate the po-
tential of metal oxide semiconductor (MOS) electronic
noses for the sensory evaluation of the freshness, geo-
graphical origin and shelf life of olive oils. An Alpha MOS
Fox 3000 nose permitted the differentiation between vir-
gin olive oils produced by milling stoned drupes and by
traditional milling techniques. The equipment also en-
abled them to identify five certified defects (e.g. rancidity,
moldiness) and oils of unknown origin.
Chemical analysis of olive oil 631
5 Quality evaluation of olive oil
The methods described here for olive oil quality analysis
are not necessarily suggested by the IOOC or by the
Commission of the European Union (EU).
5.1 Acidity determination
Acidity determination is mainly accomplished by titration
using sodium hydroxide. The method determines the
amount of free fatty acids (FFA) present in the oil, which is
expressed as percentage of oleic acid. Acidity values are
used as a basic criterion for classifying the different cate-
gories of olive oil. However, according to Kiritsakis [1],
acidity is not considered as the best criterion for evaluat-
ing olive oil quality, since one oil with relatively high acidi-
ty may have a good aroma while another one with low
acidity may not have so good a taste and aroma. For ex-
tra virgin olive oil the maximum acidity is 1%, increasing
as the quality category of the oil changes.
An automated GLC method for the determination of acid-
ity (FFA) in seed oils could be used in the field of olive oil
[2]. A sample of 200 to 400 mg is ground and dried in a
forced air oven for at least 8 h at 80 C. After drying the
sample is placed in a 10 ml test tube. Thereupon an inter-
nal FFA standard is used (1 g of heptadecanoic acid, C-
17, in 1 l heptane); 1 ml of the heptane containing the in-
ternal standard and 4 ml of solvent which is made up of 40
parts isopropyl alcohol and 1 part 1N H2SO4 are added
to the test tube. After the mixture is shaken vigorously for
15 min, 2 ml of heptane and 3 ml deionized and distilled
water are added and the test tube is shaken vigorously
again. The FFA are dissolved in the heptane, in the top
layer. To isolate them a sample from the top layer is re-
moved and the FFA are measured in a capillary-GC with
a flame ionization detector. The relationship between
GLC method and the titration method is described as fol-
lows:
Ytitration method = 1.090 GLCmethod + 0.095
This procedure permits the analysis of a large number of
samples in a short time. As an example a gas liquid chro-
matogram of the FFA determination is given in Fig. 4.
High-resolution (HR) GC can also be used for the deter-
mination of the FFA in olive oils. Besides, a rapid quanti-
tative determination of the FFA of oils can be achieved by
Fourier transform infrared spectroscopy (FT-IR). The de-
termination is based on both transmission and attenuated
total reflectance approaches, covering an analytical
range of 0.2-0.8% FFA.
Furthermore, a new pH-metric method for the determina-
tion of acidity without titration has been developed [2]. For
632 Kiritsakis et al. Eur. J. Lipid Sci. Technol. 104 (2002) 628638

that purpose, FFA have to be extracted from the oil into a

reagent containing triethanolamine (TEA), isopropanol
and water. Then the oil is mixed with the reagent and the
pH is measured before (pH1) and after (pH2) the addition
of hydrochloric acid using a commercial pH-meter with an
aqueous reference electrode.

A method for determining FFA in olive oil based on spec-

troscopic Fourier transform IR-attenuated total re-
flectance spectroscopy measurements was described by
Bertran et al. [19]. This method is rapid with a total analy-
sis time of 5 min; it is environmentally friendly and can be
applied to samples of different categories (extra virgin,
virgin, pure and pomace oil).

5.2 Peroxide value determination

Peroxide value (PV) determination is mainly accom-
plished by titration. Thereto a saturated solution of potas-
sium iodine is used which reacts with hydroperoxides.
The liberated iodine is titrated with a standardized sodium
thiosulphate solution [1].

Moreover, a colorimetric method for measuring hydroper-
oxides in photooxidized oils has been proposed [2]. Cad-
mium acetate was used and the absorbance of the
formed color was measured at 350 nm. Hydroperoxide
concentrations were calibrated using a standard solution
of benzyl peroxide in ethanol. High-performance size-ex-
clusion chromatography can also be used for the determi-
nation of the amounts of oligopolymers formed during the
oxidation of triacylglycerols. Small amounts of oligopoly-
mer compounds can be determined by this technique. Ac-
cording to Kiritsakis [1], the absence of oligopolymers in
virgin olive oils is a quality indicator.
Van de Voort et al. [20] proposed the determination of the
degree of oxidation of oils by FT-IR spectroscopy. FT-IR
analysis provides a rapid means of evaluating the oxida-
tive state of oil or of monitoring changes in oils undergo-
ing thermal stress. However, there is a need to character-
ize the spectral changes which occur with proceeding oil
oxidation, assign wavelengths to more common molecu-
lar species produced and access potential spectral cross
interferences. Recent results received with this method
are in good agreement with those obtained by the EU
method [21].
5.3 Absorption coefficients
The determination of the specific absorption coefficients
(specific extinction) in the ultraviolet region, as described
by Kiritsakis [1], is needed for estimating the oxidation
stage of olive oil. The absorption at specified wavelengths
in the ultra violet region is related to the formation of con-
Fig. 4. A gas liquid chromatogram of FFA (P Palmitic, C-
17, Internal standard, S Stearic, O Oleic, L Linoleic,
Ln Linolenic) [2].
jugated diene and triene in the olive oil system, due to ox-
idation or refining processes [1].
5.4 Accelerated oxidation tests
In order to evaluate olive oil resistance to oxidation, ac-
celerated conditions are used to facilitate the oxidation
process. Accelerated tests are only useful for comparing
the storage stability of different oils and they cannot pre-
dict the exact time at which a given oil will deteriorate.
Based on the accelerated tests, olive oils of different
stability should be stored separately and those with high-
er susceptibility should be marketed earlier.
Accelerated oxidation tests are also used to evaluate the
effects of natural or chemical antioxidants. All accelerated
methods use elevated temperatures. Thus, when mea-
suring the effect of antioxidants one should consider their
volatility and the loss of these compounds at elevated
temperatures.
Procedures and equipment mostly used are Oven test,
Rancimat and OSI equipment. The Rancimat and Oven
are known as conductivity methods.
5.4.1 Oven test
A 50 g sample of olive oil is placed in an oven at a con-
stant temperature (usually 63 C) and oxidation is fol-
Eur. J. Lipid Sci. Technol. 104 (2002) 628638
lowed by chemical and sensory tests. The time in days,
needed for the initiation stage of oxidation, indicates the
resistance of olive oil to oxidation.
5.4.2 Conductivity methods (Rancimat-OSI)
Oxidation leads to the formation of hydroperoxides pro-
ducing a series of secondary products. The exact nature
of the latter is still under investigation [1]. These sec-
ondary products include alcohols and carbonyl com-
pounds which can be further oxidized to carboxylic acids
and formic acid [1]. Formic and acetic acid are produced
in the largest quantity.
Volatile acids formed during accelerated oxidation can be
trapped in distilled water. The Rancimat procedure is
based on the conductivity change of the distillate collect-
ed from an oil sample subjected to accelerated oxidation.
The change of conductivity is plotted automatically and
the induction period of the oil is determined. Another ac-
celerated oxidation method for determining the oxidative
stability of oils is known as the oxidative stability index
(OSI). It operates on the same principle (change in con-
ductivity) as the Rancimat [1]. The OSI has big advan-
tages; up to 24 samples can be run simultaneously. The
OSI method was subjected to a fifteen laboratory collabo-
rative study in order to provide data to support its ap-
proval as an Official Method of the American Oil
Chemists Society. The overall average coefficient of vari-
ation was 11.3% for samples subjected to stability testing
at 110 C for 7 to 80 h [1].
6 New trends and recent developments in
olive oil analysis
The methods for olive oil analysis have always been sub-
ject to improvement and a lot of new studies have demon-
strated the usefulness of novel techniques for analytical
purposes [2]. Following is a brief review of the newly in-
troduced techniques in the olive oil quality analysis.
6.1 Differential scanning calorimetry
Differential scanning calorimetry (DSC) is a solventless,
simple and relatively inexpensive method used to exam-
ine the physical state and properties of food components.
Kanavouras et al. [11] applied modulated DSC to extra
virgin olive oil and to oil subjected to various accelerated
oxidation treatments. They found a very good correlation
between thermograph parameters selected for the main
crystallization peak and the various off-flavors deriving
mainly from the oleic acid degradation. Modified DSC can
adequately describe alterations related to the level of oxi-
dation due to structural conformations of the triacylglyc-
erols and their oxidation derivatives inside the olive oil
Chemical analysis of olive oil 633
mass. Thus, modified DSC can provide very useful infor-
mation regarding the degree of oxidation of any edible oil.
Since this technique is sensitive to composition changes
resulting from oxidation, it could be used as a rapid and
effective method to characterize the quality of olive oil at
different degrees of oxidation.
6.2 Nuclear magnetic resonance
One or two dimensional high-resolution NMR spec-
troscopy has been recognized as a viable technique to
analyze oils for their fatty acid composition, adulteration
and nature of unsaturation. The 13C NMR shifts of the acyl
carbon atoms depend on the functional group, molecular
conformation and environmental effects meaning that
atoms in a distance of six or more atomic centers influ-
ence the shift. Proton NMR offers an approximation of the
percentage of mono-, di- and tri-acylglycerol molecules in
oil samples while 13C NMR allows to characterize the tria-
cylglycerol mixtures. The analysis of the 13C NMR data of
numerous oil samples allowed Zamora et al. [22], to dis-
tinguish among virgin olive oils, oils with high oleic acid
content, and oils with high linoleic acid content. They ap-
plied a stepwise discriminate analysis. Based on this
method it would be possible to classify hazelnut oil
samples and determine their mixtures in virgin olive oil.
NMR studies together with statistical methods were im-
plemented to identify the geographical characterization of
olive oils (1H high-field NMR) from different areas. Linear
discrimination analysis applied to selected NMR parame-
ters of olive oils produced in the same year allowed the
grouping of samples according to their geographical ori-
gin with only very few errors. Moreover, a satisfactory
grouping was reached by combining the NMR data of
olive oils from two different years (1996 and 1997). Ac-
cording to Mannina et al. [23], the results of this method
suggest that the location of olive production could be
used as a distinctive criterion regardless of the cultivars
from which the olive oils were derived. To discriminate be-
tween olive oils by cultivar and geographical origin, Vla-
chov et al. [5], implemented the distortionless enhance-
ment by polarization transfer (DEPT) pulse sequence to
set up a quantitative high-resolution 13C NMR method for
olive oil analysis. 13C NMR is among the most powerful
techniques yet described for the analysis of olive oils.
This technique may also be used satisfactorily for dis-
criminating some specific groups of oils. However, in or-
der to obtain 100% correct classifications of the different
oils and mixtures, more information than that obtained
from the direct spectra of the oils is needed.
Mavromoustakos et al. [24] studied the olefinic region of
the 13C NMR spectrum of virgin olive oil to determine its
adulterations by seed oils. They found that the virgin olive
634 Kiritsakis et al.
oil showed 12 NMR signals and the intensities of these
were affected when olive oil was mixed with seed oil.
Squalene also present in olive oil was investigated by
NMR as well. Its site-specific deuterium distribution was
determined. The areas depicting the NMR signals of the
olefinic, methylene and methyl sites were carefully deter-
mined, and the deuterium distribution among the various
Fig. 5. HPLC chromatograms of phenolic compounds iso-
lated from Manzanilla virgin olive oil by SPE (detection at
280 nm): (a) diol phase eluted with methanol {peaks: (1)
hydroxytyrosol; (2) tyrosol; (IS1) p-hydroxyphenylacetic
acid; (3) vanillic acid; (4) vanillin; (5) p-coumaric acid; (6)
ferulic acid; (7) hydroxytyrosyl acetate; (IS2) o-coumaric
acid; (9) dialdehydic form of decarboxymethyl oleuropein
aglycon; (10) dialdehydic form of decarboxymethyl
ligstroside aglycon; (11) pinoresinol; (13) 1-acetoxy-
pinoresinol; (14) luteolin; (15) aldehydic form of oleu-
ropein aglycon; (16) apigenin; (17) aldehydic form of
ligstrosideaglycon}; (b) amino phase eluted with methanol
{peaks: (1) hydroxytyrosol; (2) tyrosol; (7) hydroxytyrosyl
acetate; (11) pinoresinol; (13) 1-acetoxypinoresinol; (16)
apigenin; (18) tyrosyl acetate}; (c) amino phase eluted
with methanol/HCl (98:2, v/v) subsequent to methanol
elution {peaks: (2) tyrosol; (IS1) p-hydroxyphenylacetic
acid; (3) vanillic acid; (4) vanillin; (5) p-coumaric acid; (6)
ferulic acid; (b) artifact; (IS2) o-coumaric acid; (c) artifact}
[10].
Eur. J. Lipid Sci. Technol. 104 (2002) 628638
sites was shown to differ for squalene from different
sources.
6.3 Fourier transform infrared technique
FT-IR studies the infrared region of the electromagnetic
spectrum. FT-IR and principal component analysis (PCA)
were used to classify oil from different origins. Unsaturat-
ed fatty ester show a peak at 3003 cm1. Sunflower oil
could be distinguished from olive oil by the high concen-
tration of polyunsaturated fatty acid esters [2]. As stated
previously, FT-IR is used for peroxide and acidity determi-
nation.
6.4 Solid-phase micro-extraction for olive oil
volatile analysis
Solid-phase micro-extraction (SPME) technique is based
on the ability of certain fibers to extract volatile com-
pounds from the food matrix or from the headspace
above it. The extracted volatiles are consequently trans-
ferred to a GC port for separation and identification.
Kanavouras et al. [11] attempted to compare and evalu-
ate the conditions of the SPME technique applied for the
analysis of virgin olive oil volatile compounds. Fig. 5 pre-
sents HPLC chromatograms of phenolic compounds iso-
lated from virgin olive oil by a conventional SPE. Fig. 6 on
the other hand shows the volatile analysis of virgin olive
oil which was obtained by applying the SPME technique
using polydimethylsiloxane (PDMS) which is a non-polar
fiber and polydimethylsiloxane-divinylbenzene (PDMS-
DVB), a bipolar fiber. The latter shows better sensitivity
for olive oil volatiles, thus a greater number of peaks are
observed (Fig. 6). Proper application of this technique
could allow efficient extraction and identification of volatile
compounds, facilitating the detection of the most signifi-
cant contributors of olive oil quality. SPME was also used
to extract volatile compounds from oils as a way to evalu-
ate its efficiency to describe the oxidation level of the oil
by portraying the profile of the various off-flavors deriving
from oxidation.
7 Packaging materials
Physical characteristics of the packaging material may af-
fect the quality of oil significantly depending on the exten-
sion of interactions. Permeability, migration, and scalping
are interactions that occur between the olive oil and the
packaging material which affect the quality and safety as-
pects of the oil further [1]. Materials used for bottling and
packaging olive oil include plastic, glass, tinplate, alu-
minum, stainless steel, fiber glass and plastic-coated card
board. The most common containers are tinplate, plastic
and glass bottles.
Eur. J. Lipid Sci. Technol. 104 (2002) 628638
Fig. 6. Solid-phase micro extraction volatile analysis of virgin olive oil using nonpolar (polydimethylsiloxane, PDMS) and
bipolar fibers (polydimethylsiloxane-divinylbenzene, PDMS-DVB), different collection and equilibrium period and amount of
olive oil sample [11].
7.1 Plastic
Plastic bottles are used extensively for packing and
bottling of vegetable oils, but they are not always suitable
for this purpose. Polyethylene (PE) and polyvinylchoride
(PVC) are materials used widely [1]. PVC is the most dif-
fused plastic industrial material in the world. PVC con-
tains various commercial additives, such as plasticizers,
stabilizers, lubricants etc., which improve its physical and
mechanical properties. It also contains some unpolymer-
ized monomers, catalysts and oligomers. Kondyli et al.
[25] tested the migration behavior of the plasticizers
dioctylphthalate (DOP) and dioctyladibate (DOA) into
olive oil. They found that after 100 h of exposure DOA had
migrated in higher amounts into the oil than DOP.
Polyethylene tetraphthalate (PET) is widely used as a
packaging material for edible oils, including olive oil. PET is
a better plastic than PVC and PE for olive oil. It is becoming
a popular plastic because of its excellent barrier, visual and
mechanical properties. Among the factors that play a role in
the migration of compounds from PET into the oil are its
acidity, time and temperature of exposure [26].
7.2 Glass
Glass is one of the most inert materials for bottles and
demijohns. Glass is easily cleaned and this helps to dis-
Chemical analysis of olive oil 635
lodge microbes adhering to the container walls. Transpar-
ent glass is widely used for bottling olive oil. This practice
leads to the photooxidation of olive oil and reduction of its
shelf life. The use of colored glass bottles, however, pre-
vents or slows down the oxidation process. Green bottles
protect oil from light rays with a wavelength of 300-
500 nm.
7.3 Metal
Various kinds of new metal containers have been devel-
oped for oil packaging since the mid-1960s. Tin-plated
containers are resistant to mechanical handling. They al-
so protect the oil from light and are suitable for litho-
graphic labeling. Furthermore, they are not very expen-
sive. Aluminum is also employed as packaging material. It
is resistant to rusting and corrosion. In order to increase
the mechanical resistance, the combination of aluminum
with other metals (Al/Mg, Al/Mn, Al/Si/Mg) is recommend-
ed.
Stainless steel material is mostly used for storage tanks
and oil tankers which are used for the transportation of
olive oil. They are highly resistant to mechanical damage
and corrosion. These iron material are less susceptible to
be attacked by the oil and can be considered as inert ma-
terial [27].
636 Kiritsakis et al. Eur. J. Lipid Sci. Technol. 104 (2002) 628638

Tab. 2. Types of containers used for bottling and packaging of olive oil and their characteristics.
Impermeability of Container Protection Resistance
against light to impact
to fat to gases
Tinplate E E E Sg
Colorless glass E E B B
Topaz glass E E A B
Light glass E E B/A B
Polyvinylchloride (PVC) E G B Sg
Polypropylene (PP) Sg Sg A G
Polyethylene (PE) A A B/A G
Polyethylene (PE) Sg Sg A G
Polyethylene tetraphthalate (PET) G G Sg G
E Excellent, G Good, Sg Semi-good, A Average, B Bad [1].
Low-density polymer.
High-density polymer.

7.4 Other material Supercritical fluid extraction/gas chromatography has

Tetra brick cartons have been used in Spain, in Brazil and
in other countries. Tetra brick packaging is inexpensive
and is considered more suitable for packaging olive oil.
Olive oil also appears in the form of spray, in the interna-
tional market. Kiritsakis and Hernandez [28] describe in
detail packaging and bottling of olive oil.
Tab. 2 shows some types of containers used for bottling
and packaging of olive oil and their characteristics.
been used to study the absorption of olive oil aroma com-
pounds into low-density-PE. The aroma compounds test-
ed were ethyl butyrate, butyl acetate, ethyl 2-methylbu-
tyrate, butyl propionate, hexyl acetate, hexanal, trans-2-
hexenal, isopentanol and hexanol. Flavors dissolved in
the oil were readily absorbed by the low-density PE. Olive
oil is nonpolar and is absorbed easily by the polymer. Fla-
vor concentration and storage temperature can affect the
absorption of aroma compounds when plastic packaging
material is used [28].

Sorption of oil into packaging materials, especially olefins,

8 Oil packaging interactions
8.1 Scalping (sorption)
Plastic packaging materials can absorb different com-
pounds from the food [29]. This phenomenon is called
scalping (sorption). Both, volatile and non-volatile com-
pounds may be absorbed. The absorption of non-volatile
constituents affects the packaging itself, while sorption of
volatiles (flavor and aroma constituents) affects the oil
quality more directly. Thus, sorption of flavors into the
plastic and subsequent diffusion through the plastic can
remove the characteristic flavors from olive oil. Removal
of flavor components can occur broadly across all the fla-
vor components or lead selectively to a flavor imbalance.
causes swelling of the polymer, which in turn increases
migration. The sorption of fatty acids increases with in-
creased chain length due to increased Van Der Waals
bonds between polymer and fatty acid. The absorption of
the oil is strongly influenced by convection and diffusion
coefficient of the oil [28].
The sorption of flavor olive oil compounds by polymeric
plastic materials during storage can result in a consider-
able decrease in the oil quality due to losses of potential-
ly acceptable organoleptic characteristics. The combined
understanding of mass transfer and industrial interest in
aroma scalping has driven the development of new
packaging structures and new polymeric materials, which
reduce flavor losses.

Kanavouras and Hernandez [30] studied the role of plas-

tic materials as potential flavor sorbents. Flavor com-
pounds might be indirectly related to changes of the ma-
terial properties due to swelling effects. That in addition to
the oil sorbed might be the reason for the changes of per-
meability and thermal analysis parameters.
8.2 Migration
Migration is the term used for the transfer of additives,
monomers and other compounds from packaging materi-
al into packaged products. Migration is of great concern in
the packaging community.
Eur. J. Lipid Sci. Technol. 104 (2002) 628638
Possible migrants from plastic packaging material are:
monomers, oligomers, catalyst residues or carrier sol-
vents, plasticizers, antioxidants, antifogging agents, col-
orants, emulsifiers, residual solvents and degradation
products. The migration of various components can affect
the safety and acceptability of olive oil [28].
9 Effect of packaging and bottling
material on olive oil quality
The nature of the packaging material such as PE,
polypropylene (PP) and PVC, has a notable influence on
olive oil quality [28]. Oil bottles permeable to air (PE, PP)
should be sold within four weeks. Air impermeable PVC
bottles can hold olive oil for three months. If the bottles
are colored, olive oil quality is retained due to the reduc-
tion in light transmittance at 670-680 nm. There are signi-
ficant changes in the oil quality stored in transparent glass
bottles and exposed to light. The decomposition of chloro-
phyll-a was used to determine the degree of oxidation.
Colorless oil bottled in white glass showed the lowest
deterioration.
A study on the relative effect of plastic and glass bottles
on the oxidation of olive oil exposed to diffused light
showed the following [1]; Oil samples stored in PE bottles
developed higher PV than those in glass bottles. Oil
stored in glass bottles wrapped in aluminum foil had low-
er PV than oil stored in plastic bottles which were also
wrapped in aluminum foil. Samples in plastic bottles
wrapped in aluminum foil displayed lower PV than those
in uncovered ones. Also, olive oil stored in polyethylene
bottles and exposed to diffused light for three months had
developed an off-taste and had lost most of its original
color. The decrease in tocopherols, -carotene and
chlorophylls is greater in illuminated samples of olive oil
than that in samples stored in darkness. Samples stored
in glass or PVC bottles in light undergo greater changes
in their sensory characteristics than those stored in dark-
ness [1]. Olive oil stored in colorless glass bottles and ex-
posed to diffused light lost all of its chlorophyll and about
70% of its carotene content. Oils are less deteriorated
when stored in glass recipients, especially when the latter
are made of colored glass (Tab. 2).
It should be noted that the quality of olive oil is highly af-
fected by the ability of the container to exclude oxygen
and light while the maintenance at relatively low tempera-
tures can further retard oxidative alterations. PVC and
PET proved to be quite adequate in preserving the quali-
ty of the extra virgin olive oil and avoid oxidation up to one
year when stored in the dark and at low temperatures.
Light and elevated temperatures have been shown to al-
ter the flavor profile of the oil.
Chemical analysis of olive oil 637
Regulations for packaged foods including oils have a long
history dating back to Roman times. Nowadays, great
care is taken because of the potential toxicity of some of
the monomers and other substances used to produce
packaging materials. Studies of the interactions and ab-
sorption of olive oil and packaging materials are needed
for the development of new packaging materials. Further
information about the migration of packaging material
components into olive oil is also needed. Additional stud-
ies are required to correlate the rate of oxidation of olive
oil when using colored glass bottles, which are known to
prevent or just slow down the rate of oxidation. It should
be noted that the EU has implemented and is still improv-
ing codes and regulations on materials for packaging.
10 Conclusion
In the previous years, olive oil received a wide recognition
throughout the world for its superior quality compared to
other oils and because of its impact on human health.
Several chemical analyses and quality tests were applied
to characterize its composition and to evaluate its quality
and detect adulteration. Interchange of research informa-
tion and knowledge in the field of chemical analysis, qual-
ity and packaging issues will further assist to establish a
worldwide acceptable product which will satisfy the con-
sumer preferences and demands.
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[Received: February 19, 2002; accepted: July 17, 2002]