Ela Ekie
Ca men Ga cía-Ruiz
Ma ia A. Ga cía
Ma ia L. Ma ina
Depa amen o de Química
Analí ica, Facul ad de Química,
Uni e sidad de Alcalá,
Alcalá de Hena es (Mad id),
Spain
Rapid de e mina ion o salbu amol in
pha maceu ical p epa a ions by chi al capilla y
elec opho esis
A as and simple me hod o chi al capilla y elec opho esis (CE) has been applied o
he analysis o salbu amol in di e en pha maceu ical p epa a ions. Using o a 25 mM
ace a e bu e (pH 5.0), con aining 13.1 mg/mL ca boxyme hyl-b-cyclodex in (CM-b-
CD), an applied ol age o 20 kV and a empe a u e o 257C, he enan iome s o salbu-
amol could be sepa a ed in abou 2 min. Th ee di e en pha maceu ical p epa a ions
( wo sy ups, one o al solu ion, and wo kind o able s) con aining a acema e o salbu-
amol we e injec ed di ec ly in he CE sys em, ollowing dilu ion in dime hyl sul oxide
(DMSO). App eciable di e ences in he e en ion imes we e obse ed o salbu amol
enan iome s in he di e en o mula ions s udied, which we e a ibu ed o he e ec o
he ma ix componen s on he elec opho e ic mobili y. The s anda d addi ion me hod
was used o he calib a ion due o he exis ence o ma ix in e e ences. Finally, he
s abili y o he enan iome s o salbu amol in he o al solu ion was s udied calcula ing
he enan iome ic a io alues when he solu ion was injec ed immedia ely a e being
opened in he i s case and a e being opened and s o ed in he idge o wo mon hs
in he second case.
Keywo ds: Chi al capilla y elec opho esis / Pha maceu ical p epa a ion / Salbu amol
DOI 10.1002/elps.200305490
1 In oduc ion
Salbu amol is a b2-ad enocep o agonis clinically used
as b onchodila o o he ea men o pa ien s wi h
as hma, b onchi is, emphysema and, in gene al, b ea h-
ing diseases wi h b onchocons ic ion [1]. The pha maco-
logical ac ion o salbu amol esides in he R-(2)-enan io-
me , which mo eo e was ound o unde go a as e me-
abolism in men han he S-(1)-enan iome [2]. Howe e ,
no in o ma ion is cu en ly a ailable on he oxici y o inac-
i e S-(1)-enan iome and salbu amol is s ill comme cial-
ized and adminis a ed as a acema e [3].
Capilla y elec opho esis (CE) is an e ec i e ool o he
esolu ion o enan iome s, which is accomplished by
supplying he backg ound elec oly e wi h a chi al selec-
o capable o disc imina ing be ween he enan iome s
conce ned. In ac , chi al CE has been he subjec o
much a en ion and has been applied wi h success o
he enan iome ic sepa a ion o di e en chi al com-
pounds du ing he las decade [4, 5]. The chi al selec o s
used o his pu pose include cyclodex ins (CDs) and
de i a i es, chi al c own e he s, noncyclic oligosaccha -
ides and polysaccha ides, mac ocyclic an ibio ics, p o-
eins and pep ides, me al complexes, and chi al su ac-
an s [4, 6–10].
The alida ion o chi al me hods o analysis is o ob ious
impo ance in pha maceu ical science o de e mining
enan iome ic pu i y o p oduc s, o analyze d ugs o mu-
la ions o o pe om o mula ion s abili y s udies as i is
shown by he g ea numbe o wo ks published on his
subjec [5, 6, 11–18]. In he case o he quan i a ion o
chi al b onchodila o s by CE, he de eloped me hods
used b-CD de i a i es as chi al selec o s. Thus, Esquisa-
bel and co-wo ke s [16] ob ained he chi al sepa a ion o
salbu amol using 2,6-di-O-me hyl-b-CD as chi al selec o
in 14 min app oxima ely. This chi al me hod was applied
o he s udy o salbu amol enan iome elease om ma ix
able s ha con ained he acemic d ug and a chi al exci-
pien as hyd oxyp opylme hylcellulose. No enan ioselec-
i e elease o he salbu amol om hese ma ices was
obse ed. The quan i a i e de e mina ion o salbu amol,
clenbu e ol, and ulobu e ol enan iome s om a spiked
sample by CE using sul a ed b-CD as chi al selec o has
been pe o med by Vela and co-wo ke s in abou 20 min
Co espondence: D . M. L. Ma ina, Depa amen o de Química
Analí ica, Facul ad de Química, Uni e sidad de Alcalá, C a.
Mad id-Ba celona Km. 33.600, E-28871 Alcalá de Hena es
(Mad id), Spain
E-mail: [email p o ec ed]
Fax: +34-91-8854971
Abb e ia ions: ANOVA, analysis o a iance; CM-â-CD, ca -
boxyme hyla ed b-cyclodex in
2680 Elec opho esis 2003, 24, 2680–2686
2003 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 0173-0835/03/1508–2680 $17.501.50/0
Elec opho esis 2003, 24, 2680–2686 De e mina ion o salbu amol by chi al CE 2681
[17]. Finally, a as enan iome ic sepa a ion, in abou
2 min, o salbu amol using a neu al b-CD de i a i e (pe -
me hyla ed b-CD) o a nega i ely cha ged b-CD de i a i e
(ca boxyme hyl-b-CD) as chi al selec o was pe o med
ecen ly by ou esea ch eam [18].
The main objec i e o his wo k was he applica ion o he
as and simple chi al CE me hod de eloped by ou e-
sea ch eam o he chi al sepa a ion o salbu amol [18]
o he de e mina ion o his compound in di e en pha -
maceu ical p epa a ions whe e i was used as a ace-
ma e. Fu he mo e, since he e is no in o ma ion conce n-
ing he uns abili y o one enan iome o salbu amol wi h
espec o he o he one in pha maceu ical o mula ions,
he e alua ion o he s abili y o hese enan iome s in an
o al solu ion was also pe o med.
2 Ma e ials and me hods
2.1 Chemicals and samples
All eagen s we e o analy ical g ade. Dime hyl sul oxide
(DMSO), and sodium hyd oxide we e supplied om
Me ck (Da ms ad , Ge many); hyd ochlo ic acid was pu -
chased om Pan eac (Ba celona, Spain); CM-b-CD
(deg ee o subs i u ion 3) was ob ained om Cyclolab
(Budapes , Hunga y). Wa e used o p epa e solu ions
was pu i ied h ough a Milli-Q sys em om Millipo e (Bed-
o d, MA, USA). All solu ions we e il e ed h ough 45 mm
po e size disposable nylon il e s om Scien i ic
Resou ces (Ea on own, NJ, USA). Salbu amol was pu -
chased om Sigma (S . Louis, MO, USA). The s uc u e
o he enan iome s o his basic d ug is shown in Fig. 1.
Di e en pha maceu ical p epa a ions con aining salbu-
amol sul a e we e acqui ed in pha macy shops a Alcalá
de Hena es (Mad id, Spain). Table 1 shows he composi-
ion o he di e en pha maceu ical p epa a ions s udied
in his wo k.
Figu e 1. S uc u e o he enan iome s o salbu amol.
Table 1. Composi ion o he di e en pha maceu ical
p epa a ions s udied in his wo k
Pha maceu ical
p epa a ion Composi ion
Sy up (A) Salbu amol sul a e, sodium saccha ine and exci-
pien s (sodium ci a e, ci ic acid monohyd a e,
hyd oxyp opylme hylcellulose, sodium benzoa e,
o ange la o , sodium chlo ide, and pu i ied
wa e )
Sy up (B) Salbu amol sul a e and excipien s (sodium ben-
zoa e, hyp omelose, sodium saccha ine, ci ic
acid, sodium ci a e, o ange la o , and pu i ied
wa e )
O al solu ion (C) Salbu amol sul a e and excipien s (sodium ben-
zoa e, ci ic acid monohyd a e, sodium sac-
cha ine, sodium ci a e, sodium chlo ide, a oma
o aspbe y, and wa e )
Table (D) Salbu amol sul a e and excipien s (co n s a ch,
poly inylpy olidone, cellulose, sodium s a ch
caboxyme hyla ed, silicium dioxide, alc,
magnesium es ea a e)
Table (E) Salbu amol sul a e and excipien s (sodium chlo ide,
po idone, sodium c osca mellosa, silicagel,
magnesium es ea a e, cellulose ace a e,
me hylhyd oxyp opylcellulose, i anium dioxide,
hyd oxyp opylcellulose, lacas ubbe , indus ial
me hyla ed spi i , 2-e oxyie hanol, n-bu yl alco-
hol, aluminium laca o lips ick, and polydime-
hylsiloxano)
2.2 Appa a us
An HP3D CE sys em (Hewle -Packa d, Waldb onn, Ge -
many) equipped wi h an on-column diode a ay de ec o
(DAD) and an HP 3D-CE Chems a ion so wa e was used.
An uncoa ed used-silica capilla y om Composi e Me al
Se ices (Wo ces e , England) wi h 50 mm inne diame e
(ID) and 375 mm ou e diame e (OD) wi h an e ec i e
leng h o 25 cm (33.5 cm o al leng h) was employed.
Capilla y empe a u e was se o 257C and UV de ec ion
was pe o med a 230 nm. Sepa a ion ol age was 20 kV.
Elec oly ic solu ions we e degassed in an ul asonic ba h
KM om Raypa (Ba celona, Spain). A 654 pH me e om
Me ohm (He isau, Swi ze land) was employed o adjus
he pH o he sepa a ion bu e .
2.3 P ocedu e
Elec oly ic solu ions we e p epa ed weighing and dis-
sol ing he app op ia e amoun o bu e and CM-b-CD
in Milli-Q wa e o ob ain he equi ed concen a ion and
CE and CEC
2682 E. Ekie e al. Elec opho esis 2003, 24, 2680–2686
Figu e 2. Sepa a ion o salbu amol enan iome s in a
s anda d solu ion (0.045 mg/mL) and in dilu ions o he
i e pha maceu ical p epa a ions s udied in his wo k
(0.045 mg/mL in salbu amol) using he chi al CE me hod.
Expe imen al condi ions: 25 mMace a e bu e (pH 5) wi h
13.1 mg/mL CM-b-CD; empe a u e, 257C; applied ol -
age, 20 kV; UV de ec ion a 230 nm; injec ion by p essu e,
30 mba o 2 s sample ollowed by 30 mba o 2 s bu e ;
50 mm ID, 375 mm OD capilla y o 33.5 cm leng h (25 cm o
he de ec o ).
adjus ing he pH o he desi ed alue wi h a 1 Mhyd o-
chlo ic acid solu ion and 0.1 Msodium hyd oxide. S an-
da d solu ions o salbu amol used o he calib a ion by
he ex e nal s anda d me hod we e p epa ed by dilu ing
wi h DMSO a s ock solu ion o 10 mg/mL o salbu amol
in DMSO o ob ain inal concen a ions comp ised om
0.005 o 0.120 mg/mL (0.005, 0.010, 0.020, 0.030, 0.045,
0.060, 0.080, 0.100, and 0.120 mg/mL). The de e mina-
ion o he salbu amol con en in he di e en pha maceu-
ical p epa a ions ( wo sy ups, an o al solu ion, and wo
able s) equi ed a dilu ion o each o mula ion in a inal
olume o DMSO o achie e a inal concen a ion o
0.015 mg/mL in salbu amol. Fo he calib a ion by he
s anda d addi ions me hod, h ee inc easing concen-
a ions o salbu amol s anda d (0.010, 0.030, and
0.060 mg/mL) we e added o a solu ion o he pha ma-
ceu ical p epa a ion wi h a concen a ion o 0.015 mg/
mL o salbu amol. The ou esul ing solu ions we e di-
ec ly injec ed in he elec opho e ic sys em. Ne e he-
less, in he case o he able s i was necessa y o include
a cen i uga ion s ep o sepa a e he insoluble excipien s
om he solu ion be o e he injec ion o he solu ion in he
CE sys em. Be ween injec ions, he capilla y was washed
wi h 0.1 Msodium hyd oxide (10 ba o 0.2 min), ollowed
by he unning bu e (10 ba o 0.4 min). The injec ion
was made by p essu e: 30 mba o 2 s o sample ol-
lowed by 30 mba o 2 s o sepa a ion bu e .
2.4 Da a ea men
All he expe imen al da a we e manipula ed using Excel
7.0 om Mic oso O ice so wa e [19]. Reg ession analy-
sis, analysis o a iance (ANOVA) and compa ison o
eg ession lines o signi ican ly di e en slopes and in e -
cep s we e made using S a g aphics Plus so wa e [20].
3 Resul s and discussion
3.1 Sepa a ion o he enan iome s o
salbu amol in di e en pha maceu ical
p epa a ions
In a p e ious wo k i was epo ed ha he as enan io-
me ic sepa a ion o salbu amol, in abou 2 min is possi-
ble, using 25 mMace a e bu e (pH 5) con aining
13.1 mg/mL CM-b-CD a a empe a u e o 257C and an
applied ol age o 20 kV in a capilla y o 25 cm o e ec i e
leng h [18]. These condi ions ha e been applied in his
wo k o he as chi al analysis o salbu amol in di e en
pha maceu ical p epa a ions: wo sy ups, an o al solu-
ion, and wo kind o able s. Figu e 2 shows he elec o-
phe og ams co esponding o a s anda d solu ion o sal-
bu amol (0.045 mg/mL) and o i e dilu ions in DMSO
o he di e en pha maceu ical p epa a ions s udied
con aining 0.045 mg/mL o salbu amol sul a e. I was
obse ed ha a mix u e o he wo enan iome s o salbu-
amol was de ec ed in all cases and no in e e ing peaks
we e obse ed. In addi ion, a simila enan iome ic esolu-
ion o he salbu amol s anda d and he salbu amol con-
ained in he di e en pha maceu ical p epa a ions was
obse ed. Howe e , an inc ease in he mig a ion imes o
he enan iome s o salbu amol de ec ed in some o he
di e en pha maceu ical p epa a ions was obse ed wi h
espec o he mig a ion imes o he enan iome s o he
salbu amol s anda d. This esul could be a ibu ed o
he e ec o ma ix componen s on he elec oosmo ic
low (EOF). Acco ding o Table 1 bo h sy ups ha e a simi-
la composi ion, being he di e ence ha sy up (A) con-
ain hyd oxyp opylme hylcellulose and sodium chlo ide
ins ead o he hyp omelose con ained in sy up (B).
Fu he mo e, he mig a ion imes obse ed o he salbu-
amol enan iome s o he o al solu ion a e mo e simila o
he s anda d solu ion han hose o he sy ups, p obably
due o he absence o compounds such as hyd oxyp o-
pylme hylcellulose o hyp omelose which can in e ac
wi h he inne wall o he capilla y when hey a e injec ed
in he CE sys em [21]. On he o he hand, al hough bo h
kind o able s s udied ha e a mo e complex composi ion
han he sy ups o he o al solu ion s udied, mig a ion
imes o he enan iome s o salbu amol in hese pha ma-
ceu ical p epa a ions a e simila o he mig a ion imes o
Elec opho esis 2003, 24, 2680–2686 De e mina ion o salbu amol by chi al CE 2683
he enan iome s o he salbu amol s anda d and simila
be ween hem, al hough hei composi ion is also e y di -
e en . These esul s could be explained aking in o
accoun he insolubili y obse ed o some excipien s
when hese able s we e dissol ed in DMSO and which
we e sepa a ed om he solu ions injec ed in he CE sys-
em by cen i uga ion. A e hese esul s, we checked i
he chi al CE me hod was use ul o he de e mina ion o
he salbu amol con en in he di e en pha maceu ical
p epa a ions conside ed.
3.2 Quan i a ion o salbu amol in
pha maceu ical p epa a ions
The quan i a ion o salbu amol by he chi al CE me hod
was pe o med in di e en pha maceu ical p epa a ions:
wo sy ups, an o al solu ion, and wo kind o able s. Fo
he calib a ion, co ec ed o al peak a eas, calcula ed as
he addi ion o he co ec ed a eas co esponding o he
i s and he second mig a ing enan iome s we e used
( he co ec ed a ea o each enan iome was ob ained by
di iding he a ea o each elec opho e ic peak by i s co -
esponding mig a ion ime). Al hough he use o co ec ed
a eas in CE is equen [22], in his case i was o ally
necessa y in o de o inc ease ep oducibili y o calib a-
ion da a and also o compensa e luc ua ions in elec o-
pho e ic condi ions due mainly o he composi ions o he
di e en samples injec ed and also due o he pH o he
sepa a ion bu e (pH 5.0 is a c i ical alue whe e small
a ia ions o pH alues p oduce big di e ences in he
elec opho e ic mobili y) [23].
Solu ions o salbu amol s anda d wi h concen a ions
anging om 0.005 o 0.400 mg/mL we e injec ed by
iplica e in o de o de e mine he linea concen a ion
ange. I was obse ed a linea ela ionship be ween he
o al co ec ed a ea and he concen a ion o salbu amol
in he concen a ion ange be ween 0.005 and 0.120 mg/
mL. The e o e, solu ions o salbu amol s anda d com-
p ised om 0.005 o 0.120 mg/mL we e p epa ed and
injec ed by iplica e du ing h ee di e en days in he CE
sys em in o de o alida e he calib a ion cu e ob ained
by plo ing co ec ed o al peak a ea e sus concen a-
ion. The equa ion ob ained when he a e age o he co -
ec ed o al peak a ea o he h ee lines conside ed e -
sus he concen a ion was plo ed is y= 0.187110.0362x
being he s anda d e o associa ed o he in e cep
0.0289, o he slope 0.0004, and o he calib a ion cu e
0.0515. The ANOVA o he a e age o he h ee calib a ion
lines conside ed e ealed ha he lack o i was always
s a is ically smalle han he pu e e o , which con i med
ha a s aigh line was a sui able model in he concen a-
ion ange employed. In addi ion, a co ela ion coe icien
equal o 0.9995 indica ed a ela i ely s ong ela ionship
be ween he a iables. On he o he hand, he sensi i i y
o his chi al CE me hod, co esponding o he slope o
he calib a ion line, was 0.0362 mL?mg21(0.036261023
mL?mg21).
The slope and he s anda d e o o he calib a ion cu e
we e used o calcula e he limi o de ec ion (LOD) and he
limi o quan i a ion (LOQ). LOD and LOQ we e de ined as
he analy e concen a ions gi en signals exceeding ha
o he in e cep by 3 and 10 imes, espec i ely, he s an-
da d e o o he calib a ion cu e [24]. The LOD calcu-
la ed o salbu amol was abou 4 mg/mL and he LOQ
was abou 14 mg/mL.
In o de o e alua e he p ecision o he CE me hod (see
Table 2), epea abili y and ep oducibili y we e s udied.
The epea abili y in mig a ion ime and peak a ea o he
enan iome s and in he co ec ed o al peak a ea, was
de e mined (as RSD) o en consecu i e injec ions o a
dilu ion in DMSO o each pha maceu ical p epa a ion
wi h a inal concen a ion o 0.045 mg/mL in salbu amol.
RSD was less han 5.0% o he mig a ion imes o he
enan iome s, less han 5.4% o peak a ea o he enan io-
me s, and less han 4.8% o co ec ed o al peak a ea.
On he o he hand, ep oducibili y in peak a ea and mig a-
ion ime o he enan iome s and in he co ec ed o al
peak a ea was measu ed as he RSD ob ained in ou di -
e en days (injec ions by iplica e) o sample dilu ions o
0.045 mg/mL in salbu amol. As i can be obse ed in
Table 2, he RSD alues ob ained we e less han 5.5%
o he mig a ion imes o he enan iome s, less han
6.5% o he peak a ea o he enan iome s, and o he
co ec ed o al peak a ea RSD was less han 6.0%. I
should be emphasized ha RSD alues o he co ec ed
o al peak a ea a e lowe han hose ob ained o he peak
a ea, ha is, he p ecision ob ained o he co ec ed o al
peak a eas is be e han hose ob ained wi h nonco -
ec ed peak a eas, he e o e, co ec ed peak a eas we e
used o he calib a ion.
P io o he quan i a i e analysis, he p esence o
absence o ma ix in e e ences was s udied. Then, he
slopes o he eg ession lines ob ained by he ex e nal
s anda d me hod and he s anda d addi ion me hod,
which was applied o he di e en pha maceu ical p ep-
a a ions, we e compa ed. Table 3 shows he calib a ion
lines ob ained o he a ia ion o he co ec ed o al
peak a ea as a unc ion o he concen a ion o salbu a-
mol s anda d by he ex e nal s anda d me hod and as a
unc ion o he added concen a ion o salbu amol o he
sample by he s anda d addi ion me hod. In addi ion,
each calib a ion line was ob ained as he a e age o h ee
calib a ion lines ob ained in h ee di e en days and each
one alida ed by ANOVA. The compa ison o hese wo
eg ession lines ob ained as explained abo e o each
2684 E. Ekie e al. Elec opho esis 2003, 24, 2680–2686
Table 2. P ecision in peak a eas and mig a ion imes o salbu amol enan iome s and in co ec ed o al peak a ea o
salbu amol in i e di e en pha maceu ical p epa a ions by he CE me hoda)
P ecision Pha maceu ical
p epa a ion
Csb) 1(RSD) 2(RSD) A1(RSD) A2(RSD) A (RSD)
Sy up (A) 0.045 2.62 (3.06) 2.69 (3.13) 1.40 (3.24) 1.38 (3.50) 1.05 (3.39)
Sy up (B) 0.045 2.64 (4.97) 2.71 (4.28) 1.17 (3.78) 1.16 (3.79) 0.87 (2.41)
Repea abili yc) O al solu ion (C) 0.045 2.13 (1.48) 2.18 (1.51) 1.24 (3.49) 1.17 (3.76) 1.12 (4.76)
Table (D) 0.045 2.02 (2.82) 2.06 (2.86) 0.91 (5.43) 0.91 (4.90) 0.89 (3.73)
Table (E) 0.045 1.92 (1.74) 1.97 (1.77) 1.00 (3.58) 1.01 (4.12) 1.03 (3.10)
Sy up (A) 0.045 2.48 (3.87) 2.54 (3.94) 1.33 (4.13) 1.30 (5.99) 1.05 (4.76)
Sy up (B) 0.045 2.60 (4.42) 2.67 (4.48) 1.25 (6.49) 1.20 (6.16) 0.93 (3.63)
Rep oducibili yd) O al solu ion (C) 0.045 2.14 (1.65) 2.18 (1.52) 1.17 (6.19) 1.20 (4.01) 1.10 (6.00)
Table (D) 0.045 1.99 (5.45) 2.02 (5.17) 0.93 (4.46) 0.93 (5.67) 0.93 (4.83)
Table (E) 0.045 1.89 (2.98) 1.94 (3.35) 0.95 (4.37) 0.94 (4.79) 0.99 (3.88)
1and 2a e he mig a ion imes co esponding o he i s and he second mig a ing enan iome s, espec i ely; A1and A2
a e he peak a eas o he i s and he second mig a ing enan iome s, espec i ely; A is he co ec ed o al peak a ea ((A1/
1)1(A2/ 2)) and he RSD alue was calcula ed wi h he unce ain y o his ma hema ic ope a ion [21].
a) Expe imen al condi ions speci ied in Sec ion 2.3
b) Concen a ion o salbu amol in a solu ion p epa ed by dilu ion o he comme cial p epa a ion (mg/mL)
c) RSD alues de e mined o en consecu i e injec ions
d) RSD alues de e mined o ou di e en days (each injec ion was made by iplica e)
Table 3. Compa ison o he calib a ion linesa) ob ained by
he ex e nal s anda d me hod and he s anda d
addi ion me hod o he di e en pha maceu ical
p epa a ions s udied
Pha maceu ical
p epa a ion
Ex e nal s anda d
me hodb)
S anda d addi ion
me hodc)
Sy up (A)
y
= 0.187110.0362
x
(
n
=9,
= 0.9995)
y
= 0.735610.0420
x
(
n
=4,
= 0.9997)
Sy up (B)
y
= 0.187110.0362
x
(
n
=9,
= 0.9995)
y
= 0.760610.0497
x
(
n
=4,
= 0.9991)
O al solu ion (C)
y
= 0.187110.0362
x
(
n = 9,
= 0.9995)
y
= 0.709910.0473
x
(
n
=4,
= 0.9999)
Table (D)
y
= 0.187110.0362
x
(
n
=9,
= 0.9995)
y
= 0.691310.0404
x
(
n
=4,
= 0.9999)
Table (E)
y
= 0.187110.0362
x
(
n
=9,
= 0.9995)
y
= 0.683310.0417
x
(
n
=4,
= 0.9995)
a) Calib a ion lines ob ained as he a e age o h ee cali-
b a ion cu es ob ained in h ee di e en days (
n
, num-
be o poin s conside ed o he calib a ion cu e;
,
co ela ion coe icien )
b) Concen a ion ange o he salbu amol s anda d:
0.005–0.120 mg/mL
c) Concen a ion ange o he salbu amol s anda d
added o he pha maceu ical p epa a ions (dilu ion
con aining 0.015 mg/mL o salbu amol sul a e): 0–
0.06 mg/mL
calib a ion me hod and o each pha maceu ical p epa a-
ion e ealed ha s a is ically signi ican di e ences
(P,0.05) among he slopes we e obse ed o all he
pha maceu ical p epa a ions s udied. Then, he p esence
o ma ix in e e ences was de ec ed being necessa y o
use he s anda d addi ion me hod o he quan i a ion o
salbu amol.
The salbu amol con en in he di e en pha maceu ical
p epa a ions s udied was de e mined om a s anda d
addi ion line ob ained as a e age o h ee calib a ion lines
ob ained in h ee di e en days and alida ed by ANOVA
o each pha maceu ical p epa a ion. Table 4 g oups he
salbu amol con en de e mined by he chi al CE me hod
and he salbu amol con en calcula ed aking in o
accoun he salbu amol con en declea ed in he label o
he p oduc and he dilu ion in DMSO. Resul s show ha
di e ences in bo h alues ob ained o he sy up (B) and
he o al solu ion (C) we e 2 and 0%, espec i ely, whe eas
o he o he h ee pha maceu ical p epa a ions s udied
(sy up (A) and able s (D) and (E)) hese di e ences we e
abou 17, 14, and 9%, espec i ely.
Finally, as he in e con e sion o enan iome s o a chi al
compound can occu wi h ime [11] and hei s abili y
can be di e en , i seems in e es ing o es he change
in he p opo ion o he enan iome s in s o ed pha maceu-

Elec opho esis 2003, 24, 2680–2686 De e mina ion o salbu amol by chi al CE 2685
Table 4. Quan i a i e analysis o salbu amol in di e en
pha maceu ical p epa a ions by he chi al CE
me hoda)
Salbu amol con en
by CE (mg/mL)b)
Pha maceu ical
p epa a ion
Declea ed salbu a-
mol con en in pha -
maceu ical p epa a-
ion (mg/mL)c)
17.561023Sy up (A) 15.061023
15.361023Sy up (B) 15.061023
15.061023O al solu ion (C) 15.061023
17.161023Table (D) 15.061023
16.461023Table (E) 15.061023
a) Expe imen al condi ions speci ied in Sec ion 2.2
b) Concen a ion o salbu amol de e mined by he chi al
CE me hod
c) Concen a ion o salbu amol ob ained by dilu ion o he
pha maceu ical p epa a ions conside ing he salbu a-
mol con en indica ed in he label o each p epa a ion
ical p epa a ions wi h a apid and simple me hod as used
in his wo k. The o al solu ion (C) s udied in his wo k is
p epa ed by he pa ien om a powde by dissol ing i in
wa e and s o ing i in a idge o consump ion o a max-
imum o en days. The chi al me hod was applied o s udy
he s abili y o he salbu amol enan iomes in his o al so-
lu ion. In his case, he enan iome ic a io alues o he
o al solu ion (C) we e calcula ed in o de o s udy i he e
was a ia ion in he p opo ion o he enan iome s o sal-
bu amol when he o al solu ion was injec ed a e being
opened and a e being opened and s o ed in he idge
o wo mon hs. The enan iome ic a io was calcula ed
as he co ec ed a ea o he second mig a ing enan iome
di ided by he co ec ed a ea o he i s mig a ing enan-
iome . An enan iome ic a io alue o 0.92 was ob ained
when dilu ions in DMSO o he o al solu ion (C) con aining
0.040 mg/mL in salbu amol was opened and immedia ely
injec ed in he CE sys em, and a alue o 0.98 was
ob ained when dilu ions we e analyzed a e he o mula-
ion was opened and s o ed o wo mon hs. Since he
di e ences be ween bo h alues o enan iome ic a io
can be conside ed wi hin he expe imen al e o , he
esul s indica ed ha he acema e has been emained
a e he o al solu ion (C) was opened and s o ed du ing
a long ime.
4 Concluding ema ks
The applica ion o a as and simple chi al CE me hod
based on he use o a 25 mMace a e bu e a pH 5 wi h
13.1 mg/mL CM-b-CD a 257C and 20 kV as he sepa a-
ion ol age o he quan i a ion o salbu amol in di e en
pha maceu ical p epa a ions ( wo sy ups, an o al solu-
ion, and wo kinds o able s) con aining a acema e o
salbu amol was pe o med in his wo k. Al hough di e -
ences in he mig a ion imes we e obse ed o he di e -
en o mula ions s udied, which we e a ibu ed o he
in luence o hei di e en composi ion on he elec opho-
e ic mobili y, he use o co ec ed o al peak a ea enabled
o compensa e he luc ua ions in he elec opho e ic
condi ions. The chi al CE me hod was cha ac e ized by a
linea concen a ion ange om 0.005 o 0.120 mg/mL o
salbu amol, LOD o salbu amol abou 4 mg/mL, and
accep able alues o p ecision ( epea abili y and ep o-
ducibili y) in e ms o mig a ion ime, peak a ea, and co -
ec ed o al peak a ea. Fo he quan i a ion o salbu amol
in he di e en pha maceu ical p epa a ions s udied he
s anda d addi ions me hod was used o he calib a ion
due o he exis ence o ma ix in e e ences o a 95%
con idence le el. Finally, he s abili y o he enan iome s
in he o al solu ion was s udied calcula ing he enan io-
me ic a io alues ob ained when i was injec ed a e
being opened and a e being opened and s o ed in he
idge o wo mon hs ob aining no signi ican di e ences.
The au ho s hank he Comisión In e minis e ial de Cien-
cia y Tecnología (Spain) o p ojec PB98–0709. D . M. A.
Ga cía hanks he Uni e sidad de Alcalá (Mad id, Spain)
o p ojec E027/2001.
Recei ed Ma ch 3, 2003
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