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Abstract

Extra virgin olive oil (EVOO) has a long history of economic adulteration, the detection of which presents significant challenges due to the diverse composition of cultivars grown around the world and the limitations of existing methods for detecting adulteration. In this study, using Method COI/T.20/Doc. No. 30/Rev. 1 of the International Olive Council, the authenticity of 88 market samples of EVOO was evaluated by comparing total sterol contents, desmethylsterol composition, and contents of triterpene dialcohols (erythrodiol and uvaol) with purity criteria specified in the United States Standards for grades of olive oil and olive-pomace oil. Three of the 88 samples labeled as EVOO failed to meet purity criteria, indicating possible adulteration with commodity oil and/or solvent-extracted olive oil. Detection of adulteration was also evaluated by spiking an EVOO sample with commodity oil at the 10 % level. As expected, eight of the spiked samples (canola, corn, hazelnut, peanut, safflower, soybean, and sunflower oils, and palm olein) failed to meet purity criteria. Two of the three samples spiked with 10 % hazelnut oil went undetected for adulteration. Overall, a low occurrence rate of adulteration (<5 %), based on purity criteria for desmethylsterols and triterpene dialcohols, was detected for the 88 products labeled as EVOO.
J Am Oil Chem Soc
DOI 10.1007/s11746-015-2759-4
1 3
ORIGINAL PAPER
Authenticity Assessment of Extra Virgin Olive Oil: Evaluation
of Desmethylsterols and Triterpene Dialcohols
Cynthia T. Srigley1 · Carolyn J. Oles1 · Ali Reza Fardin Kia1 · Magdi M. Mossoba1
Received: 29 June 2015 / Revised: 15 October 2015 / Accepted: 13 November 2015
© AOCS (outside the USA) 2015
Keywords Adulteration · Authenticity · Desmethylsterol ·
Erythrodiol · Extra virgin olive oil · Gas chromatography ·
Purity · Sterol · Triterpene dialcohol · Uvaol
Abbreviations
ANOVA Analysis of variance
AOCS American Oil Chemists’ Society
BSTFA N-O-bis(trimethylsilyl)trifluoroacetamide
CAS Chemical Abstracts Service
EVOO Extra virgin olive oil
FDA United States Food and Drug Administration
GC-FID Gas chromatography with flame ionization
detector
IOC International Olive Council
IS Internal standard
SD Standard deviation
SRM Standard reference material
TCF Theoretical correction factor
TLC Thin layer chromatography
TMSE Trimethylsilyl ether
USDA United States Department of Agriculture
Introduction
Extra virgin olive oil (EVOO) is highly regarded for its
nutritive value and potential health benefits [1]. These oils
sell at a premium for their desirable organoleptic properties
and rich concentration of bioactive constituents [1]. How-
ever, the discrepancy in pricing between EVOO and other
commodity oils has rendered this product a primary target
for fraudulent activities, namely economic adulteration and
deliberate mislabeling. The detection of economic adul-
teration poses significant challenges for EVOO due to the
diverse composition of cultivars grown around the world
Abstract Extra virgin olive oil (EVOO) has a long his-
tory of economic adulteration, the detection of which pre-
sents significant challenges due to the diverse composition
of cultivars grown around the world and the limitations of
existing methods for detecting adulteration. In this study,
using Method COI/T.20/Doc. No. 30/Rev. 1 of the Interna-
tional Olive Council, the authenticity of 88 market samples
of EVOO was evaluated by comparing total sterol con-
tents, desmethylsterol composition, and contents of triter-
pene dialcohols (erythrodiol and uvaol) with purity criteria
specified in the United States Standards for grades of olive
oil and olive-pomace oil. Three of the 88 samples labeled
as EVOO failed to meet purity criteria, indicating possible
adulteration with commodity oil and/or solvent-extracted
olive oil. Detection of adulteration was also evaluated by
spiking an EVOO sample with commodity oil at the 10 %
level. As expected, eight of the spiked samples (canola,
corn, hazelnut, peanut, safflower, soybean, and sunflower
oils, and palm olein) failed to meet purity criteria. Two of
the three samples spiked with 10 % hazelnut oil went unde-
tected for adulteration. Overall, a low occurrence rate of
adulteration (<5 %), based on purity criteria for desmethyl-
sterols and triterpene dialcohols, was detected for the 88
products labeled as EVOO.
This manuscript is based on work presented at the 106th AOCS
Annual Meeting and Industry Showcases (Abstract # 62537)
* Cynthia T. Srigley
cynthia.srigley@fda.hhs.gov
1 Center for Food Safety and Applied Nutrition, United States
Food and Drug Administration, 5100 Paint Branch Parkway,
College Park, MD 20740, USA
J Am Oil Chem Soc
1 3
and the limitations of existing official methods for detect-
ing adulteration. Moreover, technological advances for
detecting adulteration have been paralleled by increasingly
sophisticated strategies for masking fraudulent activity [2].
In 2010, the Agricultural Marketing Service of the
United States Department of Agriculture (USDA), in
response to a petition submitted by the California Olive Oil
Council, revised its voluntary US Standards for grades of
olive oil and olivepomace oil (effective October 25, 2010)
to reflect current industry standards commonly accepted
in the US and abroad for determining quality and purity
among grades of olive and olive-pomace oils [3]. The
revised US grades standards, which replaced the first edi-
tion that took effect on March 22, 1948, were designed to
provide definitions for olive oil and olive-pomace oil, to
facilitate marketing of such oils, to promote truthfulness in
labeling, and to serve as a basis for enforcement by State
and Federal agencies if such oils were found to be misla-
beled [3]. The revised US grade standards were based on
trade standards of the IOC, with exceptions for limitations
on the concentrations of alpha-linolenic acid (C18:3n-3)
and campesterol to more accurately reflect variability
among cultivars grown in the US and other areas outside
of the Mediterranean region. A total of 22 tests are recom-
mended to fully evaluate the quality and purity of an olive
oil product. Chemical tests to ascertain purity include total
sterol content, desmethylsterol composition, fatty acid
composition, trans fatty acid content, and stigmastadiene
content. Secondary or confirmatory tests, such as contents
of triterpene dialcohols (erythrodiol and uvaol), waxes or
2-glyceryl monopalmitate, or the calculated difference
between actual and theoretical contents of triacylglycer-
ols with equivalent carbon number 42, are to be performed
when analyzed values for alpha-linolenic acid and camp-
esterol fall within specified ranges (i.e., 1.0–1.5 % of total
fatty acids and 4.0–4.5 % of total sterols, respectively) [3].
The unsaponifiable fraction of EVOO, which consists
of sterols (desmethylsterols, monomethylsterols, dimethyl-
sterols), hydrocarbons, aliphatic alcohols, and tocophe-
rols, serves as a valuable tool, or fingerprint, for evaluating
authenticity [2]. The US grades standards specify threshold
values for total sterol content (calculated as the sum of the
contents of all desmethylsterols) and desmethylsterol com-
position that may be used in the authentication of olive
oil [3]. Oils failing to meet purity criteria for desmethyl-
sterol composition would be suspected of adulteration with
commodity oil due to differences in the total sterol con-
tent and desmethylsterol composition among oil types [4].
However, the desmethylsterol composition of EVOO has
also been shown to vary by cultivar, growing region, and
ripeness of the olive fruit [2], thus complicating the role
of desmethylsterols for evaluating authenticity. For exam-
ple, several recent studies have reported concentrations of
desmethylsterols in monovarietals grown in Argentina [5],
Australia [6], Tunisia [7], Turkey [8], and the US [9] that
exceeded threshold values for purity specified in the US
grade standards.
The US Food and Drug Administration (FDA) is tasked
with, among others, protecting the US food supply against
deceptive labeling and intentional adulteration (Federal
Food, Drug, and Cosmetics Act, FD&C Act; Sec. 402–
403). This mission is carried out, in part, by laboratory
analysis of domestic and import products and by system-
atic evaluation of the performance of analytical methods
and accuracy of acceptance criteria for determining authen-
ticity. The objective of the present study was to evaluate,
based on IOC methodology, the authenticity of 88 market
samples of EVOO by comparing analyzed values for total
sterol content, desmethylsterol composition, and content of
triterpene dialcohols with purity criteria specified in the US
grades standards [3]. A secondary objective was to iden-
tify commodity oils that could potentially be detected as
adulterants at the 10 % level of adulteration when analyzed
using Method COI/T.20/Doc. No. 30/Rev. 1 “Determina-
tion of the Composition and Content of Sterols and Trit-
erpene Dialcohols by Capillary Column Gas Chromatogra-
phy” [10] and compared with the US grades standards for
contents of desmethylsterols and triterpene dialcohols. This
work is part of a larger study by FDA’s Center for Food
Safety and Applied Nutrition to evaluate the performance
of official chemical methods for testing olive oil quality
and purity, and to detect adulterants in EVOO by develop-
ing new, rapid spectral methods.
Experimental Procedures
Samples
Commercial EVOO products, refined olive oil, olive oil
blends, and commodity oils (i.e., canola, corn, hazelnut,
peanut, safflower, soybean, and sunflower oils, and palm
olein) were purchased between August and October of
2013 from retail stores in the Washington, DC metropolitan
area or from online sources. A total of 93 olive oils were
acquired, of which, 88 were labeled as EVOO, two (2)
were labeled as olive oil (i.e., refined), and three (3) were
labeled as blends of EVOO and commodity oil (canola,
sunflower, or vegetable oil). Sixty-seven (67) of the oils
were labeled as single-country products (n = 1, Argentina,
1; Australia, 1; Chile, 6; France, 1; Germany, 5; Italy, 19;
Mexico, 1; Peru, 1; Portugal, 1; South Africa, 1; Spain, 17;
Tunisia, 5; United States, 8). The remaining 26 oils were
labeled as blends of oils originating from two or more
countries (e.g., Italy and Spain, or Mediterranean blend).
The average cost per 100 mL for the 88 samples labeled
J Am Oil Chem Soc
1 3
as EVOO was $3.4 ± 2.8 (range $0.5–$14.0 per 100 mL).
Intentionally-adulterated test samples were prepared by
adding 10 % commodity oil (weight/weight) to a sample of
EVOO which met purity criteria for EVOO. All oils were
stored in darkness at ambient temperature (24 ± 1 °C)
when not in use.
Reagents and Standards
ACS reagent grade chemicals (N,O-bis(trimethylsilyl)
trifluoroacetamide, BSTFA, containing 1 % trimethyl-
chlorosilane; chloroform; 2,7-dichlorofluorescein; ethyl
ether, anhydrous, stabilized with butylated hydroxytolu-
ene, 99 %; potassium hydroxide (KOH) pellets, 85 %;
and sodium sulfate, Na2SO4, anhydrous, granular, 99 %),
HPLC grade solvents (acetone, 99.7 %; ethyl acetate,
>99.8 %; and heptane, 99.5 %), and pyridine (anhydrous,
99.8 %) were purchased from Fisher Scientific (Pittsburgh,
PA, USA) or Sigma-Aldrich (St. Louis, MO, USA). Etha-
nol (200 proof) was purchased from VWR International
(Radnor, PA, USA).
The internal standard (IS), α-cholestanol (cholestan-3-ol,
(3β,5α); 95 %), and analytical standards for brassicast-
erol (98 %), erythrodiol (97.0 %), and uvaol (>95 %)
were purchased from Sigma-Aldrich. Phytosterols (mixture
of soya sterols, P/N P18680) was purchased from Pfaltz &
Bauer (Waterbury, CT). Standard Reference Material (SRM)
3251 “Serenoa repens Extract” was purchased from the
National Institute of Standards and Technology (Gaithers-
burg, MD). Test samples from the 2012–2013 AOCS Olive
Oil Laboratory Proficiency Program were acquired from the
American Oil Chemists’ Society (AOCS; Urbana, IL).
Analysis of Desmethylsterols and Triterpene Dialcohols
in Olive Oil
Oils were analyzed according to Method COI/T.20/Doc.
No. 30/Rev. 1 [10]. Briefly, sample test portions (5 g) were
saponified in 2 M ethanolic KOH solution. The unsaponi-
fiable material was extracted using diethyl ether (210 mL)
and evaporated under a stream of nitrogen gas. The unsa-
ponifiable material was then solubilized to 100 μg/μL in
chloroform, and approximately 150 μL were spotted onto
silica gel G thin layer chromatography (TLC) plates (Anal-
tech, Newark, DE, USA) that were previously treated with
0.2 M ethanolic KOH solution. Plates were developed in a
solvent system consisting of hexane/diethyl ether (50:50,
volume/volume). Individual bands were identified by
spraying with 2,7-dichlorofluorescein solution (0.1 % in
ethanol, weight/volume) and visualized under ultraviolet
light. The desmethylsterol and triterpene dialcohol frac-
tions were scraped from the TLC plates, extracted from the
silica gel with chloroform, evaporated under nitrogen gas,
and derivatized to trimethylsilyl ethers (TMSE) by addition
of BSTFA (250 μL) and pyridine (250 μL) followed by
heating at 70 °C for 15 min.
Desmethylsterol and triterpene dialcohol TMSE deriva-
tives were analyzed on a 6890 N gas chromatograph
equipped with a flame ionization detector (GC-FID; Agi-
lent Technologies, Wilmington, DE, USA) and an SE-54
column (30 m × 0.32 mm, 0.25 μm film; Agilent Technol-
ogies). The oven was maintained at 250 °C for 55 min, then
ramped at 15 °C/min to 265 °C, and held for 5 min. Post
run, the oven temperature was decreased to 250 °C and
held for 3 min for a total run time of 64 min. The flow rate
for the hydrogen (H2) carrier gas was set at 1.4 mL/min.
Flow rates for air, H2, and nitrogen gases at the FID were
400, 30, and 30 mL/min, respectively. The inlet and detec-
tor temperatures were 290 °C. The split ratio was 10:1 and
the injection volume was 1 μL, producing a total column
load of approximately 100 ng per injection.
Peaks corresponding to desmethylsterol and triterpene
dialcohol TMSE derivatives were identified by comparison
of elution profile with chromatograms presented in Method
COI/T.20/Doc. No. 30/Rev.1 [10] and the literature [11].
Further verification of peak identity was achieved by reten-
tion time comparison with neat analytical standards for
brassicasterol, uvaol, erythrodiol, and the phytosterol refer-
ence standard for which sterol/stanol structural elucidations
by electron impact mass spectrometry were previously
reported by our laboratory [12].
Calculations
Quantification of individual desmethylsterols and triterpene
dialcohols was performed according to Eq. 1. Quantifica-
tion involved the use of (1) theoretical correction factors
(TCF), which were calculated relative to α-cholestanol
TMSE [13], and (2) sterol conversion factors (FSx), which
converted sterol TMSE derivatives to their free sterol
equivalents (Table 1).
where WSTMSEx is the concentration (in mg/kg) of sterolx
(as TMSE); Ax is the peak area counts for sterolx (TMSE);
Wis is the weight (in mg) of α-cholestanol (IS); 1.1857 is
the conversion of IS to TMSE form in test; TCFx is the TCF
for sterolx; Ais is the peak area counts for α-cholestanol
TMSE (IS); WTS is the weight (in g) of test sample; WSx is
the concentration (in mg/kg) of sterolx (as free sterol equiv-
alent); FSx is the factor for conversion of sterol TMSEx to
its corresponding free sterol equivalent.
Total sterol content was calculated as the sum of the
analyzed contents of individual desmethylsterols, namely
(1)
W
STMSEx =
Ax×Wis ×
1.1857
×
TCF
x×
1000
Ais ×WTS
WSx
=W
STMSEx
×F
Sx
J Am Oil Chem Soc
1 3
cholesterol, brassicasterol, 24-methylene cholesterol, camp-
esterol, campestanol, stigmasterol, Δ7-campesterol, cleros-
terol (+Δ5,23-stigmastadienol), β-sitosterol, sitostanol,
Δ5-avenasterol, Δ5,24-stigmastadienol, Δ7-stigmastenol,
and Δ7-avenasterol [10]. Contents of individual des-
methylsterols were expressed as a percentage of the
total sterol content. The content of apparent β-sitosterol
was calculated as the sum of the contents of clerosterol,
Δ5,23-stigmastadienol, β-sitosterol, sitostanol, Δ5,24-
stigmastadienol, and Δ5-avenasterol [10]. The content of
triterpene dialcohols was calculated as the sum of the con-
tents of erythrodiol and uvaol, taken as a percentage of the
sum of the total sterol content plus the contents of erythro-
diol and uvaol [10].
Accuracy
SRM 3251 was analyzed to verify accuracy in the quan-
tification of individual desmethylsterols. The SRM was
analyzed in duplicate according to the same procedure
as described for olive oil, except that some modifications
were necessary to account for its lower abundance of
desmethylsterols (e.g., smaller volumes of IS solution and
derivatization reagents). Olive oil samples from the 2012–
2013 AOCS Laboratory Proficiency Program were also
analyzed and compared with the Proficiency test results to
further verify accuracy in quantification.
Authenticity Assessment
The assessment of authenticity was based on a subset of
purity criteria specified in the US Standards for grades
of olive oil and olive-pomace oil [3]. Eight parameters
were used to assess purity, namely total sterol content
(1000 mg/kg) and the concentrations (as % of total ster-
ols) of brassicasterol (0.1 %), campesterol (4.5 %), cho-
lesterol (0.5 %), Δ7-stigmastenol (0.5 %), stigmasterol
(relative to campesterol), apparent β-sitosterol (93.0 %),
and erythrodiol plus uvaol (4.5 %). Oils failing to meet
purity criteria for total sterol content indicated possible
adulteration with desterolized oil (i.e., edible oils in which
the sterol fraction is removed to pass undetected for adul-
teration) [4]. Oils failing to meet purity criteria for des-
methylsterol composition indicated possible adulteration
Table 1 Desmethylsterol and triterpene dialcohol peak identifications, theoretical correction factors (TCF), and free sterol conversion factors
(FSx)
a Peaks listed in order of elution on the SE-54 column
b Analyte name as shown in Chemical Abstracts Service (CAS) Registry or systematic name
c CAS numbers correspond to free desmethylsterols/triterpene dialcohols, not the TMSE derivatives
d TCF were calculated relative to α-cholestanol (IS)
e Factors for conversion of sterol TMSE derivatives to corresponding free sterol equivalents were calculated as follows: FSx = MWSx/MWSTMSEx
f Analyte also referred to as dihydrocholesterol
g Analyte also referred to as fucosterol
PeakaAnalyte (common name) Analyte (CAS registry/systematic name)bCAS numbercTCFdFe
Sx
1 Cholesterol Cholest-5-en-3ol, (3β) 57-88-5 0.9956 0.8427
2 (IS) α-CholestanolfCholestan-3-ol, (3β,5α) 80-97-7 1.0000 0.8434
3 Brassicasterol Ergosta-5,22-dien-3-ol, (3β,22E) 474-67-9 0.9887 0.8467
4 Ergosterol Ergosta-5,7,22-trien-3-ol, (3β,22E) 57-87-4 – –
5 24-Methylene cholesterol Ergosta-5,24(28)-dien-3-ol, (3β) 474-63-5 0.9887 0.8467
6 Campesterol Ergost-5-en-ol, (3β,24R) 474-62-4 0.9930 0.8474
7 Campestanol Ergostan-3-ol, (3β,5α,24R) 474-60-2 0.9972 0.8480
8 Stigmasterol Stigmasta-5,22-dien-3-ol, (3α,22E) 83-48-7 0.9864 0.8511
9Δ7-Campesterol Stigmast-22-en-3-ol, (3β,5α,22E,24Z) 65494-30-6 0.9930 0.8474
10 Clerosterol (+Δ5,23-stigmastadienol) Stigmasta-5,23-dien-3-ol 2364-23-0 0.9864 0.8511
11 β-Sitosterol Stigmast-5-en-3-ol, (3β) 83-46-5 0.9905 0.8517
12 Sitostanol 5α-Stigmastan-3β-ol 83-45-4 0.9946 0.8524
13 Δ5-Avenasterol Stigmasta-5,24(28)-dien-3-ol, (3β) 18472-36-1 0.9864 0.8511
14 Δ5,24-StigmastadienolgStigmasta-5,24(28)-dien-3-ol, (3β,24E) 17605-67-3 0.9864 0.8511
15 Δ7-Stigmastenol Stigmast-7-en-3-ol, (3β,5α) 521-03-9 0.9905 0.8517
16 Δ7-Avenasterol Stigmasta-7,24(28)-dien-3-ol, (3β,5α,24Z) 23290-26-8 0.9864 0.8511
17 Erythrodiol Olean-12-ene-3β,28-diol 545-48-2 0.9819 0.8671
18 Uvaol Urs-12-ene-3,28-diol 545-46-0 0.9819
J Am Oil Chem Soc
1 3
with a commodity oil, whereas those found to contain
erythrodiol and uvaol at concentrations exceeding 4.5 %
of total sterols indicated possible adulteration with solvent-
extracted oil (i.e., olive-pomace oil) [1].
Statistical Analysis
Statistical analysis was performed using JMP (Version
11.2.0, 2013, SAS Institute, Cary, NC). One-way analysis
of variance (ANOVA) was used to compare analyzed values
for desmethylsterols and triterpene dialcohols with those in
the AOCS Proficiency Program data reports. Unless other-
wise noted, values are reported as the means of two inde-
pendent replicate determinations. Variability in analytical
values between replicate determinations was expected to
fall within acceptable reproducibility ranges, as reported in
the collaborative study data for the IOC method [10].
Results and Discussion
Chromatography
The chromatographic separation of desmethylsterol and
triterpene dialcohol TMSE derived from a typical sample
of EVOO (EVOO-1064) is shown in Fig. 1a. Peaks corre-
sponding to the desmethylsterol TMSE derivatives eluted
between 21 and 42 min, with the peak for α-cholestanol
TMSE (IS; peak # 2) eluting at 21.7 min. Peaks corre-
sponding to the TMSE derivatives of erythrodiol and uvaol
eluted at 50.5–54.5 min, respectively. The partial gas chro-
matogram for a sample of refined olive oil (EVOO-1136;
Fig. 1b) was similar to that of the typical EVOO, except
that it also showed a characteristic peak at 32.5 min, eluting
as a shoulder on the peak for clerosterol (peak # 10). This
peak has previously been identified as the TMSE of Δ5,23-
stigmastadienol, which was found to be present in quantifi-
able abundances in refined and solvent-extracted olive oils,
but not in EVOO [11]. The peak corresponding to Δ5,23-
stigmastadienol TMSE was also observed in the gas chro-
matogram for the commercially-available blend of EVOO
and sunflower oil (EVOO-1164; Fig. 1c). The partial gas
chromatogram for the EVOO blend (EVOO-1164) also
showed significantly greater abundances of campesterol
(peak # 6), stigmasterol (peak # 8), Δ7-campesterol (peak
# 9), Δ7-stigmastenol (peak # 15), Δ7-avenasterol (peak #
16), as well as several unidentified peaks, compared with
the EVOO and refined olive oil samples.
Accuracy
SRM 3251 was analyzed to verify accuracy in the quan-
tification of individual desmethylsterols. Mean analyzed
values for campesterol, stigmasterol and β-sitosterol in
Fig. 1 Partial gas chroma-
tograms observed for des-
methylsterol and triterpene
dialcohol TMSE derived from
three commercial products that
were identified in the present
study as: a EVOO because it
met purity criteria for EVOO
(EVOO-1064); b refined olive
oil (EVOO-1136); and c a blend
of EVOO and sunflower oil
(EVOO-1164), as stated on the
product label. Peak identifica-
tions are listed in Table 1. Peak
2, α-cholestanol TMSE (IS)
20.5 25.5 30.5 35.540.545.550.555.5 min
0
2
4
6
8
10
12
pA
0
2
4
6
8
10
12
pA
0
2
4
6
8
10
12
pA
EVOO-1064 (a)
EVOO
EVOO-1136 (b)
Refined Olive Oil
EVOO-1164 (c)
EVOO + Sunflower Oil Blend
10
10
1
2
345
6
78910 12
12
12
11 13
14 15 16 17 18
1
2
345
6
8
11
13
14
16 17 18
1
2
345
6811
13
14
16
17 18
15
15
9
9
J Am Oil Chem Soc
1 3
SRM 3251 were 0.533, 0.224 and 1.635 mg/g, respectively
(standard deviation, SD < 0.0). Corresponding recoveries,
which were calculated as analyzed/certificate values ×100,
were 100, 91, and 98 %, respectively. These recoveries
were found to be within acceptable recovery limits for the
analyte concentrations observed [14].
Olive oil test samples from the 2012–2013 AOCS Labo-
ratory Proficiency Program were used as reference to further
evaluate accuracy in the quantification of total sterol content
and concentration of desmethylsterols and triterpene dialco-
hols. Analyzed values are presented in Table 2 along with
mean values reported in the Laboratory Proficiency Pro-
gram data reports. Mean recoveries across all samples were
104 ± 5, 100 ± 13, 100 ± 0, 100 ± 21, and 109 ± 7 %
for campesterol, stigmasterol, apparent β-sitosterol, trit-
erpene dialcohols, and total sterol content, respectively
(mean ± SD, n = 12). Samples with the poorest recoveries
were also those that tended to show the greatest analytical
variability among laboratories, indicating disagreement as
to the true values. However, when findings from the present
study were compared by one-way ANOVA to the corre-
sponding proficiency data, only two of the analyzed values,
namely stigmasterol in AOCS-2 and total sterol content in
AOCS-8, were found to be significantly different from the
proficiency test values. Overall, these findings verified accu-
racy in our quantification of desmethylsterols and triterpene
dialcohols and also extended the applicability of the AOCS
samples beyond conventional proficiency testing.
Table 2 Recoveries of campesterol, stigmasterol, apparent β-sitosterol, triterpene dialcohols, and total sterols from test samples prepared for the
2012–2013 AOCS Olive Oil Laboratory Proficiency Program
a Analyzed values represent the means of two independent replicate determinations; SD for desmethylsterol concentration and total sterol con-
tent were <0.1 and <30, respectively
b Reference values represent means ± SD for eight independent determinations, as reported in the Laboratory Proficiency Program data reports
c Recovery = (mean analyzed/reference value) × 100
d Analyzed values shown with an asterisk were significantly different from values reported in the Laboratory Proficiency Program data reports
when compared by using one-way ANOVA
Sample Campesterol
(% of total sterols)
Stigmasterol
(% of total sterols)
Apparent β-sitosterol
(% of total sterols)
Triterpene dialcohols
(% of total sterols)
Total sterol content
(mg/kg)
AOCS-1
Analyzed valuea3.9 0.9 93.8 2.8 1500
Reference valueb3.7 ± 0.5 0.9 ± 0.2 93.9 ± 0.3 2.6 ± 0.5 1460 ± 110
Recovery (%)c105 101 100 111 103
AOCS-2
Analyzed value 3.2 0.6*d94.6 1.8 1740
Reference value 3.2 ± 0.1 0.6 ± 0.0 95.0 ± 0.3 1.5 ± 0.4 1720 ± 180
Recovery (%) 98 115 100 121 101
AOCS-3
Analyzed value 2.9 1.7 94.4 1.3 3040
Reference value 2.9 ± 0.3 1.7 ± 0.1 94.4 ± 0.2 1.5 ± 0.6 2540 ± 380
Recovery (%) 100 100 100 86 119
AOCS-5
Analyzed value 3.3 0.6 94.6 1.6 1690
Reference value 3.3 ± 0.2 0.8 ± 0.3 94.4 ± 0.6 2.1 ± 0.5 1540 ± 180
Recovery (%) 101 79 100 75 110
AOCS-6
Analyzed value 7.5 4.5 72.7 0.9 2560
Reference value 6.6 ± 1.4 4.0 ± 1.2 73.0 ± 1.0 1.1 ± 0.3 2440 ± 490
Recovery (%) 113 113 100 84 105
AOCS-8
Analyzed value 3.2 0.6 94.6 2.0 2000*
Reference value 3.1 ± 0.4 0.6 ± 0.1 94.8 ± 0.3 1.7 ± 0.5 1740 ± 70
Recovery (%) 105 93 100 122 115
Overall recovery (%) 104 ± 5 100 ± 13 100 ± 0 100 ± 21 109 ± 7
J Am Oil Chem Soc
1 3
Authenticity Assessment of Products Labeled as EVOO
Commercially-available products labeled as blends of
EVOO and commodity oil were analyzed as positive con-
trols for detecting adulteration. As expected, each of these
EVOO-commodity oil blends failed to meet purity criteria
on multiple parameters for desmethylsterol composition
(Table 3). The canola oil blend (EVOO-1152) showed a
total sterol content of 6820 mg/kg and was found to con-
tain brassicasterol, campesterol, and apparent β-sitosterol
at 10.6, 30.0, and 56.6 % of total sterols, respectively. The
sunflower oil blend (EVOO-1164) showed a total sterol
content of 3420 mg/kg and was found to contain camp-
esterol, Δ7-stigmastenol, and apparent β-sitosterol at 9.1,
13.1, and 63.7 % of total sterols, respectively. The veg-
etable oil blend (EVOO-1201), which showed a total sterol
content of 2462 mg/kg and a sterol profile similar to that of
the sunflower oil blend, also failed authentication based on
parameters for campesterol, Δ7-stigmastenol, and apparent
β-sitosterol.
Of the 88 products that were labeled as EVOO, three
failed to meet purity criteria specified in the US grades
standards (Table 3). Two of the samples failed on param-
eters for desmethylsterol composition, whereas the third
sample failed on criteria for triterpene dialcohol concen-
tration. All of the products passed authentication based on
total sterol content, which varied from 1010 to 2560 mg/kg
(mean ± SD: 1574 ± 341 mg/kg).
Campesterol is considered a major diagnostic analyte
for detecting adulteration with commodity oil due to the
abundant concentration of this desmethylsterol in many
commodity oils [4, 15]. The IOC trade standard lists a
threshold concentration for campesterol at 4.0 % of total
sterols and it includes a decision tree for evaluating the
authenticity of samples which show campesterol concen-
trations between 4.0 and 4.5 % of total sterols [16]. Grade
standards in the US permit a higher concentration of
campesterol (i.e., 4.5 %) to reflect variability among culti-
vars grown outside of the Mediterranean region [3]. In the
present study, two monovarietal samples labeled as EVOO
were found to contain campesterol at concentrations
exceeding 4.5 % of total sterols. The first sample, which
was labeled as a Koroneiki EVOO from Chile (EVOO-
1037), was found to contain campesterol at 4.9 % of total
sterols. The second sample, which labeled as a Barnea
EVOO from Peru (EVOO-1045), was found to contain
campesterol at 4.8 % of total sterols. Previous studies have
shown that olive oils originating from South America, spe-
cifically from Argentina [5, 17], contain intrinsically high
concentrations of campesterol (i.e., >5 % of total sterols).
Similar high concentrations of campesterol have also been
observed for monovarietals from Australia [6], Turkey [8],
and the US [9].
The sample of Koroneiki (EVOO-1037) was also found
to contain apparent β-sitosterol at 92.9 % of total sterols,
which is not uncommon for some monovarietals [6, 18,
19], and a total sterol content of 1010 mg/kg. The threshold
value for total sterols is 1000 mg/kg according to both IOC
and US standards. However, samples of Koroneiki grown
in different regions of Australia [6] and the US [9] and
samples of Benizal from Spain [20] have been reported to
contain total sterols at contents less than 1000 mg/kg.
The third sample which failed to meet purity criteria was
labeled as a Kalamata EVOO from Greece (EVOO-1083,
Table 3, Fig. 2). This sample was found to contain erythro-
diol plus uvaol at 4.7 % of total sterols. The concentration
of erythrodiol plus uvaol serves as an indicator of possible
adulteration with solvent-extracted oils [21]. These triter-
pene dialcohols have also been shown to vary with culti-
var, growing region, and maturation stage of the olive fruit
[6, 18]. In addition, concentrations of triterpene dialcohols
exceeding 4.5 % of total sterols have been reported for
samples of Koroneiki grown in the US [9]. Aparicio et al.
[4] recommended that additional confirmatory tests, such
as wax or aliphatic alcohol content, be performed to further
investigate authenticity when high concentrations of triter-
pene dialcohols are observed. Moreover, Grob et al. [21]
proposed that the content of wax esters was a more suitable
marker for detecting adulteration with solvent-extracted
oil. Nevertheless, the low concentration of campesterol in
EVOO-1083 (i.e., 3.3 % of total sterols) suggests that this
sample would have passed purity criteria for EVOO based
on desmethylsterol composition alone.
Of the 88 products investigated in the present study,
EVOO-1037 was the most likely candidate for potential
adulteration with commodity oil because this sample failed
on multiple parameters for purity. However, additional pri-
mary or secondary confirmatory tests would be necessary
to ascertain adulteration. Findings from the present study
highlight the variability in desmethylsterol compositions of
EVOO grown around the world and emphasize the impor-
tance of using multiple chemical tests for assessing authen-
ticity due to the diverse compositions of such products.
Alternatively, unifying methods, such as the global method
for determining theoretical and empirical triacylglycerol
composition [22], may provide greater power in evaluat-
ing authenticity while minimizing workload and the use of
extensive resources [2].
Detecting Adulteration of Spiked Samples
Intentionally-adulterated samples were prepared by spik-
ing a sample of EVOO that met purity criteria for EVOO
(EVOO-1019; cultivar not defined) with each of ten com-
modity oils at the 10 % level (weight/weight) (Fig. 3).
EVOO-1019 was chosen as the EVOO base oil because it
J Am Oil Chem Soc
1 3
Table 3 Analysis of total sterol content, desmethylsterol composition, and content of triterpene dialcohols in commercially-available samples of olive oil and in EVOO intentionally-adulterated
with 10 % commodity oil
Analyzed values which met minimum purity criteria are indicated by “PASS”. Analyzed values for parameters which failed to meet minimum purity criteria are reported as the means of two
independent replicate determinations; SD for brassicasterol, campesterol, Δ7-stigmasterol, apparent β-sitosterol, and triterpene dialcohols were <0.0, <0.4, <0.1, <0.8, and <0.2, respectively.
Analyzed values for EVOO-1019 are presented as reference for comparison to values reported for Samples EVOO-A1–A10. Samples EVOO-A1–A10 were prepared by adding 10 % commod-
ity oil (weight/weight) to EVOO-1019
a As declared on product label
Sample ID Product
description
Country of
originaTotal sterol
content (mg/
kg)
Brassicasterol
(%)
Campesterol
(%)
Cholesterol
(%)
Δ7-Stigmastenol
(%)
Apparent
β-sitosterol
(%)
Triterpene
Dialcohols
(%)
Stigmas-
terol < camp-
esterol (%)
Authenticity
assessment
Target value 1000 0.1 4.5 0.5 0.5 93.0 4.5 – PASS
EVOO-1037 EVOO Chile PASS PASS 4.9 PASS PASS 92.9 PASS PASS FAIL
EVOO-1045 EVOO Peru PASS PASS 4.8 PASS PASS PASS PASS PASS FAIL
EVOO-1065 EVOO Tunisia PASS 0.1 PASS PASS PASS PASS PASS PASS FAIL
EVOO-1083 EVOO Greece PASS PASS PASS PASS PASS PASS 4.7 PASS FAIL
EVOO-1112 Olive Oil Italy PASS PASS PASS PASS PASS PASS PASS PASS PASS
EVOO-1136 Olive Oil Italy and Spain PASS PASS PASS PASS PASS PASS PASS PASS PASS
EVOO-1152 EVOO/canola
oil blend
Mediterranean
blend
PASS 10.6 30.0 PASS PASS 56.6 PASS PASS FAIL
EVOO-1164 EVOO/sun-
flower oil
blend
Spain PASS PASS 9.1 PASS 13.1 63.7 PASS PASS FAIL
EVOO-1201 EVOO/vegeta-
ble oil blend
Argentina PASS PASS 11.8 PASS 14.2 57.5 PASS PASS FAIL
EVOO-1019 EVOO base oil Italy 1380 0.0 3.3 0.1 0.2 94.6 2.8 PASS PASS
EVOO-A1 10 % Sunflower – PASS PASS 13.1 PASS PASS 84.6 PASS PASS FAIL
EVOO-A2 10 % Soybean PASS PASS 7.0 PASS 0.7 86.4 PASS PASS FAIL
EVOO-A3 10 % Canola PASS PASS 4.6 PASS 3.2 87.5 PASS PASS FAIL
EVOO-A4 10 % Safflower PASS PASS 5.6 PASS 3.0 87.5 PASS PASS FAIL
EVOO-A5 10 % Peanut PASS PASS 5.0 PASS PASS 91.5 PASS PASS FAIL
EVOO-A6 10 % Corn PASS PASS 9.4 PASS PASS 84.8 PASS PASS FAIL
EVOO-A7 10 % Hazelnut PASS PASS PASS PASS PASS PASS PASS PASS PASS
EVOO-A8 10 % Hazelnut PASS PASS 5.3 PASS PASS 90.7 PASS PASS FAIL
EVOO-A9 10 % Hazelnut PASS PASS PASS PASS PASS PASS PASS PASS PASS
EVOO-A10 10 % Palm olein– PASS PASS 4.6 PASS PASS 92.4 PASS PASS FAIL
J Am Oil Chem Soc
1 3
met additional quality and purity criteria for parameters of
free fatty acid content, peroxide value, absorbency in ultra-
violet, fatty acid composition, trans fatty acid content, and
maximum difference between actual and theoretical equiv-
alent carbon number 42 triacylglycerol content, which we
tested using Official Methods (data not reported). While
the unequivocal authenticity of EVOO-1019 was not estab-
lished, we felt that its use as an EVOO base oil was justi-
fied by its acceptable performance in multiple tests of qual-
ity and purity.
As expected, many of the intentionally-adulterated
samples (spiked with 10 % sunflower, soybean, canola,
safflower, peanut, and corn oils, and palm olein; EVOO-
A1-A6, and A8) failed to meet purity criteria for EVOO
(Table 3). Concentrations of campesterol and apparent
β-sitosterol were most diagnostic for detecting adulteration
at the 10 % level. This was to be expected as the concen-
tration of campesterol in common commodity oils (e.g.,
canola oil, 800–4200 mg/kg; soybean oil, 350–830 mg/kg;
corn oil, 1700–3500 mg/kg; sunflower oil, 140–450 mg/
kg; safflower oil, 215–315 mg/kg) is significantly higher
than that observed for EVOO (<150 mg/kg) [15]. High
concentrations of campesterol have been useful for detect-
ing adulteration of Chemlali EVOO with 10 % soybean oil
Fig. 2 Partial gas chromato-
gram of desmethylsterol and
triterpene dialcohol TMSE
derived from a commercial
EVOO product (EVOO-1083)
that failed to meet purity criteria
for EVOO based on the contents
of erythrodiol plus uvaol. Peak
identifications are listed in
Table 1
0
2
4
6
8
10
12
pA
20.5 25.5 30.5 35.5 40.5 45.5 50.5 min
EVOO-1083
10 12
1
2
4
6
8
11 13
14 16
15
9
7
5
3
17
18
Fig. 3 Partial gas chromato-
grams of desmethylsterol and
triterpene dialcohol TMSE
derived from a EVOO which
met purity criteria for EVOO
(EVOO-1019); b EVOO-1019
spiked with 10 % hazelnut
oil which met purity criteria
for EVOO (EVOO-A9); and
c EVOO-1019 spiked with
10 % canola oil which failed to
meet purity criteria for EVOO
(EVOO-A3). Peak identifica-
tions are listed in Table 1
0
2
4
6
8
10
12
pA
EVOO-1019 (a)
0
2
4
6
8
10
12
pA
0
2
4
6
8
10
12
pA
EVOO-A9 (b)
EVOO-1019 + 10% Hazelnut Oil
EVOO-A3 (c)
EVOO-1019 + 10% Canola Oil
10
10
1
2
345
6
7
8
9
10 12
12
12
11 13
14 15 16
17 18
1
2
345
6
8
11 13
14 16 17 18
1
2
345
6
8
11 13
14
16 17 18
15
15
9
9
7
7
20.5 25.5 30.5 35.5 40.545.550.555.5
min
J Am Oil Chem Soc
1 3
and 10 % corn oil [23]. Likewise, in a study of virgin olive
oils from Jordan, high concentrations of campesterol were
important for detecting intentional adulteration with 10 %
cottonseed, soybean, and sunflower oils, and also corn oil
at the 5 % level [24]. Moreover, the increased concentra-
tion of campesterol, among others, was likely the primary
cause for the concurrent reduction in apparent β-sitosterol
concentration.
The concentration of Δ7-stigmastenol was also found
to be indicative of adulteration with soybean, canola, and
safflower oils. Concentrations of Δ7-stigmastenol are espe-
cially high in sunflower (180–500 mg/kg) and safflower
oils (325–540 mg/kg) oils, whereas in EVOO the concen-
tration falls below 10 mg/kg [15]. Jabeur et al. [23] found
that the concentration of Δ7-stigmasterol exceeded the
threshold value of 0.5 % when as little as 1 % safflower
oil was added to Chemlali EVOO. Differences in findings
between the present study and those of Jabeur et al. [23]
highlight the importance of the desmethylsterol composi-
tion of the EVOO base oil, to which adulterant is added,
for masking adulteration. For example, the concentration
of Δ7-stigmastenol in EVOO-1019 was 0.2 %, whereas
in the Chemlali EVOO it was 0.3 % [23]. The addition
of 1 % safflower oil to the Chemlali EVOO increased the
concentration of Δ7-stigmastenol to 0.5 % of total sterols,
marginally exceeding the threshold value of 0.5 %. Addi-
tion of 1 % safflower oil to EVOO-1019 in the present
study may have gone undetected for adulteration based on
Δ7-stigmastenol concentration.
Detecting the adulteration of olive oil with hazelnut oil
has proven to be a major challenge due to compositional
similarities between the two oils (e.g., triacylglycerols,
fatty acids, desmethylsterols) [25]. In the present study, two
of the three samples intentionally-adulterated with 10 %
hazelnut oil (EVOO-A7, A9) went undetected for adul-
teration (Table 3). The adulterated sample which failed to
meet purity criteria (EVOO-A8) contained campesterol
and apparent β-sitosterol at 5.3 and 90.7 % of total sterols,
respectively. Azadmard-Damirchi [25] noted difficulties in
detecting the presence of hazelnut oil in olive oil at adul-
teration levels less than 20 %. Thus, we were surprised to
find that one of the three hazelnut oils was detected at the
10 % level of adulteration, suggesting that this oil, labeled
as an organic virgin hazelnut oil, may have itself been mis-
labeled. In contrast, Cercaci et al. [26] confirmed the find-
ings of Mariani et al. [27] that concentrations of esterified
campesterol, Δ7-stigmastenol, and Δ7-avenasterol are use-
ful for detecting adulteration with crude hazelnut at con-
centrations below the 10 % level. Azadmard-Damirchi [25]
proposed that the dimethylsterol fraction may be a more
suitable marker for adulteration with crude hazelnut oil due
to its contents of lupeol and an unidentified compound with
lupine skeleton that are not observed in neat virgin olive oil.
The 10 % level of adulteration was selected in this study
because it represented a level which the authors deemed to
be potentially high enough to be economically motivating/
profitable but low enough to pass undetected for adultera-
tion. Others have investigated the detection of adulteration
at lower concentrations, e.g., 1–5 %. In Jabeur et al. [23],
the addition of 2 % sunflower oil to EVOO was detected by
an increased concentration of Δ7-stigmastenol, whereas in
Al-Ismail et al. [24], the addition of 5 % corn oil to EVOO
was found to increase the concentration of campesterol
beyond the threshold value for detection. In both of those
studies, adulteration with soybean oil at the 10 % level was
the lowest concentration at which the presence of adulter-
ant could be detected. Thus, compositional differences
among commodity oils affect the minimum concentration
of adulterant that may be detected. Nevertheless, when con-
sidering the practicality of such low levels of adulteration,
Cercaci et al. [26] noted that the addition of 5 % hazelnut
oil to EVOO is unlikely to be performed since it yields lit-
tle profit.
Commentary on Method COI/T.20/Doc. No. 30/Rev. 1
The analysis according to Method COI/T.20/Doc. No. 30/
Rev. 1 [10] has been associated with several methodologi-
cal limitations. This procedure is arguably time-consuming
and laborious. It also shows the potential for inaccuracies
associated with improper performance of the TLC proce-
dure (e.g., inadequate plate development or defective scrap-
ing of the desmethylsterol and triterpene dialcohol bands
[2] ), loss of low abundance analytes during sample prepa-
ration, and incomplete gas chromatographic resolution of
desmethylsterol TMSE derivatives [4]. The TLC proce-
dure is performed to separate desmethylsterols from other
unsaponifiable fractions, namely the monomethylsterols
and dimethylsterols, which would otherwise coelute chro-
matographically with the desmethylsterol peaks of interest.
Alternative methods for separating such fractions, such as
online normal phase liquid chromatography-GC [28] and
solid phase extraction [29], have shown promising advan-
tages for improving accuracy and sample throughput.
Conclusions
In the present study, an evaluation of the authenticity of
EVOO products available in the US market was success-
fully carried out based on the determination of desmethyl-
sterols and triterpene dialcohols. However, the variability
in desmethylsterol composition among cultivars grown
around the world may have rendered some of the samples
false positives for adulteration. Method COI/T.20/Doc.
No. 30/Rev. 1 was found to be appropriate for detecting
J Am Oil Chem Soc
1 3
adulteration of EVOO with many commodity oils at the
10 % level, including canola, corn, peanut, safflower, soy-
bean, and sunflower oils and palm olein. However, only one
EVOO base oil was investigated and the potential to detect
adulteration in other EVOO samples may vary around the
10 % level of adulteration due to compositional differ-
ences of EVOO grown around the world. This method is
not appropriate for detecting adulteration with 10 % hazel-
nut oil due to similarities in desmethylsterol content and
composition with EVOO. Overall, a low occurrence rate
of adulteration (<5 %) was found for market samples of
EVOO based on purity criteria for total sterol content, des-
methylsterol composition, and content of triterpene dialco-
hols, as specified in the US Standards for grades of olive oil
and olive-pomace oil. These findings are consistent with a
previous report from the UC Davis Olive Center [30] which
also found a low occurrence rate of EVOO economic adul-
teration with refined nut, seed, or vegetable oils.
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... Another study confirmed this usage of Δ7-stigmastenol as well as campesterol as a marker of adulteration with soybean oil (Youseff et al., 2014). The combination of several parameters, namely total sterol content, desmethylsterol composition, and triterpene dialcohols (erythrodiol and uvaol), was successfully used to identify EVOO adulteration with canola, corn, peanut, safflower, soybean, and sunflower oils, but this strategy failed to detect hazelnut oil (Srigley et al., 2016). The mean values of each sterol allowed EVOOs to be differentiated according to the olive variety and oleaster cultivar, including hybrids (Manai-Djebali et al., 2021). ...
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The growing demand for extra virgin olive oil (EVOO), appreciated for its unique organoleptic properties and health benefits, has led to various fraudulent practices to maximize profits, including dilution with lower value edible oils. The adulterated oils would be of poor nutritional quality, more readily oxidized, and may contain unhealthy substances formed during processing. Nevertheless, the range of available techniques to detect fraud in EVOO production has been growing. Reliable markers of EVOO adulteration include fatty acids and minor components such as sterols, tocopherols, triterpene alcohols, phenolic compounds, phospholipids, volatile compounds, and pigments. Additionally, increasing consumer interest in high‐quality EVOO has led to the development of robust scientific methods for its traceability. This review focuses on (i) the usefulness of certain compounds as markers of EVOO adulteration; (ii) the potential health risks of consuming adulterated EVOO; and (iii) reliable methods for the geographical traceability of olive oil. In conclusion, fraudulent production practices need to be detected to preserve the beneficial health effects of EVOO and to avoid the potential risks associated with ingesting substandard oil. In this work, as EVOO certification and regulatory framework limitations have already been extensively reviewed, we focus our attention on biomarkers that guarantee both the authenticity and traceability of oil, and consequently its health properties. When it is unavailable to obtain a high‐resolution mass spectrometry full fingerprint, stigmastadienes and the sterolic profile are proposed as reliable markers.
... However, given the limited literature data on the majority of the studied monovarietal virgin olive oils, and in fact a relatively high frequency of the occurrence of non-compliant results for sterols and triterpene diols for particular varieties, further research is needed to determine whether these are varietal characteristics or random deviations. It is assumed that the adaptation of olives to new growing areas, such as the USA, Australia, and Argentina [142]; the trend of very early harvesting dates in some areas; and the global problem of climate change are among the possible causes of such 'anomalies'. ...
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... However, the discrepancy in pricing between EVOO and other commodity oils has rendered this product a primary target for fraudulent activities, namely economic adulteration and deliberate mislabeling (Srigley, et al., 2016). Some reports estimate that adulteration of EVOO with hazelnut oil in the European Union (EU) is a multimillion euro operation annually (Azadmard-Damirchi, et al., 2005). ...
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... However, the discrepancy in pricing between EVOO and other commodity oils has rendered this product a primary target for fraudulent activities, namely economic adulteration and deliberate mislabeling (Srigley, et al., 2016). Some reports estimate that adulteration of EVOO with hazelnut oil in the European Union (EU) is a multimillion euro operation annually (Azadmard-Damirchi, et al., 2005). ...
Chapter
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