J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Pawel L. U ban
Ca men Ga ca-Ruiz
Mangeles Ga ca
Ma. Luisa Ma ina
Depa amen o de Qumica
Anal ica, Facul ad de Qumica,
Uni e sidad de Alcal, Alcal de
Hena es, Mad id, Spain
Sepa a ion and online p econcen a ion by
mul is ep s acking wi h la ge- olume injec ion o
anabolic s e oids by capilla y elec okine ic
ch oma og aphy using cha ged cyclodex ins and
UV-abso p ion de ec ion
The sepa a ion o h ee common anabolic s e oids (me hyl es os e one, me hand o-
s enolone and es os e one) was pe o med o he i s ime by capilla y EKC. Di e -
en cha ged CD de i a i es and bile sal s we e es ed as dispe sed phases in o de o
achie e he sepa a ion. A mix u e o 10 mmol/L succinyla ed-b-CD wi h 1 mmol/L b-
CD in a 50 mmol/L bo a e bu e (pH 9) enabled he sepa a ion o he h ee anabolic
s e oids in less han 9 min. Concen a ion LODs, ob ained o hese compounds wi h
low abso p ion o UV ligh , we e ~5610–5 mol/L. The use o online e e se mig a ing
sample s acking wi h la ge- olume injec ion ( he e ec i e leng h o he capilla y) ena-
bled o imp o e he de ec ion sensi i i y. Sensi i i y enhancemen ac o s (SEFs) an-
ging om 95 ( o es os e one) o 149 ( o me hyl es os e one) we e achie ed by sin-
gle s acking p econcen a ion. Then, he possibili ies o mul is ep s acking o imp o e
he sensi i i y o hese analy es we e in es iga ed. SEFs ob ained by double s acking
p econcen a ion anged om 138 o 185, enabling concen a ion LODs o 2.79610–7
mol/L ( o me hyl es os e one), 3.47610–7 mol/L ( o es os e one) and 3.56610–7
mol/L ( o me hand os enolone). Al hough online iple s acking p econcen a ion was
achie ed, i s epea abili y was e y poo and SEFs o he s udied analy es we e no
calcula ed.
Key Wo ds: Anabolic s e oids; Capilla y elec okine ic ch oma og aphy; Online mul is ep s acking;
Recei ed: Ma ch 10, 2005; e ised: Ap il 13, 2005; accep ed:May 11, 2005
DOI 10.1002/jssc.200500113
2200 U ban, Ga ca-Ruiz, Ga ca, Ma ina
1 In oduc ion
Al hough CE shows impo an ad an ages (small quan i y
o injec ed sample, high speed and esolu ion and low
expendi u e o chemicals) o he analysis o many com-
pounds in a g ea a ie y o samples [1], he concen a ion
LODs ob ained wi h UV-abso p ion de ec ion a e s ill, in
many cases, unsa is ac o y. They can be imp o ed by
hyphena ion o CE wi h mo e sensi i e de ec o s, such as
LIF [2] o elec ochemical de ec ion [3]. Howe e , he
cheapes and he mos popula one is he UV-abso p ion
de ec o . Al hough he sho diame e o he capilla y (25–
100 lm) does no allow o measu e abso bance o some
compounds which do no possess good ch omopho es,
he use o special de ec ion windows (e.g. bubble cells,
ze a cells) [4, 5] may sligh ly imp o e sensi i i y wi h op i-
cal de ec ion. In addi ion, CE o e s a g ea possibili y o
online sample p econcen a ion and enables he au oma-
isa ion o he p ocess, which always is desi able in analy-
ical chemis y, as well as in o he ields.
Online sample concen a ion ep esen s an e ec i e and
e sa ile way o enhance concen a ion sensi i i y in CE
[6]. The high elec ic ield and unable elec opho e ic
mobili y o he analy es can be used o induce elec oki-
ne ic ocussing wi hin la ge-injec ion olumes o sample
di ec ly on-capilla y p io o de ec ion. Online ocussing is
no mally pe o med by selec ing di e en bu e p ope ies
o modi y analy e eloci y in wo o mo e sec ions in he
capilla y, such as sample and BGE.
The e a e ou majo modes o online sample concen a-
ion in CE [6]: sample s acking, ansien ITP, sweeping
and dynamic pH junc ion. Each me hod elies on a dis inc
Co espondence: P o esso Ma. Luisa Ma ina, Depa amen o
de Qumica Anal ica, Facul ad de Qumica, Uni e sidad de Al-
cal, C a. Mad id Ba celona Km. 33.600, 28871 Alcal de He-
na es, Mad id, Spain. Fax: +34-91-8854971.
E-mail: [email p o ec ed].
Abb e ia ions: CE-b-CD (DS ~ 3), ca boxye hyla ed-b-CD (de-
g ee o subs i u ion ~ 3); CM-b-CD, ca boxyme hyla ed-b-CD;
MTD,17b-hyd oxy-17a-me hyl-and os a-1,4-dien-3-one (me-
hand os enolone); MTS,17b-hyd oxy-17a-me hyl-4-and os en-
3-one (me hyl es os e one); SEFs, sensi i i y enhancemen ac-
o s; Suc-b-CD, succinyla ed-b-CD; TST,17b-hyd oxy-4-and os-
en-3-one ( es os e one)
EKC Sepa a ion/on-line p econcen a ion by mul i-s ep s acking o s e oids 2201
ocussing mechanism based on di e en elec oly e p op-
e ies be ween sample and BGE zones, such as conduc-
i i y (ionic s eng h), elec oly e coion mobili y, addi i e
concen a ion (analy e–addi i e in e ac ions) and bu e
pH, espec i ely. Applica ion o one o hese me hods e-
quen ly o e s good sensi i i y enhancemen , a imes,
wi hin se e al o de s o magni ude.
Sample s acking (in no mal and e e sed pola i y modes,
wi h e e se mig a ing micelles wi hou and wi h wa e
plug, and head column ield-enhanced sample injec ion
wi hou and wi h e e se mig a ing micelles) occu s as
ions ac oss a bounda y ha sepa a es egions o he high
elec ic ield sample zone and he low elec ic ield back-
g ound solu ion zone [7].
Some imes sensi i i y enhancemen ob ained wi h single
s acking p ocedu e is limi ed and he e is a need o ob ain
lowe concen a ion LODs. The e a e some s udies
desc ibing success ul applica ion o double s acking p o-
cedu e [8–11], showing ha mul is ep s acking can be an
in e es ing al e na i e o be used as online p econcen a-
ion me hod. Hence, he possibili ies o mul is ep s acking
and he lexibili y o his online p econcen a ion me hod o
adjus he desi ed concen a ion LODs, depending on he
numbe o s acking s eps and making a comp omise
be ween eco e y and pe o mance ( esolu ion and anal-
ysis ime), should be in es iga ed.
Me hyl es os e one, me hand os enolone and es os e -
one a e popula s e oids (see Fig. 1), audly used in
spo s. These anabolic s e oids a e also used as medi-
cines in he ea men o anaemia, enal insu iciency,
endome iosis, he edi a y angioedema and inope able
b eas cance [12], which e eals a huge necessi y o
de elop new analy ical me hods o hei quan i ica ion.
Nowadays, hese anabolic s e oids a e mainly analysed
by HPLC [13, 14] o GC [14, 15]. Wi h GC-MS, e y low
LODs can be achie ed. Howe e , since some anabolic
s e oids and hei me aboli es possess hyd oxyl and ca -
bonyl g oups in hei s uc u e, de i a isa ion is neces-
sa y, as in gene al sample p e ea men is labou ious and
ime-consuming [16]. HPLC and CE a e highly p omising
me hods o as s e oid sc eening, CE being much less
employed han HPLC [17, 18]. CE has been employed o
he analysis o di e en s e oids gene ally wi h MS, luo -
escence o UV de ec ion [14, 19–21]. Howe e , he in e -
es in using he mos widely employed de ec ion sys em in
CE (UV abso bance de ec ion) o he analysis o s e oids
has p omo ed he use o p econcen a ion echniques p e-
ious o hei analysis due o he ela i ely low expec ed
sensi i i y (s e oids show low abso p ion in he UV-Vis
egion). In ac , o line p econcen a ion echniques as
SPE [22, 23] and online p econcen a ion echniques [6,
24–27] ha e been employed. In spi e o he esul s
ob ained o he analysis o s e oids by CE, sepa a ion o
any h ee anabolic s e oids s udied in his wo k has p e-
iously been epo ed. Only he sepa a ion o es os e one
and/o me hyl es os e one om o he s e oids has been
epo ed [6, 16, 24, 28–30]. Due o he neu al na u e o
hese analy es hey need o be sepa a ed by capilla y
EKC, which is based on he di e en dis ibu ion o ana-
ly es be ween a dispe sed phase (CDs, micelles, e c.) and
he mobile phase (bu e solu ion).
The aim o his wo k was o op imize he expe imen al
condi ions enabling he sepa a ion o he h ee abo e-
men ioned anabolic s e oids by EKC, and o de elop a
me hod o hei online p econcen a ion in EKC in o de
o achie e a sensi i e UV-abso p ion de ec ion o hese
low abso ben analy es, his la e pu pose in ol ing he
s udy o he possibili ies o mul is ep s acking.
2 Ma e ials and me hods
2.1 Chemicals and samples
The s anda ds o 17b-hyd oxy-17a-me hyl-4-and os en-
3-one (me hyl es os e one, MTS), 17b-hyd oxy-17a-
me hyl-and os a-1,4-dien-3-one (me hand os enolone,
J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Figu e 1. S uc u es and names o he h ee anabolic s e -
oids s udied in his wo k.
O iginal Pape
2202 U ban, Ga ca-Ruiz, Ga ca, Ma ina
MTD) and 17b-hyd oxy-4-and os en-3-one ( es os e one,
TST) we e pu chased om Fluka (Buchs, Swi ze land)
and s o ed a 48C. b-CD and succinyla ed-b-CD (Suc-b-
CD (deg ee o subs i u ion (DS) ~3)) we e also supplied
by Fluka. Ca boxyme hyla ed-b-CD (CM-b-CD (DS ~3))
and ca boxye hyla ed-b-CD (CE-b-CD (DS ~3)) we e
om Cyclolab (Budapes , Hunga y). Sodium bile sal s o
cholic acid, au ocholic acid and au odeoxycholic acid
we e pu chased om Sigma (S . Louis, MO, USA) and
ha o deoxycholic acid om Fluka. Bo ic acid (Fluka),
sodium dihyd ogen phospha e (Me ck, Da ms ad , Ge -
many) and sodium hyd oxide (Pan eac, Ba celona,
Spain) we e used o p epa e he bu e s. HPLC-g adien
g ade me hanol was om Pan eac. Dis illed wa e used
h oughou his wo k was also pu i ied wi h he Milli-Q sys-
em (Millipo e, Bed o d, USA).
The 50 mmol/L bo a e bu e (pH 9) was p epa ed by dis-
sol ing he app op ia e amoun o bo ic acid in wa e and
by adding 1 mol/L sodium hyd oxide o he esul ing solu-
ion in o de o ob ain he desi ed pH alue. Then, he sui-
able amoun s o CDs we e dissol ed in he bu e solu-
ion. The s anda ds o he s udied compounds we e dis-
sol ed in me hanol, and hen dilu ed in wa e o ob ain he
desi ed concen a ion.
Dissolu ion o solids was acili a ed by he use o an ul a-
sonic ba h (Raypa, Ba celona, Spain). The pH o each
bu e was adjus ed using a 654 pH me e (Me ohm, He -
isau, Swi ze land). Bo h, samples and bu e s, we e il-
e ed p io o he analysis using sy inge il e s (Ti an Fil a-
ion Sys ems; nylon, po e size 0.45 lm, il e size 13 mm).
2.2 Ins umen a ion
A CE ins umen (Agilen Technologies, Waldb onn, Ge -
many) equipped wi h an oncolumn diode a ay de ec o
was used. The con ol o he ins umen and da a acquisi-
ion we e achie ed by a PC wi h he 3D-CE ChemS a ion
so wa e (A.09.03, Agilen ).
Uncoa ed used-silica capilla y was employed: e ec i e
capilla y leng h, 50 cm; o al capilla y leng h, 58.5 cm; ID,
75 lm; OD, 375 lm (Sugelabo S.A., Mad id, Spain). The
sample and ope a ing ials we e se ed om a 48- ial
ca ousel, designed o ypical 1 mL snap polyp opylene
ials.
2.3 Analysis p ocedu e
Expe imen s wi hou s acking we e pe o med by injec ing
he sample (50 mba o 3 s) ollowed by he sepa a ion
bu e (50 mba o 3 s). The applied ope a ion scheme
used o achie e single, double and iple online s acking is
shown in Table 1. The i s s age o each un is capilla y
condi ioning wi h 0.1 mol/L NaOH (1 ba o 5 min), Milli-Q
wa e (1 ba o 2 min) and unning bu e (1 ba o 5 min).
Then, he sample was injec ed o ill 50 cm o he capilla y
leng h ( om he inle o he de ec ion window). The injec-
ion ime was calcula ed om he ea anged Poiseuille’s
equa ion:
¼3200 Ll
d2Pð1Þ
whe e Pis he injec ion p essu e (mba ), he ime du a-
ion o he p essu e (s), d he ID o he capilla y (lm), l he
J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Table 1. Analysis s eps in he online mul is ep s acking and sepa a ion o MTS, MTD and TST. Running bu e : 10 mmol/L Suc-
b-CD, 1 mmol/L b-CD, 50 mmol/L bo a e bu e a pH 9. Du ing sample concen a ion, –20 kV indica es nega i e pola i y and s1,
s2 and s3 a e imes o he successi e concen a ion s eps (176, 209 and 211 s). Used ials: S, sample; B1–B5, unning bu e ; N,
0.1 mol/L NaOH; Q, Milli-Q wa e and W1, W2, was e
S eps Inle Ou le Single s acking Double s acking T iple s acking
P econdi ioning N
Q
B1
W1
W1
W2
5 min 0.1 mol/L NaOH
2 min wa e
5 min unning bu e
(1 ba )
5 min 0.1 mol/L NaOH
2 min wa e
5 min unning bu e
(1 ba )
5 min 0.1 mol/L NaOH
2 min wa e
5 min unning bu e
(1 ba )
Sample injec ion S B3 50 mba , 332 s
(50 cm o capilla y) 50 mba , 332 s
(50 cm o capilla y) 50 mba , 332 s
(50 cm o capilla y)
Bu e injec ion B2 B3 50 mba , 5 s 50 mba , 5 s 50 mba , 5 s
Zone concen a ion B2 B3 –20 kV,
s1
–20 kV,
s1
–20 kV,
s1
Sample injec ion S B3 – 50 mba , 332 s 50 mba , 332 s
Bu e injec ion B2 B3 – 50 mba , 5 s 50 mba , 5 s
Zone concen a ion B2 B3 – –20 kV,
s2
–20 kV,
s2
Sample injec ion S B3 – – 50 mba , 332 s
Bu e injec ion B2 B3 – – 50 mba , 5 s
Zone concen a ion B2 B3 – – –20 kV,
s3
Sepa a ion B4 B5 20 kV 20 kV 20 kV
EKC Sepa a ion/on-line p econcen a ion by mul i-s ep s acking o s e oids 2203
leng h o he sample injec ion plug (mm), g he iscosi y o
he bu e (cP) and L he capilla y leng h (cm). The injec-
ion ime necessa y o ill he e ec i e leng h o he capil-
la y (50 cm) calcula ed by means o Eq. (1) was 332 s.
This calcula ed ime was e y simila o he expe imen al
ime es ima ed by illing he capilla y wi h a solu ion o a
mix u e o he s e oids (~3610–4 mol/L), a e illing he
capilla y wi h bu e solu ion, and egis e ing he signal a
245 nm which inc eased when he s e oids achie ed he
de ec ion window. A e sample injec ion, a sho zone o
he unning bu e was injec ed o p e en loss o he ana-
ly e. Then, e e se pola i y ol age (20 kV) was applied.
These h ee s eps we e applied once, wice o h ice,
depending on he numbe o s acking s eps. Sepa a ion o
he mix u e was achie ed in he las s acking s ep, when
no mal (posi i e) pola i y ol age (20 kV) was applied.
Be o e he i s use, he capilla y was condi ioned by
washing i wi h 1 mol/L NaOH (1 ba o abou 1 h) and
hen wi h Milli-Q wa e (1 ba o 10 min). Du ing he whole
analysis, a ious ials we e used (see Table 1). Di e en
bu e ials we e employed o s acking and di e en o
sepa a ion (see Table 1) so as o a oid con amina ion o
he sepa a ion bu e which could inc ease he back-
g ound noise. The bu e ial B1 (used o capilla y p e-
condi ioning) was o 2 mL olume since his ial does no
equi e equen eplacemen . Two-millili e ials we e
also employed o 0.1 mol/L NaOH (N) and wa e s o e
(Q) and o he was e deposi ion (W1, W2). One-millili e
s anda d plas ic ials we e used o he samples/s an-
da ds and bu e solu ions. A empe a u e o 158C was
used in his wo k. Mos o he ope a ion s eps we e p o-
g ammed using he p econdi ioning able in he ChemS a-
ion so wa e. Howe e , he “CE TimeTable” was used o
he acquisi ion o he cu en in ensi y cu es du ing he
s acking p econcen a ion.
Since he s udied compounds sligh ly di e in hei UV-
abso p ion maxima, a single wa eleng h was chosen o
de ec all o hem (245 nm € 5 nm), and he e e ence
alue was swi ched o since i did no gi e any imp o e-
men .
2.4 Da a ea men
The cu es co esponding o he a ia ion o he cu en
in ensi y as a unc ion o he ime o applica ion o e e se
pola i y we e ob ained o mix u es o he h ee s e oids in
which each componen was a concen a ions anging
om 5610–7 o 1610–5 mol/L. Then, he 4 h o de polyno-
mial cu es (ax4+bx3+cx2+dx +e) we e adjus ed and
he in lexion poin x- alue was calcula ed making he 2nd
o de de i a i e om he 4 h o de i ed polynomial cu e
(12ax2+6bx +2c) equal o 0. One o he wo solu ions o
he 2nd o de de i a i e was conside ed and he ollowing
equa ion was used:
x¼6bþ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
36b296ac
p
24að2Þ
whe e a,band ca e he coe icien s a x4,x3and x2in he
4 h o de i ed polynomial cu e.
LODs and LOQs in concen a ion (mol/L) o he s e oids in
a 1% / me hanol/wa e solu ion we e expe imen ally cal-
cula ed acco ding o he ollowing equa ions [31]:
LOD ¼3Sa0=b0ð3Þ
LOQ ¼10Sa0=b0ð4Þ
whe e Sa0is he s anda d e o o he in e cep and b9, he
slope o he s aigh line, bo h ob ained by ANOVA.
Expe imen al da a we e ea ed using O igin 7.0 (O igin-
Lab Co po a ion) and Excel 7.0 (Mic oso ) so wa e.
Theo e ical es ima ion o LOD wi h mul is ep s acking was
achie ed using Eq. (5):
LODn¼LOD0
n ð5Þ
whe e LOD0is he LOD ob ained wi hou s acking, n he
numbe o s acking s eps (1, 2, 3…) and he s acking
ampli ica ion ac o exp essed as:
¼ s
0ð6Þ
whe e sis he sample injec ion ime in he single s acking
s ep and 0 he injec ion ime in he analysis wi hou s ack-
ing.
3 Resul s and discussion
3.1 Sepa a ion condi ions
The sepa a ion o a mix u e o neu al MTS, MTD and TST
was achie ed a e a se o ials wi h a ious dispe sed
phases (CDs o bile sal s) added o bo a e bu e a pH 9,
which enabled he e e se mig a ing selec o s o pass
h ough he de ec ion window when no mal pola i y is
applied. Th ee anionic CDs (CM-b-CD, Suc-b-CD and
CE-b-CD) and ou bile sal s (sodium chola e, sodium
deoxychola e, sodium au ochola e and sodium au o-
deoxychola e) we e in es iga ed. Figu e 2 shows he elec-
ophe og ams ob ained o a mix u e o MTS, MTD and
TST a a concen a ion o ~3610–4 mol/L in each compo-
nen when adding CM-b-CD, Suc-b-CD o CE-b-CD o he
elec oly ic solu ion. I can be obse ed ha CM-b-CD and
Suc-b-CD a a 10 mmol/L concen a ion enabled he sep-
a a ion o he h ee neu al compounds (wi h CM-b-CD,
he esolu ion be ween he i s and he second mig a ing
peaks (Rs12) was 1.2 and he esolu ion be ween he sec-
J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
2204 U ban, Ga ca-Ruiz, Ga ca, Ma ina
ond and he hi d mig a ing peaks (Rs23) was 1.3, whe eas
wi h Suc-b-CD, Rs12 = 1.2 and Rs23 = 1.5). The anabolic
s e oids passed h ough he de ec o in he ollowing
o de : TST, MTS and MTD. Howe e , unde he expe i-
men al condi ions ied, CE-b-CD did no enable he sep-
a a ion o he mix u e (Rs12 =Rs23 = 0). On he o he
hand, om he ou bile sal s es ed, only sodium au o-
deoxychola e enabled he sepa a ion o he s udied com-
pounds (Rs12 = 1.9 and Rs23 = 2.0) gi ing ise o a di e en
mig a ion o de wi h espec o he use o CDs: TST, MTD
and MTS, as i is also shown in Fig. 2.
The e ec o using mix u es o dispe sed phases was
in es iga ed in o de o imp o e he sepa a ion selec i i y
ob ained unde he abo e condi ions. Fo his eason, a
second CD, he neu al b-CD, was added o he elec oly ic
solu ion. Figu e 3 shows ha he p esence o b-CD a a
1 mmol/L concen a ion signi ican ly imp o ed he sepa a-
ion o he h ee s e oids. The mechanism h ough which
he sepa a ion o he anabolic s e oids was imp o ed by
addi ion o a neu al CD is based on he simul aneous
in e ac ion o hese compounds wi h he anionic and he
neu al dispe sed phases. Thus, hese neu al compounds
acqui e mobili y due o hei in e ac ion wi h he anionic
dispe sed phase (anionic CD o bile sal ) which, unde he
no mal pola i y mode, mig a es owa d he anode. On he
o he hand, hei in e ac ion wi h he neu al CD, which
mig a es wi h he EOF due o i s elec oneu al na u e,
enables hei mig a ion owa ds he ca hode gi ing ise o
peaks loca ed in he elec ophe og am be ween he sig-
nals co esponding o he EOF and he anionic CDs. Since
he in e ac ion o each s e oid wi h he anionic and/o neu-
al dispe sed phase is di e en , he disc imina ion
inc eases wi h he addi ion o he neu al dispe sed phase
imp o ing hei sepa a ion. Thus, he addi ion o b-CD o
he CE-b-CD sys em enabled o obse e he sepa a ion o
he analy es (Rs12 = 1.3 and Rs23 = 1.0) while he addi ion
o b-CD o CM-b-CD, Suc-b-CD o he bile sal ga e ise o
an inc ease in he esolu ion. In ac , he use o 1 mmol/L
b-CD wi h 25 mmol/L au odeoxychola e enabled o ob ain
esolu ions o 2.5 and 2.0 o he i s –second and sec-
ond– hi d peaks, espec i ely, al hough he b oades
peaks o he analy es we e ob ained unde hese condi-
ions (see Fig. 3). Al hough he mix u e o 10 mmol/L CM-
b-CD wi h 1 mmol/L b-CD o e ed an excellen sepa a ion
o he analy es (Rs12 = 17.0 and Rs23 = 4.1), he mix u e o
10 mmol/L Suc-b-CD and 1 mmol/L b-CD (Rs12 = 7.8 and
Rs23 = 4.8) was chosen o u he wo k because i p o-
ided simila shape and esolu ion o he peaks o he
h ee analy es. Unde hese expe imen al condi ions, con-
cen a ion LODs o 4.79610–5 mol/L, 5.17610–5 mol/L
and 6.17610–5 mol/L we e ob ained o TST, MTS and
MTD espec i ely. In o de o imp o e he UV-abso p ion
de ec ion sensi i i y, ob ained o hese h ee s e oids,
online sample p econcen a ion by s acking wi h la ge-
olume injec ion was applied.
J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Figu e 2. Sepa a ion o a mix u e o MTS, MTD and TST (~3610–4 mol/L in each componen in 5% / me hanol/wa e ) wi h a
50 mmol/L bo a e bu e a pH 9 con aining an anionic subs i u ed b-CD o he bile sal au odeoxycholic acid. Fused-silica capil-
la y, 58.5 cm (50 cm o he de ec ion window), 75 lm ID (375 lm OD); ol age: 20 kV (cu en in ensi y o each sepa a ion bu e
is speci ied in he igu e in pa en hesis); injec ion: 50 mba , 3 s and bu e injec ion du ing 3 s; empe a u e: 158C; UV-abso p ion
de ec ion a 245 nm (€5 nm).

EKC Sepa a ion/on-line p econcen a ion by mul i-s ep s acking o s e oids 2205
3.2 Online p econcen a ion by single s acking
Since he selec ed sepa a ion bu e con ained anionic
CDs wi h e e se mig a ion in he no mal pola i y mode,
he ollowing s eps we e conside ed o pe o m a e e se
pola i y s acking mode (see Table 1): (i) sample injec ion
o ill he e ec i e leng h o he capilla y, (ii) injec ion o he
unning bu e o a oid di usion o he analy es in he ea ly
s acking s age, (iii) zone concen a ion applying a ime o
e e se pola i y ol age (–20 kV) and (i ) analy e sepa a-
ion wi h no mal pola i y ol age (20 kV).
The key pa ame e o he p econcen a ion p ocess was
obse ed o be he ime o applica ion o he e e se pola -
i y ol age (s acking ime). I could be ound in he li e a-
u e ha he s acking ime should be ha which allows
eaching om 70 o 99% o he cu en in ensi y o he bu -
e solu ion [32–36]. The ini ial c i e ion o choose he
s acking ime alue was o selec he ime co esponding
o he in lexion poin o he cu en in ensi y e sus ime o
applica ion o e e se pola i y cu e. This poin was calcu-
la ed a e i ing he expe imen al cu en in ensi y da a o
a 4 h o de polynomial cu e and making he second
de i a i e o he i ed unc ion equal o 0 (see Sec ion
2.4). Al hough i ing he expe imen al cu en in ensi y
da a o a 3 d o de polynomial was also achie ed, he i -
ing was much be e in he case o he 4 h o de unc ions
( >0.999) being addi ionally less dependen on he ange
o he expe imen al cu en in ensi y alues selec ed o be
adjus ed. The e o e, he 4 h o de polynomial was used
o u he op imisa ion o he s acking ime. In addi ion,
due o he s ong dependence o he in lexion poin o he
i ed cu e wi h he sample sol en , he use o wa e as
sample sol en was desi able du ing he s acking p econ-
cen a ion, as i is gene ally known. Howe e , due o he
insolubili y o he s udied s e oids in wa e , he use o 1%
me hanol was necessa y o solubilise hem be o e he
p econcen a ion s age. Unde all hese condi ions, a
s acking ime o 150 s was ob ained o a mix u e o MTS,
MTD and TST, 10–6 mol/L o each componen . Howe e ,
di e en imes (calcula ed as men ioned abo e) we e
ob ained o di e en concen a ions o he analy es
(155 s o 5610–7 mol/L, 150 s o 1610–6 mol/L and
136 s o 5610–6 mol/L). Then, in o de o apply he same
s acking ime o mix u es wi h di e en concen a ions o
analy es, he a e age o he in lexion poin imes ob ained
o mix u es o concen a ions anging om 5610–7 o
5610–6 mol/L, was aken (147 s). This ime co esponded
o app oxima ely 75% o he cu en in ensi y o he bu e
solu ion. The elec ophe og am co esponding o he
injec ion o a mix u e o he h ee anabolic s e oids using
his alue as s acking ime enabled o obse e ha when
his selec ed s acking ime was applied in a single s ack-
ing un, he analy es we e no sepa a ed and pa ially
o e lapped wi h he EOF peak ( esul s no shown). Sub-
sequen ly, he applica ion o highe s acking imes co e-
sponding o pe cen ages o cu en in ensi y highe han
75% was a emp ed. In o de o achie e ha he p e-
iously selec ed ime was mul iplied by he ac o s o 1.1
(~80% o he cu en in ensi y o he bu e solu ion), 1.2
(~90%), 1.3 (~95%) and 1.4 (~100%). In all hese cases,
he sepa a ion o he h ee peaks co esponding o TST,
MTS and MTD could be obse ed enabling o ob ain he
calib a ion plo s o he s udied analy es. Since a simila
beha iou was obse ed o he h ee analy es, as an
example, Fig. 4 shows he calib a ion plo s o TST ( he
1s peak in he elec ophe og am) when mix u es o he
J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Figu e 3. Sepa a ion o a mix-
u e o MTS, MTD and TST
(~3610–4 mol/L in each com-
ponen in 5% / me hanol/
wa e ) wi h a 50 mmol/L bo a e
bu e a pH 9 adding he na i e
b-CD o he anionic CDs o he
sodium au odeoxychola e.
Expe imen al condi ions as in
Fig. 1.
2206 U ban, Ga ca-Ruiz, Ga ca, Ma ina
h ee s e oids we e injec ed using, as s acking ime, he
abo e alue (147 s) mul iplied by he ac o s om 1.1 o
1.4 (s acking imes anged om 162 o 206 s). I can be
clea ly seen ha he ac o s 1.1 ( 2= 0.984), 1.2
( 2= 0.995) and 1.3 ( 2= 0.975) showed good linea i y
and simila sensi i i y (slope o he calib a ion lines),
which means ha he e is no loss o he analy es when
e e se pola i y is applied. Howe e , he use o he long-
es s acking ime (206 s) caused losses o he analy es by
he inle posi ion. Since he use o he sho es s acking
ime (162 s) p oduced b oad peaks, caused by a oo long
s acked zone, a ac o o 1.2 ( = 0.997) was chosen o
u he expe imen s because he esul an s acking ime,
176 s, p oduced he highes sensi i i y and we ensu ed
ha any pa o he analy e zone is no escaping om he
capilla y.
Using he selec ed s acking ime (176 s, i.e. he ime o
each ~90% cu en in ensi y o he bu e solu ion) o
online single s acking p econcen a ion, he LOD and
LOQ we e calcula ed om he calib a ion lines ob ained
plo ing co ec ed peak a eas (a ea/mig a ion ime) e sus
concen a ion ( i e alues anging om 5610–7 o 5610–6
mol/L). The co ec ion o he peak a eas was made o
compensa e he a ia ions o mig a ion ime o di e en
concen a ions o he analy es [37]. Table 2 shows he
sensi i i y enhancemen ac o s (SEFs) ob ained o he
h ee analy es s udied unde hese condi ions. The lowes
SEF was ob ained o he i s peak (TST, SEF 95)
whe eas he second and he hi d peaks exhibi ed highe
SEF alues (149 o MTS and 139 o MTD), which we e
highe han he heo e ical signal ampli ica ion alue cal-
cula ed om Eq. (6), which is 111. Howe e , he ob ained
SEF alues a e close o 170- old analy e concen a ion
achie ed by Chun and Chung [38] when illing he whole
capilla y wi h he sample (only he e ec i e leng h o he
capilla y was illed wi h he sample in his wo k).
3.3 Online p econcen a ion by mul is ep s acking
The heo e ical LODs es ima ed o TST, MTS and MTD,
when using mul is ep s acking p econcen a ion, a e
shown in Fig. 5. These alues we e ob ained om he
LODs o hese compounds wi hou p econcen a ion (see
Table 2) and he expec ed ampli ica ion ac o ( om Eq.
(6), 332/3 = 111). I can be obse ed ha he e is a signi i-
J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Figu e 4. Calib a ion cu es
ob ained plo ing he peak a ea
as a unc ion o TST concen a-
ion ( om 5610–7 o 5610–6
mol/L) o a ious ac o s ( om
1.1 o 1.4) employed o mul iply
he a e age in lexion poin o he
cu en in ensi y cu es. Vol age:
20 kV; injec ion: 50 mba , 332 s
and bu e injec ion du ing 5 s;
empe a u e: 158C; UV-abso p-
ion de ec ion a 245 nm
(€5 nm).
Table 2. LODs and LOQs exp essed in mol/L, and SEF o LODs when he sepa a ion o he h ee anabolic s e oids was pe -
o med wi hou s acking and wi h single and double s acking
TST MTS MTD
LOD LOQ SEF LOD LOQ SEF LOD LOQ SEF
Wi hou s acking
n=4
4.79610–5
(€1.12610–5)
1.60610–4
(€0.37610–4)
– 5.17610–5
(€1.51610–5)
1.72610–4
(€0.50610–4)
– 6.17610–5
(€1.94610–5)
2.06610–4
(€0.65610–4)
–
Single s acking
n=5
5.02610 –7
(€4.84610 –7
)
1.67610–6
(€1.62610 –6
)
95 3.47610–7
(€0.81610–7
)
1.16610 –6
(€0.27610 –6
)
149 4.42610–7
(€2.60610 –7
)
1.47610 –6
(€0.87610 –6
)
139
Double s acking
n=5
3.47610 –7
(€1.53610 –7
)
1.16610–6
(€0.51610 –6
)
138 2.79610–7
(€1.64610–7
)
9.30610 –7
(€5.46610 –7
)
185 3.56610–7
(€1.56610 –7
)
1.19610 –6
(€0.52610 –6
)
173
EKC Sepa a ion/on-line p econcen a ion by mul i-s ep s acking o s e oids 2207
can dec ease in he heo e ical LODs o single and dou-
ble s acking, while iple and mo e exhaus i e s acking
s eps seem o be less e ec i e o imp o ing he sensi i -
i y.
Table 1 also shows he s eps ollowed o achie e double
and iple s acking p econcen a ions. Again, he s acking
ime o each s acking s ep was he c ucial pa ame e o
be calcula ed o online mul is ep s acking p econcen a-
ion. The s acking imes o he second and hi d s acking
s eps we e calcula ed by mul iplying he espec i e in lex-
ion poin x- alues, ob ained om cu en in ensi y cu es,
by he ac o o 1.2 (op imised o he i s s acking s ep).
Thus, successi e s acking imes o 176, 209 and 211 s
we e calcula ed o single, double and iple s acking,
espec i ely. Al hough he s acking ime inc eased wi h
he numbe o s acking s eps, i seemed o s abilize wi h
he numbe o s acking s eps.
LOD and LOQ o he h ee anabolic s e oids when using
online double s acking p econcen a ion (see Table 2)
we e also calcula ed using co ec ed peak a eas. In his
case, SEF alues ob ained (138, 185 and 173 o TST,
MTS and MTD, espec i ely) we e lowe han he
expec ed heo e ical ampli ica ion o he double s acking,
which is 222. This esul could be due o he e o s asso-
cia ed wi h he in eg a ion o ailing peaks and he loss o
esolu ion which became conside able when using double
s acking p econcen a ion (see Fig. 6). The compa ison o
he heo e ically es ima ed LOD o TST (Fig. 5) wi h he
co esponding expe imen al alue (Table 2), con i med
his disc epancy which can be jus i ied conside ing ha
Eq. (5) does no ake in o accoun he numbe o heo e i-
cal pla es (N) ha usually dec eases wi h he numbe o
s acking s eps. In ac , N o he las mig a ing compound
(MTD), which co esponds o he peak mo e a ec ed by
b oadening e ec s, was 11 000, 11 500 and 7000 o he
single, double and iple s acking espec i ely.
Quan i ica ion o he iple s acking was no possible
because o i s poo epea abili y, especially o he lowes
concen a ions. Peak symme y and esolu ion dec eased
in his case, as i can be obse ed in Fig. 6, causing huge
e o s du ing in eg a ion o peaks. In addi ion, online p e-
concen a ion by iple s acking seems o be e y suscep-
ible o changes in he expe imen al condi ions. Tempe a-
u e a ia ions, especially in he un he mos a ised pa o
he capilla y, in luence he iscosi y o bu e and sample
a ec ing he analy e eco e y. This makes he iple s ack-
ing p econcen a ion a doub ul way o imp o e sensi i i y
o he analysis. In addi ion, he analysis wi h mul is ep p e-
concen a ion is much longe , almos 1 h in he case o he
iple s acking.
4 Concluding ema ks
The sepa a ion o he h ee s uc u ally ela ed s e oids,
me hyl es os e one, me hand os enolone and es os e -
one, has been pe o med o he i s ime by EKC using a
dual CD sys em based on an anionic CD and a neu al
one (i.e., Suc-b-CD + b-CD) as dispe sed phases. Due o
he low abso p ion o UV ligh o hese anabolic s e oids,
hey we e used as model compounds o s udy he possibi-
li ies o online mul is ep s acking p econcen a ion wi h
la ge- olume injec ion in o de o imp o e hei sensi i i y
using UV-abso p ion de ec ion. I has been demons a ed
J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Figu e 5. Theo e ically es i-
ma ed LOD wi h mul is ep
s acking. LOD o he analysis
wi hou s acking co esponds o
he alue: 4.79610–5 mol/L.
Ampli ica ion o he signal: 111
imes (332/3 s).
2208 U ban, Ga ca-Ruiz, Ga ca, Ma ina
ha single and double s acking allow SEFs om 100 o
150 o single s acking and om 140 o 190 o double
s acking. In spi e o his, iple s acking was no epea able
due o mul iplica ion o e o s associa ed wi h li le
changes o expe imen al condi ions.
The au ho s hank he Minis e io de Ciencia y Tecnologa
(Spain) o p ojec BQU2003-03638. D . C. Ga ca-Ruiz
hanks o he same ins i u ion o he con ac om he
Ramn y Cajal P og am (RYC-2003-001). P. L. U ban
hanks he Eu opean Union o his E asmus ellowship.
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J. Sep. Sci. 2005, 28, 2200–2209 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim
Figu e 6. Compa ison o he
elec ophe og ams ob ained by
single, double and iple s ack-
ing o a mix u e o MTS, MTD
and TST (2.5610–6 mol/L o
each one in 1% / in me ha-
nol:wa e ). Expe imen al condi-
ions as in Fig. 4.