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
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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
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