Belén Góma a1
Ca men Ga cía-Ruiz2
Ma ía Luisa Ma ina1, 2
1Cen o de Tecnología de los
Alimen os y Se icios
Biosani a ios,
Uni e sidad de Alcalá,
Alcalá de Hena es,
Mad id, Spain
2Depa amen o de Química
Analí ica, Facul ad de Química,
Uni e sidad de Alcalá,
Alcalá de Hena es,
Mad id, Spain
Enan ioselec i e sepa a ion o he sunsc een agen
3-(4-me hylbenzylidene)-campho by elec okine ic
ch oma og aphy: Quan i a i e analysis in cosme ic
o mula ions
3-(4-Me hylbenzylidene)-campho (MBC) is a chi al sunsc een agen used in cosme ic
p oduc s. In his wo k, he enan iosepa a ion o MBC has been pe o med by EKC and
applied o he analysis o he MBC enan iome s in cosme ic c eams. Di e en expe i-
men al condi ions ( ype and concen a ion o he chi al selec o , empe a u e, and
sample sol en ) ha e been op imized. Due o he neu al na u e o his compound,
anionic CD de i a i es we e in es iga ed as chi al selec o s. Ca boxyme hyla ed-b-CD
(CM-b-CD) showed he highes chi al sepa a ion powe , obse ing ha a 15 mMcon-
cen a ion o his CD a a wo king empe a u e o 157C enabled o ob ain he highes
enan io esolu ion. Howe e , unde hese condi ions, ailing o peaks ob ained o he
enan iome s was obse ed. The addi ion o inc easing concen a ions o he neu al
a-CD o CM-b-CD a a 15 mMconcen a ion in a 100 mMbo a e bu e a pH 9.0
imp o ed he enan iome ic sepa a ion and dec eased peak ailing. The use o DMF o
he o al dissolu ion o he cosme ic c eams, and me hanol:wa e (1:1 / ) o app o-
p ia e dilu ion enabled o obse e good shape and size o he peaks o he MBC
enan iome s. A e op imizing a me hod o he p econdi ioning o he capilla y, he
analy ical cha ac e is ics o he chi al sepa a ion me hod o he analysis o MBC we e
in es iga ed. Linea i y, LODs and LOQs, p ecision (ins umen al epea abili y, me hod
epea abili y, in e media e p ecision), accu acy, and selec i i y we e e alua ed. The
me hod was applied o analyze MBC enan iome s con ained in wo comme cial cos-
me ic c eams con aining acemic MBC and o s udy he skin abso p ion o his com-
pound wi h ime.
Keywo ds: Cosme ic c eams; Cyclodex ins; Elec okine ic ch oma og aphy; Enan ioselec i e
analysis; 3-(4-Me hylbenzylidene)-campho DOI 10.1002/elps.200500080
1 In oduc ion
3-(4-Me hylbenzylidene)-campho (MBC) is a chi al com-
pound added as a acemic mix u e o i s wo enan iome s
(see Fig. 1) in comme cial sun p o ec ion p oduc s, such
as cosme ic c eams. This compound ac s as UV il e in
hese p oduc s. Howe e , his sunsc een chemical may
cause heal h p oblems, since i can in e e e wi h he
Figu e 1. S uc u e o MBC enan iome s.
no mal unc ions o human ho mone es ogen [1] o p o-
duce some alle gic eac ions [2]. Thisiswhy hepe cen age
o MBC, as well as o o he cosme ic chemicals, in cos-
me ic o mula ions is egula ed in he Eu opean Union. In
Co espondence: P o esso Ma ía Luisa Ma ina, Depa amen o
de Química Analí ica, Facul ad de Química, Uni e sidad de
Alcalá, C a. Mad id-Ba celona Km. 33.600, E-28871 Alcalá de
Hena es, Mad id, Spain
E-mail: [email p o ec ed]
Fax: 134-91-885-4971
Abb e ia ions: ANOVA, analysis o a iance; CE--CD, ca box-
ye hyla ed-b-CD; CE-ª-CD, ca boxye hyla ed-g-CD; CM-Æ-CD,
ca boxyme hyla ed-a-CD; CM--CD, ca boxyme hyla ed-b-CD;
CM-ª-CD, ca boxyme hyla ed-g-CD; MBC, 3-(4-me hylbenzyli-
dene)-campho ; Succ--CD, succinyla ed-b-CD; Succ-ª-CD,
succinyla ed-g-CD
3952 Elec opho esis 2005, 26, 3952–3959
©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Elec opho esis 2005, 26, 3952–3959 Enan ioselec i e analysis o 3-(4-me hylbenzylidene)-campho by EKC 3953
he Spanish legisla ion, he MBC concen a ion in cosme ic
p oduc s mus be lowe han 4% [3]. As a consequence,
analy ical me hodologies o con ol he le els o his com-
pound in cosme ic p oduc s a e needed. Howe e , in he
li e a u e, he e a e only a ew pape s dealing wi h he
analysis o sunsc een agen s in cosme ic o mula ions. UV-
il e s ha e been analyzed in cosme ic p oduc s using CE
[4–7], HPLC [6–14], o UV-Vis spec opho ome y [15, 16].
These wo ks we e ocused on he iden i ica ion and/o
quan i a ion o he o al con en o he s udied UV- il e s in
mix u es om 2 o 24 compounds. In he case o CE, un il
now only he mic oemulsion EKC (MEEKC) mode [4, 5] has
been applied o he sepa a ion [4] and he quan i a i e
de e mina ion [5] in sunsc een lo ions o nine UV- il e s,
including MBC among hem. Ne e heless, as a as we
know he e is no e e ence in he li e a u e on he de elop-
men and applica ion o an analy ical me hod o he enan-
ioselec i e sepa a ion o he chi al UV il e MBC.
Enan ioselec i e analysis o chi al compounds is impo -
an because na u e is able o disc imina e among he
enan iome s o a chi al compound, ha is, each enan io-
me o a chi al compound may ha e a di e en biological
ac i i y. As an example, he case o an MBC s uc u ally
ela ed compound, campho quinone, can be ci ed. In
ac , deg ada ion o campho quinone by yeas was
ecen ly epo ed o be enan ioselec i e [17]. Then, he
de elopmen o chi al me hods o analysis o MBC has a
high in e es in o de o s udy he bioac i i y o his
sunsc een agen in na u e. CE is a sepa a ion echnique
wi h a high po en ial in he ield o chi al analysis which
has been epo ed in hund eds o scien i ic pape s, a wide
numbe o e iews, se e al issues o in e na ional jou nals
dedica ed o enan iosepa a ions [18–22], and e en a
book [23]. Fo his eason, he CE wo king mode o EKC
was used in his wo k o ca y ou he chi al sepa a ion o
MBC using CDs as chi al selec o s. Ou goal was o
de elop a chi al sepa a ion me hod by EKC o achie e he
enan iome ic sepa a ion o MBC and o apply i o he
quan i a ion o his chi al compound in comme cial sun
c eams and o s udy he MBC skin abso p ion wi h ime.
2 Ma e ials and me hods
2.1 Reagen s and samples
All eagen s employed o he p epa a ion o he sepa a-
ion bu e s we e o analy ical g ade. DMF was supplied
om Sigma (Mad id, Spain) and me hanol was om Lab-
Scan (Dublin, I eland). Bo ic acid and sodium hyd oxide
we e supplied om Pan eac (Ba celona, Spain). MBC
s anda d was pu chased om ABCR GmbH & Co. (Ka ls-
uhe, Ge many). Ca boxyme hyla ed-b-CD (CM-b-CD,
DS ,3) and a-CD we e pu chased om Fluka (Buchs,
Swi ze land). Ca boxyme hyla ed-g-CD (CM-g-CD,
DS ,3), ca boxyme hyla ed-a-CD (CM-a-CD, DS ,3),
ca boxye hyla ed-b-CD (CE-b-CD, DS ,3), ca boxy-
e hyla ed-g-CD (CE-g-CD, DS ,4), succinyla ed-b-CD
(Succ-b-CD, DS ,3), and succinyla ed-g-CD (Succ-g-
CD, DS ,3) we e om Cyclolab (Budapes , Hunga y).
Wa e used o p epa e solu ions was pu i ied h ough a
Milli-Q sys em om Millipo e (Bed o d, MA, USA). Solu-
ions we e il e ed p io use h ough 0.45 mm po e size
disposable nylon il e s om Sugelabo (Mad id, Spain).
The cosme ic c eams analyzed we e comme cially a ail-
able and acqui ed om a chemis in Ams e dam, The
Ne he lands (cosme ic c eam A, composed o MBC (3 g
pe 100 g sample), 4- e -bu yl-4’-me hoxy-dibenzoyl-
me hane, sodium 2-phenyl-benzidimazol-5-sul ona e,
and b onopol) and om a ma ke in Mad id, Spain (cos-
me ic c eam B, composed o MBC, isop opyl s ea a e,
cap ylic/cap ic/ iglyce ide, oc yl me oxycinnama e, pa -
a inum liquidum, ce yl dime hicone copolyol, C12–C15
alkyl benzoa e, phenylbenzidimazole sul onic acid, bu yl
me oxidibenzoyl me hane, i anium dioxide, polyglice yl-
4-isos ea a e, sodium chlo ide, phenoxye hanol, hyd o-
gena ed cas o oil, me hylpa aben, e hylpa aben, p o-
pylpa aben, bu ylpa aben, ocophe yl ace a e, pan he-
nol, and e inyl palmi a e).
2.2 Appa a us
All expe imen s we e pe o med on an HP3DCE sys em
(Hewle -Packa d, Waldb onn, Ge many) equipped wi h
an on-column diode a ay de ec o (DAD). Ins umen
con ol and da a acquisi ion we e pe o med wi h
he HP3DCE ChemS a ion so wa e (Hewle Packa d).
Sepa a ions we e pe o med on un ea ed used-silica
capilla ies o 50 mm ID and 375 mm OD, pu chased om
Composi e Me al Se ices (Wo ces e , England). Capil-
la ies had a o al leng h o 58.5 cm and an e ec i e leng h
o 50 cm. Capilla y empe a u e was 157C and UV de ec-
ion was pe o med a 300 nm wi h a bandwid h (wa e-
leng h ange whe e compounds a e de ec ed) o 50 nm
(300 625 nm) and a esponse ime o 0.1 s. A PHM-93
pH-me e om Radiome e (Copenhagen, Denma k) was
used o adjus he pH o he sepa a ion bu e s.
2.3 P ocedu e
Be o e i s use, he capilla y was lushed (4 ba ) wi h
1MNaOH o 30 min, ollowed by 20 min wi h wa e , and
20 min wi h he sepa a ion bu e . Be ween injec ions o
samples, he capilla y was condi ioned (4 ba ) wi h
me hanol:wa e (1:1, / ) o 2 min ollowed by 0.1 M
©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
CE and CEC
3954 B. Góma a e al. Elec opho esis 2005, 26, 3952–3959
NaOH, Milli-Q wa e , and he sepa a ion bu e o 2 min
each. Injec ions we e made by p essu e (50 mba o 3 s),
and he applied ol age was 20 kV.
Sepa a ion bu e solu ions we e p epa ed by dissol ing
he app op ia e amoun o bo ic acid (100 mM) wi h he
app op ia e amoun o each CD in Milli-Q wa e . The pH
alue was adjus ed a 9.0 by addi ion o NaOH (1 M).
These solu ions we e il e ed h ough 0.45 mm po e size
nylon il e memb ane be o e use in he CE sys em.
S ock solu ions we e p epa ed by dissol ing he app o-
p ia e amoun o MBC in DMF o achie e a inal con-
cen a ion o 10 000 mg/L. This solu ion was dilu ed in
me hanol:wa e (1:1 / ) o ge dilu ed solu ions wi h di -
e en concen a ions o MBC.
Sample solu ions o he wo comme cial cosme ic c eams
analyzed we e p epa ed by weighing abou 0.5 g c eam
and dissol ing i in 15 mL DMF. These sample solu ions
we e dilu ed en imes in me hanol:wa e (1:1 / ) o ge
he injec ion solu ions.
Sample solu ions o he skin abso p ion s udy we e p e-
pa ed by applying abou 0.5 g o he cosme ic c eam A o
an a ea o 20 cm66 cm o skin (120 cm2), wai ing o 0, 1,
3, 5, and 10 min, aking he esidual c eam wi h a spa ula,
and dissol ing i in 15 mL DMF. These sample solu ions
we e dilu ed en imes in me hanol:wa e (1:1 / ) be o e
injec ing in he CE sys em.
2.4 Da a ea men
Resolu ion o he MBC enan iome s was ob ained
om he ChemS a ion so wa e using he equa ion
Rs¼1:18 2 1
w1=2;1þw1=2;2
, whe e 1and 2a e he mig a-
ion imes o he i s - and second-mig a ing enan iome s,
espec i ely, and w1/2,1,w1/2,2 a e hei peak wid hs a hal -
heigh .
Co ec ed peak a eas (Ac) and co ec ed mig a ion imes
( c) ha e been used o compensa e luc ua ions in elec-
opho e ic condi ions and o ob ain a good ep oducibil-
i y o da a [24]. They ha e been calcula ed by di iding he
peak a ea (A) by he co esponding mig a ion ime ( ) and
he mig a ion imes ( ) by he mig a ion ime o he EOF
( EOF), which was measu ed as he mig a ion ime co e-
sponding o he sol en peak (me hanol peak), ha is,
Ac=A/ and c= / EOF
.
Concen a ion LODs (3sa/b) and LOQs (10sa/b) we e
de e mined om he s anda d e o o he in e cep (sa)
and he slope (b) o he calib a ion cu e ob ained by
analysis o a iance (ANOVA) [25].
Expe imen al da a analysis and pa ame e s we e calcu-
la ed using Excel Mic oso XP®and O igin 7.0. Compa -
ison o he slopes o eg ession lines was made using he
F- and - es s [25]. G aphs wi h di e en elec o-
phe og ams we e composed in O igin 7.0.
3 Resul s and discussion
3.1 De elopmen o an enan ioselec i e
analy ical me hod o MBC by EKC
The i s s ep, and he mos impo an one, was he
selec ion o a chi al selec o o MBC. Since CDs ha e
shown o be chi al selec o s in EKC wi h a high dis-
c imina ion powe , and he analy e unde s udy was neu-
al, di e en anionic CDs (CM-b-CD, CM-g-CD, CE-b-
CD, CE-g-CD, Succ-b-CD, and Succ-g-CD) a a 10 mM
concen a ion we e in es iga ed. These expe imen s
we e pe o med using a 100 mMbo a e bu e a pH 9.0
and a wo king empe a u e o 257C. Bo a e bu e was
chosen since a basic pH enabled o ha e an EOF enough
o mo e he anionic CDs o he de ec o . Among hese
CDs, only CM-b-CD and Succ-b-CD enabled he dis-
c imina ion o he wo enan iome s o MBC, CM-b-CD
being he chi al selec o gi ing ise o he highes enan-
iome ic esolu ion (Rs,1.4). As a consequence, CM-b-
CD a a 10 mMconcen a ion was selec ed in o de o
s udy he e ec o he empe a u e since i s in luence on
he enan iome ic esolu ion may be conside able as i has
been demons a ed o o he analy es [26, 27]. The a ia-
ion o he enan iome ic esolu ion and he mig a ion ime
o he wo MBC enan iome s as a unc ion o he em-
pe a u e o he capilla y was de e mined ( esul s no
shown). I could be obse ed ha , as in he case o o he
chi al analy es enan iome ically sepa a ed by EKC wi h
CDs as chi al selec o s [23], he enan iome ic esolu ion
inc eased when dec easing he empe a u e, al hough
enan iome mig a ion imes inc eased when dec easing
he empe a u e due o he inc emen in solu ion iscosi y.
As a esul , a empe a u e o 157C was chosen in o de o
s udy he e ec o CM-b-CD concen a ion (in he ange
om 5 o 20 mM) on he enan iome ic esolu ion o MBC.
The esul s ob ained enabled o obse e ha 15 mMCM-
b-CD p o ided he highes enan iome ic disc imina ion.
Howe e , unde hese condi ions an impo an dis o ion
o he peak co esponding o he i s -mig a ing enan io-
me was obse ed. In o de o imp o e peak shape and
esolu ion, 1 Mu ea was added o 15 mMCM-b-CD in
100 mMbo a e bu e a pH 9.0. In ac , u ea has p e-
iously been shown o imp o e peak shape o o he
neu al a oma ic compounds p obably because i enables
he be e solubiliza ion o hem and CDs in he aqueous
bu e [28]. Howe e , in he case o MBC enan iome s he
©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Elec opho esis 2005, 26, 3952–3959 Enan ioselec i e analysis o 3-(4-me hylbenzylidene)-campho by EKC 3955
addi ion o u ea did no imp o e he enan iome ic esolu-
ion. In addi ion, a ca boxyme hyla ed CD de i a i e wi h
a smalle inne co e, CM-a-CD, was used as chi al selec-
o a wo usual and di e en concen a ions (30 and
60 mM) bu he esolu ion o MBC enan iome s was no
obse ed ( esul s no shown). Since a-CD has al eady
shown good enan io esolu ion powe o a bicyclic com-
pound as i is campho quinone [17], we s udied he e ec
o his CD on he peak shape and enan io esolu ion o
MBC, which is a compound wi h a bicyclic ing in addi ion
o an a oma ic ing (see Fig. 1). Then, inc easing con-
cen a ions o a-CD (30, 60, 90, and 120 mM) we e added
o 15 mMCM-b-CD in 100 mMbo a e bu e a pH 9.0 in
o de o dec ease he peak ailing obse ed. I could be
obse ed ( esul s no shown) ha he enan iome ic eso-
lu ion o MBC inc eased when inc easing a-CD con-
cen a ion. In addi ion, he use o a-CD in he sepa a ion
medium also con ibu ed o ob ain di e en sepa a ion
selec i i ies wi h espec o he peaks co esponding o
he o he componen s o he cosme ic c eams s udied
when hese samples we e injec ed in he elec opho e ic
sys em. Figu e 2 shows he elec ophe og ams ob ained
o he wo sunsc een c eams s udied (A and B) when
using 15 mMCM-b-CD in 100 mMbo a e bu e a pH 9.0
in absence and in p esence o 120 mMa-CD. I is in e -
es ing o obse e ha peaks co esponding o o he
componen s o he c eams mig a ed a e MBC enan io-
me s peak in absence o a-CD whe eas a highe sepa a-
ion o he sample componen s, which appea ed a
Figu e 2. Elec ophe og ams co esponding o he com-
me cial cosme ic o mula ions A and B (100 mg/L, abou
0.5 g dissol ed in 15 mL DMF and dilu ed 1:10 wi h DMF)
in 100 mMbo a e bu e a pH 9.0 wi h 15 mMCM-b-CD
wi hou and wi h 120 mMa-CD. Un ea ed used-silica
capilla y, 58.5 cm (50 cm o he de ec o window)6
50 mm ID; sepa a ion empe a u e, 157C; applied ol age,
20 kV; injec ion, 50 mba 63 s. UV-de ec ion a
300 625 nm.
sho e mig a ion imes han MBC enan iome s, was
obse ed in p esence o 120 mMa-CD. Ne e heless,
peak ailing did no disappea unde hese condi ions.
This undesi able e ec was elimina ed by changing he
sol en o he s anda d and sample solu ions. Figu e 3
shows he e ec o di e en sol en s on he shape and
size o he MBC peaks using he dual CD sys em based
on 15 mMCM-b-CD/120 mMa-CD, which p o ided he
highes enan iome ic disc imina ion. I can be obse ed
ha a mix u e o me hanol:wa e (1:1 / ) as sol en
enabled he bes peak shape and size. The e o e, we
chose his mix u e o p epa e he adequa e dilu ions o
he s anda d solu ions and samples ini ially dissol ed in
DMF because i enabled o dissol e o ally he cosme ic
c eams s udied in his wo k.
Figu e 3. Elec ophe og ams co esponding o he com-
me cial cosme ic p oduc A (p e iously dissol ed in DMF)
dilu ed in di e en sol en s. One hund ed millimola
bo a e bu e a pH 9.0 wi h 15 mMCM-b-CD and 120 mM
a-CD. O he expe imen al condi ions as in Fig. 2.
3.2 Quan i a i e analysis o MBC enan iome s
by EKC
Be o e s udying he analy ical cha ac e is ics o he chi al
me hod de eloped o he analysis o MBC enan iome s,
h ee di e en me hods o p econdi ioning he capilla y
we e es ed. Table 1 shows he s eps ollowed o each
p econdi ioning me hod and he RSD alues ob ained o
co ec ed peak a ea, co ec ed mig a ion ime, and
enan iome ic esolu ion o i e duplica ed injec ions o
sample A (each duplica e was injec ed wi h he same
bu e solu ion which was eplaced o each one o he i e
duplica ed injec ions). I can be obse ed ha he p e-
condi ioning me hod III enabled o ob ain he lowes RSD
alues o co ec ed mig a ion ime (0.19%) and enan io-
esolu ion (2.8%). As a consequence, his me hod was
©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
3956 B. Góma a e al. Elec opho esis 2005, 26, 3952–3959
Table 1. Me hods es ed o p econdi ioning he capilla y o he analysis o MBC
P econdi ioning
me hods
S eps RSD in% (n=5)
Fi s -mig a ing
enan iome
Second-
mig a ing
enan iome
Rs
Ac cAc c
I 0.1 MNaOH (4 ba ) o 2 min
Milli-Q wa e (4 ba ) o 2 min
sepa a ion bu e (4 ba ) o 2 min
4.9 0.24 4.6 0.27 5.3
II DMF (4 ba ) o 2 min
0.1 MNaOH (4 ba ) o 2 min
Milli-Q wa e (4 ba ) o 2 min
sepa a ion bu e (4 ba ) o 2 min
8.0a) 1.82 8.4a) 2.74 6.6a)
III Me hanol:wa e (1:1, / ) (4 ba )
o 2 min
0.1 MNaOH (4 ba ) o 2 min
Milli-Q wa e (4 ba ) o 2 min
sepa a ion bu e (4 ba ) o 2 min
3.8 0.18 5.2 0.19 2.8
Ac: co ec ed peak a ea; c: co ec ed mig a ion ime
a) n=4
selec ed o p econdi ioning he capilla y walls be o e
injec ions. In addi ion, p econdi ioning me hod using
DMF as i s s ep (me hod II) p oduced b oad peaks and
sensi i i y losses, p obably due o he e ec o DMF on
he MBC/CD in e ac ions.
The analy ical cha ac e is ics o he enan ioselec i e
me hod de eloped we e s udied in o de o e alua e he
quali y, eliabili y, and consis ency o i s esul s. Linea i y,
LODs and LOQs, p ecision, accu acy, and selec i i y
we e e alua ed.
The linea i y was s udied by plo ing he co ec ed peak
a ea (Ac) as a unc ion o he analy e concen a ion in
mg/L. Six s anda d solu ions con aining acemic MBC
om 10 o 1000 mg/L (each one injec ed in iplica e)
we e employed. The chi al me hod enabled o s udy
sepa a ely each enan iome , whose concen a ions
anged om 5 o 500 mg/L. ANOVA analysis enabled o
con i m ha expe imen al da a i p ope ly o linea
models o bo h enan iome s (p- alues o 0.071 and
0.076 o he i s - and second-mig a ing enan iome s,
espec i ely (a= 0.05)). The linea wo king concen a ion
ange o he acema e and MBC enan iome s as well as
he linea equa ions o each enan iome a e shown in
Table 2. This able also shows he concen a ion LODs
and LOQs o MBC enan iome s calcula ed om he
calib a ion line pa ame e s. An LOD o 10 mg/L and LOQ
o 27 mg/L o he i s -mig a ing enan iome , and LOD o
12 mg/L and LOQ o 31 mg/L o he second-mig a ing
enan iome we e ob ained. These concen a ion alues
we e adequa e o quan i a e MBC in cosme ic o mula-
ions.
P ecision was e alua ed conside ing he ins umen al and
me hod epea abili y as well as he in e media e p eci-
sion. Ins umen al epea abili y was de e mined om six
epea ed injec ions o wo s anda d solu ions a wo di -
e en concen a ion le els (50 and 100 mg/L o MBC). In
bo h cases, RSD alues lowe han 4.3% o co ec ed
peak a ea and lowe han 0.6% o co ec ed mig a ion
ime we e ob ained (see Table 2). The me hod epea -
abili y was assessed wi h six eplica e s anda d solu ions
o 50 and 100 mg/L o MBC as well as six eplica es o a
sample solu ion (cosme ic c eam A) injec ed in iplica e
on he same day. RSD alues o bo h s anda d solu ions
we e lowe han 8.7% o co ec ed peak a ea and lowe
han 0.8% o co ec ed mig a ion ime. RSD alues o
he sample solu ion we e lowe han 7.1% o co ec ed
peak a ea and lowe han 1.3% o co ec ed mig a ion
ime (see Table 2). The in e media e p ecision o he
me hod was calcula ed o h ee eshly p epa ed s and-
a d solu ions o MBC con aining 50, 100, and 500 mg/L
and injec ed in iplica e o i e di e en days and wi h
wo di e en capilla ies. In his case, RSD alues we e
lowe han 2.4% o co ec ed mig a ion ime and lowe
han 11.0% o co ec ed peak a ea.
Accu acy was es ima ed as he eco e y ob ained o
MBC enan iome s when spiking he cosme ic c eam A
wi h known concen a ions o MBC (25, 62.5, and
©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim

Elec opho esis 2005, 26, 3952–3959 Enan ioselec i e analysis o 3-(4-me hylbenzylidene)-campho by EKC 3957
Table 2. Analy ical cha ac e is ics o he me hod de eloped o he analysis o MBC enan iome s
Linea wo king concen a ion ange Racema e: 10–1000 mg/L
Enan iome s: 5–500 mg/L
Linea equa ion i s -mig a ing enan iome Ac= 0.0382c–0.1026
(sb= 0.0004; sa= 0.0947; = 0.9998; n=6)
Linea equa ion second-mig a ing enan iome Ac= 0.0404c–0.1383
(sb= 0.0005; sa= 0.1119; = 0.9997; n=6)
De ec ion limi s Fi s -mig a ing
enan iome
10 mg/L
Second-mig a ing
enan iome
12 mg/L
Quan i a ion limi s Fi s -mig a ing
enan iome
27 mg/L
Second-mig a ing
enan iome
31 mg/L
P ecision Fi s -mig a ing
enan iome
Second-mig a ing
enan iome
Ins umen al epea abili y (n=6)
50 mg/L MBC Ac, RSD in%
c, RSD in%
4.3
0.5
3.4
0.6
100 mg/L MBC Ac, RSD in%
c, RSD in%
3.0
0.3
3.2
0.3
Me hod epea abili y (n=6)
50 mg/L MBC Ac, RSD in%
c, RSD in%
6.5
0.5
8.7
0.6
100 mg/L MBC Ac, RSD in%
c, RSD in%
4.3
0.7
6.0
0.8
Cosme ic c eam A
(,100 mg/L MBC)
Ac, RSD in%
c, RSD in%
6.9
1.2
7.1
1.3
In e media e p ecision (n= 15)
50 mg/L MBC Ac, RSD in%
c, RSD in%
6.4
1.4
8.6
1.5
100 mg/L MBC Ac, RSD in%
c, RSD in%
8.5
2.2
8.9
2.4
500 mg/L MBC Ac, RSD in%
c, RSD in%
10.0
2.1
11.0
2.3
Accu acy Fi s -mig a ing
enan iome
Second-mig a ing
enan iome
Reco e y (%)
25 mg/L o he MBC enan iome added
o he c eam A
104 61 103 62
62.5 mg/L o he MBC enan iome added
o he c eam A
90 611 89 611
100 mg/L o he MBC enan iome added
o he c eam A
104 66 102 66
Mean eco e y (%) 99 699869
c, concen a ion; sa, s anda d e o o he in e cep ; sb, s anda d e o o he slope; , co ela ion
coe icien
©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
3958 B. Góma a e al. Elec opho esis 2005, 26, 3952–3959
100 mg/L o each enan iome ) and compa ing hese con-
cen a ion alues wi h hose de e mined by he EKC
me hod using he ex e nal s anda d me hod o calib a-
ion. Each s anda d addi ion le el was pe o med in ipli-
ca e. Table 2 shows ha eco e y alues anged om
90 611 o 104 66% o he i s -mig a ing enan iome
and om 89 611 o 103 62% o he second-mig a ing
enan iome wi h mean eco e ies o 99 69 and 98 69,
espec i ely.
App op ia e sepa a ion selec i i y was obse ed o he
chi al me hod de eloped since he peaks obse ed o
o he componen s o he c eams did no in e e e wi h he
MBC peaks. Thus, all he peaks co esponding o he
o he componen s o cosme ic c eams A and B mig a ed
be o e he peaks co esponding o MBC enan iome s no
p oducing any in e e ence wi h he analy e (see Figs. 2,
3). In addi ion, he exis ence o possible ma ix in e -
e ences was in es iga ed by compa ing he calib a ion
slopes ob ained by he ex e nal s anda d and he s and-
a d addi ion calib a ion me hods. The esul s ob ained by
F- and - es s (p,0.05) [25] showed ha he e we e no
s a is ically signi ican di e ences be ween he slopes
ob ained by he ex e nal s anda d and he s anda d
addi ion calib a ion me hods o bo h enan iome s. This
ac showed ha he ex e nal s anda d calib a ion me h-
od can be used o achie e he quan i a ion o MBC
enan iome s in cosme ic c eam samples.
3.3 Applica ions
The chi al me hod de eloped o he analysis o MBC
enan iome s was applied o wo di e en pu poses: (i) o
quan i a e he MBC enan iome s in wo comme cial cos-
me ic c eams con aining his compound as acema e in
hei o mula ion and (ii) o s udy he skin abso p ion o
one cosme ic c eam (comme cial cosme ic A) wi h ime.
The amoun o MBC enan iome s de e mined in he wo
comme cial o mula ions analyzed in his wo k is shown in
Table 3. The quan i a ion o MBC enan iome s enabled o
de e mine he o al MBC con en in he cosme ic c eams
analyzed. In he case o he comme cial c eam A, he
amoun de e mined by he de eloped me hod was
13.1 60.6 mg o MBC in 0.5 g o sample, being he
amoun indica ed in he label o he p oduc 15 mg o
MBC in 0.5 g o sample. Fo he comme cial c eam B, o
which he label o he p oduc did no speci y he amoun
o MBC added, an amoun o 8.4 60.5 mg o MBC in
0.5 g o sample was de e mined.
Finally, he me hod de eloped was applied o pe o m a
p elimina y s udy o he skin abso p ion o he cosme ic
c eam A wi h ime. Figu e 4 shows he a ia ion in he
a eas o he peaks co esponding o he MBC enan io-
me s and o he componen s o he cosme ic c eam A
when his c eam was applied o 120 cm2o skin o 0, 5,
and 10 min. F om his igu e, i can be obse ed ha
unde hese expe imen al condi ions he abso p ion o
bo h enan iome s by he skin was simila .
Figu e 4. Elec ophe og ams co esponding o he com-
me cial cosme ic p oduc A (dilu ed in me hanol:wa e
(1:1 / )) applied o an a ea o skin o 120 cm2 o di e en
imes. Sepa a ion bu e as in Fig. 3. O he expe imen al
condi ions as in Fig. 2.
Table 3. A e aged amoun o MBC enan iome s and o al MBC measu ed in he wo di e en com-
me cial cosme ic c eams analyzeda)
Cosme ic c eam Amoun o i s
enan iome
(mg) 6 s/n1/2
Amoun o second
enan iome
(mg) 6 s/n1/2
To al amoun
de e mined
(mg) 6 s/n1/2
To al label-
ed weigh ,
mg
A 6.6 60.3 6.5 60.3 13.1 60.6 15
B 4.1 60.3 4.3 60.2 8.4 60.5 –
a) sis he SD, = 3.18 ( o a con idence linea ange o 95%), and n=3.
©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Elec opho esis 2005, 26, 3952–3959 Enan ioselec i e analysis o 3-(4-me hylbenzylidene)-campho by EKC 3959
4 Concluding ema ks
This wo k shows ha he enan iome ic sepa a ion o he
sunsc een agen MBC is possible by EKC using a dual CD
sys em cons i u ed by he anionic CM-b-CD (15 mM) and
he neu al a-CD (120 mM) in 100 mMbo a e bu e a
pH 9.0. Al hough he selec ion o he chi al selec o was
he mos di icul s ep, he chi al selec o concen a ion
and he empe a u e a e wo e y impo an ac o s o be
aken in o conside a ion du ing he de elopmen o he
chi al me hod. In addi ion, he use o a me hanol:wa e
(1:1 / ) mix u e o dilu e s anda ds and samples (p e-
iously dissol ed in DMF) was necessa y in o de o a oid
peak ailing.
The analy ical cha ac e is ics o he chi al EKC me hod
de eloped o he sepa a ion o MBC enan iome s we e
s udied and he me hod was applied o he quan i a ion o
MBC enan iome s in cosme ic c eams. A e he op imi-
za ion o he capilla y p econdi ioning, good pe o mance
o he me hod wi h ega ds o linea i y, p ecision (ins u-
men al epea abili y, me hod epea abili y, in e media e
p ecision), accu acy, and selec i i y was achie ed. LODs
and LOQs de e mined o MBC enan iome s enabled he
quan i a ion o his compound in comme cial cosme ic
c eams. In addi ion, his me hod was also use ul o s udy
he skin abso p ion o MBC om one cosme ic c eam (A)
wi h ime.
Au ho s hank he Minis y o Science and Technology
(Spain) o he esea ch p ojec BQU2003-03638. C.G.-R.
hanks he Minis y o Science and Technology o he
Ramón y Cajal p og am (RYC-2003-001). B.G. hanks
CSIC and he RYC-2003-001 p ojec o he g an s.
Au ho s also hank Cyclolab (Budapes , Hunga y) o he
kind gi o CE-b-CD used in his wo k.
Recei ed Janua y 31, 2005
Re ised Ap il 22, 2005
Accep ed Ap il 26, 2005
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©2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim