1
Analysis o ola ile compounds in glu en- ee b ead c us s wi h an op imised and
alida ed SPME-GC/QTOF me hodology
Joana Pico a*, Bea iz An olín a, Lau a Román b, Manuel Gómez b, José Be nal a
a I.U.Cinquima, Analy ical Chemis y G oup, Uni e si y o Valladolid, Paseo de Belén
S ee 7, E-47011 Valladolid, Spain.
b Food Technology A ea, E.T.S. Ingenie ías Ag a ias, Uni e si y o Valladolid, Mad id
A enue 57, E- 34071 Palencia, Spain.
*Co esponding au ho : Joana Pico e-mail: [email p o ec ed]
Highligh s
 A SPME-GC/QTOF me hod was de eloped and op imised o analysing b ead
c us a oma.
 SPME condi ions implied 0.75 g o c us sample ex ac ed a 60°C o 51 min.
 The p oposed SPME-GC/QTOF me hodology was sensible, p ecise, accu a e
and linea .
 Fu u al was he mos abundan compound in comme cial whea b ead c us .
 Te and whea s a ch c us s showed con en s o 2-ACPY close o whea c us .
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Abs ac
The a oma o b ead c us , as one o he i s cha ac e is ics pe cei ed, is essen ial o
b ead accep ance. Howe e , glu en- ee b ead c us s exhibi weak a oma. A SPME-
GC/QTOF me hodology was op imised wi h PCA and RSM and alida ed o he
quan i ica ion o 44 ola ile compounds in b ead c us , ex ac ing 0.75 g o c us a
60°C o 51 min. LODs anged be ween 3.60-1760 µgKg-1, all he R2 we e highe han
0.99 and %RSD o p ecision and %E o accu acy we e lowe han 9% and 12%,
espec i ely. A comme cial whea b ead c us was quan i ied, and u u al was he mos
abundan compound. B ead c us s o whea s a ch and o japonica ice, basma i ice and
e lou s we e also quan i ied. Te lou and whea s a ch c us s we e e y sui able o
imp o ing glu en- ee b ead c us a oma, due o hei simila con en in 2-ace yl-1-
py oline and 4-hyd oxy-2,5-dime hyl-3(2H)- u anone compa ed o whea lou c us
and also o hei high con en in py azines.
Keywo ds: ola ile compounds; SPME-GC/QTOF; b ead c us ; glu en- ee b ead;
PCA; RSM.
Abb e ia ions: 2-ACPY (2-ace yl-1-py oline); CAR (ca boxen); CCD (cen al
composi e design); D (desi abili y unc ion); DVB (di inylbenzene); DOE (design o
expe imen s); FD ( la ou dilu ion ac o ); GC/QTOF (gas ch oma og aphy/quad upole-
ime o ligh ); HPMC (hyd oxyl p opyl me hyl cellulose); LOD (limi o de ec ion);
LOQ (limi o quan i ica ion); MSA (me hod o s anda d addi ion); OT (odou
h eshold); PA (polyac yla e); PC (p incipal componen ); PCA (p incipal componen
analysis); PDMS (polydime hylsiloxane); R2 (coe icien o de e mina ion); Re ( ela i e
e o ); RSD ( ela i e s anda d de ia ion); RSM ( esponse su ace me hod); SPME
(solid-phase mic oex ac ion).
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1. In oduc ion
The a oma o b ead c us is one o he i s a ibu es sensed when en e ing a bake y
shop. I has been cha ac e ised by ola ile compounds om Mailla d eac ions,
ca amelisa ion and he mal deg ada ion (Pico, Be nal, & Gómez, 2015), al hough he e
can be ola ile compounds om lipid oxida ion in smalle p opo ions (Moskowi z, Bin,
Elias, & Pe e son, 2012). 2-Ace yl-1-py oline, gene a ed by Mailla d eac ions, has
been conside ed he key ola ile compound o whea lou b ead c us . O he impo an
ola ile compounds include 3-me hylbu anal, 2,3-bu anedione and 4-hyd oxy-2,5-
dime hyl-3(2H)- u anone, also om Mailla d eac ions, along wi h 2-(E)-nonenal and
2,4-(E,E)-decadienal om lipid oxida ion (Zehen baue & G osch, 1998).
In he case o glu en- ee b ead, he senso y quali y is ba ely accep able, almos no ably
he ex u e and he a oma (Pacyński, Woj asiak, & Mildne -Szkudla z, 2015). Quali y
pa ame e s such as nu i ional alue, heology o he dough, ex u e, olume and colou
ha e been widely s udied in glu en- ee b ead (Houben, Höchs ö e , & Becke , 2012;
Masu e, Fie ens, & Delcou , 2016). Howe e , he e is li le knowledge ega ding he
a oma o glu en- ee b ead c us s. To ou knowledge, only Pacyński e al. (2015) ha e
s udied he ola ile compounds o glu en- ee b ead c us s wi h amino acid – suga pai s
added wi h he aim o p omo ing he gene a ion o Mailla d compounds and imp o ing
he a oma o he c us .
The e o e, he analysis o ola ile compounds o b ead c us becomes essen ial in o de
o imp o e b ead quali y, abo e all o glu en- ee b ead c us s. In he las decade, solid
phase mic oex ac ion (SPME) combined wi h GC/MS has been p e e ed because i is
a quick, simple and sol en - ee echnique (Thompson-Wi ick e al., 2015). Mo eo e ,
i only equi es a minimal amoun o sample, which is impo an in he case o glu en-
ee b eads ha p esen a poo c us . Focusing on SPME-GC/MS ola ile compounds
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analyses, mos esea che s ha e s udied he c umb and c us oge he (Pa aske opoulou,
Ch ysan hou, & Kou idou, 2012; Plessas e al., 2008, 2011; Poino e al., 2007, 2008a).
The s udy o he ola ile compounds om he c us sepa a ely om he c umb is e y
impo an in o de o unde s and i s ola ile p o ile. To ou knowledge, only Ra o e al.
(2015) and Pacyński e al. (2015) ha e s udied he ola ile compounds o b ead c us by
SPME-GC/MS, he la e examining glu en- ee b ead c us . On he o he hand,
unde s anding he pe o mance cha ac e is ics o he analy ical me hodology is c ucial
in o de o achie e eliable esul s, bu his in o ma ion has only been epo ed o
SPME-GC/MS analyses o b ead by Ra o e al. (2015). They s udied he epea abili y,
in e media e p ecision, linea i y as well as LOD and LOQ o ola ile compounds
analyses in whea b ead c us . Howe e , o he bes o ou knowledge, he accu acy has
no been s udied o any SPME-GC/MS me hodology; e i ying he accu acy is e y
impo an o in e p e ing he quan i ica ions made om hese me hodologies, since i
exp esses he closeness o he expe imen al esul o he accep ed alue (AOAC
guideline, 2002). Finally, he op imisa ion o he me hodology be o e i s alida ion is
also impe a i e so as o ensu e ha he maximum amoun o analy e is ex ac ed, bu
any op imisa ion was ca ied ou by Ra o e al. (2015) o he analysis o he ola ile
compounds o he c us by SPME-GC/QTOF. Mo eo e , as a as we know, he use o
s a is ical ools such as he Response Su ace Me hod (RSM) has no been epo ed o
he op imisa ion o SPME me hodologies o b ead ola ile compounds analyses.
The e o e, he i s aim o his s udy was o op imise and alida e a SPME-
GC/quad upole- ime-o - ligh (QTOF) me hodology o he semi-quan i ica ion (lowe
limi s o de ec ion, since i wo ks in spli less mode) and quan i ica ion (highe limi s o
de ec ion, since i wo ks in spli mode) o 44 ola ile compounds in b ead c us ,
employing a comme cial b ead c us sample o his pu pose. The quan i ica ion o he
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comme cial sample was made using he Me hod o S anda d Addi ion (MSA). I mus
be no ed ha his is he i s ime ha a SPME me hodology has been op imised h ough
he use o Design o Expe imen s (DOE) in he analysis o ola ile compounds in b ead,
speci ically wi h P incipal Componen Analysis (PCA) ollowed by RSM. The second
goal was o quan i y ola ile compounds h ough he MSA o e , basma i ice,
japonica ice and whea s a ch b ead c us s o he selec ion o he mos sui able glu en-
ee lou o s a ch o he imp o emen o he inal a oma o glu en- ee b ead c us ,
using whea b ead as a con ol sample. The choice o he quan i ied glu en- ee b ead
c us s was made using he semi-quan i ica ion me hod as sc eening p ocess o oa ,
quinoa, e , basma i ice, japonica ice and co n and whea s a ch.
2. Ma e ials and me hods
2.1. Ma e ials, eagen s and s anda ds
Fo he analy ical cha ac e isa ion o he me hod, 2-ace yl-1-py oline (2-ACPY) was
pu chased om Ep es (Ve ey, Swi ze land) and he o he 43 pu e s anda ds ound in
Table S1 we e pu chased om Sigma-Ald ich (S einheim, Ge many). Dichlo ome hane
was ob ained om Scha lab (Ba celona, Spain) and me hanol was om VWR
In e na ional (Fon enay-sous-Bois, F ance). A gon, ni ogen and helium we e acqui ed
om Ca bu os Me álicos (Ba celona, Spain).
2.2. P epa a ion o s anda d solu ions
2-ACPY solu ions we e p epa ed in dichlo ome hane, as 2-ACPY dime ises in
me hanol and wa e . I was necessa y o wo k unde ine a mosphe e o a gon a all
imes due o he compound’s lack o s abili y o oxygen and mois u e. Fo his eason,
dichlo ome hane was d ied in a SDS PS-MD-5 pu i ica ion sys em om Düpe hal
Siche hei s echnik (Ka ls ein am Main, Ge many). Fo he o he 43 ola ile compounds

6
included in Table 1, wo king solu ions o each ola ile compound we e p epa ed in
me hanol. All he solu ions we e s o ed in a eeze a -20°C.
2.3. Sample employed o he de elopmen o he SPME-GC/QTOF me hod
The de elopmen and cha ac e isa ion o he me hodology we e ca ied ou wi h he
c us o whea b ead pu chased om Fo asa (Puçol, Spain). The label indica ed ha he
ing edien s we e whea lou , wa e , sal , yeas and lou imp o e (whea lou , an i-
caking agen (E-170), emulsi ie (E-472e), an ioxidan (E-300) and enzymes).
Loa es o b ead we e cu in o slices o 5 cm wid h, including he ends. The c us was
sc a ched wi h a kni e, aking ca e no o emo e pieces o c umb. Once all he c us
was emo ed, i was ozen wi h liquid ni ogen and inally i was g ounded in an Ika
g inde model M20 (S au en, Ge many) o 10 seconds.
2.4. Glu en- ee b ead o mula ion: lou s, s a ches, hyd ocolloid and yeas
Whea s a ch was supplied by Roque e Laisa (Valencia, Spain), co n s a ch by Miwon
Daesang (Seul, Ko ea) and whea lou by Ha ine a Cas ellana (Medina del Campo,
España). Japanica ice lou was pu chased om Molendum ing edien s (Zamo a,
Spain), oa lou om Emilio Es eban (Valladolid, Spain), quinoa lou om El G ane o
In eg al (Mad id, Spain) and e lou om Salu e (Palencia, Spain). Basma i lou was
milled om basma i ice om Dacsa (Lisboa, Po ugal), employing a g inde model
Pe en 3300 (Häge s en, Sweden). Hyd oxyl p opyl me hyl cellulose (HPMC) K4M was
supplied by Dow Chemicals (Michigan, USA) and he d y bake ’s yeas
(Saccha omyces ce e isiae) by Lesa e (Ce ences, F ance). All yeas s belonged o he
same ba ch o dec ease he isk o di e en cell coun o yeas and di e en con aminan
bac e ia.
2.5. Glu en- ee b ead making
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The ollowing ing edien s, as g/100g o lou o s a ch, we e used in all he o mulas:
sun lowe oil (6 g/100 g), suc ose (5 g/100 g), sal (1.8 g/100g), yeas (3 g/100 g),
HPMC (2 g/100 g) and wa e (100 g/100 g). They we e mixed using a Ki chen-Aid
P o essional mixe (KPM5, Ki chenAid, S . Joseph, Michigan, USA) o 8 min a speed
o 56 pm. The e men a ion was ca ied ou o 90 min in a chambe a 30°C wi h 90%
o ela i e humidi y, eaching a e age speci ic olumes be ween 1.91 ml/g and 6.89
ml/g (da a no shown). The doughs we e baked, in ows o wo, a 190°C o 40 min in a
con ec ion o en model Sal a 5 g id (Guipuzcoa, Spain). A e baking, he glu en- ee
b eads we e le a oom empe a u e o 30 min and cu as desc ibed in sub-sec ion 2.3.
Each sample was p epa ed in duplica e (n=2).
2.6. Solid-phase mic oex ac ion
Fou ib es we e es ed, including polydime hysiloxane / di inylbenzene (PDMS/DVB)
(65 µm), ca boxen / polydime hysiloxane (CAR/PDMS) (85 µm), di inylbenzene /
ca boxen / polydime hysiloxane (DVB/CAR/PDMS) (50/30 µm) and polyac yla e (85
µm), all o hem om Sigma Ald ich (Gillingham, UK). The selec ed ib e was
DVB/CAR/PDMS and an au osample was employed o he ex ac ion o he ola ile
compounds. An amoun o 0.75 g (± 0.0050 g) o whea b ead c us was weighed in o a
20 mL ial and sealed wi h a magne ic sc ew cap p o ided wi h PTFE/silicone sep a.
The sample was incuba ed in he o en o 5 min a 60°C (wi hou he ib e) and hen he
ola ile compounds we e ex ac ed in he same o en o 51 min a 60°C, wi hou
agi a ion. A e ha , he ib e was inse ed in o he GC injec o po o he mal
deso p ion o 5 min a 270°C, wi h an injec ion olume o 1 µL. Finally, he ib e was
condi ioned o 30 min a 270°C a e each analysis.
2.7. GC/QTOF ch oma og aphic condi ions
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GC/QTOF analyses we e pe o med on a 7890A gas ch oma og aph coupled o a 7200
Quad upole-Time o ligh (QTOF) mass spec ome e de ec o and MassHun e
B.07.00 so wa e, all om Agilen Technologies (San a Cla a, Cali o nia, USA). The
GC was equipped wi h a CombiPAL RSI 85 au osample om CTC Analy ics AG
(Zwingen, Swi ze land). The sepa a ion was achie ed on a pola Innowax column
(100% polye hylene glycol, 30 m × 0.25 mm ID × 0.25 μm) ob ained om J&W
Scien i ic (Agilen Technologies, Cali o nia, USA). The ch oma og aphic condi ions
we e p e iously op imised by he esea ch g oup using s anda d solu ions (Pico, del
Nozal, Be nal, & Gómez, 2017). The GC was ope a ed unde p og ammed empe a u e
condi ions: om 45°C (1.5 min) o 100°C (0 min) a 7°C/min, hen he empe a u e was
inc eased o 114°C (6.7 min) a 1°C/min, a e wa ds i was inc eased o 136°C (0 min)
a 2.5°C/min and inally i was inc eased o 245°C (5 min) a 85°C/min. To al un ime
was 43 min. The ca ie gas was helium a a low a e o 1.1 mL/min. The injec o
empe a u e was 270°C, wo king in spli less mode o semi-quan i a i e analyses and in
spli mode o quan i a i e analyses. When he sample was spiked in he quan i a i e
analysis using MSA, he mos abundan compounds sa u a ed he de ec o , hus i was
compulso y o dilu e he sample wo king in spli mode. I he sample was spiked wi h
less concen a ion, he inc ease in he signal was no su icien o achie e good
quan i ica ion. Howe e , when he sample was no spiked he e was no sa u a ion and i
was possible o wo k in spli less mode, inc easing he sensi i i y. The use o wo
wo king modes o di e en compounds was possible because he same ola ile
compound was s udied in all he samples, which we e injec ed in he same mode.
Howe e , di e en compounds injec ed in di e en modes we e no compa ed. The
in e ace, ion sou ce and quad upole empe a u es we e 250°C, 230°C and 150°C,
espec i ely. Analyses we e pe o med in SCAN mode and included a mass ange o
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20–350 m/z, ope a ing in elec on ioniza ion mode wi h ene gy o 70 eV. All he 44
ola ile compounds shown in Table S1 we e iden i ied by compa ison o hei e en ion
imes and accu a e mass spec a (wi h ou decimal places) wi h s anda ds as well as
using hei Ko a s Index (Table S1) and hei Mass Spec a Lib a y (NIST MS Sea ch
2.2 & MS In e p e e ).
2.8. Valida ion o he SPME-GC/QTOF me hod
The analy ical pa ame e s we e e alua ed ollowing he AOAC guidelines (2002).
2.8.1. Limi s o de ec ion (LODs) and quan i ica ion (LOQs)
These pa ame e s we e calcula ed compa ing he a ea o analy e peaks om a spiked
c us sample and he a ea o he noise om a blank ( he ai o an emp y ial) a he same
e en ion ime as ha o he analy e peaks. Injec ions we e made in quin uplica e (n=5).
LODs we e calcula ed as 3 imes he signal o noise a io (S/N), while LOQs we e
calcula ed as 10 imes he S/N.
2.8.2. P ecision: in a-day epea abili y and in e -day epea abili y
Fo in a-day epea abili y, c us samples we e injec ed in quin uplica e and he RSD
(%) o each compound was calcula ed (n=5). In e ms o in e -day epea abili y, c us
samples we e injec ed in quin uplica e on h ee al e na e days and RSD (%) was
calcula ed (n=5). Following he AOAC guidelines (2002), maximum RSDs o 15%
we e accep ed o he epea abili y.
2.8.3. Quan i ica ion o ola ile compounds o he comme cial whea b ead c us
sample: linea i y and accu acy.
The quan i ica ion was made using he MSA. A ma ix-ma ched calib a ion cu e was
made spiking six aliquo s o he comme cial c us sample wi h inc easing concen a ion
o he s anda d mix u e (which con ains he 44 ola ile compounds) wi hin he ange o
0.150 – 1.30 mg Kg-1. Six poin s we e included in he calib a ion cu e. The
16
highe han 1 mg Kg-1 we e conside ed. Then, he PCA was cons uc ed wi h he 30
ola ile compounds epo ed in Table S3, in peak a eas, as shown in Figu e 1.
Mo eo e , in o de o a oid ha he highes a eas had mo e impo ance in he weigh o
he PCs, he h ee i s PCs we e no malised as a co ela ion ma ix (Table S3). Then,
only hose compounds wi h no malised PCs highe han 0.700 we e aken in o
conside a ion in his discussion, which we e calcula ed as he PC mul iplied by he
squa e oo o he co esponding eigen alue. These ola ile compounds we e: py azine,
2-me hylpy azine, 2,5,-dime hylpy azine, 2,3,5- ime hylpy azine, 2-e hyl-3-
me hylpy azine, all om he Mailla d eac ion, and 2-(E)-nonenal, 2,4-(E,E)-decadienal
and benzaldehyde, om lipid oxida ion.
As an o e iew o he sco es plo o he PCA (Figu e 1), ice b ead c us as well as e
b ead c us we e he samples wi h ola ile p o iles mo e simila o whea b ead c us . 4-
Hyd oxy-2,5-dime hyl-3(2H)- u anone, 4- inylguaiacol, 2-(E)-nonenal and 2,4-(E,E)-
decadienal we e he ola ile compounds ha con ibu ed mo e o he posi i e PC1
(Table S3); hey we e ound in highe p opo ion in quinoa c us , whea c us , e c us
and ice c us and in lowe p opo ion in oa c us , basma i c us , co n s a ch c us and
whea s a ch c us . On he o he hand, he nega i e PC1 was cha ac e ised by he highes
con ibu ions o py azines, including py azine, 2-me hylpy azine, 2,5-dime hylpy azine,
2,3,5- ime hylpy azine and 2-e hyl-3-me hylpy azine. Whea s a ch c us p esen ed he
highes p opo ions o all o hem, being expec ed ha his would lead o a da ke c us
colou , bu i was one o he ligh es c us s (da a no shown). Py azines ha e been
epo ed as impo an Mailla d compounds in b ead c us (Pa aske opoulou,
Ch ysan hou, & Kou idou, 2012), ha should con ibu e o i s colou (Cho & Pe e son,
2010); howe e , whea lou c us was he da kes c us . This sugges ed ha hese
py azines we e no he esponsible o he c us colou om compounds o Mailla d

17
eac ions. Addi ionally, u an de i a i es ha e been epo ed o con ibu e o he colou
o he hea ed ood (Ho mann, 1998). Conc e ely, u u yl alcohol has been epo ed o
polyme ise in acidic condi ions o alipha ic polyme s ha gi e a b own colou a ion o
he b ead (Oka u & Lachenmeie , 2017). Whea lou c us p esen ed he highes
abundance o u u yl alcohol, which can explain i da kes colou .
Mo eo e , he simila i y be ween whea s a ch c us and whea lou c us , mos no ably
ega ding he nega i e PC2, was ela ed o he high con en o py azines. Finally, he
highes p opo ion o 2-ACPY was ound in oa c us , al hough i was loca ed opposi e
o whea b ead and i s use was dis ega ded.
As a consequence, he c us s we e dis inguished mainly due o hei con en in
py azines, 2-ACPY, 2-(E)-nonenal and 2,4-(E,E)-decadienal, as i was explained in sub-
sec ion 3.3. The e o e, since ice c us and e c us we e loca ed nea o whea c us ,
hey we e selec ed o be quan i ied. Due o he high con en o py azines, whea s a ch
c us was also selec ed o be quan i ied and basma i c us was chosen in o de o s udy
he e ec o o he a ie ies o ice.
3.5. Quan i ica ion o he ola ile compounds o he selec ed glu en- ee b ead c us s:
imp o emen o glu en- ee b ead c us a oma
Fo y- wo ola ile compounds om whea s a ch b ead c us as well as e , japonica
ice and basma i ice b ead c us s we e quan i ied (Table 2). Whea b ead c us se ed
as a con ol sample and ace ic acid and u u yl alcohol we e excluded om he
quan i ica ion, since hei R2 alues we e lowe han 0.99 and hey did no pass he - es
o linea i y.
As o he semi-quan i a i e analysis, only hose ola ile compounds wi h OTs highe
han 1 mg Kg-1 we e aken in o conside a ion. Then, he PCA was cons uc ed wi h he
ola ile compounds labelled in Table S3 om numbe 1 o 28. The PCA o he
18
concen a ion o each ola ile compound, in µg Kg-1, is shown in Figu e 2. Rega ding
he sco es plo , basma i b ead c us and whea b ead c us we e loca ed in he nega i e
PC1 while whea s a ch, ice and e b ead c us s we e ound in he posi i e PC1. In he
nega i e PC1 o he loadings plo he e we e only 4 ola ile compounds, which mean
ha bo h b eads p esen ed a less complex ola ile p o ile in he c us . Basma ic c us
was cha ac e ised by he highes con en in 2-(E)-nonenal and limonene while whea
c us was cha ac e ised by he highes con en in 4-hyd oxy-2,5-dime hyl-3(2H)-
u anone (simila o he con en in whea s a ch). The con en o 2-(E)-nonenal in whea
c us was he second highes , almos 3 imes highe han he hi d one ( e c us ). Thus,
he main di e ence be ween whea c us and ice, e and whea s a ch c us s should be
ound in he con en o 2-(E)-nonenal. The e a e some con o e sies abou he impac
o 2-(E)-nonenal on he inal a oma o b ead, since i has been epo ed as co ela ing
posi i ely wi h g een no es (Hansen & Hansen, 1996; Salim-u -Rehman, Pa e son, &
Piggo , 2006), bu also nega i ely wi h a y no es (Quílez, Ruiz, & Rome o, 2006). In
ac , i has been epo ed as one o he ola ile compounds esponsible o he s aling o
b ead (Zehen baue & G osch, 1998). Rega ding he nega i e no es gene a ed du ing
he s aling o b ead, he lowe concen a ion o 2-(E)-nonenal in whea s a ch, e and
ice c us s could be conside ed a posi i e a ibu e o glu en- ee b eads. Finally,
al hough i con ibu ed minimally o he co ela ion ma ix (Table S3), 4-hyd oxy-2,5-
dime hyl-3(2H)- u anone om Mailla d eac ions (Moskowi z e al., 2012) has been
epo ed as an impo an con ibu o o c us a oma (Zehen baue & G osch, 1998), wi h
a ca amel-like smell (Moskowi z e al., 2012).
In he posi i e PC1 o he loadings plo , 1-oc en-3-ol, py azine, 2-me hylpy azine and
2,3,5- ime hylpy azine we e he ola ile compounds in highes abundance; hey we e
common o he ice, whea s a ch and e b ead c us s. Mo eo e , all o hem p esen ed
19
co ela ion alues highe han 0.75, hus hey con ibu ed o he o e all la ou o he
c us . 1-Oc en-3-ol is a ola ile compound om lipid oxida ion ha has been epo ed o
co ela e nega i ely wi h he inal a oma o b ead (Pa aske opoulou e al., 2012),
p esen ing he highes concen a ion in he ice b ead c us . Al hough he con en o
lipids is highe in e han in ice and whea s a ch (USDA Da abase, 2009), he amoun
o lipoxygenases is highe in ice (Wongdechsa ekul & Kongkia ikajo n, 2010) and he
concen a ion o an ioxidan s, such as la onoids and i amin E, is lowe in ice (Ingle ,
Chen, & Liu, 2015). Then, he oxida ion o lipids is encou aged in ice c us , jus i ying
he highes amoun o 1-oc en-3-ol in ice c us (lowe amoun o lipids bu highe
lipoxygenase ac i i y and lowe an ioxidan ac ion) and in he second place in e c us
(highe amoun o lipids bu lowe lipoxygenase ac i i y and highe an ioxidan ac ion).
In ac , ice c us and e c us p esen ed simila amoun s o hexanal and nonanal,
ola ile compounds o lipid oxida ion (Pico e al., 2015), which explained he balance
be ween he con en o lipids and he amoun o lipoxygenases and an ioxidan s. The
h ee py azines we e in simila concen a ions in ice, whea s a ch and e b ead c us s,
which could be one o he easons o hei sepa a ion om basma i and whea c us s.
2,3,5-T ime hylpy azine was he mos abundan py azine o e all and, in whea s a ch
c us , i was in highes concen a ion. In gene al, py azines ha e been epo ed as
impo an Mailla d compounds in b ead c us (Pa aske opoulou e al., 2012),
con ibu ing g ea ly o i s colou (Cho & Pe e son, 2010). The da kes c us was ha o
e , ollowed by ice and hen whea s a ch (da a no shown), bu he concen a ion o
hese py azines was no e y di e en (see Table 2). This sugges ed, as in he semi-
quan i a i e sec ion, ha py azine, 2-me hylpy azine and 2,3,5- ime hylpy azine we e
no esponsible o c us colou . The same easoning could be applied o he highes
con en o 2,6-dime hylpy azine in whea s a ch c us .
20
Wi hin he posi i e PC1, e c us (nega i e componen o he PC2) was sepa a ed om
ice and whea s a ch c us (posi i e componen o he PC2). Highe con en s o
hep anal, 2,4-decadienal, 1-me hylpy ol and 2,5-dime hylpy azine ound in ice and
whea s a ch c us s compa ed o e c us could explain his sepa a ion; meanwhile, e
was cha ac e ised by he highes con en in e men a ion ola ile compounds like
ace oin, phenylace aldehyde and 3-me hylbu anoic acid. Howe e , hese h ee
e men a ion ola ile compounds did no show high alues o co ela ion (Table S3),
p obably because hei p esence depended on he mig a ion om he c umb o he c us
and no on hei homogeneous gene a ion in he c us . The same occu ed wi h he
highes con en o 3-me hyl-1-bu anol in whea s a ch c us , which had a con en simila
o ha o whea lou c us . Hep anal and 2,4-decadienal, wi h co ela ion alues highe
han 0.70, a e lipid oxida ion ola ile compounds (Bi ch e al., 2014) wi h he highes
concen a ion in ice, p obably due o he same easons explained o 1-oc en-3-ol. 1-
Me hylpy ol as well as 2,5-dime hylpy azine, bo h ola ile compounds om he
Mailla d eac ion (Poino e al., 2008b), had high alues o co ela ion, al hough hey
ha e no been epo ed as impo an con ibu o s o he c us a oma.
The e o e, he glu en- ee b ead c us s we e mainly dis inguished by hei con en s o
ola ile compounds om lipid oxida ion and Mailla d eac ions, which ha e been
epo ed as he main compounds in he c us o whea b ead (Moskowi z e al., 2012), as
i was explained in sub-sec ion 3.4. Fo whea b ead c us , he con ol sample, he mos
abundan ola ile compound was 4-hyd oxy-2,5-dime hyl-3(2H)- u anone (26.8 µg Kg-
1). This could be he eason o he simila i y be ween whea lou c us and e and
whea s a ch c us s, as he con en s o 4-hyd oxy-2,5-dime hyl-3(2H)- u anone we e
20.0 µg Kg-1 and 22.6 µg Kg-1, espec i ely. Mo eo e , he con en s o he key a oma 2-
ACPY in whea lou b ead c us and in e and whea s a ch b ead c us s we e also
21
simila (0.0459 µg Kg-1, 0.0398 µg Kg-1 and 0.0321 µg Kg-1, espec i ely), explaining
he likeness o e and whea s a ch c us s ega ding whea lou c us . In he case o
e c us , 2-ACPY was he mos abundan compound ollowed by 2-e hyl-3-
me hylpy azine (32.1 µg Kg-1), while in he case o whea s a ch c us 2,3,5-
ime hylpy azine was he mos abundan (48.1 µg Kg-1) and 2-ACPY was second in
abundance. The e o e, a sui able mix u e be ween whea s a ch and e lou was
sugges ed in o de o imp o e he inal a oma o glu en- ee b ead.
By con as , Pacyński e al. (2015) epo ed ha hei glu en- ee b eads we e
cha ac e ised by a lack o py azines and 2-ace yl-1-py oline compa ed o he con ol
whea b ead. Howe e , we ound con en s o py azines ha a ied be ween 0.440 and
48.1 µg Kg-1 among he ou glu en- ee b eads and con en s o 2-ACPY ha a ied
be ween 0.210 and 39.8 µg Kg-1. The di e ences a e su p ising since Pacyński e al.
(2015) added, besides co n and whea s a ches, sou ces o amino acids and suga s ha
encou age he Mailla d eac ion, like glucose, milk powde and egg. The e o e, i
would be expec ed ha py azines and o he compounds om Mailla d eac ion we e
ound in he glu en- ee b eads s udied by Pacyński e al. (2015).
4. Conclusions
A SPME-GC/QTOF me hodology o he analyses o 44 ola ile compounds in b ead
c us s has been de eloped, op imised and alida ed. The op imisa ion was accomplished
wi h he applica ion o he Response Su ace Me hod (RSM), wi h p e ious educ ion o
he dimensionali y employing P incipal Componen Analysis (PCA). The inal SPME
condi ions we e 0.75 g o c us ex ac ed a 60°C o 51 min. The SPME-GC/QTOF
me hodology was alida ed in e ms o LOD and LOQ, p ecision, accu acy and
linea i y, p o ing ha i was sensible, p ecise, accu a e and linea . The me hodology
was applied o quan i ica ion h ough he Me hod o S anda d Addi ion (MSA) o a

22
comme cial whea b ead c us . Fu u al, which comes om Mailla d eac ions and
ca amelisa ion p ocesses, was he mos abundan compound in he comme cial whea
lou b ead c us , co esponding wi h he li e a u e. Fou selec ed glu en- ee b ead
c us s ( ice, basma i, e and whea s a ch) we e also quan i ied and compa ed wi h a
whea b ead c us con ol sample. I was concluded ha whea s a ch c us as well as
e c us we e he closes o he con ol whea c us due o hei simila con en s in 2-
ace yl-1-py oline (2-ACPY), 4-hyd oxy-2,5-dime hyl-3(2H)- u anone and py azines,
which ha e been epo ed as main compounds in whea b ead c us .
Acknowledgemen s
Joana Pico and Lau a Román would like o hank he Uni e si y o Valladolid o he
PhD ellowships. The au ho s acknowledge he inancial suppo o he Spanish
Minis y o Economy and Compe i i eness (P ojec AGL2014-52928-C2-2-R) and he
Eu opean Regional De elopmen Fund (FEDER). The au ho s would like o hank Anna
Hayes o he e ision o he English scien i ic language. Finally, he au ho s wan also
o hank Ep es (Ve ey, Swi ze land) o hei kindness when we pu chased he 2-ace yl-
1-py oline s anda d.
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32
Table 3. Peak a eas, di ided by 106, o he 44 s udied ola ile compounds in he c us s o co n s a ch, whea s a ch, basma i, ice, oa , e , quinoa and whea
b eads as well as in he comme cial b ead. Values a e means o h ee de e mina ions ± SD. Di e en le e s in he same ow show he signi ican di e ences.
Vola ile
compound
Co n s a ch
c us
Whea s a ch
c us
Basma i ice
c us
Japonica ice
c us
Oa
c us
Te
C us
Quinoa
c us
Whea lou
c us
2,3-Bu anedione
0.876 ab ±
0.0449
0.826 ab ±
0.0991
1.11 b ± 0.0983
0.991 b ±
0.0987
0.605 a ±
0.0710
0.919 ab ±
0.231
0.960 b ±
0.251
1.05 b ± 0.138
Hexanal
3.78 a ± 0.112
6.31 e ± 0.535
4.76 b ± 0.362
7.46 ± 0.408
5.84 de ±
0.294
5.37 bcd ±
0.200
5.66 cde ±
0.140
5.11 bc ±
0.0208
2-Me hyl-1-
p opanol
0.100 ab ±
0.0115
0.190 d ±
0.00213
0.183 cd ±
0.0209
0.252 e ±
0.0346
0.0613 a ±
0.00648
0.252 e ±
0.0375
0.0611 a ±
0.00857
0.141 bc ±
0.00215
1-Me hylpy ol
0.0374 a ±
0.00513
0.116 c ±
0.0123
0.102 c ±
0.00139
0.0658 b ±
0.00300
0.0476 ab ±
0.00512
0.160 d ±
0.0208
0.124 c ±
0.0139
0.116 c ±
0.00460
Hep anal
0.949 a ± 0.0531
1.41 bc ±
0.0771
1.08 ab ± 0.0489
2.22 d ±
0.0214
1.23 ab ±
0.0316
3.17 ± 0.178
2.65 e ±
0.124
1.74 c ± 0.355
R-Limonene
0.416 a ± 0.0570
2.82c ± 0.214
1.88 b ± 0.311
2.32 c ± 0.314
0.773 a ±
0.216
1.88 b ± 0.232
2.54 c ±
0.334
2.36 c ± 0.291
Py azine
2.62 bc ± 0.0211
3.60 d ± 0.132
3.59 d ± 0.296
3.02 c ± 0.115
2.26 b ±
0.216
2.38 b ± 0.284
0.636 a ±
0.0489
3.01 c ±
0.0482
2-Me hyl-1-
bu anol
1.38 a ± 0.218
3.30 e ± 0.186
2.53 cd ± 0.191
2.84 d ± 0.295
1.01 a ±
0.0368
2.06 b ± 0.224
1.04 a ±
0.0546
2.19 bc ±
0.218

33
Table 3. (con inued)
Vola ile
compound
Co n s a ch
c us
Whea s a ch
c us
Basma i ice
c us
Japonica ice
c us
Oa
c us
Te
C us
Quinoa
c us
Whea lou
c us
3-Me hyl-1-
bu anol
2.63 b ± 0.254
3.89 d ± 0.342
3.81 cd ± 0.0323
3.75 cd ±
0.0501
2.28 ab ±
0.113
3.50 c ±
0.0440
2.16 a ±
0.000333
3.65 cd ±
0.132
1-Pen anol
0.216 a ± 0.0319
0.793 bc ±
0.258
0.799 bc ±
0.0745
1.80 d ± 0.305
1.07 c ±
0.0309
0.731 b ±
0.0450
1.07 c ±
0.0225
0.817 bc ±
0.00182
2-Me hylpy azine
0.856 c ±
0.00643
2.14 ±
0.00514
1.26 e ± 0.0352
1.09 d ±
0.00524
1.22 e ±
0.00658
1.15 d ±
0.0351
0.158 a ±
0.00877
0.773 b ±
0.0589
Ace oin
4.43 ab ± 0.0937
4.18 a ± 0.172
5.19 c ± 0.0738
4.64 b ±
0.0573
5.74 d ± 0.231
4.61 b ±
0.0342
6.32 e ±
0.176
5.97 de ±
0.291
2-Oc anone
0.542 ab ±
0.0680
1.42 e ±
0.0662
0.629 c ± 0.0212
0.894 d ±
0.0256
0.366 a ±
0.00951
1.05 d ±
0.150
1.33 e ±
0.137
1.02 d ±
0.140
2,5-
Dime hylpy azine
0.185 b ±
0.0118
0.715 ±
0.0414
0.338 d ±
0.00568
0.289 c ±
0.0211
0.667 e ±
0.00713
0.358 d ±
0.0166
0.0432 a ±
0.00512
0.179 b ±
0.0199
2,6-
Dime hylpi azine
0.190 b ±
0.0154
0.299 d ±
0.00149
0.186 b ±
0.00286
0.184 b ±
0.00158
0.416 e ±
0.0129
0.199 bc ±
0.00468
0.0622 a ±
0.00162
0.221 c ±
0.138
2-E hylpy azine
0.0941 b ±
0.00348
0.258 ±
0.00484
0.157 d ±
0.00361
0.152 d ±
0.00768
0.157 d ±
0.00767
0.210 e ±
0.00940
0.0442 a ±
0.00424
0.129 c ±
0.0208
34
Table 3. (con inued)
Vola ile
compound
Co n s a ch
c us
Whea s a ch
c us
Basma i ice
c us
Japonica ice
c us
Oa
c us
Te
C us
Quinoa
c us
Whea lou
c us
2-Ace yl-1-
py oline
0.772 c ±
0.0411
1.07 d± 0.0759
0.690 bc ± 0.105
0.318 a ±
0.00103
1.37 e ±
0.0309
0.366 a ±
0.0878
0.498 ab ±
0.0838
0.317 a ±
0.0518
2,3-
Dime hylpy azine
3.55 b ±
0.00348
5.81 e ±
0.0737
3.65 b ± 0.262
3.48 b ± 0.0415
5.13 d ±
0.0641
3.53 b ±
0.215
0.817 a ±
0.0294
4.18 c ±
0.00460
1-Hexanol
0.591 a ±
0.0934
1.46 c ± 0.212
1.29 c ± 0.129
2.81 d ± 0.106
0.918 b ±
0.0738
3.66 e ±
0.203
3.95 e ±
0.0109
1.53 c ±
0.355
Nonanal
1.64 a ± 0.296
1.57 a ± 0.691
1.31 a ± 0.281
1.83 a ± 0.401
1.80 a ±
0.239
1.72 a ±
0.497
1.93 a ±
0.401
1.75 a ±
0.291
2,3,5-
ime hylpy azine
0.111 b ±
0.00363
0.567 ±
0.0370
0.162 c ± 0.0150
0.144 bc ±
0.00613
0.333 e ±
0.00146
0.200 d ±
0.000373
0.0445 a ±
0.00381
0.127 b ±
0.0482
2-E hyl-3-
me hylpi azine
1.84 b ± 0.0624
4.90 g ±
0.0361
2.20 c ± 0.102
2.44 cd ± 0.187
3.48 e ±
0.242
3.80 ±
0.128
0.777 a ±
0.0109
2.61 d ±
0.218
E hyl oc anoa e
2.88 ab ± 0.458
4.40 cd ±
0.485
3.34 bc ± 0.517
3.12 ab ± 0.482
1.97 a ±
0.235
4.75 d ±
0.317
4.05 bcd ±
0.840
3.32 bc ±
0.132
1-Oc en-3-ol
0.0523 c
±0.0000341
0.0293 ab ±
0.00329
0.0319 b ±
0.00522
0.0323 b ±
0.00702
0.0214 a ±
0.00223
0.0500 c ±
0.00724
0.028
ab±0.00446
0.0270ab±0.
00182
35
Table 3. (con inued)
Vola ile
compound
Co n s a ch
c us
Whea s a ch
c us
Basma i ice
c us
Japonica ice
c us
Oa
c us
Te
C us
Quinoa
c us
Whea lou
c us
Ace ic acid
0.961 a ± 0.114
1.62 bc ±
0.0788
1.55 bc ± 0.130
1.49 b ± 0.126
1.49 b ±
0.0580
2.06 d ±
0.178
2.98 e ±
0.286
1.88 cd ±
0.131
Fu u al
0.436 b ±
0.0367
0.437 b ±
0.0255
0.482 bc ±
0.0248
0.541 c ± 0.0185
0.232 a ±
0.00130
2.27 e ±
0.0704
4.58 ±
0.0714
2.17 d ±
0.0272
2-E hyl-1-hexanol
0.598 b ±
0.0629
0.602 b ±
0.113
0.536 ab ±
0.0442
0.562 b ±
0.0388
0.515 ab ±
0.00605
0.421 a ±
0.00181
0.425 a ±
0.00735
0.535 ab ±
0.00725
Benzaldehyde
0.0514 a ±
0.00504
0.083 ab ±
0.00903
0.138 bc ±
0.0216
0.141 bc ±
0.00224
0.101 abc ±
0.0123
0.122 abc ±
0.00890
0.166 c ±
0.0710
0.147 bc ±
0.0536
2-(E)-Nonenal
0.0519 bc ±
0.00477
0.023 a ±
0.00167
0.0473 b ±
0.00677
0.0630 c ±
0.00108
0.0463 b ±
0.00374
0.0796 d ±
0.00711
0.0853d ±
0.00922
0.0619 c ±
0.00744
5-Me hyl-2-
u aldehyde
0.0719 a ±
0.00133
0.088 a ±
0.000283
0.0860 a ±
0.00667
0.0896 a ±
0.00323
0.0760 a ±
0.00461
0.735 c ±
0.0229
1.89 d ±
0.0363
0.414 b ±
0.00920
Bu y olac one
2.59 b ± 0.308
2.08 ab ±
0.409
2.08 ab ± 0.117
1.79 a ± 0.249
2.59 b ±
0.221
4.40 d ±
0.381
4.01 cd ±
0.175
3.54 c ±
0.151
2-Ace ilpy azine
0.0986 a ±
0.00782
0.131 ab ±
0.0320
0.233 cd ±
0.0284
0.140 ab ±
0.0339
0.268 d ±
0.0189
0.198 bc ±
0.0334
0.0953a
±0.000390
0.246 cd ±
0.0487
Bu y ic acid
0.0507 a ±
0.00442
0.527 b ±
0.0572
0.0515 a ±
0.00144
0.0586 a ±
0.00337
0.0329 a ±
0.00164
0.0550 a ±
0.00586
0.0500±0.0
00718
0.0460 a ±
0.00252
Phenylace aldehyde
1.58 e ± 0.174
1.55 e ±
0.0777
0.739 cd ±
0.00331
0.917 d ±
0.0896
0.321 a ±
0.0467
0.662 bc ±
0.0554
0.507 ab ±
0.0534
0.430 a ±
0.0339
Fu u yl alcohol
0.906 c ± 0.0472
1.08 d ±
0.0303
0.845 bc ±
0.00720
0.922 c ±
0.00571
0.538 a ±
0.0101
0.753 b ±
0.0505
2.15 e ±
0.0782
3.24 ±
0.0649
2-Me hylbu y ic
acid
2.08 a ± 0.323
3.25 b ± 0.677
2.99 ab ± 0.485
3.22 b ± 0.663
2.01 a ±
0.283
3.31 b ±
0.439
2.42 ab ±
0.217
3.38 b ±
0.351
36
Table 3. (con inued)
Vola ile
compound
Co n s a ch
c us
Whea s a ch
c us
Basma i ice
c us
Japonica ice
c us
Oa
c us
Te
C us
Quinoa
c us
Whea
lou
c us
3-Me hylbu y ic
acid
2.29 ab ± 0.374
2.94 bc ± 0.518
2.90 bc ± 0.545
3.16 c ± 0.421
1.73 a ±
0.153
3.35 c ±
0.229
2.67 bc ±
0.171
3.36 c ±
0.270
2,4-(E,E)-
Decadienal
0.0381a± 0.00138
0.147b± 0.0198
0.0395a±
0.00303
0.152b± 0.0195
0.0432a±
0.00147
0.0521a±0.0
0427
0.525c±
0.0568
0.0602 a ±
0.00822
Hexanoic acid
0.0312ab±
0.000241
0.147c± 0.00238
0.0543ab±
0.00689
0.0831b±
0.00375
0.0249a±
0.00144
0.445e±
0.0637
0.271d±
0.0201
0.0480 ab ±
0.000601
Benzyl alcohol
0.853 a ± 0.250
1.20 a ± 0.338
1.44 a ± 0.284
8.01 c ± 0.664
4.66 b ±
0.431
1.070 a ±
0.261
4.91 b ±
0.356
0.827 a ±
0.104
Phenyle hyl
alcohol
0.772 a ± 0.0742
1.79 c ± 0.108
1.62 c ± 0.0223
1.20 b ± 0.115
0.653 a ±
0.0478
1.10 b ±
0.0265
0.806 a ±
0.0570
2.02 c ±
0.183
2-Ace ylpy ol
2.28 b ± 0.165
1.68 a ± 0.237
2.44 b ± 0.0961
2.57 bc ± 0.260
2.55 bc ±
0.160
3.44 d ±
0.209
4.31 e ±
0.267
2.94 c ±
0.00399
4-Hyd oxy-2,5-
dime hyl-3(2H)-
u anone
0.0484 a ± 0.0127
0.0238 a ±
0.00371
0.0853ab ±
0.00224
0.0719ab ±
0.00324
0.116 bc ±
0.0102
0.160c±
0.000359
0.495 e±
0.0652
0.289 d±
0.0424
4-Vinylguaiacol
0.108 cd± 0.0154
0.0200a ±
0.000557
0.102 bcd±
0.00686
0.0688 abc±
0.00524
0.0487ab ±
0.00266
0.155 d ±
0.00776
0.765 e ±
0.0661
0.0714abc
± 0.00766
37
Table S1. Peak a eas, di ided in o 106, o he 44 s udied ola ile compounds ound wi h he ou ib es es ed. The Ko a s index (KI) calcula ed o each ola ile
compound as well as he KI ound in he li e a u e a e also gi en. Di e en le e s in he same ow show he signi ican di e ences.
Vola ile compounds
KI
calcula ed
KI
li e a u e
DVB/CAR/PDMS
CAR/PDMS
PDMS/DVB
Polyac yla e
2,3-Bu anedione
978
984
1.59 d
1.42 c
0.270 b
nd* a
Hexanal
1060
1080
3.84 c
4.84 d
0.853 b
0.115 a
2-Me hyl-1-p opanol
1073
1052
0.657 b
1.18 c
0.0659 a
0.0456 a
1-Me hylpy ol
1013
1140
0.0664 c
0.115 c
0.0179 b
nd* a
Hep anal
1141
1168
0.117 b
0.122 b
0.186 c
nd* a
R-Limonene
1154
1202
0.803 c
0.442 b
0.917 d
0.0139 a
Py azine
1207
1216
2.80 c
4.61 d
0.685 b
0.103 a
2-Me hyl-1-bu anol
1207
1218
1.32 b
1.32 b
0.116 a
0.0315 a
3-Me hyl-1-bu anol
1207
1218
2.94 b
3.31 c
0.288 a
0.0766 a
1-Pen anol
1251
1257
0.449 c
0.697 d
0.141 b
0.0261 a
2-Me hylpy azine
1259
1268
2.66 c
5.83 d
1.83 b
0.175 a
Ace oin
1279
1286
9.29 c
17.9 d
3.74 b
1.96 a
2-Oc anone
1279
1283
0.0364 b
0.0945 c
nd* a
nd* a
2,5-Dime hylpy azine
1315
1316
0.152 b
0.143 b
0.214 c
0.0123 a
2,6-Dime hylpi azine
1321
1319
0.192 b
0.190 b
0.304 c
0.0181 a
2-E hylpy azine
1326
1323
0.402 b
0.300 c
0.381 c
0.0261 a
2-Ace yl-1-py oline
1326
1325
0.468 c
0.306 b
0.773 d
0.0495 a
2,3-Dime hylpy azine
1331
1330
0.277 b
0.327 c
0.311 c
nd* a
1-Hexanol
1353
1359
0.182 b
0.416 c
0.198 b
0.0316 a
Nonanal
1388
1396
0.185 c
0.0606 b
0.508 d
0.0315 a
2,3,5- ime hylpy azine
1395
1396
0.240 c
0.100 b
0.224 c
0.0171 a
2-E hyl-3-me hylpi azine
1395
1400
0.110 c
0.0720 b
0.114 c
0.00862 a
E hyl oc anoa e
1432
1437
0.120 b
0.0360 a
0.285 c
0.0277 a
1-Oc en-3-ol
1452
1456
2.64 d
1.82 b
2.22 c
0.289 a

38
Table S1. (con inued)
Vola ile compounds
KI
calcula ed
KI
li e a u e
DVB/CAR/PDMS
CAR/PDMS
PDMS/DVB
Polyac yla e
Ace ic acid
1445
1465
15.4 b
22.0 c
3.95 a
4.80 a
Fu u al
1461
1467
6.03 c
7.44 d
2.58 b
0.753 a
2-E hyl-1-hexanol
1490
1489
0.544 c
0.579 c
0.307 b
0.0396 a
Benzaldehyde
1511
1521
0.722 b
0.829 c
1.29 d
0.106 a
2-(E)-Nonenal
1526
1546
1.01 b
0.271 a
2.13 c
0.185 a
5-Me hyl-2- u aldehyde
1566
1574
0.251 b
0.304 c
0.348 d
0.0414 a
Bu y olac one
1610
1622
2.09 c
2.45 d
0.697 b
0.432 a
2-Ace ilpy azine
1613
1614
0.120 d
0.0433 b
0.0834 c
0.0263 a
Bu y ic acid
1623
1636
2.59 c
3.49 d
0.606 b
0.356 a
Phenylace aldehyde
1630
1642
2.66 b
0.956 a
4.51 c
0.774 a
Fu u yl alcohol
1657
1666
5.75 c
6.92 d
4.21 b
3.15 a
2-Me hylbu y ic acid
1663
1674
0.943 c
1.19 d
0.351 b
0.150 a
3-Me hylbu y ic acid
1663
1679
0.768 c
0.900 d
0.210 b
0.104 a
2,4-(E,E)-Decadienal
1797
1797
0.614 c
0.0539 a
1.68 d
0.332 b
Hexanoic acid
1904
1880
4.51 c
3.75 b
4.14 bc
2.10 a
Benzyl alcohol
1962
1893
0.393 c
0.314 b
0.705 d
0.255 a
Phenyle hyl alcohol
2041
1942
4.90 c
2.86 a
6.05 d
3.42 b
2-Ace ylpy ol
2168
1950
0.884 b
0.673 a
1.40 c
0.647 a
4-Hyd oxy-2,5-dime hyl-3(2H)- u anone
2205
2020
0.500 c
0.0542 a
0.788 d
0.308 b
4-Vinylguaiacol
2253
2230
0.544 b
0.126 a
0.786 c
0.601 b
*nd = no de ec ed
39
Table S2. Op imisa ion pa ame e s o he p oposed SPME me hod. The ma ix o
expe imen a ion as well as he p incipal componen s (PC1, PC2) and he desi abili y o he
mul iple RSM o each expe ience a e gi en.
Se
Weigh (g)
Time (min)
Tempe a u e (T)
PC1
PC2
Desi abili y
1
0.500
51.0
50.0
-1.95
1.51
0.542
2
0.750
51.0
50.0
0.21
3.08
0.709
3
0.250
51.0
50.0
-5.44
-1.23
0.259
4
0.500
51.0
70.0
7.36
-4.58
0.000
5
0.500
51.0
40.0
-7.92
-1.08
0.000
6
0.500
75.0
50.0
-0.76
0.42
0.538
7
0.500
30.0
50.0
-4.39
-0.29
0.350
8
0.500
51.0
60.0
4.64
-0.58
0.637
9
0.750
51.0
60.0
8.25
2.73
0.977
40
Table S3. Co ela ion pa ame e s ound o he ola ile compounds ha p esen ed odou h esholds (OT) lowe han 1 mg Kg-1 in he quan i a i e
me hodology. The OT lowe han 1 mg Kg-1 a e also gi en.
Vola ile compound
OT (µg Kg-1)
PC1* √EV 1
Quan i a i
e
PC2* √EV 2
Quan i a i e
PC3* √EV 3
Quan i a i e
2,3-Bu anedione (1)
6.50
0.831
0.047
-0.553
Hexanal (2)
4.50
0.511
-0.583
-0.608
1-Me hylpy ol (3)
37.0
0.921
0.101
0.336
Hep anal (4)
3.00
0.809
0.554
0.195
R-Limonene (5)
10.0
-0.850
-0.053
-0.467
Py azine (6)
100
0.848
-0.474
0.162
3-Me hyl-1-bu anol (7)
250
0.293
0.932
0.215
2-Me hylpy azine (8)
105
0.948
0.122
0.075
Ace oin (9)
800
0.316
-0.915
0.251
2-Oc anone (10)
50.0
0.550
0.215
0.525
2,5-Dime hylpy azine (11)
800
0.746
0.626
-0.191
2,6-Dime hylpi azine (12)
200
0.049
0.324
0.801
2-Ace yl-1-py oline (13)
0.0530
0.222
-0.311
0.889
Nonanal (14)
1.00
0.922
-0.329
0.127
2,3,5- ime hylpy azine (15)
400
0.731
0.020
0.088
2-E hyl-3-me hylpi azine (16)
0.400
0.661
-0.630
-0.404
E hyl oc anoa e (17)
92.0
0.730
0.390
-0.561
1-Oc en-3-ol (18)
1.00
0.928
-0.152
-0.141
41
Table S3. (con inued)
Vola ile compound
OT (µg K-1)
PC1* √EV 1
Quan i a i e
PC2* √EV 2
Quan i a i e
PC3* √EV 3
Quan i a i e
2-E hyl-1-hexanol (19)
138
0.106
0.333
0.535
Benzaldehyde (20)
350
0.682
0.321
-0.646
2-(E)-Nonenal (21)
0.0800
-0.894
0.053
-0.441
2-Ace ylpy azine (22)
62.0
0.164
0.378
0.280
Bu y ic acid (23)
240
0.851
-0.243
-0.105
Phenylace aldehyde (24)
4.00
0.091
-0.979
0.162
3-Me hylbu y ic acid (25)
120
0.490
-0.661
0.420
2,4-(E,E)-Decadienal (26)
0.100
0.714
0.228
-0.554
4-Hyd oxy-2,5-dime hyl-3(2H)- u anone (27)
30
-0.046
-0.151
0.979
4-Vinylguaiacol (28)
3.00
-0.202
-0.939
-0.252
Ace ic acid (29)
30
nq*
nq*
nq*
Fu u yl alcohol (30)
1000
nq*
nq*
nq*
* nq = no quan i ied