Sensitive chiral analysis by CE: An update. Review

Lau a Sánchez-He nández An onio Luis C ego Ma ía Luisa Ma ina Ca men Ga cía-Ruiz Depa amen o de Química Analí ica, Facul ad de Química, Uni e sidad de Alcalá, Alcalá de Hena es, Mad id, Spain Recei ed July 20, 2007 Re ised Sep embe 19, 2007 Accep ed Sep embe 20, 2007 Re iew Sensi i e chi al analysis by CE: An upda e A gene al iew o he di e en s a egies used in he las yea s o enhance he de ec ion sensi i i y in chi al analysis by CE is p o ided in his a icle. Wi h his pu pose and in o de o upda e he p e ious e iew by Ga cía-Ruiz e al., he a icles appea ed on his subjec om Janua y 2005 o Ma ch 2007 a e conside ed. Th ee we e he main s a egies employed o inc ease he de ec ion sensi i i y in chi al analysis by CE: (i) he use o o -line sample ea men echniques, (ii) he employmen o in-capilla y p econcen a ion echniques based on elec opho e ic p inciples, and (iii) he use o al e na i e de ec ion sys ems o he widely employed on-column UV–Vis abso p ion de ec ion. Combina ions o wo o h ee o he abo e-men ioned s a egies ga e ise o adequa e concen a ion de ec ion limi s up o 10210 M enabling enan iome analysis in a a ie y o eal samples including complex bio- logical ma ices. Keywo ds: CE / Chi al analysis / De ec ion sensi i i y / In-capilla y p econcen a ion DOI 10.1002/elps.200700531 Elec opho esis 2008, 29, 237–251 237 1 In oduc ion Due o he di e en biological ac i i y ha he enan iome s o a chi al compound may ha e, chi al analysis has nowadays an inc easing in e es in a a ie y o disciplines such as pha ma- ceu ical, en i onmen al, o ood analysis, among o he s. The indi idual de e mina ion o he enan iome s o a chi al com- pound may equi e a high sensi i i y o a g ea numbe o applica ions. Thus, he analysis o low concen a ed and lim- i ed amoun s o biological samples, he analysis o en i on- men al samples whe e analy es a e usually p esen a ace le el o he de e mina ion o ood componen s, ing edien s, o esidues a e examples o his kind o applica ions. Mo e- o e , he de e mina ion o enan iome ic impu i ies in d ugs can equi e sensi i e analy ical me hodologies due o he low pe cen ages o he impu i y ha should be de e mined acco ding o he ICHguidelines ( o impu i y con en shighe han 0.05% he impu i ies ha e o be epo ed) [1]. Since CE has shown in he las yea s a g ea po en ial o achie e chi al sepa a ions, he de elopmen o analy ical me hodologies enabling he sensi i e de e mina ion o enan iome s has been he aim o a conside able numbe o a icles. The use o a leas one chi al selec o in he sepa a- ion media is necessa y o enable a chi al sepa a ion by CE being CDs he a o i e chi al selec o s employed. Al hough he e a e some au ho s ha conside as CZE he sepa a ion mode in CE when neu al CDs a e used, we will conside in his e iew ha when CDs a e employed as chi al selec o s, and independen ly o hei na u e, he sepa a ion mode is EKC. In ac , he enan iome ic disc imina ion in EKC is p oduced by a ch oma og aphic mechanism whe e in e ac- ions a e es ablished be ween each one o he enan iome s and he chi al selec o [2–4]. Al hough much less used, CEC and NACE can also be employed o chi al analysis. CEC is a hyb id echnique be ween CE and HPLC cha ac e ized by a high sepa a ion e iciency due o he plug p o ile o he mobile phase d i en by he EOF. A wide ange o HPLC chi al s a iona y phases (CSPs) ans e able o CEC o monoli hic columns can be used o p o ide adequa e enan- ioselec i i y. Mo eo e , an impo an aspec om he poin o iew o he sensi i i y and selec i i y in chi al analysis is he easy coupling o CEC o MS de ec ion, when he chi al selec o in CEC is immobilized [5]. On he o he hand, he Co espondence: D . Ca men Ga cía-Ruiz, Depa amen o de Quí- mica 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: 134-91-8854971 Abb e ia ions: CBI, cyanobenz[ ]isoindole; CSP, chi al s a iona y phase; DAS--CD, hep akis(2,6-diace hyl-6-sul a o)-b-CD; DIM, dime hindene; DIO, dioxop ome hazine; ECL, elec ochemilumi- nescence; FASS, ield-ampli ied sample s acking; HS--CD, highly sul a ed-b-CD; HS-ª-CD, highly sul a ed-g-CD; HDAS-- CD, hep akis(2,3-di-O-ace yl-6-O-sul o)-b-CD; LE, leading elec o- ly e; LLE, liquid–liquid ex ac ion; MA, me hamphe amine; MDA, me hylenedioxyamphe amine; MDMA, me hylenedioxy- me hamphe amine; MTD, me hadone; NAC, N-ace yl L-cys eine; NE, no ephed ine; OPA, o hoph halaldehyde; PHM, pheni a- mine; poly-L-SUCL, poly(sodium N-undecenoxy ca bonyl-L-leuci- na e); poly-LL-SUCLV, poly (sodium N-undecenoxyca bonyl-LL- leucyl- alina e); SPCD, sample p econcen a ion wi h chemical de i a iza ion; TEA, ie hylamine; TE, e mina ing elec oly e; TMD, amadol ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 238 L. Sánchez-He nández e al. Elec opho esis 2008, 29, 237–251 aqueous bu e employed in CE is eplaced in NACE by an o ganic sol en con aining an elec oly e. The use o a sol- en ins ead o an aqueous bu e has p o ided addi ional selec i i ies o hose ob ained in aqueous CE sys ems and may be ad an ageous o he hyphena ion o his sepa a ion mode wi h MS, i ola ile sol en s a e used [6]. A a ie y o s a egies ha e been employed in CE in o de o ob ain he de ec ion sensi i i y needed o a gi en applica- ion. These s a egies included he use o o -line o on-line sample ea men echniques, sample p econcen a ion in he capilla y using echniques based on elec opho e ic p inciples, and/o he use o al e na i e de ec ion sys ems o he UV–Vis abso p ion de ec ion [7]. In addi ion, o he s a - egies such as pa ial illing o he capilla y o he use o CDs wi h coun e cu en mig a ion can be necessa y, i MS de ec ion is employed when chi al selec o s a e in he sepa a ion media. These s a egies a oid he in oduc ion o hese compounds in o he MS de ec o which causes a dam- age in elec osp ay e iciency and inc eases backg ound noise dec easing he sensi i i y o de ec ion. Some e iews ha e been published in he las yea s co - e ing he chi al sepa a ion o d ugs [8–10] o pollu an s and hei me aboli es [11]. The aim o his e iew is o p o ide a gene al iew o he di e en s a egies ha ha e been used in he las yea s in o de o enhance he sensi i i y o de ec ion in chi al analysis by CE wi h applica ion o he analysis o d ugs o biological, en i onmen al, o ood samples. A icles appea ed om he publica ion o he p e ious e iew by Ga cía-Ruiz and Ma ina [7] ha e been conside ed co e ing he pe iod o ime om Janua y 2005 o Ma ch 2007. 2 Enhancemen o he sensi i i y in chi al analysis by CE An impo an aspec o ake in o accoun in CE is he e y low olumes injec ed in he sys em (in he nL ange) ha imply ha he de ec ion o a concen a ion 1025M leads o he de ec ion o ,10214 mol o analy e. Fo his eason, all hose wo ks whe e a leas one s a egy has been employed o imp o e he de ec ion sensi i i y in chi al analysis by CE enabling he de e mina ion o enan iome s a leas a mola concen a ions o 1025ha e been included in his e iew. Nex sec ions will desc ibe he di e en app oaches used in he las yea s o enhance he de ec ion sensi i i y in chi al analysis by CE: (i) o -line sample ea men echniques, (ii) in-capilla y p econcen a ion echniques, and (iii) al e na i e de ec ion sys ems o he widely employed on-column UV– Vis abso p ion de ec ion. 2.1 O -line sample ea men echniques in sensi i e chi al analysis by CE Table 1 g oups he di e en sample ea men s employed in he pe iod o ime e iewed in his a icle p io o chi al analysis by CE. Analy es, samples, sepa a ion bu - e , de ec ion sys em, and de ec ion limi s (LODs) ob ained a e also gi en in his able. Sample ea men s can be aimed o elimina e some componen s o he sam- ple ma ix in addi ion o enhance he de ec ion sensi i i y h ough sample p econcen a ion. SPE, liquid–liquid ex ac ion (LLE), solid-phase mic oex ac ion (SPME), o mic odialysis we e used. In all cases, o -line s a egies we e used. SPE and LLE we e he main ex ac i e echniques employed. P econcen a ion by bo h echniques implies he econs i u ion o he esidue ob ained a e sample ea men in a olume smalle han he ini ial sample olume. The e- o e, he imp o emen in he concen a ion sensi i i y will depend on he sample olume a ailable. Howe e , p e- concen a ion possibili ies by SPE a e usually be e han by LLE, because he olume ac o (sample olume/ esidue ol- ume) is mo e a o able in SPE han in LLE. SPE is one o he mos popula and widely used ex ac- i e echniques used o liquid samples due o i s high selec i i y and also p econcen a ion possibili ies. SPE was only employed as sample ea men o isola e chi al d ugs in biological samples as u ine [12, 13, 15, 17] and o selec- i ely p econcen a e chi al he bicides in spiked wa e sam- ples [14, 16]. Mos o hese wo ks we e pe o med wi h UV de ec ion [12–14, 17] achie ing LODs in he 1028–1027M ange. These LODs we e imp o ed by one o wo o de s o magni ude using SPE as sample ea men depending on he sample olume a ailable (p econcen a ion ac o s anging om 3 o 250 we e achie ed). Howe e , o he de ec ion sys ems such as ESI-MS we e also used enabling o de ec up o 361028and 661028M o each salbu amol enan iome [15]. In his case, he SPE s ep enabled an en ichmen o he ini ial sample concen a ion o ou imes. The bes LODs (261029M) we e eached using lu- o escence de ec ion o he analysis o glu osina e enan io- me s [16]. The high sensi i i y achie ed in he la e wo k was due o he combina ion o an SPE s ep wi h an in-cap- illa y p econcen a ion s a egy. Classical LLE was also employed o he ex ac ion and p econcen a ion o chi al compounds p io o CE chi al analysis. Thus, Table 1 shows he use o LLE o he de e - mina ion o d ugs in biological samples as plasma [20–23], human se um [17, 19], human u ine [17], and a mic osomal ac ion o li e homogena es [18]. P econcen a ion ac o s up o 21 we e achie ed by his ex ac ion p ocedu e. A e his sample ea men s ep, LODs om 1026 o 361028M we e epo ed when UV de ec ion was used [17–20] and LODs anging o m 361026 o 461029M we e eached wi h ESI-MS de ec ion [21, 22]. As example, Schapple e al. [22] assessed wo app oaches o enhance he sensi i i y in CE. The o me consis ed o a p o ein p ecipi a ion using ACN ollowed by hyd odynamic injec ion o he supe na an . The second was he combina ion o LLE, which p oduces a sample cleanup and en ichmen minimizing any ma ix e ec , wi h elec okine ic injec ion. In spi e o he ac ha he o me me hod was apidly achie ed wi h minimal ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2008, 29, 237–251 CE and CEC 239 Table 1. O -line sample ea men echniques employed o he enhancemen o he sensi i i y in chi al analysis by CE Sample ea men Analy e and sample Sepa a ion bu e De ec ion LOD (M) Re . SPE Lo azepam in human u ine 6 mM bo a e/10 mM phospha e (pH 9.1) 160 mM HP-b-CD 175 mM SDS UV-200 nm ,261025[12] SPE CIT, DCIT, DDCIT, CIT-NO, and CIT-PA in human u ine 20 mM phospha e (pH 5) 10.2% CM-g-CD 10.05% HPMC UV-205 nm 661028– 361027 [13] SPE Mala hion in spiked ap wa e 25 mM T is (pH 7.0) 120 mM CM-b-CD UV-230 nm 661027[14] SPE Salbu amol in human u ine 10 mM ammonium o ma e (acidi ied wi h 0.75 M o mic acid) 115 mM HDAS-b-CD MS 361028, 661028 [15] SPE DNS-DL-Glu osina e in spiked i e wa e 2 mM phospha e (pH 6.5) 117 mM g-CD Fluo escence (lexc = 327 nm, lem = 557 nm) 261029[16] SPE/LLE Ibup o en in human se um and u ine 200 mM o hophospho ic acid 1200 mM ie hanolamine (pH 5.0) 150 mM TM-b-CD UV-220 nm ,261027 (se um), ,1026 (u ine) [17] LLE Hyd oxychlo oquine and i s me aboli es (DCQ, DHCQ, BDCQ) in mic osomal ac ion o li e homogena es 100 mM T is/phospha e (pH 9.0) 11% HS-b-CD 130 mg/mL HP-b-CD UV-220 nm ,1027[18] LLE P opa enone in human se um 100 mM phospha e (pH 2.0) 10.6% HS-b-CD. UV-195 nm ,361028[19] LLE Ke amine and no ke amine in equine plasma 50 mM T is (pH 2.5) 110 mg/mL HS-b-CD UV-195 nm ,461028[20] LLE Amphe amine de i a i es (A, MA, MDA, MDMA, MDEA, E, NE) in plasma 20 mM ammonium o ma e (pH 2.5) 10.15% HS-g-CD MS ,761027– 361026 [21] LLE Amphe amine de i a es (A, MA, MDA, MDMA, MDEA, TMD, MTD) in human plasma 20 mM ammonium o ma e (pH 2.5) 10.15% HS-g-CD MS 461029[22] LLE Disopy amide in spiked plasma sample 40 mM ace a e (pH 4.5) 13 mg/mL HS-b-CD ECL ,1027[23] SPME (1R,2S)-Ephed ine, (1R,2R)- pseudoephed ine, (1S,2S)- pseudoephed ine in wa e and human u ine 150 mM phospha e (pH 2.5) 117.5 mM b-CD UV-192 nm 2–361028[24] A, amphe amine; BDCQ, bisdese hylchlo oquine; CIT, ci alop am; CIT-NO, ci alop am N-oxide; CIT-PA, ci alop am p opionic acid; CM-g-CD, ca boxyme hyl-g-CD; DCIT, deme hylci alop am; DCQ, dese hylchlo oquine; DDCIT, dideme hylci alop am; DHCQ, dese hylhyd oxy- chlo oquine; DNS, dansyl cho ide; E, ephed ine; HDAS-b-CD, hep akis(2,3-di-O-ace yl-6-O-sul o)-b-CD; HP-b-CD, 2-hyd oxyp opyl-b-CD; HPMC, hyd oxyp opylme hylcellulose; MDEA, me hylenedioxye hylamphe amine; NDA, naph halene-2,3-dica boxaldehyde; TM-b-CD, hep akis 2,3,6- i-O-me hyl-b-CD. sample manipula ion, a concen a ion ac o o 1000- old was ob ained wi h he second one al hough i was mo e ime consuming. Finally, he MS signal supp ession e ec was in es iga ed on he complex ma ices o biological samples (plasma) wi h a con en ional CE-ESI-MS se up. Supp ession occu ed in he mig a ion window o analy es o in e es in con en ional analysis. Howe e , when LLE was employed, no ma ix e ec s we e e idenced leading o he conclusion ha his sample p epa a ion me hod emains o u mos in e es o he analysis o biological samples by CE-MS. The combi- na ion o LLE-elec okine ic injec ion in CE-MS enabled o ob ain he bes LODs (,1029M) indica ed in Table 1, and his was o amphe amine de i a i es in human plasma. Figu e 1 shows he sensi i e de e mina ion o se en am- ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 240 L. Sánchez-He nández e al. Elec opho esis 2008, 29, 237–251 Figu e1. Chi alanalysiso aplasmasamplespikedwi h0.5 ppbo each enan iome o se en di e en amphe amine de i a i es, a e LLE and elec okine ic injec ion in CE-ESI-MS. Elec opho- e ic condi ions: used-silica capilla y, 75 cm o al leng h and 50 mm id; sepa a ion bu e , 20 mM ammonium o ma e (pH 2.5) con aining 0.15% HS-g-CD; sepa a ion empe a u e, 257C; applied ol age, 25 kV; elec okine ic injec ion, 10 kV610 s. MS condi- ions: shea h liquid, isop opanol/H2O (50:50 / ) con aining 0.5% o mic acid; sy inge low a e, 3 mL/min. ESI capilla y a 4.5 kV. The nebulizing p essu e and he d ying gas low a e we e se a 4 psi and 4 L/min, espec i ely. Gas empe a u e, 2007C; agmen- o 70 V. Iden i ica ion o peaks: A, amphe amine; MA, me ham- phe amine; MDA, me hylenedioxyamphe amine; MDMA, me hylenedioxyme hamphe amine; MDEA, me hylenedioxy- e hylamphe amine; TMD, amadol; MTD, me hadone ( ep in ed om e . [22] wi h pe mission). phe amine de i a i es in a plasma sample spiked wi h 0.5 ppb o each enan iome a e LLE and elec okine ic injec ion in CE-ESI-MS. On he o he hand, Fang e al. [24] p oposed an SPME sample ea men p e ious o he injec ion in o a CE sys em wi h UV de ec ion as a chi al sensi i e me hod o doping con ol o ephed ine de i a i es. Al hough his ex ac i e echnique is mainly used in GC whe e compounds a e de- so bed om he ibe by applying high empe a u es, in his wo k he analy es (ephed ine de i a i es) we e ex ac ed om 5 mL o he liquid samples. This was ca ied ou by exposing he coa ed ibe end o he headspace o he ial o an app op ia e ime and hen deso bing he analy es in 80 mL o back-ex ac ion sol en con aining an op imized con- cen a ion o ACN. Figu e 2 shows a ,160- old in ensi y imp o emen a e SPME o ephed ine de i a i es om an u ine sample. In e es ingly, SPME no only enhanced he sensi i i y o de ec ion by concen a ing he sample, also supplied he possibili y o employ an in-capilla y sample p econcen a ion s a egy p o iding an app op ia e sample ma ix o be injec ed in he CE sys em. Figu e 2. Sepa a ion o ephed ine de i a i es in a spiked u ine sample (a) di ec ly injec ed in CE (concen a ion o each analy e: 5.00 mg/mL), and (b) a e o -line SPME (concen a ion o each analy e: 0.25 mg/mL) ollowed by CE. Elec opho e ic condi ions: used-silica capilla y, 60.5 cm o al leng h and 75 mm id; sepa a- ion bu e , 150 mM phospha e bu e (pH 2.5) con aining 17.5 mM b-CD; sepa a ion empe a u e, 207C; applied ol age, 25 kV; elec okine ic injec ion, 7 kV610 s. UV de ec ion a 192 nm. Iden i ica ion o peaks: (2)-PE, (1R,2R)-pseudoephe- d ine; (2)-E, (1R,2S)-ephed ine; (1)-PE, (1S,2S)-ephed ine ( e- p in ed om [24] wi h pe mission). 2.2 In-capilla y p econcen a ion echniques based on elec opho e ic p inciples in chi al analysis by CE Ano he way o inc ease he sensi i i y in chi al CE is by using in-capilla y p econcen a ion echniques based on elec opho e ic p inciples. Table 2 summa izes he main cha ac e is ics o wo ks whe e wo di e en in-capilla y p e- concen a ion s a egies we e used. On one hand, classical in-capilla y p econcen a ion echniques, such as ITP, s ack- ing, and/o sweeping we e employed, and on he o he hand, in-capilla y sample p econcen a ion wi h chemical de i a i- za ion (SPCD) is included in his able as an inno a i e s a egy. A b ie desc ip ion o he di e en p econcen a ion echniques will be made p e iously o indica e he applica- ions pe o med by CE in he chi al ield. In ITP, ionic analy es a e concen a ed and sepa a ed on he basis o hei elec opho e ic mobili ies using a dis- con inuous bu e sys em (leading elec oly e (LE) and ail- ing elec oly e (TE). This echnique may be pe o med in he same capilla y whe e he elec opho e ic sepa a ion is ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2008, 29, 237–251 CE and CEC 241 Table 2. In-capilla y p econcen a ion echniques employed o he enhancemen o he sensi i i y in chi al analysis by CE P econ- cen a ion echnique Analy e and sample Sepa a ion bu e De ec ion LOD (M) Re . cITP An ihis aminic d ugs (PHM, DIM, DIO) in u ine 25 mM ace ic acid (pH 3.2–4.5) 12.5 mg/mL (PHM, DIM) and 5.5 mg/mL (DIO) CE-b-CD UV-265 and 240 nm 461029– 261028, 1028 [25] cITP D ugs s anda ds (S-alp enolol, S-a enolol, R-p op anolol, R-salbu amol, S- e bu aline) LE: 160 mM ace a e (pD 4.7) 110 mM b-CD TE: 160 mM ace ic acid (pH 2.4) 110 mM b-CD NMR-600 MHz ,2.561024[26] ITP R,S-Timolol in s anda d 40 mM KOH in me hanol/e hanol (40:60 / ) 1100 mM (1)-KPA UV-220 461026[27] FASS 561026 FASS (1R,2S)-Ephed ine, (1R,2R)- pseudoephed ine, (1S,2S)- pseudoephed ine in wa e and human u ine 150 mM phospha e (pH 2.5) 117.5 mM b-CD UV-192 nm 2–361028[24] LVSS DNS-DL-glu osina e in spiked i e wa e 2.0 mM phospha e (pH 6.5) 117 mM g-CD Fluo escence (lexc = 327 nm, lem = 557 nm) 261029[16] LVSS 1 sweeping CBI-Se , CBI-Glu in biological samples 25 mM phospha e (pH 2.0) 12% HS-b-CD LIF (lexc = 420 nm) 2610210, 3610210 [28] Sweeping Lo azepam in human u ine 6 mM bo a e/10 mM phospha e (pH 9.1) 160 mM HP-b-CD 175 mM SDS UV-200 nm ,261025[12] In-capilla y SPCD OPA/NAC-amino acids (Ala, Glu) in E. coli bac e ial cul u e 140 mM bo a e (pH 9.5) 11mMb-CD UV-340 nm 461027, 661027 [29] In-capilla y SPCD OPA/NAC-mu amic and diaminopimelic acids in E. coli bac e ial cul u e 140 mM bo a e (pH 9.5) UV-214 and 340 nm 261026, 261027 [30] CE-b-CD, ca boxye hyl-b-CD; DIKGA, di-O-isop opylidene-2-ke o-L-gulonic acid; DIM, dime hindene; DIO, dioxop ome hazine; KPA, ke o- pinic acid; PHM, pheni amine. achie ed, which is denomina ed ansien ITP ( ITP), o in wo di e en capilla ies, which is named capilla y ITP (cITP). Table 2 shows ha cITP was used as p econcen a ion echnique o de e mine di e en an ihis aminic d ugs in a biological sample as u ine [25] and o de ec se e al d ugs in s anda d solu ions by CE-NMR [26]. Likewise, his able also shows he use o ITP o he p econcen a ion o R,S- imolol [27]. Thus, Mikus e al. [25] p oposed a highly sensi i e cITP- EKC me hod combining wo coupled capilla ies: in he i s capilla y, ITP was ca ied ou o achie e a p esepa a ion and a sample p econcen a ion, and in he second capilla y, whe e he sample was ans e ed on-line, he EKC sepa a ion occu ed. These expe imen s showed a o able condi ions o he sepa a ion and de e mina ion o aces (ng/mL) o an ihis aminic d ugs (dioxop ome hazine (DIO), dime hin- dene (DIM), and pheni amine (PHM)) enan iome s p esen in u ine samples dilu ed wi h wa e . By using UV de ec ion, LODs achie ed in his wo k we e om 461029 o 261028M depending on he compound analyzed. Ano he example o he use o cITP sample s acking p ocess is he sepa a ion and concen a ion o analy es o NMR measu emen s [26]. I is impo an o emphasize ha he coupling o cITP o NMR enabled o de ec small amoun s (,2 nmol) o he basic d ugs s udied, bu he concen a ions de ec ed (,70 mM) we e a away o a sensi i e de ec ion. Finally, a apid ana- ly ical me hod by using ITP o he de e mina ion o he enan iome ic impu i y o S- imolol was de eloped by Hede- land e al. [27]. In his me hod, he combina ion o 1S,4R-(1)- ke opinic acid (KPA) as chi al selec o in NACE and p e- concen a ion by ITP p o ided LODs as low as 0.2% o R- imolol in S- imolol samples. Wi h espec o s acking p econcen a ion, i is p oduced because ions mig a e elec opho e ically h ough a low-con- duc i i y sample ma ix in o a high-conduc i i y bu e solu- ion and hey a e ocused in a hick zone be ween he bounda ies o bo h solu ions. Mos s acking modes use ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 242 L. Sánchez-He nández e al. Elec opho esis 2008, 29, 237–251 hyd odynamic injec ion o la ge- olume sample being named la ge- olume sample s acking (LVSS). Howe e , when elec okine ic injec ion is used o ocus he analy es o he sample, he s a egy is denomina ed ield-ampli ied sample s acking (FASS). As shown in Table 2, highly sensi- i e chi al me hods we e de eloped using his p econcen a- ion echnique based on he s acking o he analy es in na - ow bands p e iously o he elec opho e ic sepa a ion. The possibili y o p econcen a ion by FASS was e al- ua ed and compa ed wi h ITP by Hedeland e al. [27]. FASS was pe o med using an elec okine ic injec ion o 8 kV o 10 s. Simila LODs, in he 1026M ange, we e ob ained by bo h in-capilla y p econcen a ion echniques. In addi ion, FASS was applied o in-capilla y sample concen a ion o ephed ine de i a i es in wa e and u ine samples [24]. In his wo k, a s udy o he pe o mance o he FASS p ocedu e (7 kV o 10 s) using he samples p epa ed in wa e enabled o ob ain a concen a ion ac o o up o 80- old compa ed wi h he con en ional elec okine ic sample injec ion (7 kV o 10 s), whe e he samples we e dissol ed in he sepa a ion bu e . On he o he hand, he concen a ion en ichmen achie ed by LVSS allowed he analysis o aces o he enan- iome s o he he bicide glu osina e in spiked i e wa e (model sample) [16]. The combina ion o his in-capilla y p econcen a ion s a egy wi h SPE enabled o ob ain LODs o D- and L-glu osina e enan iome s as low as 261029M. Mo eo e , Ki schne e al. [28] ob ained a e y high sensi i i y wi h a CE-LIF me hod in ol ing a combina ion o LVSS and sweeping which is a p econcen a ion echnique enabling a signi ican inc ease in he de ec ion sensi i i y o hose analy es wi h a high solu e-pseudos a iona y phase associa- ion cons an [31]. The combined p econcen a ion mechan- ism p oposed by Ki schne e al. is illus a ed in Fig. 3A. Fi s , LVSS in ol es a combina ion o ield-ampli ied s ack- ing and pH-media ed s acking. Then, sweeping using he anionic highly sul a ed-b-CD (HS-b-CD) as pseudophase in e ac ing wi h he analy es was pe o med. A solu ion con aining he analy es was injec ed illing 1/3 o he capil- la y. Then, e e se pola i y was applied and he anionic cya- nobenz[ ]isoindole (CBI)–amino acids mig a ed owa d he pH junc ion a he ou le side o he injec ion plug, whe e hey we e subs an ially neu alized and s acked by he low pH bu e . The mig a ion o he analy e anions p oduced a ield-ampli ied s acking in he dilu e wa e . Simul aneously, EOF began o pump wa e ou o he capilla y and mo ed he s acked analy es band owa d he inle . Du ing his pe iod, mos elec oly es s ayed wi hin he capilla y, since he cu - en was nea ze o o a ew mic oampe es. Finally, once he analy es we e nea ly ejec ed om he capilla y, he cu en inc eased as he HS-b-CD mig a ed apidly h ough he s acked band o he analy e, sweeping i o he ou le . In e - es ingly, LODs up o 10210 M o he baseline esol ed CBI– amino acid enan iome s we e eached by his way. This p e- concen a ion echnique was also applied o mo e complex mix u es o amino acids wi hou loss o esolu ion, as i can be seen in Fig. 3B. Ano he example o sweeping is he sen- Figu e 3. (A) Schema ic diag am o he s acking/sweeping: (i) hyd odynamic injec ion o la ge olume (1/3 o he capilla y) o he CBI–amino acids in wa e a pH 6.0; (ii) mig a ion owa d he pH junc ion a he ou le side o he injec ion plug o he anionic CBI–amino acids; (iii) pumping wa e ou o he capilla y and mo emen o he s acked analy e band owa d he inle by he EOF; (i ) sweeping o he HS-b-CD h ough he s acked band o analy e. (B) Elec ophe og am showing he po en ial o s acking/ sweeping-EKC combina ion o enan iosepa a ion o a complex sample o CBI–amino acids (,0.5 mM each). Elec opho e ic con- di ions: used-silica capilla y, 70 cm o al leng h (45 cm de ec o leng h) and 25 mm id; sepa a ion bu e , 25 mM phospha e bu e (pH 2.0) con aining 2% HS-b-CD; applied ol age, 230 kV; hyd o- dynamic injec ion, 380 mba o 180 s. LIF de ec ion wi h lexc a 420 nm. Peak iden i ica ion: 1, CBI-D-a ginine (A g); 2, CBI-L-A g; 3, CBI-D-his idine (His); 4, CBI-L-His; 5, CBI-glycine (Gly); 6, CBI-L- y osine (Ty ); 7, CBI-L-glu amine (Glu); 8, CBI-D-Se ; 9, CBI-L-Se ; 10, CBI-L-Glu; 11, CBI-D-Glu (adap ed om e . [28] wi h pe mis- sion). si i e de e mina ion o lo azepam in human u ine, a d ug used o he ea men o anxie y and wi h seda i e and hyp- no ic p ope ies. In his wo k, he sweeping o he sample was achie ed wi h a bu e con aining SDS micelles and a ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2008, 29, 237–251 CE and CEC 243 neu al CD as chi al selec o [12]. Sweeping was pe - o med by p epa ing he sample in a ma ix wi hou micelles bu o he wise simila o he unning bu e , and p olonging he sample zone by applying longe injec ion imes. In his way, when ol age was applied, micelles sweep he analy e in a na ow band p oducing a sample p econcen a ion. Table 2 also includes wo wo ks whe e a single-s ep me hod ha combines in-capilla y SPCD was de eloped o he enan ioselec i e de e mina ion o amino acids [29] and he bac e ial biobac e s mu amic acid and diaminopimelic acid [30] in Esche ichia coli bac e ial cul u e using CE wi h UV de ec ion. The e ec i e sepa a ion o eigh amino acid enan io- me s using in-capilla y de i a iza ion wi h o hoph halalde- hyde (OPA)/N-ace yl L-cys eine (NAC) was pe o med [29]. In compa ison wi h con en ional CE, SPCD p o ided a 40- old imp o emen in concen a ion sensi i i y and pe - mi ed sho e o al analysis imes main aining chi al eso- lu ion due o he lowe a e age mobili y o zwi e ionic amino acids in he weakly acidic sample plug. Figu e 4A shows he gene al p inciple o SPCD in CE o he sepa a- ion o he enan iome s o he wo amino acids, alanine (Ala) and glu amic acid (Glu). B ie ly, he injec ion sequence was ca ied ou by i s insing he capilla y wi h bo a e bu e ha se ed as an op imal alkaline medium o analy es and OPA and NAC as de i a izing agen s. A mul- iple sample hyd odynamic injec ion sequence was pe - o med consis ing o sho concen a ed plugs o NAC and OPA eagen s posi ioned in be ween a long plug o dilu e sample. OPA was injec ed a he back end o he sample plug because i is neu al and comig a es wi h he EOF, unlike he anionic chi al NAC co eagen ha mig a es wi h a slowe appa en mobili y. The sample was injec ed wi h low p essu e dissol ed in phospha e bu e o educe he local elec opho e ic mobili y o weakly ionic me aboli es in he sample o induce elec okine ic ocusing. Consecu i ely, OPA and NAC zones comig a ed and p econcen a ion sample zones esul ed in he o ma ion o dias e eome ic isoindole adduc s. A e wa d, enan iome ic esolu ion o he adduc s was achie ed along wi h inc easing band sepa a ion o all species. Al hough he sepa a ion o he dias e eome s o he amino acids was possible wi hou chi al addi i es (Fig. 4B), he addi ion o 1 mM b-CD allowed he esolu ion o he las amino acid wi hou de e io ing sample en ich- men p ope ies, bu dec easing esolu ion o he wo i s mig a ing amino acids (Fig. 4C). The SPCD me hod de el- oped in eg a ed sample en ichmen wi h chemical labeling s eps di ec ly wi hin a single capilla y du ing elec omig a- ion imp o ing he concen a ion sensi i i y bu e aining high- esolu ion chi al sepa a ions. LODs o 261027M o mu amic acid, and 261026M o diaminopimelic acid, we e achie ed by his me hod. Ne e heless, in his case, he use o inc easing concen a ions o b-CD as a neu al chi al selec o o he un bu e was unsuccess ul o u he imp o ing o diaminopimelic acid esolu ion [30]. Figu e 4. (A) Gene al p inciple o SPCD–CE o single-s ep enan- ioselec i e analysis o submic omola le els o amino acids: (i) mul iple hyd odynamic injec ion sequence; (ii) on-line sample p econcen a ion; (iii) in-capilla y chemical labeling by zone pas- sing o OPA/NAC; (i ) chi al sepa a ion o dias e eome ic amino acid adduc s o med. (B) Elec ophe og am o eigh amino acid enan iome s using in-capilla y OPA/NAC de i a iza ion o amino acid adduc s by SPCD–CE. (C) Elec ophe og am demons a ing he use o 1 mM b-CD as a chi al addi i e o enhance he enan- ioselec i i y by dynamic inclusion complexa ion o amino acid adduc s by SPCD–CE. Elec opho e ic condi ions: used-silica capilla y, 65 cm o al leng h and 50 mm id; sepa a ion bu e , 140 mM bo a e bu e (pH 9.5) con aining 1 mM b-CD o Fig. (C); sepa a ion empe a u e, 257C; applied ol age, 25 kV; hyd o- dynamic injec ion, 35 mba 6100 s; UV de ec ion a 340 nm. Sample solu ions con ained 25 and 50 mMo heD-andL-amino acids, espec i ely. Analy e peak numbe s co espond o amino acid–isoindole adduc s: 1a, D-Se ; 1b, L-Se ; 2a, D-Ala; 2b, L-Ala; 3a, D-Glu; 3b, L-Glu; 4a, D-Asp; 4b, L-Asp; *, OPA hyd olysis p od- uc s (adap ed om e . [29] wi h pe mission). ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 244 L. Sánchez-He nández e al. Elec opho esis 2008, 29, 237–251 2.3 Al e na i e de ec ion sys ems o on-column UV–Vis abso p ion de ec ion o he enhancemen o he sensi i i y in chi al analysis by CE UV–Vis abso bance de ec ion is he i s op ion o be con- side ed in CE gi en i s in e es ing ea u es such as comme - cial a ailabili y, simplici y, e sa ili y, ela i ely low cos , and equen use as uni e sal de ec ion echnique because many o ganics can be de ec ed a 195–210 nm. This de ec o p o- duces LODs in CE co esponding o a ew em omoles o analy e (a subpicomole le els), i.e., high mass sensi i i y. Howe e , due o he need o small olumes employed in CE o a oid peak b oadening ha dec ease he e iciency o he sepa a ion (pL o nL olumes), such sensi i i y is in he mic omola ange, i.e., appea s modes when exp essed in e ms o concen a ion (LODs anging om 1025 o 1027M depending upon he analy e being analyzed). These LODs a e clea ly insu icien o sol e many analy ical p oblems. One op ion o o e come he poo concen a ion sensi i i y ob ained in CE is he selec ion o low-UV wa eleng hs (190– 205 nm), whe e his de ec ion exhibi s he bes sensi i i y, possibili y used in se e al wo ks summa ized in Table 1. Ano he op ion o imp o e de ec ion sensi i i y in CE is he use o al e na i e de ec ion sys ems. Du ing he pe iod o ime e iewed in his a icle (see Table 3), LIF and MS de ec- ion sys ems we e mainly employed. LIF sys em is con ig- u ed o make he de ec ion di ec ly on he sepa a ion capil- la y (on-column de ec ion), such as UV–Vis abso p ion de ec ion, bu he second sys em is connec ed o he end o he capilla y (end-column de ec ion). In addi ion, elec o- chemiluminescence (ECL) was also employed. 2.3.1 On-column de ec ion in chi al analysis by CE LIF de ec ion is one o he mos sensi i e on-column de ec- ion sys ems cu en ly a ailable in which i is possible o use se e al lase s, he common a gon lase (458 and 488 nm), he He-Cd lase (440 nm), o he blue diode lase (420 nm). Since only a ew chi al compounds possess na i e luo es- cence, de i a iza ion p ocedu es a e usually equi ed o de ec o he non luo escen analy es. LIF was used o he chi al de e mina ion o pho osensi ize s [32], d ugs in hu- man plasma [33], and an icance agen s in d ugs [34] achie - ing LODs a ound 1028M. Howe e , he main applica ion o his de ec ion sys em in CE was he analysis o amino acid enan iome s in s anda ds [35], oods [36], and biological samples [28, 37–42]. In all cases, he me hods in ol ed p e- capilla y chi al de i a iza ion o he amino acids wi h di e - en de i a izing eagen s, FITC [35, 36], 4- luo o-7-ni o- 2,1,3-benzoxadiazole (NBD-F) [37], CBI [28, 38–41], o luo- escamin (FA) [42]. Chi al and sensi i e analysis o amino acids is a ema k- able me hodology ha can p o ide impo an in o ma ion on adul e a ion and quali y o ood p oduc s [49]. Fo example, analysis o chi al amino acids in inega s was shown o be a powe ul me hod o de ec di e en adul e a ions including he de ec ion o syn he ic inega s [50]. Thus, se e al D-amino acids we e de ec ed and quan i ied in inega s by MEKC-LIF, obse ing in e es ing di e ences in hei L- and D-amino acid p o iles and con en s [36]. LODs lowe han ,20 nM we e achie ed using FITC as de i a izing eagen . This luo escen label has been equen ly chosen since i s exci a ion wa eleng h ma ches he 488 nm ligh o he a gon lase , and he de i a i es a e easily o med and gene a e s ong luo escence signals [51]. Se e al imp o ed me hods we e de eloped o he sensi- i e de e mina ion o D-se ine (Se ) in neu al samples [28, 37–39]. This molecule is a p ima y endogenous amino acid ha binds o he glycine si e o N-me hyl-D-aspa a e (NMDA) ecep o in ol ed in a a ie y o physiological unc- ions and diso de s including memo y, lea ning, pain, and ischemia. The sensi i e de ec ion o his amino acid is essen ial, because i is a low concen a ion in biological samples. Thus, wo di e en EKC me hods wi h LIF de ec- ion o he chi al sepa a ion o D/L-Se we e de eloped and applied o de ec D-Se in highe e eb a es, such as a s, achie ing LODs abou 1027M [37, 38]. In one o he me h- ods, a saccha ide [D-(1)-glucose] was added o he sepa a ion bu e in o de o enhance he chi al ecogni ion o 2-hy- d oxyp opyl-g-CD (HP-g-CD) [38]. On he o he hand, a p o- cedu e o he de e mina ion o D-Se in squi el b ain was p oposed o achie e a e y high sensi i i y (LODs up o 10210 M) [28]. In his case, he p ocess in ol ed wo p e- concen a ion echniques (s acking and sweeping), as has p e iously been commen ed. By his me hod, in addi ion o L-Se , app eciable le els o D-Se , L-aspa a e (Asp), and L-glu- ama e (Glu) we e obse ed in mic odialysa e om he hip- pocampus o a ic g ound squi els, while D-Asp and D-Glu we e below he LOD. The enan iosepa a ions we e accom- plished wi h HS-b-CD as chi al selec o a low pH and e e se pola i y. D-Se was also de ec ed in in e eb a es [38, 39]. Thus, Zhao e al. [39] s udied he con en s o D-Se in Aplysia cali- o nica, a sea mollusc widely used as neu onal model. The sepa a ion o CBI-D/L-Se enan iome was achie ed by using a dual chi al selec o sys em consis ing o b-CD and chi al micelles o med by DOC. Fo he i s ime, peaks co e- sponding o L-Se and D-Se we e well iden i ied in Aplysia ganglian homogena es. I was no iced ha while he le els o L-Se we e simila , D-Se le els a ied subs an ially om ani- mal o animal. In e es ingly, D-Se was no de ec ed in single neu ons isola ed om Aplysia ganglia due o D-Se migh pe haps no occu in neu ons o Aplysia o he con en s in single neu ons we e oo low o be de ec ed. This lack o sen- si i i y was epo ed also in o he wo k o he same au ho s [38]. D-Asp was also de ec ed in he cen al ne ous sys em o A. cali o nica [40, 41]. The e a e some indica ions ha D-Asp is a neu omodula o , ho mone, o e en a p ecu so o he endogenous syn hesis o he NMDA ecep o . Miao e al. [40] demons a ed an app oach o he quan i a i e in es iga ion ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2008, 29, 237–251 CE and CEC 245 Table 3. Al e na i e de ec ion sys ems o on-column UV–Vis abso p ion de ec ion employed o sensi i e chi al analysis by CE De ec ion Sys em CE Mode Analy e and sample Sepa a ion bu e LOD (M) Re . LIF (lexc = 422/488 nm, lem = 690 nm) CD-MEKC Po phy in and ph halocyanines in s anda ds 200 mM bo a e (pH 9.2) 1 10 mM HP-b-CD 110 mM SDS ,361028[32] LIF (lexc = 442 nm, lem = 500 nm) CD-EKC CBI-Baclo en in human plasma 50 mM bo a e (pH 9.5) 12% HS-b-CD 561028[33] LIF (lexc = 320 nm, lem = 380–600 nm) CD-EKC Homocamp o hecin de i a i es in d ugs 75/25 mM phospha e (pH 2.5) 17.5% w/ HS-b-CD/2.5% w/ HS-b-CD ,1028[34] LIF (lexc = 488 nm, lem = 520 nm) EKC FITC-amino acids (Glu, P o) in s anda ds 50 mM ace a e (pH 6.1) 1 1.25 mM ancomycin ,1025[35] LIF (lexc = 488 nm, lem = 520 nm) CD-MEKC FITC-amino acids (A g, P o, GABA, Ala, Glu, Asp) in inega s 100 mM bo a e (pH 9.7) 1 20 mM b-CD 130 mM SDS ,261028[36] LIF (lexc = 457.9 nm) CD-EKC NBD-F-D-Se in a b ain 100 mM bo a e (pH 10.0) 1 40 mM HP-b-CD 361027[37] LIF (lexc = 457.9 nm) CD-EKC CBI-D-Se in a b ain and mollusc neu ons 60 mM bo a e (pH 10.0) 12M u ea 115% D-glucose 1 20 mM HP-g-CD 1027[38] LIF (lexc = 420 nm) CD-EKC CBI-Se , CBI-Glu in hippocampus o squi el 25 mM phospha e (pH 2.0) 12% HS-b-CD ,10210 [28] LIF (lexc = 457.9 nm) CD-MEKC CBI-D-Se in mollusc neu ons 100 mM bo a e (pH 9.5) 1 30 mM b-CD 160 mM DOC 361028[39] LIF (lexc = 457.9 nm) CD-MEKC CBI-D-Asp in mollusc neu ons 50 mM bo a e (pH 9.4) 1 20 mM b-CD 150 mM SDS 5610210 [40] LIF (lexc = 457.9 nm) CD-MEKC CBI-D-Asp in mollusc neu ons 50 mM bo a e (pH 9.4) 1 20 mM b-CD 150 mM SDS ,561026[41] LIF (lexc = 457.9/488 nm) CD-MEKC FA-D-amino acid-con aining neu opep ides in mollusc neu ons 50 mM bo a e (pH 9.4) 1 20 mM g-CD 150 mM SDS 861028[42] Fluo escence (lexc = 327 nm, lem = 557 nm) CD-EKC DNS-DL-Glu osina e in spiked i e wa e 2.0 mM phospha e (pH 6.5) 117 mM g-CD 261029[16] MS CD-EKC Amphe amine de i a i es (A, MA, MDA, MDMA, MDEA, E, NE) in plasma 20 mM ammonium o ma e (pH 2.5) 10.15% HS-g-CD ,761027– 361026 [21] MS CD-EKC Amphe amine de i a es (A, MA, MDA, MDMA, MDEA, TMD, MTD) in plasma 20 mM ammonium o ma e (pH 2.5) 10.15% HS-g-CD 461029[22] MS CD-EKC MA, AP, DMA, E, NE, ME in human u ine 1 M o mic acid (pH 1.7) 10.85 mM DAS-b-CD ,661028– 1027 [43] MS MEKC b-Blocke s (A e, Me , Pin, Oxp, Alp, P o, Ca , Tal) in s anda ds 25 mM ammonium ace a e 125 mM TEA (pH 8.0) 115 mM poly-L-SUCL 961027– 761026 [44] MS MEKC Lo azepam, oxazepam, and ne opam in s anda ds 25 mM ammonium ace a e (pH 8.0) 115% ACN 115 mM poly-L-SUL ,661026[45] Lo azepam and oxazepam in s anda ds 25 mM ammonium ace a e (pH 8.5) 115 mM poly-LL- SUCLV ©2008 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com