Enantioselective separation of the sunscreen agent 3-(4-methylbenzylidene)-camphor by electrokinetic chromatography: Quantitative analysis in cosmetic formulations

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 5 Re e ences [1] Sunsc een chemicals cause heal h p oblems (h p:// www.coas alguide.o g/news/2001-09.h ml) checked on Jan- ua y 2005. 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