Use of vancomycin silica stationary phase in packed
Part IV: Enantiomer separation of fluoxetine and
norfluoxetine employing UV high sensitivity
Chimiche, Consiglio Nazionaledelle Ricerche, Area dellaRicerca di Roma, P.O. Box 10,
The enantiomeric separation of the antidepressant drug fluoxetine and its main
metabolite (norfluoxetine) was achieved by using capillary electrochromatography
employing packed capillaries with a vancomycin stationary phase. The capillary (21,
23, or 25 cm675 lm ID), with frits at both ends, was connected to a commercially
available high sensitivity UV detection cell to improve the detection limit. With the aim
of optimizing the separation of the two pairs of enantiomers in the same run, several
parameters were studied: these included the mobile phase composition (buffer pH,
organic solvent ratio), the capillary temperature, the length of the packed capillary,
and the packed stationary phase type. Experiments were performed using a mobile
phase containing aqueous ammonium acetate pH 6 (5 mM final concentration)
Bologna, Via Belmeloro 6,40126 Bologna, Italy
dissolved in 90% acetonitrile/methanol (MeCN/MeOH) with various organic modifierratios. Increasing MeOH concentration improved the enantioresolution for both analy-tes. However, the two compounds were not separated under these conditions. Theaqueous electrolyte of pH 6 was dissolved at the same concentration as describedabove in organic solvent (MeCN/MeOH, 70/20 v/v) and used for further studies inwhich the length of the stationary phase and its composition were changed. Capilla-ries of 21, 23, and 25 cm length were packed bed length with vancomycin-diol mixedwith silica (3:1) or with vancomycin-diol only and tested for the separation of Flx andNFlx enantiomers. Limits of detection (LOD) and limits of quantification (LOQ) as lowas 25 and 50 ng/mL, respectively, were observed for each racemic analyte.
Key Words: Electrochromatography; Enantiomers; Chiral; Packed capillaries; Drugs;High sensitivity UV detection cell;
Received: May 23, 2002; revised: June 14, 2002; accepted: June 14, 2002
chiral compounds are administered as racemic mixtures. Therefore, with the help of the regulatory authorities, con-
siderable effort is being invested in the development of
In the last decade great attention has been paid by several
chiral separation methods to control the enantiomeric pur-
research groups to the separation and quantitation of
ity of drugs as well as for pharmacokinetic/pharmacody-
chiral compounds in biomedical, pharmaceutical, environ-
namic (PK/PD) and/or clinical studies. The analytical
mental, and other sciences. Since it has been shown that
methods developed should offer good performance such
the pharmacological activity and toxicological properties
as high efficiency and resolution allowing rapid analysis at
of two enantiomers of a certain drug can be different the
demand for chiral separation methods by research andapplication groups is continuously increasing . Some
Several separation techniques possess the above men-tioned characteristics and consequently are currently
Correspondence: Salvatore Fanali, CNR-Istituto di Metodo-
employed in the field of chiral analysis; they include high
logie Chimiche, Area della Ricerca di Roma, P.O. Box 10,
performance liquid chromatography (HPLC), gas chroma-
00016 Monterotondo Scalo (Roma), Italy. E-mail: [email protected]
tography (GC), supercritical fluid chromatography (SFC),
and capillary electrophoresis (CE) [2 – 11].
Abbreviations: acetonitrile, MeCN; enantioresolution factor,Rs; fluoxetine, Flx; methanol, MeOH; norfluoxetine, NFlx; re-
In CE several separation modes have been studied in
tention time, tR; retention factor, k; supercritical fluid chroma-
order to improve the selectivity of the separation and
among them capillary electrochromatography (CEC)
i 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
seems to be very promising according to the results
evaluated in order to find the optimum experimental condi-
tions. Finally biological samples spiked with Flx and NFlxracemic mixtures were analyzed.
In CEC improvement of peak efficiency and peak resolu-tion can be easily obtained because the mobile phase iselectrodriven through the stationary phase, thus avoidingthe peak dispersion observed in pressure driven system
such as HPLC. The above mentioned properties makeCEC a very attractive separation technique also for the
analysis of enantiomers. A wide number of chiral selec-tors, most of them previously used in HPLC, have been
Electrochromatographic experiments were carried out
applied in CEC for enantiomer separations employing
using an automatic HP 3DCE capillary electrophoresis
either packed, or open or monolithic columns [11, 16 – 17].
instrument (Agilent, Waldbronn, Germany) equipped with
Several racemic analytes belonging to different classes
a diode-array UV detector operating at 195 nm. A constant
such as derivatized amino acids, herbicides, pharmaceuti-
voltage of 25 kV (unless otherwise stated) was applied and
cals, etc. have been resolved into their enantiomers
the temperature of the capillary cartridge was set in the
employing stationary phases containing cyclodextrins or
range 15 – 308C. The inlet and the outlet ends of the capil-
their derivatives, proteins, cellulose derivatives, Pirkle
lary were pressurized at 500 kPa during runs in order to
type phases, molecularly imprinted polymers (MIP), weak
avoid bubble formation. Fused silica capillaries (75 lm ID,
anion exchange type materials, polymethacrylate, glyco-
375 lm OD) were purchased from Composite Metal Ser-
peptide antibiotics (GAs), etc. [18 – 32].
vices (Hallow, UK). The packed capillary was connectedto a high sensitivity detection cell with a path length of
GAs were first used by Armstrong et al. for the enantio-
1,200 lm (Agilent Technologies, Waldbronn, Germany).
meric separation of a wide number of analytes in HPLC
The fused silica capillaries were packed by using a LC se-
 and later vancomycin and teicoplanin were success-
ries 10 pump (Perkin Elmer, Palo Alto, CA, USA). Samples
fully applied as chiral selectors in CEC using both packed
were injected at the anodic end of the capillary by pressure
and monolithic stationary phases in reversed-phase or
(unless otherwise stated) at 500 kPa60.4 min followed by
polar organic modes [18, 19, 34 – 38]. Recently we
a buffer plug injection at 500 kPa60.2 min.
showed the practical applicability of CEC to biomedicalanalysis employing a packed capillary with a silica-vanco-mycin stationary phase for the separation, in the same
run, of the two enantiomer pairs of venlafaxine and O-des-
Vancomycin hydrochloride was purchased from Sigma
methylvenlafaxine in human plasma .
(St. Louis, MO, USA). S-venlafaxine, racemic fluoxetine
(Flx), norfluoxetine (N-Flx), R-nor-fluoxetine, and S-nor-
amine, fluoxetine, a selective serotonin reuptake inhibitor
fluoxetine were gift from C.B. Eap (UnitØ de Biochimie et
drug, is widely used worldwide as an antidepressant due
Psychopharmacologie adulte, site de Cery, Switzerland).
to its efficacy and reduced side effects . It was also
For the chemical structure of Flx and NFlx, see Figure 1.
used in the therapy of alimentary disorders, bulimia andsevere obesity [40, 41]. Demethylation of fluoxetine, as aconsequence of hepatic metabolism, produces a mainactive metabolite, the norfluoxetine (NFlx). The pharma-cological activity of norfluoxetine has also been documen-ted; however, only the S-isomer exhibited similar activityto the parent drug ,
The aim of this work was to develop a rapid and sensitivemethod for the simultaneous enantiomeric separation of
Figure 1. Chemical structures of fluoxetine (Flx) and nor-
fluoxetine and its metabolite norfluoxetine by using a
fused silica capillary packed with vancomycin-derivatizedsilica stationary phase. In order to increase the detectionsensitivity the packed capillary was connected to an
LiChrospher diol silica phase and LiChrospher Si-60, both
extended path length UV detection cell. The effects of the
with 5 lm particle diameter, sodium cyanoborohydride
mobile phase composition (organic modifier type and con-
and sodium periodate were from Merck (Darmstadt, Ger-
centration), capillary effective length, and chiral stationary
many). Ammonium acetate, methanol (MeOH), acetoni-
trile (MeCN), all of HPLC analytical grade, were pur-
Use of vancomycin silica stationary phase
Figure 2. Scheme of the CEC set-up used for the experi-ments.
2.3 Vancomycin stationary phase and CEC packed
The chiral stationary phase (CSP) used in this work wassynthesized in our laboratory modifying a publishedmethod ; vancomycin was chemically bound toLiChrospher diol silica as described in our previous work. In order to prepare the packed capillaries (75 lm) infollowing procedure was adopted: first a porous temporary
Figure 3. Effect of organic modifier ratio present in the
frit was prepared by dipping the end of the capillary in a
mobile phase on a) retention factor (k) and b) enantioresolu-tion factor (Rs) of fluoxetine and norfluoxetine enantiomers.
slurry of 2 lm silica particles in water and burning with an
The capillary (23 cm effective length), was completely
electric heater. Secondly, a short zone (about 5 cm) was
packed with a stationary phase mixture of vancomycin-diol/
packed with a diol-silica slurry in water and then the van-
silica (3:1, w/w), 21 cm; the outlet end was 8.5 cm; 75 lm
comycin stationary phase or vancomycin stationary
was the ID. Mobile phase 100 mM ammonium acetate pH 6,5% (v/v)/5% water and 90% (v/v) of MeOH-MeCN at different
phase/silica (3 : 1, w/w) was packed for the desired length
ratios. Applied voltage and capillary temperature were 25 kV
(21 or 25 cm) and finally diol-silica slurry was again
packed for 5 cm. Frits were prepared as close as possible
500 kPa60.4 min of 50 lg/mL of each racemic analyte. For
to the vancomycin packed zone; the capillary was cut,
other experimental conditions see text.
equilibrated for 30 min with the mobile phase using the LCpump, connected to the high sensitivity detection cell, and
MeOH, which changed in the range 0 – 90 and 90 – 0%
positioned in the cartridge for the CEC experiments. An
respectively). As expected, modification of the ratio of the
untreated fused silica capillary (8.5 cm675 lm ID) was
organic solvents (concentration ratio) present in the
connected to the detection cell by fitting the appropriate
mobile phase caused alterations of the flow velocity due
screw (outlet), as shown in Figure 2.
to change of i) the medium viscosity, ii) the dielectric con-stant, and iii) the double layer and zeta potential on boththe stationary phase and the capillary wall. In fact the EOFincreased on decreasing the MeCN concentration from
90% (tEOF, 3.9 min) to 60% (tEOF, 3.4 min), where the vis-cosity of the methanol-acetonitrile mixture reaches a mini-
Based on our previous results achieved in CEC  by
mum, thus altering the magnitude of the EOF in the oppo-
using a packed capillary with vancomycin-modified silica
site manner. It then decreased, reaching a minimum at
particles for the enantioresolution of basic compounds, an
aqueous-polar organic solvent mixture (5 mM ammoniumacetate pH 6/acetonitrile-methanol) was selected as
Figure 3.a shows the effect of organic modifier ratio on
mobile phase in order to study the influence of the organic
the retention factor (k) of the two pairs of enantiomers. As
modifier on the enantioresolution of fluoxetine and nor-
shown in the Figure, when the mobile phase contained
fluoxetine. The overall concentration of organic modifier in
0% MeOH (90% MeCN) the retention factors of the two
the mobile phase was always 90% (v/v) (x + y = 90%,
enantiomers of NFlx were higher than those of Flx enan-
where x and y were the concentrations of MeCN and
tiomers. On increasing the concentration of MeOH to 10
and 20%, a decrease of k was recorded for the two pairsof enantiomers. Further increase of MeOH concentrationin the mobile phase resulted in an increase of k for the fourstudied compounds. However, the highest values of theretention factor were achieved when the mobile phasecontained 90% of MeOH (0% MeCN). From the abovementioned data it is possible to conclude that i) at 50%MeOH the two pairs of enantiomers were moving very
Figure 4. Electrochromatogram of the separation of the two
close to each other, exhibiting very similar retention fac-
couples of fluoxetine and norfluoxetine enantiomers usingvancomycin CSP packed capillary and extended path length
tors, ii) at MeOH concentrations higher than 50% the elu-
UV detection cell. Experimental conditions: capillary 25 cm
tion order of Flx and NFlx was reversed, with Flx being
packed with vancomycin stationary phase; mobile phase,
eluted behind NFlx. Therefore, the composition of the
5% of 100 mM ammonium acetate pH 6/5% water/MeCN
mobile phase (organic modifier ratio) plays a very impor-
(55%, v/v)- MeOH (35%, v/v); applied voltage, 27 kV; capil-
tant role in determining both the chiral and the achiral
lary temperature, 208C; vials pressurized at 400 kPa duringthe CEC run. Electrokinetic injection 15 kV610 s of 0.5 lg/
separation. This change in selectivity could be related to
mL of each racemic compounds dissolved in water; the con-
the difference in dissociation constants and solvation of
centration of S-venlafaxine (S-Vx) was 0.25 lg/mL. For other
the analytes in the solvents of various compositions.
Furthermore, changes in pKa values of the studied com-pounds could not be excluded on modification of mixture
the two enantiomers of Flx from those of its metabolite we
composition, e. g., increase of MeCN concentration
investigated the effect of a different stationary phase com-
increased the separation capability of the stationary phase
position and length of capillary. Capillaries of 21, 23, and
of Flx and NFlx; on the other hand, raising the alcohol con-
25 cm length were packed with stationary phases contain-
centration, while increasing the enantioresolution of both
ing vancomycin-modified diol/silica (3 : 1) or with vanco-
racemic analytes, caused a decrease of their achiral reso-
Increasing the length of the packed bed did not improve
Resolution of the two pairs of fluoxetine and nor-fluoxetine
the chiral resolution of either racemic analyte; however,
enantiomers was achieved at any ratio of organic modifier
longer elution times were recorded. Satisfactory results
studied. Figure 3.b shows the effect of organic solvent
were obtained on packing the capillaries with vancomycin
ratio on the enantioresolution factor.
stationary phase only. However, longer retention timeswere observed due either to the greater amount of vanco-
Rs of both Flx and NFlx enantiomers increased on raising
mycin or to the slight reduction of the electroosmotic flow.
the MeOH concentration in the mobile phase up to 45%. Rs of Flx first decreased at 50 and 60% MeOH and then
The best results (optimum chiral and achiral resolution of
rose, reaching a maximum at 90%. In the case of NFlx, the
Flx and NFlx analytes) were obtained employing the capil-
enantioresolution decreased on increasing the MeOH
lary fully packed with vancomycin-modified diol particles
concentration up to 80%, showing a higher Rs value at
of 25 cm length and using a mobile phase containing 5%
90% MeOH. Therefore the highest enantioresolution was
of 100 mM aqueous ammonium acetate pH 6/5% water
achieved for both analytes in the absence of MeCN. The
and 90% of organic modifier (55% MeCN and 35%
higher enantioresolution achieved in the presence of only
MeOH). Figure 4 shows an electrochromatogram of the
MeOH for both racemic compounds is probably due to the
CEC separation of Flx and NFlx enantiomers using the
longer time spent by the enantiomers in contact with the
above described experimental conditions.
chiral stationary phase. Although the mobile phase with
The same standard mixture was analyzed ten times under
lower content of MeOH did not allow baseline resolution of
the same conditions, recording the electrochromato-
NFlx enantiomers, higher efficiency and shorter elution
grams, measuring the retention times, peak areas, and
times (data not shown) were observed.
calculating the enantioresolution factors (Rs). The stan-dard deviation (STD%) of retention time was found to be
Further experiments were performed in order to improve
1.2 and 1.1 for Flx and NFlx enantiomers, respectively.
the selectivity of the separation of the two pairs of enantio-
The enantioresolution factor STD% was 4.1 and 6.7% for
mers, changing the capillary temperature in the range
Flx and NFlx, respectively. The limit of detection (LOD,
15 – 308C. A temperature of 208C was selected for enan-
signal to noise ratio 3 : 1) was 25 ng/mL for both racemic
tiomeric separation because no substantial differences
Flx and NFlx while the limit of quantitation (LOQ, signal to
were noticed in comparison with the data obtained at low
noise ratio 10:1) was 50 ng/mL for each racemic analytes.
temperature. However, under these experimental condi-tions no satisfactory resolution of the two pairs of enantio-
The enantiomer elution order was verified by analyzing a
mers was achieved. Thus, in order to completely separate
standard mixture containing the R isomer of NFlx at a con-
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Ascoltaci, o Signore. 1 È veramente cosa buona e giusta in noi e renderci docili ascoltatori del Per la Chiesa: segua il suo maestro renderti grazie e innalzare a te l'inno c/da Ciuccafa Porto Empedocle nella povertà, nell’amore fino al dono di benedizione e di lode Dio onnipo- Confesso a Dio onnipotente e a voi, fratelli, che ho molto peccato in Per gli ammalati e i soffer