Accep ed Manusc ip
Imp o emen in he iden i ica ion and quan i ica ion OF UV il e s and addi i es in
sunsc een cosme ic c eams by gas ch oma og aphy/mass spec ome y h ough
h ee-way calib a ion echniques
L. Rubio, L. Val e de-Som, L.A. Sa abia, M.C. O iz
PII: S0039-9140(19)30782-9
DOI: h ps://doi.o g/10.1016/j. alan a.2019.120156
A icle Numbe : 120156
Re e ence: TAL 120156
To appea in: Talan a
Recei ed Da e: 3 June 2019
Re ised Da e: 10 July 2019
Accep ed Da e: 14 July 2019
Please ci e his a icle as: L. Rubio, L. Val e de-Som, L.A. Sa abia, M.C. O iz, Imp o emen in he
iden i ica ion and quan i ica ion OF UV il e s and addi i es in sunsc een cosme ic c eams by gas
ch oma og aphy/mass spec ome y h ough h ee-way calib a ion echniques, Talan a (2019), doi:
h ps://doi.o g/10.1016/j. alan a.2019.120156.
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IMPROVEMENT IN THE IDENTIFICATION AND QUANTIFICATION OF UV FILTERS AND
ADDITIVES IN SUNSCREEN COSMETIC CREAMS BY GAS CHROMATOGRAPHY/MASS
SPECTROMETRY THROUGH THREE-WAY CALIBRATION TECHNIQUES
L. Rubio
a
, L. Val e de-Som
a
, L.A. Sa abia
b
, M.C. O iz
a,1
a
Depa men o Chemis y,
b
Depa men o Ma hema ics and Compu a ion
Facul y o Sciences, Uni e sidad de Bu gos
Plaza Misael Bañuelos s/n, 09001 Bu gos (Spain)
Abb e ia ions
2
Abs ac
The simul aneous de e mina ion o
2,6-di- e -bu yl-4-me hyl-phenol (BHT), benzophenone
(BP), benzophenone-3 (BP3) and diisobu yl ph hala e (DiBP) in se en sunsc een c eams
was ca ied ou by gas ch oma og aphy/mass spec ome y (GC/MS) using DiBP-d
4
as
in e nal s anda d. The con en o BP3, which is a UV il e , mus no exceed 6% (w/w) in
1
Co esponding au ho . Telephone numbe : +34-947-259571. E-mail add ess: mco [email protected] (M.C.
O iz).
2
2,6-di- e -bu yl-4-me hyl-phenol (BHT), benzophenone (BP), benzophenone-3 (BP3), capabili y o
de ec ion (CCβ), co e consis ency diagnos ic (CORCONDIA), decision limi (CCα), diisobu yl ph hala e
(DiBP), elec on impac (EI), ellip ical join con idence egion (EJCR), gas ch oma og aphy/mass
spec ome y (GC/MS), in e nal s anda d (IS), mul i a ia e cu e esolu ion coupled o al e na ing leas
squa es (MCR-ALS), pa allel ac o analysis (PARAFAC),
p incipal componen analysis (PCA),
p obabili y o alse posi i e (α), p obabili y o alse nega i e (β),
p og ammed empe a u e apo ize
(PTV), single ion moni o ing (SIM), sun p o ec ion ac o (SPF), o al ion ch oma og am (TIC),
ul a iole (UV).
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cosme ic p oduc s acco ding o Regula ion (EU) 2017/238 and he use o DiBP in cosme ic
p oduc s shall be p ohibi ed acco ding o Regula ion (EC) No 1223/2009.
The conclusions ob ained wi h he uni a ia e s anda d me hodology in he iden i ica ion o he
analy es con ained in he c eams we e w ong. Howe e , a calib a ion based on PARAFAC o
PARAFAC2 decomposi ions, whe e he samples o he p edic ion se we e p ojec ed on he
model ob ained p e iously wi h he calib a ion se , enabled he unequi ocal iden i ica ion and
quan i ica ion o he analy es e en in he p esence o in e e en s no conside ed in he
calib a ion model.
The PARAFAC2 decomposi ion was used o o e come he shi s in he
e en ion ime o BP and BP3. These h ee-way calib a ion echniques a e needed o a oid
alse nega i e esul s. The me hod had no p opo ional o cons an bias.
The p esence o BHT was de ec ed in he se en sunsc een c eams analysed a an amoun
o 6.48
.
10
-2
%, 8.53
.
10
-2
%, 1.70
.
10
-4
%, 1.11
.
10
-4
%, 2.51
.
10
-3
%, 3.20
.
10
-5
% and 6.35
.
10
-3
%.
The concen a ions o DiBP ound in ou c eams we e 3.49
.
10
-2
%, 3.19
.
10
-2
%, 3.26
.
10
-2
%
and 2.51
.
10
-2
%. On he o he hand, BP was only de ec ed in wo o he cosme ic c eams
analysed a an amoun o 7.84
.
10
-3
%
and 1.04
.
10
-2
%. In addi ion, BP3 was de ec ed in six o
he c eams a an amoun o 4.73%, 3.49%, 4.94
.
10
-3
%, 1.98
.
10
-3
%, 6.62
.
10
-1
%
and 1.73%.
The e o e, none o he cosme ic c eams con ained BP3 in an amoun highe han 6%.
Keywo ds: Benzophenone-3; PTV-GC/MS; PARAFAC; PARAFAC2; UV il e ; sunsc een
c eam.
1. In oduc ion
The use o sunsc een cosme ic c eams p o ec s he skin om he nega i e e ec s o
ul a iole (UV) ays such as sunbu n o skin cance . Un o una ely, some o he addi i es
con ained in hese c eams could in e e e in he ho mone le els o he human body [1,2,3].
Regula ion (EC) No 1223/2009 [4] on cosme ic p oduc s es ablishes ules o be complied
wi h by any cosme ic p oduc made a ailable on he ma ke , in o de o ensu e he
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unc ioning o he in e nal ma ke and a high le el o p o ec ion o human heal h. The con en
o oxybenzone o benzophenone-3 (BP3), wi h he chemical name 2-hyd oxy-4-
me hoxybenzophenone, was modi ied in Commission Regula ion (EU) 2017/238 [5] and
mus no exceed 6% (w/w). In addi ion, he label o he cosme ic p oduc mus include he
wo ding "con ains benzophenone-3" when he concen a ion is uppe han 0.5% (w/w) and i
is no used o p oduc p o ec ion pu poses. This ange does no pose a isk o human heal h,
apa om i s con ac which could p oduce alle gy [6].
BP3 is a sunsc een agen used o abso b UV adia ion in plas ics and in pe sonal ca e
p oduc s. In addi ion, i is used as a pho o-s abilize o minimize he colou and odou
changes o he cosme ic p oduc [7].
The concen a ion o his compound is egula ed because i is ha m ul o human heal h [7]
and causes alle gy [8,9]. Benzophenone- ype UV il e s also induce endoc ine dis up ing
e ec s [10,11,12]. BP3 pene a es he skin and 1-2% o he sunsc een is abso bed in
humans [13,14,15]. BP3 has been de ec ed in blood plasma [16,17], in human b eas milk
[18,19] and in u ine since he compound is exc e ed [1,13,14,20]. On he o he hand, mos o
he compounds p esen in c eams a e soluble in wa e so nega i e en i onmen al e ec s
appea [7] such as ish con amina ions which cause he appea ance o hese compounds in
ood [21,22]. In addi ion, he p esence o BP3 in swimming pool wa e could p oduce
haza dous p oduc s by eac ion wi h chlo ine [7,23] being a p oblem o human heal h.
O he addi i es a e added o sunsc een cosme ic c eams such as benzophenone (BP) and
2,6-di- e -bu yl-4-me hyl-phenol (BHT). BP is ano he UV il e [2,24], whe eas BHT is used
as an ioxidan o p e en ancidi y o o inhibi oxida ion in cosme ic o mula ions [2,25]. On
he o he hand, he p esence o ph hala es such as diisobu yl ph hala e (DiBP) in hese
c eams can be due o manu ac u ing p ocess o o hei mig a ion om packaging when
plas ic is used [26]. The con ol and analysis o hese compounds is impo an because hey
ha e ha m ul e ec s on heal h oo [27,28]. DiBP has been classi ied as ca cinogenic,
mu agenic o oxic subs ance o ep oduc ion (ca ego y 1B) in [29]. The e o e, he use o his

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compound in cosme ic p oduc s shall be p ohibi ed as s a ed in [4]. Howe e , he non-
in ended p esence o a small amoun o his subs ance shall be pe mi ed i i s p esence is
echnically una oidable, ha is, i i comes om impu i ies o na u al o syn he ic ing edien s,
he manu ac u ing p ocess, s o age o mig a ion om packaging.
Many analysis me hods ha e been de eloped o de ec hese compounds [2,30]. The
ex ac ion o he analy es om he cosme ic p oduc can be pe o med wi h o ganic sol en s
such as me hanol [24,31], e hanol [32,33], among o he s [26,34]. The use o an ul asonic
ba h o a o ex can accele a e he solubilisa ion o hose compounds. Then, he ex ac
could be il e ed o cen i uged o ex ac he ac ion o in e es and emo e he insoluble
ac ion o he cosme ic ma ix. O he ex ac i e app oaches a e based on solid phase
mic oex ac ion [35], o liquid-liquid mic oex ac ion [24].
The analy ical echniques employed o de e mine UV il e s in cosme ics [30] and o he
addi i es [2] a e: (i) ch oma og aphic echniques wi h di e en de ec o s; (ii) spec oscopic
echniques; and (iii) elec ochemical echniques. The wo la e ha e been less used han
ch oma og aphic echniques. The use o liquid ch oma og aphy wi h di e en de ec o s in his
de e mina ion has la ely inc eased [32,33,36]. Howe e , gas ch oma og aphy has been less
used since a de i a iza ion s ep wi h silyla ing eagen s is some imes equi ed o inc ease he
ola ili y and sensi i i y o he compounds [35,37,38]. The de e mina ion o ph hala es and
sunsc een agen s in cosme ic p oduc s is ca ied ou using a gas ch oma og aph coupled o
a mass spec ome e de ec o wi hou de i a iza ion in [26,39].
Se e al h ee-way algo i hms can be used wi h ch oma og aphic signals. P e ious wo ks
[40,41,42] ha e demons a ed he use ulness o h ee-way calib a ions based on he
PARAFAC decomposi ion using ch oma og aphic da a ob ained wi h di e en de ec o s ha
p o ide mul i a ia e signals (mass spec ome e s o diode a ay de ec o s) [43]. These wo ks
highligh he ad an age o using he abundances eco ded a all he ions selec ed (o he
abso bance spec um) when quan i ying o iden i ying he analy es acco ding o he
equi emen s s a ed in Eu opean egula ions. In addi ion, hese calib a ions a e use ul o he
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op imiza ion and e alua ion o he obus ness o analy ical me hods [42] since he second-
o de p ope y enables he iden i ica ion o he analy e o in e es as a single ac o
independen ly o he change in he ins umen al ac o s, e en in he p esence o unknown
in e e en s, which coelu e wi h he analy es.
The PARAFAC2 decomposi ion o e comes some de ia ions in he ch oma og aphic signals
[43,44]. PARAFAC2 has he second-o de p ope y i he co ela ion be ween he ime
p o iles is he same in all he samples, which is a weake condi ion han he equali y o
ch oma og aphic p o iles imposed by he PARAFAC model. I he loss o ilinea i y is
impo an , hen mul i a ia e cu e esolu ion echniques a e a use ul al e na i e, because
hei signal- ela ed equi emen s a e weake han hose demanded by PARAFAC o
PARAFAC2. Mul i a ia e cu e esolu ion coupled o al e na ing leas squa es (MCR-ALS)
has been widely applied in analy ical chemis y and i s ela ed ields [43,45,46], and i has
been used o esol e coelu ed compounds. I s majo limi a ion in iden i ying and quan i ying
an analy e is he p esence o o a ional ambigui ies and non-unique solu ions. Howe e , he
non-uniqueness p oblem can be alle ia ed o o ally a oided in some cases h ough he
in elligen use o he da a s uc u e and app op ia e cons ain s. This p oblem is discussed in
dep h in [46].
In his wo k, he simul aneous de e mina ion o BHT, BP, BP3 and DiBP in se en sunsc een
cosme ic c eams, using DiBP-d
4
as in e nal s anda d (IS), was ca ied ou by means o gas
ch oma og aphy/mass spec ome y (GC/MS) wi h a single quad upole mass analyse in
selec ed ion moni o ing (SIM) mode. Pa allel ac o analysis (PARAFAC) o PARAFAC2
decomposi ion me hods we e used o disco e i a coeluen ha sha es ions wi h he analy e
o in e es is p esen [47,48] and o iden i y unequi ocally he compounds p esen by hei
ch oma og aphic and spec al p o iles ollowing he c i e ia es ablished in Decision
2002/657/EC o esidues o e e ina y medicinal p oduc s [49] which a e s ic e han o he
o icial egula ions and guidelines [49,50,51]. In his case, a leas a minimum o 3
iden i ica ion poin s is needed o he con i ma ion o each compound; in his wo k 5 ions
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we e selec ed. The PARAFAC2 decomposi ion o e come he shi s in he e en ion ime o
BP and BP3 in he samples and he analy es we e unequi ocally iden i ied al hough he shi
o he e en ion ime is limi ed in egula ed analyses. The esul s o he iden i ica ion we e
compa ed wi h he ones ob ained wi h he uni a ia e s anda d me hodology. The
de e mina ion o he compounds in he sunsc een c eams analysed was pe o med h ough
he p ojec ion o he samples o he p edic ion se on he co esponding PARAFAC o
PARAFAC2 model.
O he ad an ages o his wo k wi h espec o analy ical me hods p e iously epo ed a e he
de e mina ion o di e en compounds, ha a ec human heal h, in sunsc een cosme ic
c eams and he de e mina ion o BP3 by GC/MS wi hou a de i a iza ion eac ion, which is
no e y usual.
2. Ma e ial and me hods
2.1. Chemicals
Benzophenone (CAS no. 119-61-9, pu i ied by sublima ion, ≥ 99% pu i y), 2-hyd oxy-4-
me hoxybenzophenone (benzophenone-3, CAS no. 131-57-7, 98% pu i y), 2,6-di- e -bu yl-4-
me hyl-phenol (CAS no. 128-37-0, ≥ 99% pu i y), diisobu yl ph hala e (CAS no. 84-69-5, 99%
pu i y) and diisobu yl ph hala e-3,4,5,6-d
4
(CAS no. 358730-88-8, analy ical s anda d, 99.7%
pu i y) we e pu chased om Sigma-Ald ich (S einheim, Ge many).
E hanol (96% ol., CAS no. 64-17-5, HiPe Sol CHROMANORM®, g adien g ade o HPLC)
was supplied by VWR In e na ional (Radno , Pennsyl ania, USA). N-hexane (CAS no. 110-
54-3) and ace one (CAS no. 67-64-1) o liquid ch oma og aphy Lich osol ® we e om
Me ck KGaA (Da ms ad , Ge many).
2.2. S anda d solu ions
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S ock solu ions o BHT a 1 g L
-1
, o BP a 5 g L
-1
, o DiBP a 2 g L
-1
, and o DiBP-d
4
a 0.45 g
L
-1
we e p epa ed indi idually in hexane. In e media e solu ions we e p epa ed om he
o me ones by dilu ion in he same sol en .
A s ock solu ion o BP3 a 30.5 g L
-1
we e p epa ed in e hanol and in e media e solu ions a
concen a ions o
1, 5, 7.5, 10, 15, 20 and 25 g L
-1
we e p epa ed om ha s ock solu ion in
e hanol. All hose solu ions o BP3 we e dilu ed 25000 imes o p epa e he co esponding
calib a ion s anda ds in hexane. The s ock and in e media e solu ions o BP3 we e s able o
15 days. The es o he solu ions o his analy e we e p epa ed daily since his compound
was no s able.
All he s ock and in e media e solu ions, which weigh was con olled o e i y ha he sol en
had no e apo a ed, we e s o ed in c imp ials a 4ºC and p o ec ed om ligh . The
labo a o y glasswa e used was ho ough cleaned and plas ic consumables we e a oided as
a as possible.
The numbe and ype o he samples analysed oge he wi h he concen a ion anges o he
sol en s anda ds (analy e s anda ds p epa ed in sol en ) o each analy e in each s age a e
collec ed in Table S1 in he Supplemen a y Ma e ial.
2.3. Sunsc een c eam samples
Se en di e en sunsc een c eams we e pu chased a local s o es and pha macies (Bu gos,
Spain). These cosme ic p oduc s we e: i) c eam 1 (sun p o ec ion ac o (SPF) 50+), ii)
c eam 2 (SPF 30), iii) c eam 3 (SPF 50+), i ) c eam 4 (SPF 30), ) c eam 5 (SPF 30), i)
c eam 6 (SPF 50+), and ii) c eam 7 (SPF 15). C eam 1 and 2 belong o he same cosme ic
b and, whe eas c eam 4 and 7 belong o ano he one. The sunsc een p oduc ’s label o
c eam 1, 2 and 7 speci ied ha BP3 was con ained in hei o mula ion and he label o c eam
1, 2, 5 and 7 speci ied ha BHT was one o hei ing edien s.
2.4. Sample p epa a ion me hod
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numbe o samples, while he second and hi d ones we e he numbe o scans and ions,
espec i ely.
3.1.3.1. Tole ance in e als (s ep ii)
To es ima e he ole ance in e als, he e e ence s anda ds measu ed in Sec ion 3.1.1 we e
used. In addi ion, h ee e e ence s anda ds ha con ained he IS a h ee concen a ion
le els and he analy es a a ixed in e media e concen a ion (see Table S1 in he
Supplemen a y Ma e ial, hi d ow) we e also conside ed.
The ch oma og ams o all hese samples we e equally agmen ed a ound he e en ion ime
o each analy e a e a baseline co ec ion. The esul ing da a ma ices we e a anged in a
h ee-way a ay, X
0
, o each analy e excep o DiBP and DiBP-d
4
peaks o which a join
a ay was conside ed o bo h compounds. The dimension o he ou h ee-way a ays buil
a e gi en in he second ow o Table 1, whe eas he samples included in hose a ays a e
de ailed in ows 1 o 3 o Table S1 in he Supplemen a y Ma e ial. The i s dimension
co esponds o he numbe o scans conside ed, he second one e e s o he numbe o ions
eco ded and he hi d one is he numbe o samples. Then, a PARAFAC decomposi ion was
pe o med o each o he a ays. Table 1 ( ows 1-7) also con ains he ea u es o he models
ob ained in each case. The co e consis ency diagnos ic (CORCONDIA) [60] measu es he
ilinea i y deg ee o he expe imen al h ee-way a ay when he e a e mo e han 2 ac o s in
he model. I he a ay is ilinea , hen he maximum CORCONDIA alue o 100 is ound.
Fo BP and BP3, a PARAFAC2 decomposi ion was ca ied ou since he e we e shi s in he
e en ion ime o hose analy es in he samples. In addi ion, he a iance explained by hese
PARAFAC2 models was highe han he one ob ained wi h a PARAFAC model. The
PARAFAC model o DiBP and DiBP-d
4
equi ed h ee ac o s whe e he hi d one was
ela ed o an in e e en (cha ac e is ic m/z a io: 223) ha elu ed be o e DiBP-d
4
.

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The ole ance in e als o he ela i e e en ion ime (see Table 2A, ou h column) and o
he ela i e ion abundances (see Table 2B, i h column) we e es ima ed om he loadings o
he ch oma og aphic and spec al p o iles, espec i ely, o he PARAFAC o PARAFAC2
models. These wo in e als we e buil ollowing he equi emen s es ablished in [49] and
we e used as e e ence o he unequi ocal iden i ica ion o he analy es. PARAFAC
decomposi ions p o ide a unique ch oma og aphic p o ile o each compound ha is
common o all he samples, whe eas PARAFAC2 decomposi ions p o ide a ch oma og aphic
p o ile o each compound o e e y sample. When a PARAFAC2 decomposi ion was
conside ed, he median o he e en ion imes o he analy e ob ained om he
ch oma og aphic p o ile was used o calcula e he ela i e e en ion ime ( he a io o he
ch oma og aphic e en ion ime o he analy e o ha o he in e nal s anda d). Table 2A
con ains he e en ion imes (second column o his able) and ela i e e en ion imes ( hi d
column o his able) o each analy e ob ained om he models es ima ed wi h he a ay ha
con ained he e e ence s anda ds. The ole ance in e als o he ela i e e en ion ime
we e buil wi h a ole ance ma gin o ± 0.5% as [49] es ablished. I has also been checked
ha he ela i e e en ion imes o BP and BP3 in all he samples ob ained h ough he
PARAFAC2 decomposi ion we e wi hin he ole ance in e als es ima ed wi h he e en ion
imes o hese analy es in each sample.
The e o e, he use o he median o es ima e a
ep esen a i e e en ion ime o BP and BP3 was adequa e. On he o he hand, PARAFAC
and PARAFAC2 decomposi ions p o ide a unique spec al p o ile o each compound
common o all he samples. The ela i e ion abundances o each m/z a io used o de e mine
he ole ance in e als acco ding o e . [49] we e calcula ed wi h he co esponding spec al
loading (see Table 2B, hi d column) wi h espec o he one o he co esponding base peak
o he analy e.
3.1.3.2. Calib a ion models and p ojec ion (s eps iii o iii)
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Then, a h ee-way da a a ay was buil o he calib a ion se (X
1
) and ano he one o he
p edic ion se (X
2
) o each analy e, excep o DiBP and DiBP-d
4
peaks o which a join
a ay was conside ed again in bo h cases. The samples ha made up he calib a ion se
we e: ou sys em blanks (emp y ials wi hou sol en ), he i s sol en blank measu ed, he
i s calib a ion ba ch oge he wi h he wo eplica es injec ed a e i and wo calib a ion
s anda ds o he second and o he hi d calib a ion ba ches. The p edic ion se was
cons i u ed wi h he es o he samples ( he es o he calib a ion s anda ds, sol en blanks,
blank ex ac s and he dilu ed ex ac s o he c eams). Some calib a ion s anda ds we e
included in he p edic ion se o gua an ee he easibili y o he p ojec ion o he samples. The
dimension o X
1
and X
2
is included in Table 1 ( ows 9 and 15, espec i ely).
Then, PARAFAC decomposi ions (o PARAFAC2 decomposi ions in he case o BP and
BP3) we e pe o med wi h he h ee-way a ay ha con ained he calib a ion se . When a
PARAFAC decomposi ion was ca ied ou , a change in he o de o he dimension o he
h ee-way a ays o he calib a ion and p edic ion se s was ca ied ou (see Table 1). In he
case o he a ay o DiBP and DiBP-d
4
, some addi ional sol en s anda ds we e added as
can be seen in Table 1 since bo h compounds we e comple ely o e lapped and he
abundance o DiBP-d
4
was much lowe han he abundance o DiBP, so PARAFAC needs a
g ea e a ia ion o DiBP-d
4
. The e o e, a s anda d con aining all he analy es and a highe
amoun o DiBP-d
4
(100 µg L
−1
) oge he wi h wo s anda ds ha only con ained 100 µg L
−1
o
DiBP-d
4
we e added o ha h ee-way a ay.
The cha ac e is ics o he PARAFAC and PARAFAC2 models ob ained in each case a e
lis ed in Table 1 ( ows 8-15, columns 2-5). An uncons ained one- ac o model was needed
o BHT, BP and BP3, whe eas he decomposi ion o he common a ay o DiBP and DiBP-
d
4
needed h ee ac o s (CORCONDIA index o 100%).
Once hese models we e ob ained, he samples o he p edic ion se we e p ojec ed on he
co esponding model. The ex ac ob ained om c eam 6 dilu ed 10 imes exceeded he
h eshold alue o he Q and Ho elling’s T
2
s a is ics a he 95% con idence le el when i was
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p ojec ed on he PARAFAC2 model o BP3. The e o e, i was conside ed an ou lie and
emo ed om he h ee-way a ay ha con ained he samples o he p edic ion se (see he
dimension o his a ay in Table 1). Fig. 3 shows he ch oma og aphic, spec al and sample
p o iles o he one- ac o PARAFAC2 model ob ained o BP3. As can be seen in Fig. 3 (c),
he sample loadings o BP3 we e ze o in he sol en blanks measu ed a e he injec ion o
each ex ac o c eam (samples numbe 23, 25, 27, 30, 33, 36, 39, 49, 51, 53, 56, 59, 62 and
65) so he cleanliness o he GC/MS sys em was gua an eed.
By way o example, he loadings o he h ee- ac o PARAFAC model o DiBP and DiBP-d
4
a e shown in Fig. 4. The sample loadings o he ac o co esponding o DiBP Fig. 4 (a))
inc eased wi h he concen a ion o he calib a ion s anda ds as expec ed and he eplica es
we e simila . The sample loadings o his analy e in he ex ac s o c eam 3 and 4 dilu ed 10
imes we e ou side he calib a ion ange (samples numbe 80 and 82 no shown in ha
igu e) so he amoun o DiBP in hose dilu ions could no be quan i ied. The loadings o he
sample p o ile o he ac o associa ed o he in e nal s anda d (Fig. 4 (b)) we e ze o in he
sol en and sys em blanks, whe eas hey emained nea ly cons an in he es o he samples
excep o he addi ional samples added o he h ee-way a ay whe e he loadings we e
highe as expec ed. On he o he hand, he sample loadings o he hi d ac o (Fig. 4 (c))
we e e y high in he ex ac s o c eam 3 and 4 dilu ed 10 imes. Some o he m/z eco ded
o DiBP we e sha ed wi h his ac o being 223 i s mos cha ac e is ic m/z (see Fig. 4 (d)).
This ac o was a ibu ed o an uniden i ied in e e en elu ing nea he beginning o he
DiBP-d
4
peak as can be seen in ed in Fig. 4 (e).
The analy es we e unequi ocally iden i ied since he ela i e e en ion imes (see Table 2A,
i h column) and he ela i e ion abundances (see Table 2B, six h column) es ima ed om
he loadings o he ch oma og aphic and spec al p o iles ob ained in his analysis,
espec i ely, we e wi hin hei co esponding ole ance in e als (see Table 2A ( ou h
column) and Table 2B ( i h column), espec i ely). As can be seen in Fig. 1, he ela i e
abundances es ima ed om he spec al PARAFAC loadings o DiBP ob ained we e wi hin
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he ole ance in e als o all he m/z a ios. The e o e, he conclusion ha DiBP was no
p esen in he c eams s a ed in Sec ion 3.1.1 was w ong since a coelu ing in e e en (see
Fig. 2 (b)) p esen in hose samples sha ed some m/z a ios wi h DiBP. This in e e en did
no appea in he PARAFAC model conside ed (see Table 1) since i was no p esen in he
samples o he calib a ion se and he ex ac s o he c eams we e p ojec ed on he model
ob ained wi h he calib a ion se . The e o e, he e was no p oblem in he iden i ica ion o
DiBP using PARAFAC. I is impo an o bea in mind ha one o he ad an ages o he
PARAFAC decomposi ion o e he uni a ia e s anda d me hodology is ha his h ee-way
echnique p o ides a unique spec al p o ile o each analy e ha is common o all he
samples.
The esul s o he s eps ix, x and xi o he p ocedu e will be shown in he ollowing sec ion
al hough many app oaches can be used o pe o m a calib a ion based on PARAFAC o
PARAFAC2 decomposi ions [61].
3.2. Quan i ica ion using h ee-way echniques
The sample loadings o each analy e we e nume ically high since hey came om he i s
mode o a PARAFAC decomposi ion o om he hi d mode o a PARAFAC2 decomposi ion
and hese modes a e no no malized in he decomposi ion. The e o e, hey we e manually
no malized p io o s anda diza ion. Once he sample loadings o each analy e we e
s anda dized by di iding each o hem by ha o he in e nal s anda d, calib a ion models
“s anda dized sample loading e sus ue concen a ion” using he s anda ds con ained in
he calib a ion se we e i ed and alida ed. The pa ame e s o he eg ession models
es ima ed o each analy e a e included in Table S2 in he Supplemen a y Ma e ial. A
quad a ic eg ession model was conside ed o all he analy es excep o DiBP. One ou lie
was de ec ed in he calib a ion models o BP and BP3 since hese da a had a s uden ized
esidual g ea e han 3 in absolu e alue. The e o e, hose ou lie s we e emo ed, and a new
i ing was ca ied ou wi h he emaining da a in bo h cases (see he calib a ion models
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ob ained in Table S2 in he Supplemen a y Ma e ial). The lowes mean o he absolu e alue
o he ela i e e o s in calib a ion was 1.97% (n = 11) o BP, whe eas he highes alue was
6.86% (n = 8) o BP3 when he samples wi h p edic ed concen a ion lowe han he
co esponding CCβ had been excluded. In addi ion, he mean o he absolu e alue o he
ela i e e o s in p edic ion o he second and hi d calib a ion ba ches we e calcula ed.
Taking hese alues in o accoun , i was concluded ha BP3 was no s able o e ime since
he e o s inc eased om he second calib a ion ba ch o he hi d one. The e o e, he
solu ions we e p epa ed daily.
Table S2 in he Supplemen a y Ma e ial also con ains he pa ame e s o he co esponding
accu acy lines buil wi h he calib a ion s anda ds, ha is he eg essions “p edic ed
concen a ion e sus ue concen a ion”. The ellip ical join con idence egion (EJCR) es
was compu ed and Fig. S2 in he Supplemen a y Ma e ial shows he con idence ellipses, a a
95% con idence le el, o he slope and he in e cep o he accu acy line es ima ed o each
analy e. All he con idence ellipses con ained he poin (0,1). In addi ion, Table S2 in he
Supplemen a y Ma e ial con ains he p- alues o his es . These p- alues we e highe han
0.05 (0.973 o BHT, 0.960 o BP, 1.000 o DiBP and 0.123 o BP3) so he in e cep and
he slope we e signi ican ly no di e en om 0 and 1, espec i ely. The e o e, he me hod
had no cons an o p opo ional bias a a 95% con idence le el.
The alues o decision limi (CCα) and capabili y o de ec ion (CCβ) o each analy e wi h he
p obabili ies o alse posi i e (α) and alse nega i e (β) ixed a 0.05 a e lis ed in he i s wo
ows o Table 3. The blank ex ac con ained DiBP in all i s dilu ions excep o he one
dilu ed 8000 and 10000 imes, whe eas he amoun o he es o he analy es in he blank
ex ac was below he co esponding CCα alues. The amoun o each analy e ound in he
sunsc een c eams oge he wi h he co esponding 95% con idence in e al a e de ailed in
Table 3. The label o c eam 1, 2, 5 and 7 speci ied ha BHT was con ained in hei
o mula ion bu BHT was de ec ed in he se en c eams analysed. On he o he hand, BP3
was ound in all he sunsc een c eams excep o c eam 4 since he con idence in e al o

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ha analy e in ha c eam con ained ze o and he alues we e below CCα as can be seen in
Table 3. The c eams 1, 2, 6 and 7 we e he ones which con ained a concen a ion o BP3
be ween 0.5% and 6%. Howe e , only he p oduc ’s label o c eams 1, 2 and 7 speci ied ha
BP3 was one o hei ing edien s as equi ed in [5]. In addi ion, DiBP and BP we e only
de ec ed in ou and wo cosme ic c eams, espec i ely. The amoun ound o DiBP (below
0.035%) may come om impu i ies o na u al o syn he ic ing edien s, he manu ac u ing
p ocess, s o age o mig a ion om packaging o he cosme ic p oduc .
4. Conclusions
The p ojec ion o he samples o he p edic ion se on he co esponding PARAFAC o
PARAFAC2 model enabled he de e mina ion o BHT, BP, DiBP and BP3 in he sunsc een
cosme ic c eams analysed e en in he p esence o in e e en s no conside ed in he
calib a ion model. PARAFAC2 decomposi ion o e came he p oblems due o he shi s in he
e en ion ime o BP and BP3.
In addi ion, he unequi ocal iden i ica ion and quan i ica ion o each analy e acco ding o he
equi emen s es ablished by EU egula ions we e possible using a PARAFAC o PARAFAC2
decomposi ion despi e some o he m/z a ios o a coelu ing in e e en we e sha ed wi h
DiBP. In ac , he unequi ocal iden i ica ion o his analy e could ne e ha e been achie ed
om he mass spec um eco ded a i s e en ion ime due o he p esence o a coelu ing
in e e en p esen in he sunsc een c eams analysed. The p esence o BHT was de ec ed in
he se en sunsc een c eams analysed, whe eas BP3, DiBP and BP we e de ec ed in some
o hese c eams. None o he cosme ic c eams con ained BP3 in an amoun highe han 6%
as es ablished in Regula ion 2017/238 [5].
5. Acknowledgmen s
The au ho s hank he inancial suppo p o ided by Spanish MINECO (AEI/FEDER, UE)
h ough p ojec CTQ2017‐88894‐R and by Jun a de Cas illa y León h ough p ojec
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BU012P17 (bo h co‐ inanced wi h Eu opean FEDER unds). L. Val e de-Som hanks JCyL
o he pos doc o al con ac h ough BU012P17 p ojec .
CONFLICT OF INTEREST
The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal
ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape .
6. Re e ences
[1] K.N. Jallad, Chemical cha ac e iza ion o sunsc eens composi ion and i s ela ed
po en ial ad e se heal h e ec s, J. Cosme . De ma ol-US. 16 (2017) 353-357.
h ps://doi.o g/10.1111/jocd.12282.
[2] M. Lo es, M. Llompa , G. Al a ez-Ri e a, E. Gue a, M. Vila, M. Celei o, J.P.
Lamas, C. Ga cia-Ja es, Posi i e lis o cosme ic ing edien s: Analy ical
me hodology o egula o y and sa e y con ols - A e iew, Anal. Chim. Ac a. 915
(2016) 1-26. h ps://doi.o g/10.1016/j.aca.2016.02.033.
[3] T. Wong, D. O on, Sunsc een alle gy and i s in es iga ion, Clin. De ma ol. 29
(2011) 306-310. h ps://doi.o g/10.1016/j.clinde ma ol.2010.11.002.
[4] Regula ion (EC) No 1223/2009 o he Eu opean Pa liamen and o he Council o 30
No embe 2009 on cosme ic p oduc s, O . J. Eu . Union, L342 (2009) 59-209.
[5] Commission Regula ion (EU) 2017/238 o 10 Feb ua y amending Annex VI o
Regula ion (EC) No 1223/2009 o he Eu opean Pa liamen and o he Council on
cosme ic p oduc s, O . J. Eu . Union, L36 (2017) 37-38.
[6] Scien i ic Commi ee on Consume P oduc s, Heal h & Consume P o ec ion
Di ec o a e-Gene al, Opinion on benzophenone-3. COLIPA Nº S38, Eu opean
Commission, 2008.
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[7] J.C. DiNa do, C.A. Downs, De ma ological and en i onmen al oxicological impac o
sunsc een ing edien oxybenzone/benzophenone-3, J. Cosme . De ma ol. 17 (2018)
15-19. h ps://doi.o g/10.1111/jocd.12449.
[8] A.R. Heu ung, S.I. Raju, E.M. Wa shaw, Benzophenones, De ma i is 25 (2014) 3-
10. h ps://doi.o g/10.1097/DER.0000000000000025.
[9] M.D. Palm, M.N. O'Donoghue, Upda e on pho op o ec ion, De ma ol. The . 20
(2007) 360-376. h ps://doi.o g/10.1111/j.1529-8019.2007.00150.x.
[10] J. Wang, L. Pan, S. Wu, L. Lu, Y. Xu, Y. Zhu, M. Guo, S. Zhuang, Recen ad ances
on endoc ine dis up ing e ec s o UV il e s, In . J. En . Res. Pub. He. 13 (2016) 1-
11. h ps://doi.o g/10.3390/ije ph13080782.
[11] M. K ause, A. Kli , M.B. Jensen, T. Søebo g, H. F ede iksen, M. Schlump , W.
Lich ens eige , N.E. Skakkebaek, K.T. D zewiecki, Sunsc eens: a e hey bene icial
o heal h? An o e iew o endoc ine dis up ing p ope ies, In . J. And ol. 35 (2012)
424-436. h ps://doi.o g/10.1111/j.1365-2605.2012.01280.x.
[12] M. Schlump , P. Schmid, S. Du e , M. Conscience, K. Mae kel, M. Hensele , M.
G ue e , I. He zog, S. Reolon, R. Cecca elli, O. Faass, E. S u z, H. Ja y, W.
Wu ke, W. Lich ens eige , Endoc ine ac i i y and de elopmen al oxici y o cosme ic
UV il e s - an upda e, Toxicology 205 (2004) 113-122.
h ps://doi.o g/10.1016/j. ox.2004.06.043.
[13] H. Gonzalez, A. Fa b o , O. La kö, A.M. Wennbe g, Pe cu aneous abso p ion o he
sunsc een benzophenone-3 a e epea ed whole-body applica ions, wi h and
wi hou ul a iole i adia ion, B i . J. De ma ol. 154 (2006) 337-340.
h ps://doi.o g/10.1111/j.1365-2133.2005.07007.x.
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[14] H.G. Gonzalez, A. Fa b o , O. La kö, Pe cu aneous abso p ion o benzophenone-3,
a common componen o opical sunsc eens, Clin. Exp. De ma ol. 27 (2002) 691-
694. h ps://doi.o g/10.1046/j.1365-2230.2002.01095.x.
[15] C.G Hayden, M.S. Robe s, H.A. Benson, Sys emic abso p ion o sunsc een a e
opical applica ion, Lance . 350 (1997) 863-864. h ps://doi.o g/10.1016/S0140-
6736(05)62032-6.
[16] N.R. Janjua, B. Kongshoj, A.M. Ande soon, H.C. Wul , Sunsc eens in human
plasma and u ine a e epea ed whole-body opical applica ion, J. Eu . Acad.
De ma ol. 22 (2008) 456-461. h ps://doi.o g/10.1111/j.1468-3083.2007.02492.x.
[17] V. Sa eiya, S. Risk, H.A.E. Beson, Liquid ch oma og aphic assay o common
sunsc een agen s: applica ion o in i o assessmen o skin pene a ion and
sys emic abso p ion in human olun ee s, J. Ch oma og . B 803 (2004) 225-231.
h ps://doi.o g/10.1016/j.jch omb.2003.12.022.
[18] D. Molins-Delgado, M.M. Olmo-Campos, G. Vale a-Juan, V. Pleguezuelos-
He nández, D. Ba celó, M.S. Díaz-C uz, De e mina ion o UV il e s in human b eas
milk using u bulen low ch oma og aphy and babies' daily in ake es ima ion,
En i on. Res. 161 (2018) 532-539. h ps://doi.o g/10.1016/j.en es.2017.11.033.
[19] R. Rod íguez-Gómez, A. Za a-Gómez, N. Do i al-Ga cía, O. Balles e os, A.
Na alón, De e mina ion o benzophenone-UV il e s in human milk samples using
ul asound-assis ed ex ac ion and clean-up wi h dispe si e so ben s ollowed by
UHPLC-MS/MS, Talan a 134 (2015) 657-664.
h ps://doi.o g/10.1016/j. alan a.2014.12.004.
[20] M. Schlump , B. Co on, M. Conscience, V. Halle , B. S einmann, W. Lich ens eige ,
In i o and in i o es ogenici y o UV sc eens, En i on. Heal h Pe sp. 109 (2001)
239-244. h ps://doi.o g/10.1289/ehp.01109239.
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FIGURE CAPTIONS
Fig. 1 Tole ance in e als o he ela i e ion abundances o DiBP es ima ed o each
m/z a io wi h h ee e e ence samples ha con ained a di e en concen a ion
le el o DiBP: 25 µg L
-1
(in blue), 75 µg L
-1
(in ed) and 125 µg L
-1
(in g een).
Rela i e abundance o each m/z a io ob ained wi h: he PARAFAC models
ob ained wi h he e e ence samples (ligh blue ci cles) and wi h all he samples
o he analysis o he c eams (black c oss), he ex ac ob ained om c eam 1
dilu ed 10000 imes (pink iangle), he ex ac ob ained om c eam 2 dilu ed
10000 imes (pu ple diamond), he ex ac ob ained om c eam 3 dilu ed 10000
imes ( ed squa e) and he ex ac ob ained om c eam 6 dilu ed 8000 imes
(ligh g een s a ).
(Fo in e p e a ion o he e e ences o colou in his igu e
legend, he eade is e e ed o he web e sion o he a icle).
Fig. 2 Ch oma og aphic p o iles o he PARAFAC models ob ained wi h he h ee-way
a ays ha con ained he calib a ion and p edic ions se s oge he in he same
a ay o : (a) BHT, (b) DiBP and DiBP-d
4
, (c) BP3 (a PARAFAC2 decomposi ion
o he a ay was ca ied ou in his las case).
Fig. 3 One- ac o PARAFAC2 model ob ained wi h he h ee-way a ay ha con ained
he calib a ion se o BP3. Loadings o he: (a) ch oma og aphic p o ile, (b)
spec al p o ile and (c) sample p o ile. The sample loadings o he 20 samples o
he calib a ion se a e ep esen ed by g ey ci cles, whe eas he sample loadings
o he 62 samples o he p edic ion se , which ha e been p ojec ed on he
PARAFAC2 model, a e ep esen ed by ed diamonds. (Fo in e p e a ion o he
e e ences o colou in his igu e legend, he eade is e e ed o he web
e sion o he a icle).

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Fig. 4 Loadings o he sample p o ile o : a) ac o 1 (DiBP), b) ac o 2 (DiBP-d
4
) and c)
ac o 3 (unknown in e e en ) o he h ee- ac o PARAFAC model i ed wi h he
common h ee-way a ay o DiBP and DiBP-d
4
ha only con ained he calib a ion
se . The sample loadings o he 24 samples o he calib a ion se a e ep esen ed
by g ey ci cles, whe eas he ones o he 63 samples o he p edic ion se , which
ha e been p ojec ed on he model, a e ep esen ed by ed diamonds. d)
Loadings o he spec al p o ile and e) loadings o he ch oma og aphic p o ile. In
igu es (d-e), ac o 1 (DiBP) is in ligh blue, ac o 2 (DiBP-d
4
) is in ligh g een,
while ac o 3 (in e e en ) is in ed. (Fo in e p e a ion o he e e ences o colou
in his igu e legend, he eade is e e ed o he web e sion o he a icle).
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Table
1
Dimensions (scans × ions × samples) o he h ee-way a ays buil o e e y analy e in each expe imen al
s age and cha ac e is ics o he PARAFAC o PARAFAC2 models (numbe o ac o s, cons ain s imposed,
explained a iance and CORCONDIA index) ob ained om he decomposi ion o each a ay.
Analy ical s age BHT BP DiBP and DiBP-d
4
BP3
Tole ance in e als
Dimension o
X
0
21 × 5 × 9 49 × 5 × 9 41 × 10 × 9 56 × 5 × 9
Model PARAFAC PARAFAC2 PARAFAC PARAFAC2
# Fac o s 1 1 3 1
Non-nega i i y cons ain s None None In modes 1 and 2 None
Expl. Va (%) 99.71 99.88 99.45 99.79
CORCONDIA (%)
a
--- --- 100 ---
Analysis o he sunsc een c eams
Dimension o
X
1
(calib a ion se ) 20 × 21 × 5
b
49 × 5 × 20 24 × 41 × 10
b
56 × 5 × 20
Model PARAFAC PARAFAC2 PARAFAC PARAFAC2
# Fac o s 1 1 3 1
Non-nega i i y cons ain s None None In he h ee modes None
Expl. Va (%) 99.74 99.87 99.08 99.82
CORCONDIA (%)
a
--- --- 100 ---
Dimension o
X
2
(p edic ion se ) 63 × 21 × 5
b
49 × 5 × 63 63 × 41 × 10
b
56 × 5 × 62
Expl. Va .: Explained a iance by he model.
a
The CORCONDIA index canno be calcula ed in he PARAFAC o PARAFAC2 decomposi ion wi h only one ac o .
b In his case, he dimension o he a ay co esponds o he numbe o samples × scans × ions.
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Table
2
Tole ance in e als o : A) he ela i e e en ion ime and o B) he ela i e ion abundances es ima ed
om he loadings o he ch oma og aphic and spec al p o iles, espec i ely. Iden i ica ion o e e y analy e in
he analysis o sunsc een c eams. In he case o BP and BP3, he median o he e en ion imes ob ained in
he co esponding PARAFAC2 decomposi ion was conside ed.
A) Re en ion ime
Analy e
R
(min) Rela i e
R
Tole ance in e al
Iden i ica ion in he analysis
o he c eams
Rela i e
R
BHT 8.229 0.806 (0.802-0.810) 0.806
BP 9.016 0.883 (0.879-0.887) 0.883
DiBP-d
4
10.211 1.000 (0.995-1.005) 1.000
DiBP 10.220 1.001 (0.996-1.006) 1.001
BP3 11.120 1.089 (1.083-1.094)
1.089
B) Diagnos ic ions
Analy e
m/z a io Spec al
loading Rela i e
abundance (%)
Tole ance in e al
(%)
b
Iden i ica ion in he analysis
o he c eams
Rela i e abundance (%)
BHT 91 6.85
.
10
-
2
7.13 (3.57-10.70) 7.08
145 1.15
.
10
-
1
11.94 (9.55-14.33) 11.94
177 7.67
.
10
-
2
7.98 (3.99-11.97) 8.03
205
a
9.61
.
10
-
1
100 - 100
220 2.29
.
10
-
1
23.81 (20.24-27.38) 23.94
BP
51 1.37
.
10
-
1
17.75 (14.20-21.30) 17.42
77 4.38
.
10
-
1
56.77 (51.09-62.45) 56.63
105
a
7.71
.
10
-
1
100 - 100
152 3.27
.
10
-
2
4.24 (2.12-6.36) 4.34
182 4.40
.
10
-
1
57.05 (51.35-62.76) 57.61
DiBP-d
4
80 5.65
.
10
-
2
5.68 (2.84-8.52) 5.70
153
a
9.94
.
10
-
1
100 - 100
171 2.48
.
10
-
2
2.50 (1.25-3.75) 2.57
209 1.17
.
10
-
2
1.18 (0.59-1.77) 1.23
227 4.95
.
10
-
2
4.98 (2.49-7.47) 5.14
DiBP 104 7.82
.
10
-
2
7.86 (3.93-11.79) 7.66
149
a
9.95
.
10
-
1
100 - 100
167 2.75
.
10
-
2
2.76 (1.38-4.14) 2.75
205 1.37
.
10
-
2
1.38 (0.69-2.07) 1.39
223 5.24
.
10
-
2
5.27 (2.64-7.91) 5.27
BP3 77 1.56
.
10
-
1
22.86 (19.43-26.29) 22.33
105 8.16
.
10
-
2
11.99 (9.59-14.39) 11.74
151 5.73
.
10
-
1
84.19 (75.77-92.61) 83.43
227
a
6.81
.
10
-
1
100 - 100
228 4.21
.
10
-
1
61.86 (55.67-68.05) 61.80
a
Base peak.
b
Acco ding o e . [42], he es ima ion o he ole ance in e al is di e en depending on he alue o he ela i e
abundance o he co esponding m/z a io. The ole ance ma gin was ±50% o ela i e in ensi ies lowe o equal o 10%,
a ± 20% o ela i e in ensi ies om 10% o 20%, a ± 15% o ela i e in ensi ies om 20% o 50% and ± 10% o ela i e
in ensi ies highe han 50%.
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
Table
3
Decision limi (CCα), capabili y o de ec ion (CCβ) a x
0
= 0 and concen a ion o each analy e ound in he sunsc een c eams oge he
wi h he co esponding con idence in e als o he p edic ed concen a ion a a 95% con idence le el.
BHT BP DiBP BP3
CCα (x
0
= 0) (µg L
−
1
) 4.03 6.32 5.93 142.67
CCβ (x
0
= 0) (µg L
−
1
)
a
7.93 12.40 11.65 279.80
Sunsc een c eam 1
Concen a ion (% w/w) 6.48
.
10
-
2
< CCα
b
3.49
.
10
-
2
4.73
In e al a a 95% con idence le el (% w/w) (4.62
.
10
-
2
, 8.21
.
10
-
2
)
b
(6.75
.
10
-
3
, 6.20
.
10
-
2
)
b
(4.17, 5.52)
b
Sunsc een c eam 2
Concen a ion (% w/w) 8.53
.
10
-
2
< CCα
b
3.19
.
10
-
2
3.49
In e al a a 95% con idence le el (% w/w) (6.70
.
10
-
2
, 1.02
.
10
-
1
)
b
(3.75
.
10
-
3
, 5.91
.
10
-
2
)
b
(2.90, 4.17)
b
Sunsc een c eam 3
Concen a ion (% w/w) 1.70
.
10
-
4
< CCα
b
3.26
.
10
-
2
4.94
.
10
-
3
In e al a a 95% con idence le el (% w/w) (1.46
.
10
-
4
, 1.92
.
10
-
4
)
c
(4.50
.
10
-
3
, 5.98
.
10
-
2
)
b
(4.17
.
10
-
3
, 5.95
.
10
-
3
)
c
Sunsc een c eam 4
Concen a ion (% w/w) 1.11
.
10
-
4
7.84
.
10
-
3
< CCα
d
8.41
.
10
-
4
In e al a a 95% con idence le el (% w/w) (8.68
.
10
-
5
, 1.34
.
10
-
4
)
c
(3.92
.
10
-
3
, 1.14
.
10
-
2
)
d
(-1.56
.
10
-
4
, 1.56
.
10
-
3
)
c
Sunsc een c eam 5
Concen a ion (% w/w) 2.51
.
10
-
3
1.04
.
10
-
2
< CCα
e
1.98
.
10
-
3
In e al a a 95% con idence le el (% w/w) (5.75
.
10
-
4
, 4.27
.
10
-
3
)
e
(7.49
.
10
-
3
, 1.31
.
10
-
2
)
e
(1.08
.
10
-
3
, 2.75
.
10
-
3
)
c
Sunsc een c eam 6
Concen a ion (% w/w) 3.20
.
10
-
5
< CCα
2.51
.
10
-
2
6.62
.
10
-
1
In e al a a 95% con idence le el (% w/w) (6.87
.
10
-
6
, 5.56
.
10
-
5
)
c
(2.40
.
10
-
3
, 4.67
.
10
-
2
)
(3.84
.
10
-
2
, 1.06)
Sunsc een c eam 7
Concen a ion (% w/w) 6.35
.
10
-
3
< CCα
g
< CCα
g
1.73
In e al a a 95% con idence le el (% w/w) (5.40
.
10
-
4
, 1.17
.
10
-
2
)
g
(1.56, 2.00)
g
a
α = β = 0.05.
b
The concen a ion alue was calcula ed using he ex ac dilu ed 10000 imes (n = 2).
c
The concen a ion alue was calcula ed using he ex ac dilu ed 10 imes (n = 1).
d
The concen a ion alue was calcula ed using he ex ac dilu ed 1300 imes (n = 2).
e
The concen a ion alue was calcula ed using he ex ac dilu ed 1000 imes (n = 2).
The concen a ion alue was calcula ed using he ex ac dilu ed 8000 imes (n = 2).
g
The concen a ion alue was calcula ed using he ex ac dilu ed 3000 imes (n = 2).
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
Fig. 1
Rela i e abundance (%)

MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
Fig. 2
8.20 8.21 8.22 8.23 8.24 8.25 8.26
Time (min)
0
2
4
6
8
10
12 104
(a)
BHT
In e e en 1
In e e en 2
10.18 10.19 10.20 10.21 10.22 10.23 10.24 10.25
Time (min)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5 106
(b)
DiBP
IS
In e e en
Ch oma og aphic loading
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
Fig. 3
Ch oma og aphic loading
77 105 151 228
m/z a io
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
(b)
227
10 20 30 40 50 60 70 80
Sample numbe
0
2
4
6
8
10
12
14 104
(c)
PREDICTION SET
CALIBRATION SET
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
Fig. 4