Beyond cu en quali y indices: Quan i a i e ola ilomics un e ealed
cul i a ai s, ha es ing p ac ices impac , and a oma bluep in o
ex a- i gin oli e oils
And ea Ca a i
a
, Angelica Fina
a
, Ful ia T apani
a
, E ica Libe o
a
, B ígida Jim´
enez-He e a
b
,
Lou des A ce
c
, Raquel M. Callej´
on
d,*
, Chia a Co de o
a,*
a
Dipa imen o di Scienza e Tecnologia del Fa maco, Uni e si `
a di To ino, Via Pie o Giu ia 9, Tu in 10125, I aly
b
Jun a de Andalucía, Ins i u o Andaluz de In es igaci´
on y Fo maci´
on Ag a ia, Pesque a, Alimen a ia y de la P oducci´
on Ecol´
ogica (IFAPA), Cen o de Cab a, , An igua
C a. Cab a-Do˜
na Mencía, km. 2.5 Cab a, C´
o doba, Spain
c
Depa men o Analy ical Chemis y, Uni e si y o Co doba, Ins i u e o Fine Chemis y and Nanochemis y. In e na ional Ag i ood Campus o Excellence (ceiA3), Ma ie
Cu ie Annex Building. Campus de Rabanales, Co doba 14071, Spain
d
Depa amen o de Nu ici´
on y B oma ología, Toxicología y Medicina Legal. Facul ad de Fa macia. Uni e si y o Se illa, C/ P o eso Ga cía Gonz´
alez, 2, Se illa 41012,
Spain
ARTICLE INFO
Keywo ds:
O ganic Ex a- i gin oli e oil
Comp ehensi e wo-dimensional gas ch oma-
og aphy
Pa allel de ec ion MS/FID
P edic ed ela i e esponse ac o s
A oma bluep in
Accu a e quan i ica ion
ABSTRACT
Vi gin oli e oil, de i ed om Olea eu opaea L., is a s aple in he Medi e anean die and is classi ied in o quali y
ca ego ies by EU egula ions and he In e na ional Oli e Council. Ex a Vi gin Oli e Oil (EVOO), he highes
quali y, is alued o i s nu i ional and senso y a ibu es, d i ing consume willingness o pay a p emium.
Howe e , his makes EVOO suscep ible o aud, necessi a ing obus quali y con ol me hods. This s udy
combines ad anced un a ge ed inge p in ing and quan i a i e ola ilomics using headspace solid-phase
mic oex ac ion (HS-SPME) coupled o comp ehensi e wo-dimensional gas ch oma og aphy wi h pa allel
mass spec ome y and lame ioniza ion de ec ion (GC×GC-MS/FID) on EVOOs om Hojiblanca and Picual
cul i a s o de ine obus ma ke s o quali y, named cul i a ma ke s and cul i a ion p ac ice indica o s. The
comp ehensi e analysis iden i ied o e 190 peak ea u es and 84 compounds, e ealing dis inc ola ile p o iles
in luenced by cul i a , cul i a ion me hods, and ipening s ages. O he 125 diagnos ic ola ile ea u es (Fishe
alue >4) o cul i a disc imina ion, alkenes, ca bonyls, and alcohols p edomina e. Key compounds, such as
(E)-2-oc enal, (E)-2-hexenal, and (Z)-3-hexen-1-ol, e lec lipoxygenase pa hway ac i i y and ma u a ion s ages
ac oss cul i a s. Quan i ica ion con i med dis inc a oma bluep in s be ween cul i a s, d i en by di e ences in
OAVs o key-odo an s. Howe e , minimal di e ences be ween odo ac i e ma ke s o o ganic and con en ional
cul i a ion p ac ices, sugges consume s a e unlikely o pe cei e a ia ions in a oma be ween he wo.
1. In oduc ion
Vi gin oli e oil, de i ed om Olea eu opaea L., is cen al o he
Medi e anean die and p ima ily p oduced in he Medi e anean egion
(Lioupi e al., 2022; MAPAMA, n.d.). Classi ied by o ganiza ions like
Codex Alimen a ius, In e na ional Oli e Council, an subjec ed o EU
Regula ions (“Eu opean Commission Regula ion (EC) Reg. 2568/91,”
1991), i s quali y ca ego ies depend on senso y and physicochemical
pa ame e s. Ex a Vi gin Oli e Oil (EVOO), he highes quali y ca ego y,
is p ized o i s nu i ional ai s and senso y a ibu es (Capo aso e al.,
2015; Cecchi e al., 2021), d i ing consume demand and willingness o
pay p emiums o speci ic quali ies, such as heal h bene i s, o ganic
ce i ica ion, o geog aphic o igin (A oyo-Manzana es e al., 2019).
Howe e , his high demand also inc eases he isk o aud, necessi a ing
s ingen quali y con ol measu es o main ain consume con idence
(Con e e al., 2020; M. P. Segu a-Bo ego e al., 2022a; M. Pila Segu -
a-Bo ego e al., 2022b).
Cha ac e izing EVOO in ol es analyzing a wide ange o senso y and
physicochemical pa ame e s, wi h a g owing need o ad anced
analy ical me hods due o he limi ed e ec i eness o cu en echniques
* Co esponding au ho s.
E-mail add esses: [email p o ec ed] (A. Ca a i), [email p o ec ed] (A. Fina), [email p o ec ed] (F. T apani), [email p o ec ed] (E. Libe o),
[email p o ec ed] (B. Jim´
enez-He e a), [email p o ec ed] (L. A ce), [email p o ec ed] (R.M. Callej´
on), [email p o ec ed] (C. Co de o).
Con en s lis s a ailable a ScienceDi ec
Jou nal o Food Composi ion and Analysis
jou nal homepage: www.else ie .com/loca e/j ca
h ps://doi.o g/10.1016/j.j ca.2024.106975
Recei ed 18 July 2024; Recei ed in e ised o m 19 Sep embe 2024; Accep ed 11 No embe 2024
Jou nal o Food Composi ion and Analysis 137 (2025) 106975
A ailable online 14 No embe 2024
0889-1575/© 2024 The Au ho (s). Published by Else ie Inc. This is an open access a icle unde he CC BY license (
h p://c ea i ecommons.o g/licenses/by/4.0/ ).
and e ol ing adul e a ion me hods (Con e e al., 2020). Technological
ad ancemen s ha e been emb aced by he oli e sec o o mee quali y
demands, wi h p oduce s and esea che s ocusing on op imizing p o-
duc ion unde a ious ag onomic and en i onmen al condi ions. E o s
aim o ensu e he highes quali y o oli e oils despi e hese a iables
(Cecchi e al., 2022; Hong e al., 2017; Quin anilla-Casas e al., 2020b).
A oma is a c ucial quali y c i e ion o EVOO, signi ican ly in lu-
enced by ac o s such as oli e a ie y/cul i a , geog aphic o igin, ui
ma u a ion, i iga ion, c op season, ag onomic p ac ices, and oil
ex ac ion condi ions (Bel ´
an e al., 2005; Beni o e al., 2012; Jim´
enez
e al., 2017; Mansou i e al., 2015; Mo ell´
o e al., 2004; Rome o e al.,
2003; Vidal e al., 2019). The ola ile p o ile and o e all a oma a e
especially a ec ed by oli e a ie y/cul i a and ipening s age, d i ing
in e es in mono a ie al oils. Despi e p omising analy ical me hods, no
o icial app oach exis s o au hen ica e mono a ie al EVOOs, high-
ligh ing he need o obus au hen ica ion me hods (He e a e al.,
2012; Jimenez e al., 2015, 2014; Jim´
enez e al., 2017).
The ype o a ming, pa icula ly o ganic e sus con en ional
me hods, is ano he signi ican ac o due o consume demand o
o ganic p oduc s and s ic ag icul u al egula ions (Jim´
enez-He e a
e al., 2019). While o ganic oli e a ming has g own, s udies compa ing
o ganic and con en ional EVOOs a e limi ed and o en inconclusi e due
o uncon olled a iables. Fu u e esea ch needs con olled samples o
ad ance knowledge on o ganic EVOOs, p e en aud, and p o ide
aluable in o ma ion o he indus y, p oduce s, and consume s.
Gas ch oma og aphy-mass spec ome y (GC-MS) is widely used o
p o ile oli e oil ola ile compounds. Combined wi h headspace (HS)
app oaches like solid-phase mic oex ac ion (SPME) o dynamic head-
space (D-HS), i enables high- h oughpu p o iling and moni o ing o
many a oma-ac i e compounds (Cecchi e al., 2022; Co de o e al.,
2015; Quin anilla-Casas e al., 2020b). The in oduc ion o comp e-
hensi e wo-dimensional GC (GC×GC) has signi ican ly imp o ed he
unde s anding o EVOO’s chemical complexi y, p o iding highe peak
capaci y, esolu ion, lowe de ec ion h esholds, and mo e con iden
analy e iden i ica ion (Luki´
c e al., 2019; Ma io and Nol achai, 2021;
Pu ca o e al., 2014; Ryan and Ma io , 2003). Mo eo e , adop ing
non- a ge ed da a p ocessing [e.g., combined un a ge ed and a ge ed
inge p in ing UT inge p in ing (Magagna e al., 2016)], he in es iga-
ion o complex ac ion o ola ile o ganic compounds (VOCs) by
GC×GC has opened o ola ilomics, he discipline ha connec s he
chemical in o ma ion enc yp ed in he ola ilome (o ola ome) wi h
biological p ope ies (B oza e al., 2015; Phillips e al., 2013; S ilo e al.,
2021a). The ola ilome “con ains all o he ola ile me aboli es as well as
o he ola ile o ganic and ino ganic compounds ha o igina e om an o -
ganism”(Amann e al., 2014) supe -o ganism, o ecosys em; as pa o
he me abolome i b ings in o ma ion wo hy o be in es iga ed o
p oduc alo iza ion and eaciabili y.
Analy ical pla o ms ha implemen pa allel de ec ion, combining
mass spec ome y (MS) and lame ioniza ion de ec o (FID), o e g ea
oppo uni ies allowing o eliable iden i ica ion and accu a e quan i-
ica ion o analy es wi hou ex e nal calib a ion. This enables ue
quan i a i e esul s o obus ma ke selec ion and co ela ion wi h
quali y ai s (S ilo e al., 2023; S ilo e al., 2021). The use o
GC×GC-MS/FID in oli e oil esea ch in pa icula , allows o a
comp ehensi e mapping o de ec able VOCs, p o ides accu a e amoun s
on selec ed analy es, and enables causal co ela ion wi h biological
a iables such as oli e cul i a , ipening s ages, geog aphical o igin,
ag icul u al p ac ices, and echnological p ocessing (Magagna e al.,
2016; Reichenbach e al., 2019; S ilo e al., 2023; S ilo e al., 2021). The
quan i a i e mapping o key-a oma compounds delinea es he a oma
bluep in o EVOOs, i.e., he unique pa e n o odo ac i e compounds
esponsible o he a oma iden i y o he p oduc (G an ogl and Schie-
be le, 2022)
This s udy, o he i s ime, combines ad anced un a ge ed
inge p in ing and quan i a i e ola ilomics using HS-SPME-GC×GC-
MS/FID on a selec ion o monocul i a EVOOs o de ine bo h obus
quali y ace s and dis inc i e chemical bluep in s p edic ing a oma
ea u es. Samples we e ob ained om Hojiblanca and Picual oli e cul i-
a s ha es ed in he Co doba egion (Spain) unde di e en a ming
p ocedu es (o ganic and con en ional) and ipening s ages om
No embe 2021 o Janua y 2022.
2. Ma e ials and me hods
2.1. Ex a i gin oli e oil samples
Ex a i gin oli e oils (EVOOs) we e ob ained om Picual and Hoji-
blanca oli e cul i a s ha es ed in 2021. Oli e ees we e subjec ed o
wo di e en cul i a ion me hods, o ganic and con en ional. T ees we e
loca ed a he Ag icul u al Resea ch T aining Cen e in “Cab a”in he
p o ince o Co doba (Spain) [plo coo dina es 37◦29’36.4"N
4◦25’45.8"W], which has a con inen al Medi e anean clima e wi h d y
summe s and mild win e s. Rain all occu s om au umn o sp ing, wi h
a mean annual ain all o 400 mm. The a e age annual empe a u e is 17
◦C, eaching −1.3 ◦C in win e and 43 ◦C in summe . Soils ha e low
dep hs, being mainly o e limes one and siliceous s ones. They ha e a
loamy ex u e and an alkaline pH (8–8.5 ange). The elec ical con-
duc i i y is low, as is he o ganic ma e con en and he ca bon/ni o-
gen a io. Twen y oli e ees we e andomly selec ed (always among he
mos loaded o gua an ee sampling) o he Picual and Hojiblanca cul i-
a s om con en ional cul i a ion and wen y ees o he wo cul i a s
om o ganic cul i a ion. O ganic cul i a ion was conduc ed acco ding
o EU Regula ions and ce i ied by Ins i u o Andaluz de In es igaci´
on y
Fo maci´
on Ag a ia, Pesque a, Alimen a ia y de la P oducci´
on Ecol´
ogica
(IFAPA) (Jun a de Andalucía) (“Regula ion (EU) 2018/848 o he Eu-
opean Pa liamen and o he Council o 30 May 2018 on o ganic p o-
duc ion and labelling o o ganic p oduc s and epealing Council
Regula ion (EC) No 834/2007,”2018).
Oli e ha es ing was done by andomly hand-picking heal hy ui s
(wi hou any ype o in ec ion o physical damage), beginning in
No embe 2021 and ending in Janua y 2022, and comp ising di e en
ha es pe iods. I con o ms o a o al o 3 ipening s ages: 15 o
No embe (s age I), 13 o Decembe (s age II), and 1 o Janua y (s age
III). The ipening index (RI) o each ha es was de e mined acco ding o
he me hodology p oposed by Uceda and F ias (Uceda and F ias, 1975).
Table 1
EVOO samples cha ac e is ics and coding.
Sample
coding
Cul i a Cul i a ion
me hod
Oli es
ha es ing
Ma u i y
s age
Ripening
Index
EVOO_1 Picual Con en ional No embe
15
I 3.5
EVOO_2 Picual Con en ional Decembe
13
II 4.8
EVOO_3 Picual Con en ional Janua y 1 III 5.7
EVOO_4*Picual Con en ional Decembe
13
II 4.8
EVOO_5 Picual O ganic No embe
15
I 4.1
EVOO_6 Picual O ganic Decembe
13
II 4.8
EVOO_7 Picual O ganic Janua y 1 III 5.9
EVOO_9 Hojiblanca Con en ional No embe
15
I 1.4
EVOO_10 Hojiblanca Con en ional Decembe
13
II 3.8
EVOO_11 Hojiblanca Con en ional Janua y 1 III 4.0
EVOO_12*Hojiblanca Con en ional Decembe
13
II 3.8
EVOO_13 Hojiblanca O ganic No embe
15
I 1.2
EVOO_14 Hojiblanca O ganic Decembe
13
II 2.6
EVOO_15 Hojiblanca O ganic Janua y 1 III 4.7
* Technological eplica es om a di e en ba ch
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
2
The ui RI alues o each modali y a e shown in Table 1.
In each ha es ing s age, 200 kg o oli es we e collec ed wi hou any
disc iminan selec ion be o e immedia e p ocessing in he expe imen al
mill o IFAPA Ven a del Llano (Mengíba , Ja´
en, Spain) o ob ain he oils.
Thus, he ex ac ion o he oils was ca ied ou in a wo-phase semi-in-
dus ial con inuous sys em (Il Moline o, G uppo Pie alisi, I aly)
equipped wi h a hamme mill, a he mo-blende , and a ho izon al
cen i uge. The 200 kg o oli es we e g ound and hen, he esul ing
pas e was bea en o 45 min a 28 ◦C, a e which he oil was sepa a ed
by cen i uging he bea en pas e a ≈3250 G. The oils ob ained we e
decan ed and il e ed. Samples o ola ilome sc eening we e immedi-
a ely sen o he Uni e si y o Tu in labo a o y in da k-glass bo les.
Be o e analysis hey we e kep a −18◦C away om UV exposu e.
Sample desc ip ions and de ails a e epo ed in Table 1.
Oli e oils we e also subjec ed o he e alua ion o he senso y panel
o he P iego de C´
o doba P o ec ed Denomina ion o O igin (PDO),
ollowing he s anda ds o he In e na ional Oli e Council (IOC)
(Ríos-Reina e al., 2021). Acco ding o senso y esul s, all samples
conside ed in his wo k esul ed “Ex a-Vi gin”Oli e Oils.
2.2. Re e ence s anda ds and sol en s
Pu e e e ence compounds o iden i y con i ma ion o ma ke ol-
a iles and po en odo an s lis ed in Table 2,n-alkanes (n-C7 o n-C25) o
Linea Re en ion Indices (I
T
) calib a ion, and
α
/β- hujone used as in-
e nal s anda ds o esponse s abili y check we e ob ained om Me ck
(Milan, I aly).
The ex e nal s anda d o p edic ed ela i e esponse ac o s (RRF)
based on combus ion en halpies calib a ion was hexanal (99 % pu i y,
Me ck). The e e ence solu ion was p epa ed in cyclohexane (99 % pu-
i y, Me ck) a a inal concen a ion o 10.00 g/L and hen dilu ed in
dibu yl ph hala e (99 % pu i y, Me ck) o sui able concen a ions o
linea i y e alua ion.
2.3. Mul iple headspace solid phase mic oex ac ion: de ices and
condi ions
Vola iles om EVO oils we e ex ac ed by HS-SPME wi h a di inyl-
benzene/ca boxen/polydime hyl siloxane (DVB/CAR/PDMS) ibe (d
50/30
μ
m; 2 cm leng h) om Supelco (Belle on e, PA, USA) acco ding o
a p e iously op imized p ocedu e (S ilo e al., 2019). The SPME ibe
was condi ioned be o e use as ecommended by he manu ac u e .
Sampling was ca ied ou on 0.100 ±0.003 g o oil, p ecisely weighed in
a 20 mL headspace ial, and kep a 40 ◦C o 60 min unde cons an
agi a ion. The e y low amoun o sample was chosen o ma ch o HS
linea i y condi ions o mos o he cha ac e is ic analy es o he EVO oil
ola ilome (S ilo e al., 2021). A e ex ac ion, he SPME de ice was
au oma ically ans e ed o he spli /spli less injec ion po o he
GC×GC sys em, kep a 250 ◦C, and he mal deso p ion was o 5 min.
Samples we e analyzed in ou eplica es andomly dis ibu ed o e wo
weeks.
Mul iple headspace ex ac ion by SPME (MHS-SPME) o samples and
calib a ion solu ions we e conduc ed by applying he abo e-indica ed
condi ions, and he numbe o consecu i e ex ac ion s eps was se o
ou , achie ing an almos exhaus i e ex ac ion o he analy es unde
s udy (S ilo e al., 2021).
2.4. Comp ehensi e wo-dimensional gas ch oma og aphy: ins umen se -
up and condi ions
Au oma ed MHS-SPME was pe o med by a mul ipu pose sample ,
model MPS-2 (Ge s el, Mülheim a/d Ruh , Ge many), ins alled on a
GC×GC sys em equipped wi h a e e se-injec di e en ial- low modu-
la o based on capilla y low echnology™(Agilen Technologies, Li le
Falls, DE, USA). The Agilen 7890B GC uni was coupled o an Agilen
5977B equipped wi h a high-e iciency sou ce (HES) and as
quad upole MS analyze (Agilen Technologies). The MS was ope a ing
in elec on ioniza ion mode a 70 eV. Ion sou ce and ans e -line em-
pe a u es we e se a 280 ◦C, and he quad upole empe a u e was se a
240 ◦C. The scan ange was se be ween 40 and 250 m/z, achie ing a
da a acquisi ion equency o 30 Hz. Pa allel de ec ion was by a as FID
wi h base empe a u e held a 280 ◦C; H
2
low 40 mL/min, ai low
350 mL/min, and sampling equency 200 Hz.
The column se was con igu ed as ollows: i s dimension (
1
D)
Hea yWax™column (100 % polye hylene glycol - PEG; 20 m ×0.18 mm
dc ×0.18
μ
m d
) coupled wi h second dimension (
2
D) DB17 column
(50 % phenyl-me hylpolysiloxane; 1.8 m ×0.18 mm d
c
×0.18
μ
m d
),
bo h om Agilen Technologies. The connec ion be ween he
2
D column
and deac i a ed silica capilla ies (Agilen Technologies) owa d MS
(0.5 m ×0.1 mm d
c
) and FID (1.1 m x 0.18 mm d
c
) o pa allel de ec ion
was by a h ee-way unpu ged capilla y mic o luidic spli e (G3181B,
Agilen Technologies). The esul ing spli a io was 70:30 FID/MS.
The GC spli /spli less injec o po was se a 250 ◦C and ope a ed in
pulsed-spli mode (250 kPa o e p essu e applied o he injec ion po
un il 2 min) wi h a spli a io 1:20. The ca ie gas was helium a a
nominal low o 0.4 mL/min along he
1
D column and 10 mL/min along
he
2
D column. The o en empe a u e p og am was se as: om 40 ◦C
(2.29 min) o 240 ◦C (11’) a 3.06 ◦C/min. The modula ion pe iod (P
M
)
was se a 3 s and pulse ime a 250 ms. The n-alkanes liquid sample
solu ion o I
T
de e mina ion was analyzed unde he ollowing condi-
ions: spli /spli less injec o in spli mode, spli a io 1:50, injec o
empe a u e 250 ◦C, and injec ion olume 1
μ
L.
2.5. Analy es iden i ica ion c i e ia
Analy e iden i ica ion was by combining e en ion da a (ca ied ou
using expe imen al I
T
wi h ±10 uni s ole ance s. abula ed ones) and
compa ing elec on ioniza ion (EI)-MS spec al signa u e wi h e e ence
spec a in comme cial and in-house da abases by using he NIST iden i y
sea ch algo i hm wi h di ec ma ch ac o (DMF) and e e se ma ch
ac o (RMF) sco es h eshold ≥900.
2.6. Analy ical da a acquisi ion, p ocessing and mining
2.6.1. Combined un a ge ed and a ge ed (UT) inge p in ing by sma
empla es
A empla e is a pa e n o 2D peaks and/o g aphic objec s ( ea u es)
c ea ed o e a e e ence ch oma og am o image (single o cumula i e).
This empla e is used o iden i y simila pa e ns o 2D peaks in a se o
analyzed ch oma og ams o images (B essanello e al., 2018; Reich-
enbach e al., 2019). The p ocess in ol es speci ic ma ching unc ions
ha es ablish co espondences be ween ea u es ac oss mul iple ch o-
ma og ams. Speci ici y is achie ed by de ining con idence h esholds o
e en ion imes and MS spec al simila i y o accoun o a iable peak
de ec ion, and by using sui able ans o m unc ions o co ec o
e en ion ime inconsis encies be ween uns (Squa a e al., 2023b; S ilo
e al., 2019). Once he co espondences be ween ea u es a e es ab-
lished, he empla e’s me ada a (chemical name, e en ion imes, mass
spec a, in o ma i e ions, and hei ela i e a ios) a e ans e ed o
candida e peaks and/o g aphic objec s (peak egions) in he analyzed
ch oma og am. This ea u e empla e, which includes un a ge ed eli-
able peaks and peak egions, is ob ained h ough a ully au oma ed
wo k low in GC Image In es iga o ™(GC Image™, GC Image LLC).
The wo k low consis s o he ollowing s eps:
(a) Ma ch peak pa e ns be ween all ch oma og am pai s om a se
o ep esen a i e samples, using op imized pa ame e s: S/N h eshold o
50, MS cons ain o 700 o bo h he di ec ma ch ac o (DMF) and
e e se ma ch ac o (RMF) based on he NIST simila i y algo i hm
(Squa a e al., 2023b; Technology, 2005).
(b) Selec eliable peaks ac oss he analyzed ch oma og ams, using a
elaxed eliabili y c i e ion ha includes peaks ma ching a leas 50 %
+1 o he ch oma og ams (Reichenbach e al., 2013). This empla e o
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
3
Table 2
Iden i ied compounds in he ola ile ac ion o EVOOs oge he wi h analy ical in o ma ion. See ex o iden i ica ion c i e ia.
Compound Name
1
D
R
min SD
2
D
R
sec SD Exp I
T
Li . I
T
MW Fo mulae PRRF β(±SD) Odo quali y OT (ng/g)*
Analy es subjec ed o accu a e quan i ica ion
P opanal 4.83 0.073 0.42 0.019 792 786 58.1 C
3
H
6
O 1.38 0.90 (±0.07) F esh, ui y, mal y 9.4 [1]
E hyl ace a e 6.44 0.028 0.37 0.070 897 898 88.1 C
4
H
8
O
2
1.60 0.92 (±0.08) F ui y, swee , winey 940 [3]
1-Pen en−3-one 10.15 0.016 0.68 0.049 1017 1021 84.1 C
5
H
8
O 1.12 0.65 (±0.05) Pungen , spicy 1.6 [1]
α
-Pinene 10.24 0.087 1.68 0.055 1017 1017 136.2 C
10
H
16
0.79 0.93 (±0.07) He bal, woody, e penic 274 [4]
Hexanal (In e nal S anda d o FID) 12.57 0.027 0.91 0.048 1079 1080 100.2 C
6
H
12
O - 0.73 (±0.07) G een, g ass 300 [1]
β-Pinene 13.34 0.062 1.85 0.067 1097 1100 136.2 C
10
H
16
0.79 0.92 (±0.06) He bal, pine -
(E)−2-Pen enal 14.45 0.011 0.73 0.049 1123 1131 84.1 C
5
H
8
O 1.12 0.75 (±0.02) Pungen , apple-like 300 [3]
2-Pen anol 14.99 0.016 0.44 0.052 1136 1138 88.1 C
5
H
12
O 1.02 0.88 (±0.11) Mus y, e men ed 380 [2]
(Z)−3-Hexenal 15.14 0.020 0.77 0.050 1139 1133 98.1 C
6
H
10
O 1.04 0.56 (±0.03) G een, g assy 1.7 [1]
δ−3-Ca ene 15.16 0.040 1.77 0.085 1139 1144 136.2 C
10
H
16
0.79 0.9 (±0.04) Ci us, pine, he bal 770 [4]
1-Pen en−3-ol 15.75 0.006 0.42 0.052 1153 1157 86.1 C
5
H
10
O 1.07 0.57 (±0.02) Pungen , bu e 400 [3]
Hep anal 16.99 0.024 1.02 0.062 1181 1181 114.2 C
7
H
14
O 0.96 0.93 (±0.05) Ci us-like, a y 500 [1]
Limonene 17.48 0.027 1.67 0.050 1192 1195 136.2 C
10
H
16
0.79 0.92 (±0.06) Ci us, e penic 250 [2]
3-Me hyl−1-bu anol 17.80 0.006 0.45 0.050 1199 1211 88.1 C
5
H
12
O 1.02 0.63 (±0.03) Mal y, e he eal 100 [3]
Eucalyp ol 17.88 0.036 1.86 0.089 1201 1205 154.2 C
10
H
18
O 0.90 0.94 (±0.03) He bal, min y 15 [2]
(E)−2-Hexenal 18.40 0.006 0.79 0.051 1212 1216 98.1 C
6
H
10
O 1.04 0.6 (±0.02) Bi e almond, g een, ui y 320 [1]
Hexyl ace a e 21.05 0.014 1.15 0.050 1271 1275 144.2 C
8
H
16
O
2
1.09 0.7 (±0.08) F ui y, pea -like 1040 [3]
Oc anal 21.70 0.006 1.10 0.053 1285 1289 128.2 C
8
H
16
O 0.92 0.94 (±0.04) Ci us-like, a y 140 [1]
(Z)−2-Pen en−1-ol 22.50 0.006 0.42 0.052 1303 1306 86.1 C
5
H
10
O 1.07 0.57 (±0.02) G een, almond 250 [2]
(E)−2-Pen en−1-ol 22.84 0.022 0.40 0.047 1310 1313 86.1 C
5
H
10
O 1.07 0.68 (±0.03) Mush oom, ea hy 250 [2]
(Z)−3-Hexenyl ace a e 23.01 0.025 1.01 0.047 1315 1317 142.2 C
8
H
14
O
2
1.12 0.55 (±0.04) G een 200 [1]
(E)−2-Hep enal 23.17 0.025 0.86 0.031 1318 1320 112.2 C
7
H
12
O 0.99 0.81 (±0.05) G een, a y 1200 [1]
6-Me hyl−5-hep en−2-one 23.80 0.006 0.95 0.054 1332 1339 126.2 C
8
H
14
O 0.95 0.69 (±0.03) Pungen , g een, ui y-like 1000 [3]
1-Hexanol 24.30 0.044 0.48 0.054 1343 1348 102.2 C
6
H
14
O 0.97 0.66 (±0.02) F ui y, banana 400 [3]
(Z)−3-Hexen−1-ol 25.69 0.022 0.46 0.048 1374 1380 100.2 C
6
H
12
O 1.00 0.6 (±0.01) Banana, esh, g ass 1100 [5]
(E,E)−2,4-Hexadienal 26.30 0.006 0.63 0.051 1388 1397 96.1 C
6
H
8
O 1.08 0.68 (±0.12) G een, soapy 270 [2]
Nonanal 26.35 0.006 1.13 0.054 1389 1390 142.2 C
9
H
18
O 0.90 0.95 (±0.02) Fa y, waxy, pungen 610 [1]
(E)−2-Hexen−1-ol 26.55 0.011 0.62 0.050 1394 1398 100.2 C
6
H
12
O 1.00 0.7 (±0.07) G een, g ass 500 [1]
(E)−2-Oc enal 27.70 0.009 0.40 0.054 1420 1427 126.2 C
8
H
14
O 0.95 0.92 (±0.04) Fa y, nu y 120 [1]
Ace ic acid 28.51 0.023 0.24 0.051 1440 1440 60.1 C
2
H
4
O
2
3.62 0.73 (±0.09) Sou , inega y 350 [1]
(E,E)−2,4-Hep adienal 29.20 0.006 0.70 0.055 1456 1461 110.2 C
7
H
10
O 1.02 0.86 (±0.05) Fa y, g een, oily 710 [1]
α
-Copaene 30.55 0.016 2.37 0.052 1489 1491 204.4 C
15
H
24
0.78 0.94 (±0.06) Woody -
Benzaldeyde 31.59 0.022 0.66 0.051 1513 1524 106.1 C
7
H
6
O 0.92 0.91 (±0.04) Almond, bu n suga , e hy 60 [4]
Popanoic acid 32.15 0.013 0.26 0.055 1526 1534 74.1 C
3
H
6
O
2
2.06 0.81 (±0.05) Acidic, pungen 720 [4]
(E)−2-Nonenal 32.26 0.026 0.91 0.058 1530 1534 140.2 C
9
H
16
O 0.92 0.95 (±0.02) Fa y, g een, soapy 140 [1]
1-Oc anol 33.01 0.022 0.55 0.055 1548 1553 130.2 C
8
H
18
O 0.90 0.86 (±0.03) Nu , mush oom 27 [2]
Bu anoic acid 35.75 0.006 0.25 0.053 1616 1613 88.1 C
4
H
8
O
2
1.60 0.85 (±0.05) Cheesy, sou 34 [1]
(E,E)-
α
-Fa nesene 40.60 0.015 1.56 0.051 1742 1744 204.4 C
15
H
24
0.78 0.78 (±0.03) Swee , lo al -
Analy es no quan i ied
Hexane 3.58 0.135 0.45 0.049 600 600 86.2 C
6
H
14
Alkane
Oc ane 4.87 0.066 0.99 0.063 800 800 114.2 C
8
H
18
Sol en 940 [5]
Ace one 5.05 0.017 0.45 0.049 850 845 58.1 C
3
H
6
O Pungen -
2-Me hyl bu anal 7.08 0.140 0.64 0.073 924 926 86.1 C
5
H
10
O Mal y 10 [5]
E hanol 7.35 0.015 0.37 0.051 928 933 46.1 C
2
H
6
O E hanol-like -
3,4-Die hyl−1,5-hexadiene (RS/SR) 7.90 0.018 1.21 0.049 946 952 138.2 C
10
H
18
- -
3-Me hylbu anal 8.00 0.133 0.72 0.066 939 930 86.1 C
5
H
10
O Mal y 13 [5]
3,4-Die hyl−1,5-hexadiene (meso) 8.45 0.145 1.40 0.083 956 956 138.2 C
10
H
18
- -
Pen anal 8.76 0.027 0.75 0.050 975 974 86.1 C
5
H
10
O Almond-like, pungen , mal 150 [4]
(Z)−1-Me hoxy−3-hexene 9.74 0.025 1.17 0.050 1007 997 114.2 C
7
H
14
O - -
(5Z)−3-E hyl−1,5-oc adiene 9.93 0.026 1.69 0.050 1012 1006 138.2 C
10
H
18
- -
(5E)−3-E hyl−1,5-oc adiene 10.47 0.027 1.69 0.049 1026 1032 138.2 C
10
H
18
- -
Toluene 10.73 0.050 0.91 0.057 1029 1036 92.1 C
7
H
8
Chemical-like -
(E,Z)−3,7-Decadiene 12.52 0.028 1.92 0.051 1078 1068 138.2 C
10
H
18
- -
(E,E)−3,7-Decadiene 12.85 0.016 1.89 0.049 1085 1082 138.2 C
10
H
18
- -
(con inued on nex page)
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
4
Table 2 (con inued)
Compound Name
1
D
R
min SD
2
D
R
sec SD Exp I
T
Li . I
T
MW Fo mulae PRRF β(±SD) Odo quali y OT (ng/g)*
(Z)−2-Pen enal 13.49 0.017 0.74 0.049 1101 1105 84.1 C
5
H
8
O Pungen , apple-like 300 [3]
3-Me hylbu yl ace a e 14.42 0.053 0.97 0.060 1123 1126 130.2 C
7
H
14
O
2
Banana-like, ui y
2-Hep anone 16.85 0.027 0.98 0.086 1178 1171 130.2 C
7
H
14
O Swee , ui y 470 [4]
(Z)−2-Hexenal 17.66 0.022 0.82 0.048 1197 1193 98.1 C
6
H
10
O F ui y -
γ-Te pinene 19.59 0.024 1.71 0.058 1239 1247 98.1 C
6
H
10
O Pe ol-like -
ans-β-Ocimene 19.95 0.006 1.44 0.050 1246 1249 136.2 C
10
H
16
Ci us-like, soapy, ge anium-like -
o-Cymene 20.72 0.027 1.42 0.050 1264 1268 134.2 C
10
H
14
- -
3-Hyd oxy−2-bu anone 21.45 0.021 0.46 0.059 1279 1283 88.1 C
4
H
8
O
2
Bu e y -
2-Oc anone 21.50 0.014 1.07 0.052 1280 1284 128.2 C
8
H
16
O Mould, g een 510 [4]
(E)−3-Hexen−1-ol ace a e 22.49 0.020 1.57 0.050 1303 1300 142.2 C
8
H
14
O
2
F ui y -
Dioxa−1,6-spi o[4.5]decane 23.54 0.032 1.44 0.077 1326 - 142.2 C
8
H
14
O
2
- -
3-Me hyl-cyclopen anol 23.68 0.034 0.65 0.064 1329 1342 100.1 C
6
H
12
O - -
(E)−3-Hexen−1-ol 24.78 0.031 0.47 0.044 1354 1352 100.2 C
6
H
12
O g een, g assy -
Me hoxyme hyl-benzene 26.05 0.006 0.93 0.053 1382 1382 122.2 C
8
H
10
O - -
(Z)−2-Hexen−1-ol 26.62 0.035 0.43 0.029 1396 1405 101.2 C
6
H
12
O G een, g ass -
Dime hyl Sul oxide 32.60 0.015 0.49 0.051 1537 1549 78.1 C
2
H
6
OS - -
3-Me hyl−2-hexen−4-one 34.60 0.000 0.47 0.054 1587 1586 112.2 C
7
H
12
O - -
Benzoic acid, me hyl es e 35.66 0.028 0.75 0.060 1613 1614 136.1 C
8
H
8
O S a ui -like, swee -
1,4-Cyclohex−2-enedion 39.93 0.025 0.53 0.047 1725 - 110.1 C
6
H
6
O
2
- -
Pen anoic acid 39.93 0.024 0.25 0.050 1725 1730 102.1 C
5
H
10
O
2
Swea y, ui y 400 [1]
2,3-Dime hylbenzaldehyde 39.99 0.052 0.66 0.049 1725 1736 134.2 C
9
H
10
O - -
5-e hyl−2(5 H)-Fu anone 40.73 0.024 0.51 0.045 1746 1757 112.1 C
6
H
8
O
2
Swee , spicy -
Me hyl salicyla e 41.53 0.029 0.72 0.046 1768 1765 152.1 C
8
H
8
O
3
Fa y, allowy, e pene-like -
2-(2-bu oxye hoxy)E hanol 42.09 0.024 0.55 0.054 1783 1786 162.2 C
8
H
18
O
3
- -
Hexanoic acid 43.84 0.022 0.26 0.049 1831 1839 116.2 C
6
H
12
O
2
Goa -like, swea y 460 [1]
Benzyl alcohol 44.78 0.025 0.38 0.046 1858 1857 108.1 C
7
H
8
O Swee , ui y -
Phenyle hyl alcohol 46.02 0.029 0.44 0.053 1893 1904 122.2 C
8
H
10
O Flo al, honey-like -
(E)−2-Hexenoic acid 47.89 0.022 0.30 0.047 1947 1941 114.1 C
6
H
10
O
2
- -
Phenol 49.10 0.006 0.26 0.054 1983 1987 94.1 C
6
H
6
O Ink-like, phenolic -
Benzoic acid, 2-me hoxy-, me hyl es e 51.31 0.045 0.66 0.060 2049 2032 166.2 C
9
H
10
O
3
- -
*Odo h eshold e e ences: [1] (Neugebaue e al., 2020); [2] (Van Geme , 2003); [3] (Luna e al., 2006); [4] (Squa a e al., 2023a); [5] (Pu ca o e al., 2014)
Re en ion imes (
1
R
min,
2
R
sec), s anda d de ia ion (SD), expe imen al and abula ed linea e en ion indexes (I
T
), molecula weigh (MW), p edic ed FID ela i e e en ion ac o (RRF), decay unc ion (β), odo
h eshold (OT) in ng/g
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
5
eliable peaks is used o e-aligning ch oma og ams in he empo al
domain.
(c) Align ch oma og ams o ep esen a i e samples o he a e age
e en ion imes o he eliable peaks, hen sum/ use hem in o a com-
posi e ch oma og am.
(d) Gene a e a comp ehensi e un a ge ed ea u e empla e om he
composi e ch oma og am, including eliable peaks and peak egions
de ined by he oo p in o all de ec ed peaks (Reichenbach e al., 2013).
This p ocess was used o gene a e a composi e image om a selec ion
o sample ch oma og ams ep esen a i e o he di e en exp essions o
unc ional a iables. F om he composi e image, a ea u e empla e
consis ing o 190 UT ea u es (i.e., peak egions co esponding o
un a ge ed and a ge ed componen s) was de ined and applied o single
ch oma og am images (i.e, samples and eplica es) o eliable acking
and alignmen o ea u es. The lis o a ge ed ea u es is epo ed in
Table 2 oge he wi h chemical names,
1
D and
2
D e en ion imes (
1
R
;
2
R
), expe imen al I
T
, abula ed I
T
, odo quali y, and odo h esholds
(OTs) in oil as om e e ence li e a u e.
2.6.2. Da a acquisi ion and s a is ical analysis
Raw ch oma og aphic da a we e acqui ed by MassHun e Wo ks a-
ion (Agilen Technologies). Raw da a we e p ocessed by GC Image™
V2020 1.2 sui e (GC Image, LLC Lincoln, NE, USA).
S a is ical analysis and chemome ics we e pe o med using GC
In es iga o ™(GC Image), XLSTAT s a is ical and da a analysis solu ion
(Addinso 2020, New Yo k, USA), and Mic oso O ice Excel 2016
(Mic oso , Redmond, WA USA).
Fig. 1. PCA on un a ge ed and a ge ed (UT) peak ea u es (analy es) % esponses; ellipses se a 95 % o con idence le el, show cul i a na u al clus e s. In Fig. 1A
all 190 UT ea u es we e compu ed o all samples; do ed lines indica e Ma u i y S age e olu ion acco ding o sample loadings. Fig. 1B esul s om Fishe a io
ea u es educ ion conside ing cul i a disc imina ion (Picual s. Hojiblanca) he PCA e e s o 92 UT peaks wi h a F >10. Fig. 1CFishe a io ea u es educ ion
conside ing cul i a ion ype on Picual oils (o ganic s. con en ional) he PCA e e s o 49 UT peaks wi h a F >10. Fig. 1DFishe a io ea u es educ ion conside ing
cul i a ion ype on Hojiblanca oils (o ganic s. con en ional) he PCA e e s o 49 UT peaks wi h a F >10.
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
6
3. Resul s and discussion
EVO oil ola ilome enc yp s in o ma ion on many key-quali y a i-
ables (e.g., oli es cul i a and geog aphical o igin, cul i a ion me hod-
ologies, oli es ha es ing s age, oil au o-oxida ion le el, and senso y
p o ile (Luna e al., 2006; Melucci e al., 2016; Mo ales e al., 2005), by
GC×GC i was possible o consis en ly moni o mo e han 190 eliable
peak ea u es (including a ge ed and un a ge ed compounds (Magagna
e al., 2016)), o which 84 we e iden i ied acco ding o
1
D e en ion
index (I
T
±10 uni s o ole ance) combined o EI-MS spec al simila i y
(Di ec Ma ch Fac o h eshold >900) wi h comme cial and in-house
da abases. Table 2 lis s pu a i ely iden i ied compounds (i.e., a ge ed
ea u es) and po en odo an s oge he wi h e en ion da a, odo quali-
ies, odo h esholds (OTs) in oil, and FID p edic ed RRFs calcula ed
acco ding o hexanal as e e ence in e nal s anda d. Table S1, p o ided
as supplemen a y ma e ial, lis s a ge ed and un a ge ed ea u es
comp ehensi ely mapping he de ec able ola ilome o analyzed
samples.
As a i s app oach o unde s anding he exis ence o na u al sam-
ples’clus e s acco ding o ola ile pa e ns dis ibu ion, unsupe ised
explo a ion by P incipal Componen Analysis (PCA) was conduc ed.
Fig. 1Aillus a es PCA esul s ob ained by examining he da a ma ix
including un a ge ed and a ge ed (UT) peak ea u es (analy es) % e-
sponses o all Picual oils (blue indica o s) and Hojiblanca oils (g een
indica o s) (190 ×32 – ea u es ×samples eplica es). I ep esen s a
o al explained a iance o 41.62 % (PC1 and PC2) and, along he PC1
wi h 31.32 % o he o al explained a iance, he wo cul i a s o m
na u al clus e s as shown by ellipses, se a 95 % con idence. Besides
some ou lie s, he cul i a s’chemical signa u e is almos clea con-
i ming ha he ola ile ac ion in o o enc yp s in o ma ion on oli es’
chemo ype use ul o hei disc imina ion. This esul ag ees wi h o he
au ho s who obse ed ha bo anical ai s ha e he mos in luence on
he ola ile p o ile o EVOOs (Ríos-Reina e al., 2021).
Ne e heless, a ce ain deg ee o o e lap be ween he wo clus e s
sugges s ha o some samples he ola ilome has simila composi ion,
possibly due o he in luence o he ha es egion wi h i s common
pedoclima ic and soil cha ac e is ics. Howe e , he simul aneous p es-
ence o many a iables in luencing he ola iles’quali a i e and quan-
i a i e p o iles, such as he ipening s age o he ui s, migh ha e a
con ounding e ec . Acco ding o p e ious in es iga ions, he ipening
s age o he ui was, a e he a ie y/cul i a , he mos in luen ial
ac o on he ola ile ac ion (Magagna e al., 2016; Ríos-Reina e al.,
2021; S ilo e al., 2021b). A deepe obse a ion o he samples’dis i-
bu ion on he Ca esian plane (Fig. 1A), indica es a clea end along PC1
wi h oils ob ained by oli es ha es ed a S age I cha ac e ized by lowe
loadings and hose ob ained by oli es a S age III wi h highe alues.
This is in acco dance o he inc easing ipening index as epo ed in
Table 1. Analy es esponsible o his dis ibu ion a e, among he o he s:
(E,E)-
α
- a nesene, (5Z)-3-e hyl-1,5-oc adiene, oc ane, (E,Z)-3,7-deca-
diene, 3,4-die hyl-1,5-hexadiene (meso), 3,4-die hyl-1,5-hexadiene
(RS+SR), (Z)-3-hexenal, (Z)-2-hexenal, phenyle hyl alcohol, 6-me h-
yl-5-hep en-2-one, (Z)-3-hexen-1-ol, (E,E)-3,7-decadiene, 1-pen e-
n-3-one, 1-pen en-3-ol, and nonanal. Resul s a e in keeping wi h
p e ious s udies on ipening indices (Ange osa e al., 1998; Magagna
e al., 2016; S ilo e al., 2021b).
To iden i y cul i a disc imina ing a iables, he UT ea u es %
esponse da a we e il e ed by he Fishe a io (F) alue c i e ion
(Sch¨
oneich e al., 2022). Since F
c i
(1,15) wi h
α
=0.05 is 4.54, UT
a iables wi h a F
calc
>10 (a bi a ily se abo e he c i ical alue) we e
e ained (n=92 UT ea u es), and by applying his ea u es selec ion
c i e ion, he esul ing PCA shown in Fig. 1Bclea ly clus e s samples
acco ding o he cul i a ype. The o al explained a iance aises
60.74 % (PC1 and PC2) and samples a e now independen ly clus e ed
and disc imina ed along he PC1 (49.65 % o he o al explained a i-
ance). F alues e e ed o UT ea u es disc imina ing cul i a s a e e-
po ed in Table S1.
The impac o cul i a ion me hodology (i.e., con en ional s.
o ganic) was hen explo ed. The PCA was conduc ed on he da a ma ix
including UT peak ea u es (analy es) % esponses wi h an F alue >10
o o ganic s. con en ional classes. F alues disc imina ing cul i a ion
me hods a e lis ed in Table S1. PCA loadings plo based on he 35 ea-
u es wi h F>10 is shown in Fig. 2. Resul s con i m he majo ole played
by cul i a ai s on he ola ilome exp ession, wi h a o al explained
a iance o 61.58 % he i s wo PCs do no show any independen
clus e ing o o ganic and con en ional cul i a ed oli e ee samples.
Howe e , o Picual oils, a dis inc ion o he wo sub-g oups along he
PC1 appea s (pink and ligh g een indica o s).
Due o he concu en e ec o ex e nal a iables, as cul i a and
cul i a ion p ac ices, he iden i ica ion o ma ke ola iles o o ganic
cul i a ion was conduc ed on samples belonging o he same cul i a
wi h he aid o supe ised s a egies.
3.1. Picual oli e oils: o ganic s. con en ional cul i a ion diagnos ic
ola iles signa u e
Conside ing he Picual cul i a , he unsupe ised s a is ics on he wo
cul i a ion me hodologies (F
calc
>5 o o ganic s. con en ional classes)
show wo independen g oups along PC1, wi h a o al explained a i-
ance o 58.93 % (e.g., Fig. 1C). A he same ime, hea map isualiza ion
(e.g., Fig. S1A) cap u es diagnos ic pa e ns o UT ola iles and by hi-
e a chical clus e ing (HC) based on Pea son co ela ion o a iables
o m wo independen clus e s co esponding o cul i a ion me hodol-
ogies. Sub-clus e s a e cohe en wi h biological and echnical eplica es.
To selec s a is ically ele an ye in o ma i e compounds ela ed o
cul i a ion me hodologies on Picual oils, supe ised analysis by pa ial
leas squa es-disc iminan analysis (PLS-DA) was adop ed (Lee e al.,
2018). The model was de eloped on an es ima ion se consis ing o 70 %
andomly selec ed measu es/samples (20 samples o e 28) and ali-
da ed on he esidual 30 % (8 samples o e 28). Model pe o mances,
a e 10 ei e a ions, we e good e e ing o a 98 % o co ec ness. The
a iable impo ance in he p ojec ion sco es (VIPs) we e used o iden i y
meaning ul a iables, esul ing om he classi ica ion model on Picual
EVO oils con en ional s. o ganic, hey a e isualized as his og am in
Fig. 3 (o ange ba s). Table sS2 lis s VIPs alues oge he wi h ela i e
s anda d de ia ion. Disc iminan analy es be ween he cul i a ion
me hodologies belongs o he class o po en odo an s wi h 2-pen anol,
1-hexanol, (Z)-3-hexen-1-ol likely disc imina ing he Hojiblanca
cul i a om he Picual which has a cha ac e is ic pa e n o odo an s
wi h (E)-2-pen en-1-ol, hep anal, and (E)-2-pen enal. Va iables showing
Fig. 2. PCA on un a ge ed and a ge ed (UT) peak ea u es (analy es) % e-
sponses a e Fishe a io educ ion acco ding o classes o ganic s. con en-
ional and F>10 (35 UT ea u es). Red indica o s a e o Hojiblanca
con en ional, pink is o Hojiblanca o ganic, da k g een is o Picual con en-
ional, and ligh g een is o Picual o ganic.
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
7
a ole in he disc imina ion a e also alida ed by HC esul s. O in e es
a e ca bonyls [2-hep anone, 2-oc anone, hep anal, (E)-2-pen enal] and
alkenes [(5E)-3-e hyl-1,5-oc adiene, (E,E)-3,7-decadiene, 3,4-die hyl-1,
5-hexadiene (RS+SR), (5Z)-3-e hyl-1,5-oc adiene, (E,Z)-3,7-deca-
diene]; his las class o chemicals known o hei co ela ion wi h ol-
i es ipening (Ange osa e al., 1998).
The ela i e dis ibu ion o he mos in o ma i e a iables is illus-
a ed by box-plo s in Fig. 4. Analy es wi h highe % esponse in con-
en ional cul i a ion a e me hoxyme hyl-benzene [
α
-me hylbenzyl
e he al eady documen ed(da Sil a e al., 2012)], (5E)-3-e hyl-1,
5-oc adiene, (5Z)-3-e hyl-1,5-oc adiene, 3,4-die hyl-1,5-hexadiene
(RS+SR), (E,E)-3,7-decadiene, (E,Z)-3,7-decadiene, (Z)-3-hexenyl ace-
a e, (E)-3-hexen-1-ol ace a e, and
α
-copaene. Compounds wi h an
opposi e end a e 2-hep anone, 2-oc anone, and bu anoic acid.
Acco ding o he li e a u e, (5E)-3-e hyl-1,5-oc adiene, (5Z)- 3-e hyl-
1,5-oc adiene, 3,4-die hyl-1,5-hexadiene (RS o SR), (E,E)-3,7-deca-
diene and (E,Z)-3,7-decadiene ha e been ound in Picual EVOOs and
we e conside ed as ma ke s o ea ly ipening s ages, independen ly o
he a ie y/cul i a and geog aphical o igin (S ilo e al., 2021b). In
addi ion, 3-e hyl-1,5-oc adiene, a compound de i ing om he lip-
oxygenase pa hway, eco ded signi ican a ia ions acco ding o he
cul i a and/o en i onmen al condi ions on oli e ee g owing (Kosma
e al., 2020).
(Z)-3-Hexenyl ace a e is a compound esponsible o senso y a i-
bu es as g een lea es, and i was shown o be ele an in he di e en i-
a ion be ween non-de ec i e (EVO) and de ec i e (non-EVO) oli e oil
samples, being p esen in highe concen a ion in EVOO samples
(Ríos-Reina e al., 2021). Non-de ec i e (EVO) oli e oil is o he highes
quali y and ee o senso y de ec s, while de ec i e (non-EVO) oli e oil
does no mee hese s ic s anda ds and is o lowe quali y. In addi ion,
aligned wi h he cu en e idence, (Z)-3-hexenyl ace a e was di e en-
ially dis ibu ed in o ganic and con en ional samples conside ed in a
p e ious s udy (Ju ado-Campos e al., 2021).
α
-Copaene is a mono-unsa u a ed sesqui e pene ha has al eady
been de ec ed in Spanish oils ob ained om oli es o he Hojiblanca
cul i a and Picual (Bo olomeazzi e al., 2001; Guinda e al., 1996), and
oge he wi h
α
-muu olene and
α
- a nesene we e he e penes ha aided
he disc imina ion be ween ex a i gin oli e oil acco ding o he
cul i a and geog aphical o igin (Bubola e al., 2014; Luki´
c e al., 2018;
Zunin e al., 2005) and hey ha e been sugges ed as ma ke s o oli e oil
di e en ia ion (Kosma e al., 2020). Acco ding o he li e a u e, bu a-
noic acid has been associa ed wi h inega y,mus y, and ancid de ec s
(Ange osa, 2002; Cecchi e al., 2019).
3.2. Hojiblanca oli e oils: o ganic s. con en ional cul i a ion diagnos ic
ola iles signa u e
A i s explo a o y app oach was conduc ed on Hojiblanca samples
conside ing he cul i a ion p ac ice. Fig. 1Dshows he PCA esul ing
om UT ea u es il e ed by Fishe a io alue (F
calc
>10). Samples, as
expec ed, a e clea ly clus e ed acco ding o cul i a ion p ac ice and he
o al explained a iance o PC1-PC3 achie es 61.72 %. A he same
ime, hea map isualiza ion (p o ided as supplemen a y ma e ial
Fig. S1) cap u es diagnos ic pa e ns o UT ola iles and by hie a chical
clus e ing (HC) based on Pea son co ela ion o a iables o m wo in-
dependen clus e s co esponding o cul i a ion me hodologies. Sub-
clus e s a e cohe en wi h biological and echnical eplica es. Chemi-
cal a iables highligh ed by PLS-DA as dis inc i e o he cul i a ion
Fig. 3. : His og am showing he VIPs alues de i ed by he PLS-DA classi ica ion models con en ional s. o ganic classes. Blue ba s a e o Hojiblanca and o ange ba s
o Picual. The Venn diag am isualizes he numbe o a ge ed analy es ( ea u es) in common (n=6) o unique (n=10 o Picual and n=6 o Hojiblanca) o he
wo cul i a s.
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
8
Fig. 4. Box plo s o disc iminan compounds ( om PLS-DA modeling) be ween o ganic and con en ional cul i a ion on Picual cul i a samples. G een boxes
ep esen da a om o ganic a ming (mean/median) while pink boxes a e o con en ional a ming (mean/median).
A. Ca a i e al. Jou nal o Food Composi ion and Analysis 137 (2025) 106975
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