Improvement of Capillary Electrophoretic Enantioseparation of Fluoxetine by a Cationic Additive .

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by  Sattary Javid, Farin

Iranian Journal of Pharmaceutical Research (2013), 12 (supplement): 71-76 Copyright © 2013 by School of Pharmacy
Received: January 2013 Shaheed Beheshti University of Medical Sciences and Health Services
Accepted: January 2013
Original Article
Improvement of Capillary Electrophoretic Enantioseparation of
Fluoxetine by a Cationic Additive
Farin Sattary Javida,b, Alireza Shafaatia,c* and Afshin Zarghia,c
aPharmaceutical Chemistry Department, School of Pharmacy, Shahid Beheshti University
of Medical Sciences, Tehran, Iran. bStudents Research Committee, School of Pharmacy, Shahid
Beheshti University of Medical Sciences, Tehran, Iran. cPharmaceutical Sciences Research
Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Abstract
One of the problems encountered in CE separations of basic compounds is the adsorption
of analytes in negatively charged capillary wall which could lead to poor repeatability of
migration time and peak area. Additionally, separation of enantiomers of chiral of basic drugs
is commonly carried out in low pH buffer which contributes to strong ionic interaction of
the cationic drug ions with negatively charged chiral selectors. The two phenomena results in
poor enantioseparations. To overcome the problems associated with chiral separations of basic
drugs by CE, the effect of guanidine (GU) on the improvement of chiral separation of a model
basic drug, fluoxetine (FLX), was investigated. In the present study, GU was used as a cationic
additive to the running buffer containing a chiral selector, sulfated beta cyclodextrine. Better
results obtained with GU as the buffer additive in enantioseparation of FLX.
Keywords: Capillary electrophoresis; Chiral separation; Fluoxetine; Guanidine.
Introduction enantioseparations offers high efficiency and
high flexibility with regard to the separation
More than 30% of drugs in market are chiral conditions, as well as the low consumption
(1) and mostly two enantiomers of a chiral drug of chemicals and solvents. Consequently, it is
show different pharmacological, toxicological or shown that the major application of the technique
pharmacokinetic properties (2). is chiral separations (4). However, CE has some
In addition, separation methods are required limitations, especially for chiral and non-chiral
for quality control of single-enantiomer resolution of basic drugs.
pharmaceutical raw materials and products Basic analytes are positively charged over a
during their production, storage and application wide range of pH (especially in acidic solutions)
(3). Thus, different analytical techniques have and electrostatically attracted to the negatively
been developed for controlling synthesis, charged capillary walls. The irreproducible
enantiomer purity check and pharmacodynamic adsorption may cause variations to migration
studies of chiral drugs. time and peak area (5), and consequently results
Capillary electrophoresis (CE) as a in lower precision. Furthermore, impurity peaks
powerful technique, especially for analytical may be masked by the peak tailing or resolution
of enantiomers, in which two analytes migrating
* Corresponding author: closely after each other may deteriorate.
E-mail: [email protected] Additionally, analyte absorption could raise
Sattary Javid F et al. / IJPR (2013), 12 (supplement): 71-76
the limit of detection (LOD) and limit of +
H2 H2
quantification (LOQ) of analysis. O C N
Different approaches have been applied to
avoid undesirable interactions of the analytes
with the capillary wall, including the use of
F3C
high salt concentration, extremes of pH, buffer
.HCI
additives, and coated capillaries (5).
In the challenge of developing a separation
Figure 1. Chemical structure of fluoxetine HCl.
method by capillary electrophoresis (CE),
a variety of additives may be added to the
background electrolyte to improve the separation
(6) the most used additives are nonionic additive Experimental
namely organic modifiers (7-8) and urea (9-10).
The highly ionic triethylamine could optimize Materials
the resolution of separation of peptides (11). All solutions were prepared in Nanopure
Our previous work (12) has shown that the 18MΩ ultrapure water (Barnstead, Chicago,
addition of guanidine (GU) to the running IL, USA). Sodium phosphate buffer with
buffer containing sulfated-b-cyclodextrin (SB- pH range of 2-4 was prepared from sodium
CD) as a chiral selector increased the resolution monohydrogen phosphate purchased from
of enantiomers of basic chiral compounds. In Merck (Darmstadt, Germany), and adjusting the
the course of our ongoing interest in chiral pH with orthophosphoric acid purchased from
separations, the present study was conducted to the same company, sulfated β-cyclodextrin
investigate the influence of GU on the SB-CD (Sigma, Louis, MO, USA), were used as
mediated enantioseparations of fluoxetine. received. GU HCl was obtained from Merck
Fluoxetine (FLX), as an antidepressant, is a (Darmstadt, Germany), Racemic FLX (as
selective serotonin reuptake inhibitor which is HCl) was kindly gifted by Noor Research and
less sedating compared to tricylic antidepressants Education Institute (Tehran, Iran).
and has fewer antimuscarinic and cardiotoxic
effects (13). FLX (Figure 1) is a basic drug CE system
which contains tertiary amine nitrogen (pK of All experiments were performed on a
a
10.3) (14) that is positively charged in neutral Prince-C650 CE system equipped with UV/
and lower pHs. This drug includes one chiral visible detector BISCHOFF (lambda 1010). CE
center within its structures. (R)-fluoxetine and analysis was performed using untreated fused-
(S)-fluoxetine are similarly effective at blocking silica capillary of 50 μm internal diameter and
serotonin reuptake. As these enantiomers are 75-cm total length (25-cm effective length). The
metabolized differently, the use of R-enantiomer capillary was conditioned prior to the first use
was expected to result in less variable plasma by rinsing with 1 M NaOH for 15 min, followed
levels of fluoxetine and its active metabolites by 0.1 M NaOH and water for 5 min each.
compared to that observed with racemic Between runs, the capillary was flushed for 3
fluoxetine. Additionally, it is shown that (R)- min with running buffer to guarantee the good
fluoxetine and its metabolites inhibit CYP2D6, reproducibility. The electrophoretic integration
a cytochrome P450 system enzyme, to a lesser was performed by Dax Data Acquisition Analysis
extent than (S)-fluoxetine and its metabolites software (version 8.0).
(15-16).
Few reports on the application of CE methods Capillary electrophoretic conditions
were found in the literature for separation of FLX The applied voltage was - 15 kV at 25°C, and
enantiomers (17-18). These methods presented the migrant species were detected at 230 nm.
the enantioseparation in acidic condition using The running buffer was a 50 mM phosphate
combined neutral and negatively charged buffer adjusted at pH = 2-4 by 1 M sodium
cyclodextrins. hydroxide. The running buffer contained 1-4%
72
Improvement of Capillary Electrophoretic Enantioseparation of
Y
0.06
0.05
2 4 6 8 10
Time (min)
Figure 2. Separation of FLX enantiomers by CE. Experimental conditions: running buffer phosphate 25 mM (pH = 2.5) containing 3%
SB-CD; wavelength, 230 nm; voltage, -15 kV; sample FLX 0.1 mg/mL, other condition as detailed in experimental section.
sulfated beta-cyclodextrine as chiral selector of the selector led to the deterioration of the
and guanidine (40-100 mM) as buffer additive, enantioseparation.
which was freshly prepared daily and filtered
through a 0.45 µm filter membrane. Concentration of the cationic additive
Sample injection was performed To improve chiral separation of the two
hydrodynamically by applying 50 mbar of enantiomers of FLX, different concentration of
pressure for 12 s. GU over a range of 20-100 mM was added to the
running buffer. As shown in Figure 3, baseline
Sample preparations separation of the enantiomers was obtained at any
Standard stock solutions of racemic fluoxetine GU concentration. At higher GU concentrations,
containing 1 mg/mL were prepared by dissolving better peak shapes were observed. But to avoid
standard powder in water. working solutions Joule heating, GU concentration was adjusted to
were prepared by diluting stock solution of 80 mM which resulted in a reasonable separation
fluoxetine with water. and peak shapes, as well as minimum current
and Joule heating.
Results and Discussion
Running buffer pH and concentration
Optimization of the method Buffer pH is one of the most important factors
Concentration of the selector in CE as it may affect the charge of the analytes
Based on our previous works on and the chiral selector, and thus the binding
enantioseparation of basic drugs with CE (19- characteristics (21). In order to obtain the
20), the initial attempt was based on applying optimum results, the buffer pH and concentration
a reversed-mode CE with the pH adjusted was reconsidered. Figure 4 shows the effect of
to 2.5 using a 25 mM phosphate buffer as the buffer pH on the enantioseparation of FLX, with
running buffer. The SB-CD concentration was SB-CD and GU concentration kept constant at
studied over a range of 1-4% with maximum their best. Baseline resolution of the two peaks
resolution achieved at 3% of SB-CD (see Figure with acceptable peak shapes was observed at
2). Lower concentrations of the selector resulted pH of 3. At this pH, the electroosmotic flow
in very broad peak, whilst higher concentrations was low enough to allow applying carrier-mode
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Sattary Javid F et al. / IJPR (2013), 12 (supplement): 71-76
Y
0.04
0.02
10 12 14
Time (min)
Figure 3. The effect of GU concentration on resolution of the two enantiomers of FLX Experimental conditions as described in Figure 2.
separation, whilst ionization of FLX was high obtained at the optimum conditions, i.e. 80
enough to render the formation of complex with mM GU (as HCl) and 3% SB-CD in 25 mM
negatively charged SB-CD molecules. phosphate buffer at pH of 3.0 with applied
Despite better peak shapes obtained with voltage of - 15 kV.
higher buffer concentrations, to avoid Joule
heating, buffer concentration was kept at 25 GU Effects on enantioseparation
mM. Besides, GU was used as hydrochloride salt The mechanism of chiral separation using
which in turn contributed to the buffer strength. CDs is based on the different inclusion of the
hydrophobic part of the enantiomers into the
Optimized resolution CD cavity. The stereoselectivity is enhanced by
Figure 5 shows the final electropherogram secondary interactions between the functional
Y
0.07
pH=2.5
pH=3.0
0.06
pH=4.0
0.05
5 10 15
Time (min)
Figure 4. The effect of buffer pH on resolution of the two enantiomers of FLX Experimental conditions as described in Figure 2 (GU
concentration was 80 mM).
74
Improvement of Capillary Electrophoretic Enantioseparation of
6 8 10 12
Time (min)
Figure 5. Resolution of the FLX enantiomers at optimum conditions
groups of the analyte with the hydroxyl (or with the silanol groups on the capillary inner
derivetized hydroxyl) groups on the outside wall and with the negatively charged sulfate
rim of the CD (22). As shown in Figure 2, in moieties on the CD molecules.
the absence of GU, complete resolution of two
enantiomers of FLX was not achieved which Acknowledgment
was attributed to the strong interaction of FLX
cations with the sulfate groups on the CD We wish to thank the Research Deputy of
molecule. School of Pharmacy, Shahid Beheshti University
GU is a basic compound which bears positive of Medical Sciences (SBMU) for the financial
charge in acidic solutions. The positive charge on support of this work. The authors also appreciate
the molecule is dispersed and delocalized (24). Noor Research and Education Institute for kind
Thus, it is expected that the cation interacts with gift of racemic-FLX.
silanol groups on the inner surface of the bared
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This article is available online at http://www.ijpr.ir
the mechanism of activity–dependant sodium channel
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