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SEPARATION OF OXAZEPAM
ENANTIOMERS BY CYCLODEXTRIN
MODIFIED MICELLAR ELECTROKINETIC
CHROMATOGRAPHY
GABRIEL HANCU1*, ATTILA GÁSPÁR 2, ÁRPÁD GYÉRESI 1
University of Medicine and Pharmacy, Târgu Mureş, Faculty of
Pharmacy, Department of Pharmaceutical Chemistry, 540139 Târgu
Mureş, Romania
2
University of Debrecen, Department of Inorganic and Analytical
Chemistry, Debrecen, Hungary
*
corresponding author: g_hancu@yahoo.com,
1
Abstract
Oxazepam is a chiral 3-hydroxy-1,4-benzodiazepine (BZD) derivative,
frequently used in therapy as anxiolytic. The applicability of micellar electrokinetic
chromatography (MEKC) using cyclodextrines (CDs) as additive for the separation of
oxazepam enantiomers has been studied. Using a buffer solution containing 25 mM sodium
tetraborate, 50 mM sodium dodecyl sulphate (SDS), 20 mM hydroxypropyl-β-CD and 10%
methanol (pH-9.3) we managed the baseline separation of the two enantiomers. The
resolution of the separation was improved by decreasing the capillary temperature and by
adding methanol to the buffer solution.
Rezumat
Oxazepamul este un derivat chiral de 3-hidroxi-1,4-benzodiazepină (BZD),
utilizat frecvent în terapie ca anxiolitic. În lucrarea de faţă s-a studiat aplicabilitatea
electroforezei capilare micelare (ECM) folosind ca aditiv ciclodextrina (CD), în separarea
enantiomerilor oxazepamului. Am rezolvat separarea celor doi enatiomeri utilizând un
tampon format din 25 mM tetraborat de sodiu, 50 mM dodecil sulfat de sodiu, 20 mM
hidroxipropil-β-ciclodextrină şi 10% metanol (pH-9,3). Rezoluţia separării a fost
îmbunătăţită prin scăderea temperaturii şi prin adăugarea de metanol la soluţia tampon.
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oxazepam
micellar electrokinnetic capillary chromatography
cyclodextrines
enantioseparation
INTRODUCTION
The most widely used anxiolytic drugs are the benzodiazepine
(BZD) derivatives. Besides their main application as anxiolytics, some
BZDs have also been used as sedatives, hypnotics, anticonvulsants, muscle
relaxants and general anesthetics [6].
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It is well known that 3-hydroxy-1,4-BZD derivatives (lorazepam,
oxazepam, temazepam) are optically active, having a chiral center in the C3
position. Pure 3-OH-1,4-BZDs are difficult to isolate, because they are
quickly racemized in aqueous medium. BZDs are clinically used in racemic
forms, despite the fact that the pharmacological activity of the two
enantiomers is different, the activity being restricted only to the enantiomers
with (S) configuration [2, 6, 12].
Closely linked with the great therapeutic importance of the BZDs
are their analytical problems. The aim of this paper was to elaborate a
simple, sensitive and rapid method for the enantiomers separation of
oxazepam, the most frequently used 3-OH-1,4-BZD using capillary
electrophoresis (CE).
Oxazepam (fig. 1) is an officinal compound in the major modern
pharmacopoeias (European Pharmacopoeia 6th edition [14], United States
Pharmacopoeia 30 [15], British Pharmacopoeia 2007 [13]), and is a very
widely used anxiolytic, being regarded as the prototype for the 3-OH-1,4BZD derivates. Oxazepam is also the ultimate pharmacologically active
metabolite of many 1,4-BZD derivatives, and it is metabolized to the
inactive glucuronide [6].
O
H
N
OH
Cl
N
Figure 1
Oxazepam chemical structure
Capillary electrophoretic techniques have been applied in
pharmaceutical analysis with great success. Capillary electrophoresis (CE)
is an officinal method in European Pharmacopoeia 6th edtition 6 [14], which
proves to be an alternative or complementary technique for the
chromatographic separation techniques (high-performance liquid
chromatography - HPLC), playing a major role in the separation of chiral
compounds, including here the 1,4-BZD derivatives, as it is stated by
several specific reviews [3,7,10]. The approaches used in CE, are relatively
simple compared to HPLC methods in which expensive, chiral stationary
phases are frequently used.
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Oxazepam is a lipophilic (log P – 2.24) crystalline substance,
neutral from electrophoretic point of view. It possesses weak basic
properties due to the nitrogen atom in the 4 position that can be protonated
(pKa - 1.7), but exhibits also weak acid properties due to the acidifiation
(pKa – 11.6) of the unsubstituted nitrogen atom in position 1 [4,11].
Taking into consideration the aspects mentioned above, a good
separation of its enantiomers by conventional capillary zone electrophoresis
(CZE), which is based on the differences between the electrophoretic
mobilities of the analytes, cannot be expected. The best method for the
electrophoretic separation proved to be micellar electrokinetic
chromatograpy (MEKC), which extends the application range of CE to
neutral molecules, such as BZDs. MEKC is based on a micellar
“pseudostationary phase” added to the buffer solution, which interacts with
the analytes according to partitioning mechanisms, in a chromatography-like
mode. In this system, the electroosmotic flow (EOF) acts as the
chromatographic “mobile phase” [1].
The separation of enantiomers by MEKC is achieved mainly using a
direct separation method, where the chiral selector is simply added to the micellar
solution. Cyclodextrines (CDs) are by far the most frequently used chiral additive
in MEKC, as they are relatively cheap, water-soluble and have low UV activity,
which permits using low UV wavelengths for sensitive detection [5].
CDs are cyclic oligosaccharides with truncated cylindrical
molecular shapes, having an external hydrophilic surface, and a
hydrophobic cavity, in which they tend to include compounds by
hydrophobic interactions. The inclusion mechanism is sterically selective,
because analytes must fit the size of the cavity, the diameter of which
depends on the number of glucose units in the CD structure (6,7, 8 for α, β,
γ-CDs, respectively). Because of the chirality of the hydroxyls in the
glucose molecules, which form the rim of the CD cavity, the inclusion
complex formation will be chirally selective. CD will not interact with the
micelles due to the hydrophilic nature of the outer surface and will migrate
with the same velocity as the bulk solution [5, 7].
MATERIALS AND METHODS
Instrumentation
The CE instrument was a HP 3DCE model (Agilent, Waldbronn,
Germany). In all measurements hydrodynamic sample introduction was
used for injecting samples, at the anodic end of the capillary, by applying a
pressure of 50 mbar for 2 seconds. Separations were performed using a
polyimide-coated fused-silica capillary of 64,5 cm length (effective length
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56 cm) x 50μm I.D. (CS-Chromatographie, Langerwehe, Germany). The
applied voltage was + 25 kV. The detection was carried out by on-column
spectophotometric measurements at 214 nm. The electrophoregrams were
recorded and processed by Chemstation software 7.01 version (Agilent).
Chemicals and samples
Reagents of analytical grade were purchased from various
distributors: β-CD, hydroxylpropyl - β-CD (Cyclolab, Budapest, Hungary),
sodium dodecyl sulfate (SDS) (Fluka), sodium tetraborate (Reanal,
Hungary), sodium hydroxide solution 0.1 M (Fluka). Oxazepam was
purchased from Terapia, Romania.
Oxazepam is a hydrophobic substance, insoluble in water;
consequently the samples were prepared by dissolving the substance in
methanol, and then diluting the solution with water (50:50).
The capillaries were preconditioned with the buffer electrolyte for
5 minutes. After the daily work, the capillaries were flushed with NaOH 0.1
M for 5 minutes, water for 5 minutes, and buffer solution for another 5
minutes. Prior to CE measurements, all the samples and buffers were
filtered through a 0.45 m syringe filter, and the samples were stored in the
refrigerator at + 40C.
RESULTS AND DISCUSSION
During initial attempts at the MEKC analysis of a set of eight
frequently used BZD derivatives by MEKC [14] using hydroxypropyl-β-CD
(HP-β-CD) as additive, we observed in the case of oxazepam a peak
splitting. As oxazepam was the only chiral BZD analyzed it was obvious
that chiral selectivity could be the reason for the peak splitting. Using a
buffer containing 25 mM sodium tetraborate, 50 mM SDS and 20 mM
hydroxypropyl-β-CD (pH – 9.3), we managed to separate the two oxazepam
enantiomers. It is interesting that peak splitting didn’t occure when we used
β-CD as buffer additive.
HP-β-CD is a derivatized β-CD obtained by modification of the
hydroxyl groups on the rim of the CD at position 2,3 and 6 of each glucose
unit. The derivatized CDs have less rigid structures than the natural ones,
and exhibit higher stereoselectivity [5].
Since the separation factor of enantiomeric pairs is small, the
separation conditions must be carefully optimized in order to maximize the
resolution. This was achieved using a longer capillary, a higher voltage, or
by expanding the migration time window.
The migration time of oxazepam increased with the increase of the
borate concentration, because the electroosmotic flow (EOF) decreases with
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the increase of ionic strength. However, higher ionic strength is responsible
for the increase in current that can cause a reduction in efficiency. The
migration time of oxazepam also increased with increasing SDS
concentration, due to the solubilization of the solute into the micellar phase.
Increasing the temperature leads to an increase in racemization
speed, while a decrease in temperature will lead to a better chiral
recognition (fig. 2). Furthermore, a change of this parameter can influence
the stability of the inclusion complex formed during the electrophoretic run.
Figure 2
Oxazepam chiral separation by CD-MEKC
a) at 300C; b) at 250C c) at 200C;
(Buffer: 25 mM borate, 50 mM SDS, 20 mM hydroxypropyl β-CD, separation conditions:
capillary 64.5 cm x 50 μm I.D., buffer: 25 mM borate, 50 mM SDS, pH: 9.2, voltage +25
kV, detection: UV absorption at 214 nm).
The addition of methanol enhances solubility and improves the
resolution of the separation. Migration times increased with the increase in
the methanol concentration, caused by the reduction of EOF. We managed
the baseline separation of the two enantiomers by adding 10% methanol to
the buffer solution (fig. 3).
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Figure 3
Oxazepam chiral separation by CD-MEKC using a buffer containing: a) 25 mM
borate, 50 mM SDS, 20 mM hydroxypropyl β-CD; b) 25 mM borate, 50 mM SDS,
20 mM hydroxypropyl β-CD, 10% methanol
(separation conditions: capillary 64.5 cm x 50 μm I.D., buffer: 25 mM borate, 50 mM SDS,
pH: 9.2, voltage +25 kV, temperature 200C, detection: UV absorption at 214 nm)
Knowing the separation mechanism in chiral analysis by CE is of
paramount importance for the selection of the appropriate chiral selector, in
order to find the optimum experimental conditions.
As mentioned above, an important requirement for inclusion–
complexation is that the analyte fits into the CD. Having fused aromatic
rings in the molecule, α-CD cannot be selected to form inclusion complexes
with BZDs, β-CD can be selected even if not the entire BZD molecule will
fit in the cavity, and γ-CD could be the appropriate one. For the BZDs
separation by MEKC β- or γ-CD can be choose [5,8].
CDs have a relatively hydrophobic cavity, able to guest the whole
molecule or a part of it, consequently the main mechanism of the CDfunction involves inclusion-complexation of the analytes. Secondary bonds
between the analyte and the hydroxyl or modified hydroxyl groups from the
rim of the CD, can stabilize the inclusion complexes formed and
considering that the primary and secondary hydroxyl groups are bound to
asymmetric carbons, these interactions can be stereoselective and thus
responsible for the separation of enantiomeric compounds [11].
Partition of a hydrophobic analyte like oxazepam takes place
between the bulk solution, the CD and the negatively charged SDS micelle,
which migrates in the opposite direction of EOF, thus retaining the analyte
[1].
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Chiral resolution results from stereospecific interactions of the
chiral selector molecules displaying different affinities for the two
enantiomers of oxazepam generating a difference in the respective migration
velocities under the applied electric field. The migration velocity of the
complex CD-oxazepam will differ from that of the free molecule, caused by
the bigger size of the complex [11].
CONCLUSIONS
In the pharmaceutical industry, the determination of optical purity
and separation of enantiomers is becoming increasingly important. Highresolution separation methods are required to achieve chiral separations.
In summary, we studied in detail the MEKC conditions for the
separation of oxazepam. Using a buffer solution containing 25 mM sodium
tetraborate, 50 mM SDS , 20 mM hydroxypropyl-β-CD (pH – 9.3) and 10%
methanol, we managed the baseline separation of the two enantiomers. The
usefulness of CD-modified MEKC as an analytical tool for the qualitative
separation of BZD enantiomers was demonstrated. We believe that the
method may be helpful for the separation of other 3-OH-1,4-BZD
derivatives enantiomers.
Acknowledgments
Our work was supported by the CEEPUS program (CII-HU-001001-0607 network).
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