Comparison of Different Extraction Methods to Determine

Phenolic Compounds in Virgin Olive Oil

M. Luz Pizarro & Mercedes Becerra & Ana Sayago &
Mara Beltrn & Rafael Beltrn
Received: 10 February 2012 / Accepted: 10 April 2012
#Springer Science+Business Media, LLC 2012
Abstract A comparison between different extraction meth-
ods has been performed in order to assess their effectiveness
for the analysis of total phenols (liquidliquid extraction
(LLE), liquidliquid micro extraction (LLME), and ultra-
sound liquidliquid extraction (USE)) and individual phe-
nols (LLME, USE, and solid-phase extraction (SPE)) from
virgin olive oil (VOO). Statistical analysis of the analytical
data obtained for the total phenol content of a VOO, showed
that LLME and USE can represent a reliable alternative to
the traditional procedure based on LLE that needs more
amount of sample, generates more wastes, and is more time
consuming. When an olive oil spiked with phenols was used
to test the efficiency of LLME, USE, and SPE methods, the
statistical analysis of the data obtained for each individual
phenol, again proved LLME and USE methods to be the
most suitable, in terms of precision and recoveries, for this
purpose. The analysis of real samples (Arbequina and Picual
cultivars) confirmed the results obtained for the spiked oil.
Keywords Phenols
.
Liquidliquid extraction
.
Liquid
liquidmicroextraction
.
Ultrasoundliquidliquidextraction
.
Solid-phase extraction
Introduction
Virgin olive oil (VOO) is the main edible oil of Mediterranean
diet. It is directly obtained from ripe olive fruits without any
further refining process. Among the other vegetable oils,
VOO provides beneficial effects to human health because of
its antioxidant capacity (due to its content of phenols, toco-
pherols, carotenoids, and other constituents) and its excellent
organoleptic and nutritional qualities (Mndez and Falqu
2007; Kiritsakis and Christie 2000).
The possible correlation between the antioxidant activity
and the quantity of phenolic compounds has been established
(Servili and Montedoro 2002; Ninfali et al. 2001). These
compounds contribute to the stability of VOO and prolong
its shelf-life compared to other vegetable oils (Angerosa et al.
2000; Toschi et al. 2005). Besides, these antioxidant species
give virgin olive oil an important role towards treatment and
prevention of chronic diseases associated to generation of
oxidants, such as atherosclerosis, neurodegeneration, and oth-
er normal process of aging. Phenolic compounds are also
related to the organoleptic characteristics affecting flavor and
taste (bitterness and astringency), and also to the nutritional
qualities of virgin olive oil (Andrewes et al. 2003; Gutirrez-
Rosales et al. 2003).
Polyphenols is a broad term used to define substances that
possess a benzene ring bearing one or more hydroxyl groups,
including functional derivative (Harborne and Dey 1989). The
main structural feature responsible for the anti-oxidative and
free radical-scavenging activity of phenolic derivates is the
phenolic hydroxyl group. Phenols are able to donate the
hydrogen atom of the phenolic OH to the free radicals, inter-
rupting the propagation of the oxidation process.
The phenolic fraction of VOO consists of a heteroge-
neous mixture of compounds, such as phenolic acids
(hydroxybenzoic and hydroxycinnamic derivatives), phe-
nylethyl alcohols (tirosol and hydrohytyrosol), secoiridoids
(aglyconic derivatives of oleuropein), and lignans. The con-
centration of these species is influenced by many factors
(Garca-Gonzlez et al. 2010) such as the area of production
(Tura et al. 2007; Vinha et al. 2005), climate (Kalua et al.
2005), cultivars (Tura et al. 2007), cultivation techniques
M. L. Pizarro
:
M. Becerra
:
A. Sayago (*)
:
M. Beltrn
:
R. Beltrn
Department of Chemistry and Science of Materials,
Area of Analytical Chemistry, Faculty of Experimental Sciences,
University of Huelva,
Agrifood Campus of International Excellence (ceiA3),
Avd. Tres de Marzo S/N. 21071,
Huelva, Spain
e-mail: ana.sayago@dqcm.uhu.es
Food Anal. Methods
DOI 10.1007/s12161-012-9420-8
(Tovar et al. 2001), fruit ripeness (Gutierrez et al. 1999), oil
extraction process (Servili et al. 1996), and storage condi-
tions (Morell et al. 2004).
Therefore, the qualitative and quantitative determination
of phenolic compounds in VOO is of special importance and
the development of efficient and rapid analytical methods
for its extraction is needed (Tsimidou et al. 1992). Many
analytical procedures for determination of phenolic com-
pounds have been proposed within the last decade (Brenes
et al. 1999; Brenes et al. 2000; Carrasco-Pancorbo et al.
2005). Several methods for the isolation of polar phenolic
fraction of VOO have been employed based on liquidliquid
extraction (LLE) (Brenes et al. 2000; Owen et al. 2000a;
Owen et al. 2000b; Vzquez Roncero 1978; Cortesi and
Fedell 1983; Cortesi et al. 1995), and solid-phase extraction
(SPE) (Servili et al. 1999; Favati et al. 1994; Favati et al.
1995; Andreoni and Fiorentini 1995; Mateos et al. 2001).
Different solvents such as lipophilic solvents with several
portions of methanol (Owen et al. 2000a, b), methanol/water
(Vzquez Roncero 1978; Cortesi and Fedell 1983), tetrahy-
drofuran/water (Cortesi et al. 1995), or N,N-dimethylforma-
mide (Brenes et al. 2000) have been utilized for LLE methods.
In regard to SPE, different sorbents have been used for the
isolation of polar components from VOO, being the most
common C
18
-cartridges (Servili et al. 1999; Favati et al.
1994, 1995), although anionic exchange cartridges (Andreoni
and Fiorentini 1995), amino phase cartridges, and diol-bond
phase SPE cartridges (Mateos et al. 2001) have also been used.
Usually, quantitative determination of total phenolic com-
pounds in VOO is performed according to FolinCiocalteau
colorimetric method (Gutfinger and Am 1981), while separa-
tion and determination of individual phenolic compounds in
the extracts often employs HPLC-DAD (high-performance
liquid chromatography equipped with a diode-array detector).
The aim of this research work is to compare different
extraction methods based on liquidliquid and solid-phase
extraction procedures in order to determine total and individual
phenol content optimizing the ratio quality-time of analysis.
Experimental Procedures
Chemicals
All the reagents used for spectrophotometric measures, such
us methanol, n-hexane, FolinCiocalteau reagent, glacial
acetic acid, and sodium hydroxide, were of spectrophoto-
metric grade (99.9 %) (Panreac, Barcelona, Spain). Tyrosol
(2-(4-hydroxyphenyl) ethanol) (97 %), caffeic acid (3,4-
dihydroxycinnamic acid) (98 %), vanillic acid (4-hydroxy-
3-methoxybenzoic acid) (97 %), p-coumaric acid (4-hydrox-
ycinnamic acid) (98 %), o-coumaric acid (2-hydroxycin-
nami c aci d) (97 %) and vani l l i n (4-Hydroxy-3-
methoxybenzaldehyde) (98 %) were purchased from Fluka
(Steinheim, Germany). Gallic acid (3,4,5-trihydroxybenzoic
acid) (99 %), rutin (2-(3,4-dihydroxyphenyl)-5,7-dihy-
droxy-3-[-L-rhamnopyranosyl-(1-6)--D-glucopyranosy-
loxy]-4H-chromen-4-one) (95 %), and sinapic acid (4-
hydroxy-3,5-dimethoxycinamic acid) (98 %) were from
Aldrich (USA). Apigenin (5,7-dihydroxy-2-(4-hydroxy-
phenyl)-4H-1-benzopyran-4-one) (99 %), luteolin (2-(3,4-
dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) (99 %),
and oleuropein ((4S,5E,6S)-4-[2-[2-(3,4-dihydroxyphenyl)
ethoxy]-2-oxoethyl]- 5-ethylidene-6-[[(2S,3R,4S,5S,6R)-
3,4,5-trihydroxy-6-(hydroxymethyl)- 2-tetrahydropyranyl]
oxy]-4H-pyran-3-carboxylic acid, methyl ester) (99 %) were
bought from Extrasynthse (Genay, France). Hydroxytyro-
sol [(3,4-dihydroxyphenyl) ethanol] was synthesized at the
Department of Organic and Pharmaceutical Chemistry of
the Faculty of Pharmacy (Seville, Spain).
Supelclean
TM
ENVI
TM
C18 SPE Tubes 6 mL (0.5 g)
(Supelco, USA) were used for solid-phase extraction. The
ultrapure water was obtained from a system of purification
Milli-Q (Millipore, Bedford, MA, USA).
Samples
A commercial extra virgin olive oil (EVOO) was used to
study the repeatability, reproducibility, and recoveries in
total phenolic compounds extraction. Four commercial
EVOO and a refined phenolics-free olive oil were used to
study the extraction methods for individual phenols.
For the individual phenolic compounds extraction assays, a
spiked olive oil was prepared adding the studied phenols.
Thirty grams of refined olive oil was weighed and transferred
to 100-mL round bottom flask. Stock standard solutions of
each compound were prepared in methanol and added to the
refined olive oil. The concentration of the 13 phenolic com-
pounds selected (gallic acid, hydroxytyrosol, tyrosol, vanillic
acid, caffeic acid, vanillin, p-coumaric acid, sinapic acid, o-
coumaric acid, rutin, oleuropein, luteolin, and apigenin) in the
spiked olive oil were 40 g/g. For 24 h, the prepared olive oil
was stirred under nitrogen atmosphere. After this process, a
rotary vacuum evaporator was used to eliminate the solvent.
Doped olive oil and standard solutions were stored at 4C.
Methods
Three different methodologies (liquidliquid micro extrac-
tion (LLME), LLE, and ultrasound liquidliquid extraction
(USE) (Hernanz et al. 2008; Rodrigues and Pinto 2007)
were used for the extraction of phenolic fraction from com-
mercial olive oils in order to determine the total content by
colorimetry. Figure 1 shows the scheme for the three pro-
cesses. Variations of two of these methodologies, LLME
and USE, and a solid-phase extraction method were also
Food Anal. Methods
used to obtain the phenolic extract for quantifying individ-
ual species (Fig. 2).
LiquidLiquid Extraction of Total Phenolic Compounds
The extraction procedure was performed according to the
method described in Vazquez et al. method (Vzquez Roncero
et al. 1973) slightly modified. In this method, 10 g of olive oil
were dissolved with 50 mL of n-hexane in a funnel. The
mixture was shaken for 2 min and the phenolic phase
extracted with three portions of 20 mL of methanol 60 %.
The lower methanolic phase was collected. The methanolic
phases were combined and diluted in 100 mL to the mark with
ultrapure water.
LiquidLiquid Micro Extraction of Total Phenolic
Compounds
Phenolic compounds were extracted using a variation of Mur-
kovic et al. method (Murkovic et al. 2004). Olive oil (500 mg)
were extracted with 1 mL of a methanol/water mixture (80:20,
v/v) in 2-mLEppendorf reaction tubes. After vigorous shaking
for 1 min using a vortex (Heidolph relax top, JP Selecta,
Barcelona), the sample was centrifuged (Eppendorf MiniSpin
Plus Microcentrifuges, Eppendorf, Madrid) at 13,400 rpm for
5 min at room temperature. This process was performed three
times. The three extracts were combined and the volume
adjusted to 10 mL with ultrapure water.
Ultrasound LiquidLiquid Extraction of Total Phenolic
Compounds
Olive oil (500 mg) was placed into polyethylene centrifuge
tube (50 mL, conical bottom) containing 1 mL of methanol/
water mixture (80:20, v/v) and directly sonicated using an
ultrasound probe (Bandelin Sonoplus UW 2200, Progen
Scientific, London, UK) with a titanium tip probe immersed
2 cm into solution for three cycles of 10 s at 20 C. Then, the
solution was placed into an Eppendorf tube and it was
centrifuged at 13,400 rpm for 5 min at room temperature.
This process was performed three times. The upper meth-
anolic phases were transferred into 10-mL volumetric flask
and diluted to the mark with ultrapure water.
Colorimetric Determination of Total Phenolic Content
The quantitative determination of total phenols in VOO
was colorimetric assay, following the method described
for Gutfinger et al. (Gutfinger and Am 1981), with
minor modifications. The method is based on the reac-
tion of FolinCiocalteau reagent with the functional
hydroxy groups of phenolic compounds. Five milliliters
Shake for 2 minutes
Add 20 mL Methanol 60%
Extract
methanolic phase
10 g sample + 50 mL n-Hexane in funnel
Dilute methanolic phase in 100 mL
volumetric flask with ultrapure water
0.5 g sample + 1 mL MeOH 80%
in 2 mL Eppendorf tubes
Shake Eppendorf tubes in a
vortex during 1 minute
Centrifuge Eppendort tubes at
13400 rpm for 5 minutes
Dilute methanolic phase in 10 mL
volumetric flask with ultrapure water
Liquid-liquid extraction (LLE) Liquid-liquid microextraction (LLME) Ultrasound extraction (USE)
0.5 g sample + 1 mL MeOH 80%
in 50 mL centrifuge tube
Centrifuge Eppendort tube at
13400 rpm for 5 minutes
Place the tubes on ice bath
Apply ultrasound for 30
seconds in cycles of 10 seconds
Transfer the sample to a
2 mL Eppendorf tube Extract
methanolic phase
Extract
methanolic phase
Dilute methanolic phase in
10 mL volumetric flask with ultrapure water
Add 1 mL
MeOH 80%
x2
Add 1 mL
MeOH 80%
x2
Add 20 mL
MeOH 60%
x2
EXTRACTION METHODS FOR TOTAL PHENOLS DETERMINATION
Fig. 1 Scheme of the extraction methods for total phenols determination
Food Anal. Methods
of the aqueous-methanolic solution of phenolic compounds
extracted from VOO was diluted in 15 mL of ultrapure water,
followed by the addition of 1.25 mL of FolinCiocalteau
reagent and maintained for 3 min. Then, 2.5 mL of sodium
hydroxide 6 % (v/v) were added. After 5 min, the reaction
mixture was mixed and diluted with ultrapure water to 25 mL.
The absorbance of the solution was measured after 2 h against
a blank sample using a UVvis spectrophotometer (Termo
Spectronic Helios- v4.60, Cambridge, UK) at a wavelength
of 725 nm. The calibration curve was constructed using stan-
dard solutions of caffeic acid within the range 010 mg/L.
Results were expressed as milligrams of caffeic acid per
kilogram of oil.
LiquidLiquid Micro Extraction of Individual Phenols
The phenolic fraction for individual phenols determination,
was extracted following the method previously described
(LiquidLiquid Micro Extraction of Total Phenolic Com-
pounds section) with minor variations in order to avoid
sample manipulation. Five hundred milligrams of olive oil
was extracted with 1 mL of methanol/water mixture (80:20,
v/v) in 2-mL Eppendorf tubes. The mixture was vortexed for
1 min and centrifuged at 13,400 rpm for 5 min at room
temperature. The other two cycles were eliminated to avoid
an excess of methanol that would dilute the extract and should
be eliminated by rotary vacuum evaporator. Methanolic phase
was filtered through a 0.45-m pore size and 17-mm diameter
nylon filter. Of the methanolic phase, 0.150 mL was diluted
with ultrapure water up to 0.5 mL.
Ultrasound LiquidLiquid Extraction of Individual Phenols
The extraction method was a variation of the one pre-
viously described (Ultrasound LiquidLiquid Extraction of
Total Phenolic Compounds section) with minor variations for
avoiding sample manipulation. Only one extraction cycle was
performed. Methanolic phase was filtered through a 0.45-m
pore size and 17-mm diameter nylon filter. Of the methanolic
phase, 0.150 mL was diluted with ultrapure water up to
0.5 mL.
Solid-Phase Extraction of Individual Phenols
For the solid-phase extraction of the phenolic compounds, a
modification of the method described by Pirisi et al. (2000)
was used. Olive oil (500 mg) was dissolved in 7.5 mL of
hexane and the mixture was stirred for 1 min with a vortex.
Pass the diluted sample
through the activated cartridges
Eliminate apolar fraction
Wash with 5 5 mL
n-Hexane
0.5 g sample + 7.5 mL n-Hexane
Elut with 4 5 mL
MeOH
Recuperate polar fraction
Re-dissolve with 500 L
MeOH
Evaporate the extraction to dryness
with a rotavapor T<40C
Take 0.150 mL of methanolic phase and
dilute with ultrapure water up to 0.5 mL
0.5 g sample + 1 mL MeOH 80%
in 2 mL Eppendorf tubes
Shake Eppendorf tubes in a
vortex during 1 minute
Centrifuge Eppendorf tubes at
13400 rpm during 5 minutes
Filter through a 0.45 m pore size
and 17 mm diameter nylon filter
Take 0.150 mL of methanolic phase and
dilute with ultrapure water up to 0.5 mL
Solid-phase extraction (SPE) Liquid-liquid microextraction (LLME) Ultrasound extraction (USE)
0.5 g sample + 1 mL MeOH 80%
in 2 mL Eppendorf tubes
Take 0.150 mL of methanolic phase and
dilute with ultrapure water up to 0.5 mL
Filter through a 0.45 m pore size
and 17 mm diameter nylon filter
Centrifuge Eppendorf tubes at
13400 rpm during 5 minutes
Introduce the sample in an ice bath
Apply ultrasound for 30
seconds in cycles of 10 seconds
Transfer the sample to a 2 mL
Eppendorf tube
EXTRACTION METHODS FOR INDIVIDUAL PHENOLS DETERMINATION
Filter through a 0.45 m pore size
and 17 mm diameter nylon filter
Fig. 2 Scheme of the extraction methods for individual phenols determination
Food Anal. Methods
An octadecyl C18 phase cartridge was placed in a vacuum
elution apparatus and conditioned by consecutive addition of
25 mL of methanol and 25 mL of hexane. The vacuum
was then released to prevent the column from drying. The oil
solution was applied to the column, and the solvent was pulled
through, leaving the sample on the solid phase. To eliminate
the apolar fraction, the cartridge was eluted with 55 mL of
hexane, and the polar compounds retained were recovered
with 45 mL of methanol. Methanolic phase was evaporated
under vacuum and dissolved with 0.5 mL of methanol. Meth-
anolic phase was filtered through a 0.45-m pore size and 17-
mm diameter nylon filter. Of the methanolic phase, 0.150 mL
was diluted with ultrapure water up to 0.5 mL.
HPLC
All individual phenols were analyzed by high-performance
liquid chromatography. An Agilent 1100 series (Palo Alto,
CA, USA) chromatograph equipped with a diode-array de-
tector was used. A gradient of solvent A (watermethanol
acetic acid, 93:5:2, v/v/v) and solvent B (methanolacetic
acid, 98:2, v/v) was applied to a reversed-phase Nova-pack
C18 column (4 m, 3003.9 mm I.D, Waters Co., Milford,
Massachusetts, USA) as follows: from 0 to 5 min,
100 %A:0 %B; from 5 to 50 min, 60 %A:40 %B; from 50
t o 60 mi n, 40 %A: 60 %B; from 60 t o 65 mi n,
25 %A:75 %B; from 65 to 70 min, 0 %A:100 %B; and
from 70 to 80 min, 100 %A:0 %B. The flow rate was
1 mL min
1
and the column was termostatized at 20 C.
The volume injected was 100 L. Detection was performed
by scanning from 190 to 700 nm. Quantification of phenolic
compounds was carried out using the area values measure-
ments at 280 nm for gallic acid, hydroxytyrosol, tyrosol,
vanillic acid, caffeic acid, vanillin, o-coumaric acid, and
oleuropein, at 320 nm for p-coumaric acid and sinapic acid
and at 360 nm for rutin, luteolin, and apigenin. Figure 3
shows the elution phenolic profiles of a standard mixture (a
c) and Picual virgin olive oil extract (df) at 280 nm (a, d),
320 nm (b, e), and 360 nm (c, f). The chromatograms show
that phenylethyl alcohols and simple phenols eluted in first
place before 45 min, while flavonoids due to their greater
hydrophobicity, elute after 60 min increasing the concentra-
tion of methanol in the mobile phase. Identification of
chromatographic peaks was carried out by comparing their
retention times and spectra with those of standards.
Quantitative assays were achieved using external calibra-
tion curves for all standard phenols. The extraction methods
were applied to the refined oil in order to use the results as
blank. All analyses were carried out in triplicate and the
results were expressed as mean values.
Statistics
Significant differences between extraction techniques were
tested by one-way analysis of variance (ANOVA). Statistical
differences with p values under 0.05 were considered sig-
nificant and means were compared by 95 % least squares
differences test (LSD).
Two multivariate methods were used for the data analy-
sis: cluster analysis (CA) and principal component analysis
Fig. 3 Elution phenolic profiles of a standard mixture (a, b, c) and
Picual virgin olive oil extract (d, e, f) at 280 nm (a, d), 320 nm (b, e),
and 360 nm (c, f). 1: Gallic acid; 2: hydroxytyrosol; 2: hydroxytyrosol
derivate; 3: tyrosol; 4: vanillic acid; 5: caffeic acid; 6: vanillin; 7: p-
coumaric acid; 8: sinapic acid; 9: o-coumaric acid; 10: rutin; 11:
oleuropein; 12: luteolin; 13: apigenin; 13: apigenin derivate
Food Anal. Methods
(PCA). Cluster analysis provides means for classifying a
given population into groups (clusters), based on similarity
or closeness measures. PCA is one of the most powerful and
common techniques used for reducing the dimensionality of
large sets of data without loss of information. New variables
obtained as linear combination of the original ones are
calculated in such a way as to keep most of the information
present in the original data set in the least possible number
of new variables or principal components (PCs). PCA was
applied for visualizing data trends.
All the statistical analyses were performed using Statis-
tica v.6.0 software (StatSoft Inc., Statistica 6.0 for Win-
dows Computer Program Manual, Tulsa, OK, 2001).
Results and Discussion
Precision and Recoveries
The precision of the method was evaluated by determining the
repeatability and internal reproducibility of the recoveries; the
precision was estimated by means of the averages of the
coefficient of variation (%RSD). The information of the re-
peatability was attained by analyzing five replicates of the
same oil sample (EVOO var. Picual for total phenol content
and prepared oil for individual phenol extraction methods) in a
work session, while the internal reproducibility was studied by
five independent assays performed under the same analytical
conditions in five different days by two different analysts.
Each day five replicates of the same oil sample were analyzed.
Concerning the precision of the total phenols extraction
methods (Table 1), the coefficients of variation (%RSD)
were fine for the three methods, displaying values lower
than 9.4 % for repeatability and 7.9 % for reproducibility,
being LLE the method that showed better values. In the
other hand, USE method showed the highest values for
precision (9.4 % for repeatability and 7.9 % for reproduc-
ibility); this fact could be attributed to the difficulty on
maintaining the temperature of the sample during the pro-
cess. Finally, LLME method presents good values of RSD
for repeatability (5.1 %) and reproducibility (7.4 %) and has
the advantage of needing less amount of sample, generating
less waste and being less time consuming.
Concerning the values obtained for total phenols (Ta-
ble 1), a one-way ANOVA test was applied, significant
differences (p<0.05) were observed as a function of factor
extraction method. When LSD test was applied to deter-
mine the significant differences between the methods, no
significant differences were found among the LLME and
USE methods, whereas they presented average values sig-
nificantly different compared to those obtained by LLE.
Although the best results for precision were obtained by
LLE method, this technique showed the lowest values for
total phenols extracted from olive oil. This method also
needs more amount of sample, generates more wastes, and
is more time consuming than the other studied methods.
Table 1 Repeatability, reproducibility, and mean value of total phenol
extraction methods
Method r (%) R (%) Mean value
LLE 2.7 6.8 427.08 a
LLME 5.1 7.4 449.58b
USE 9.4 7.9 459.57b
r repeatability, R reproducibility expressed as relative standard
deviation
Different letters (a,b) in the same column imply significant differences
Table 2 Repeatability and re-
producibility of individual phe-
nol extraction methods
r repeatability, R reproducibility
expressed as relative standard
deviation, Re recoveries
SPE LLME USE
r (%) R (%) Re (%) r (%) R (%) Re (%) r (%) R (%) Re (%)
Gallic acid 7.3 11.3 53.6 0.8 1.2 77.3 1.3 3.5 82.3
Hydroxytyrosol 7.7 9.3 73.0 4.7 3.7 83.6 2.7 4.4 93.0
Tyrosol 6.6 7.5 96.6 2.4 3.0 84.0 2.3 3.0 94.9
Vanillic acid 5.9 7.5 96.6 2.5 3.5 84.3 2.7 3.2 95.7
Caffeic acid 4.7 9.0 72.4 1.3 3.8 80.1 1.6 2.5 92.6
Vanillin 6.3 6.7 93.8 1.6 3.3 78.3 2.1 2.7 85.9
p-Coumaric acid 5.3 7.9 92.8 2.7 2.7 84.4 1.9 2.3 95.8
Sinapic acid 5.5 8.0 74.3 0.7 3.7 82.6 4.4 4.6 88.5
o-Coumaric acid 5.8 11.9 90.9 3.2 4.6 82.1 3.5 4.2 95.9
Rutin 6.3 10.7 40.6 0.9 3.2 81.6 8.3 6.6 82.2
Oleuropein 4.9 6.2 40.8 3.2 4.8 85.0 7.2 5.4 89.7
Luteolin 6.1 8.5 61.7 1.8 3.2 74.4 1.4 2.9 86.8
Apigenin 4.9 7.3 74.2 1.6 2.9 72.0 1.6 2.5 82.1
Food Anal. Methods
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Food Anal. Methods
Therefore, and also in order to avoid the high generation of
wastes from LLE method, this method was excluded for the
following experience of determining individual phenols
compounds, and a new method based on solid-phase extrac-
tion was applied.
Concerning the precision of individual phenols ex-
traction methods, the recoveries and the coefficients of
variation (%RSD) for repeatability and reproducibility of
the individual compounds are displayed in Table 2. In
relation to SPE method, the repeatability shows values
ranging from 4.7 to 7.7 % of RSD while reproducibility
presents higher values, being greater than 10 % for
gallic acid, o-coumaric acid, and rutin, the lowest RSD
were obtained for oleuropein (6.3 %). With regard to
the repeatability of LLME extraction the RSD values
were lower than 4.7 % for all phenolic compounds.
Likewise reproducibility values show relative standard
deviations (%RSD) ranging from 1.2 to 4.8 %. Repeat-
ability of USE method showed RSD values around
1.5 % for gallic acid, caffeic acid, p-coumaric acid,
luteolin, and apigenin being in the 24 % range for
the rest of the compounds except to oleuropein (7 %)
and rutin (8 %). However reproducibility, displays val-
ues lower than 6.6 % for all the phenols studied.
Taking into consideration the 13 phenolic compounds
studied, LLME was the best method in terms of precision
and hence the method providing highest repeatability and
reproducibility (Table 2).
To determine recoveries, an average value of the consec-
utive extractions performed on each method was applied to
the spiked oil. For each phenolic compound, the recovery
rate was calculated by the ratio (C1/C2)100, where C1 is
the means of measured concentrations in sample, and C2 is
the amount of the analyte added to prepared oil. When the
SPE extraction procedure was applied, recoveries ranging
from 40 to 96 % (73.9 % in average) were obtained. The
lowest recoveries (<60 %) were obtained for rutin, oleuro-
pein, and gallic acid whereas the highest ones (>90 %)
corresponded to tyrosol, vanillic acid, and o-coumaric acid.
Recoveries of gallic acid, flavonoids (luteolin, apigenin, and
rutin), and secoroids (oleuropein) obtained with SPE meth-
od showed smaller values than the other extraction method.
With regards to the LLME method, recoveries varying from
72 % (apigenin) to 85 % (oleuropein) were obtained with an
averaged recovery of 80.7 %. When USE method was
applied, the best recoveries were found, achieving values
between 82 % (gallic acid, apigenin, and rutin) and 95 %
(vanillin acid, p-coumaric acid, and o-coumaric acid)
Fig. 4 Cluster analysis obtained from the extraction methods for individual phenols. (A: Arbequina samples; P: Picual samples)
Food Anal. Methods
(89.6 % in average). The existence or not of significant
differences in the levels of phenols detected, as function of
the extraction method was evaluated by the analysis of
variance (ANOVA). The result obtained when the values
of all the phenols were considered, showed that there were
statistically significant differences (p<0.05) between the
methods applied. When the univariate results were ana-
lyzed, the existence of significant differences for individual
phenols extracted was obtained except for tyrosol, vanillin,
vanillic acid, and o-coumaric acid.
When LSD test was applied, similar behavior was ob-
served for phenols belonging to the group of acids and
phenylethyl alcohols, in which USE and LLME showed
no significant differences, and the percentage of extraction
was higher than for the SPE method, while phenols belong-
ing to the group of flavonoids presented significant differ-
ences between the three methods being the USE method
which offers higher rates of extraction.
Analysis of Real Samples
The phenolic profile of four commercial extra virgin olive
oils of two different varieties (Arbequina and Picual) was
assessed by applying the three extraction methods. The
values obtained have been summarized in Table 3.
Analyzing the information provided by the three extrac-
tion methods for individual phenols, it was found that for
Arbequina samples the predominant phenolic compounds
were phenylethyl alcohols, representing 84 % of total phe-
nolic compounds analyzed, followed by flavonoid com-
pounds representing 12 %, benzoic acids 1.3 %, phenolic
aldehydes 1 %, and the last one were cinamic acids, 0.8 % of
total phenolic analyzed. A similar profile was obtained for
Picual samples with 88, 8.7, 0.9, 0.8, and 0.6 % for phenyl-
ethyl alcohols, flavonoids, benzoic acids, cinamic acids, and
phenolic aldehydes, respectively, of total phenolic com-
pounds analyzed. When ANOVA was applied, significant
differences (p00.037) were observed as a function of vari-
ety. The univariate results showed that there were significant
differences (p<0.05) in levels of phenylethyl alcohols, ben-
zoic acids, o-coumaric acid, luteolin, and apigenin derivate.
The data set obtained from the three extraction methods
for individual phenols was employed to perform a cluster
analysis, applying city-block Manhattan distance algorithm
and Ward's method as linkage rule. The results were
reported as a dendrogram, shown in Fig. 4. On the basis of
the connecting distances, two main clusters were defined,
one for each of the varieties of olive oil. Each of the clusters
is subdivided into two groups, one including the samples
taken with a solid-phase extraction method (SPE) and the
Fig. 5 Principal component analysis from the extraction methods for individual phenols. (A: Arbequina samples; P: Picual samples)
Food Anal. Methods
other, those extracted by liquidliquid methods (LLME and
USE). This confirms the results obtained for the spiked oil.
Similar results were obtained when principal component
analysis (PCA) was applied to the matrix of data. Three PCs
with eigenvalues higher than 1.0 were extracted (Kaiser's
criterion), which accounted for 95.6 % of the total variance.
Figure 5 shows the PC1/PC2 score plot, in which the first
two principal components summarize more variation in the
data than any other principal components, accounting for
84.73 % of the data variance. PC1 (52.95 % explained
variance) allowed the distinction between varieties of olive
oil. Hydroxytyrosol, tyrosol, and vanillic acid with positive
loading values and o-coumaric acid with negative loading
values were the variables corresponding to PC1. PC2
(31.78 % explained variance) led to differentiate the liq-
uidliquid extraction method from the solid-phase extrac-
tion method. Luteolin, apigenin, and derivates of apigenin
and hydroxytyrosol with negative loading values, were the
variables correlated with PC2.
Conclusions
It was noticed that the method leading to higher mean values
for total phenols was USE. However, the existence of no
significant differences between this and LLME, added to the
latter has better values for repeatability and reproducibility,
and its inherent advantages such as lower solvent and time
consuming, makes LLME method of choice for total phenol
extraction from olive oil. When LLME and USE methods
were compared with SPE method for the extraction of
individual phenols, again it was proved that liquidliquid
extraction methods were the most suitable for this purpose.
Acknowledgments The authors gratefully acknowledge Daniel
Sanchez Rodas (University of Huelva) for their helpful assis-
tance. We also thank industrial olive oil mills from Beas, San
Bartolom, Trigueros, and Gibralen (Huelva, Spain) for provid-
ing us with the samples for this work. This investigation has
been supported by RISE (0042_RISE_5_E) grant.
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