BIOMEDICAL CHROMATOGRAPHY
`Biomed. Chromatogr. 17: 113–117 (2003)
`Published online in Wiley InterScience (www.interscience.wiley.com).
`DOI: 10.1002/bmc.220
`
`ORIGINAL RESEARCH
`
`Enantioseparation of some clinically used drugs by HPLC
`using cellulose Tris (3,5-dichlorophenylcarbamate) chiral
`stationary phase
`
`Imran Ali and Hassan Y. Aboul-Enein*
`
`Pharmaceutical Analysis Laboratory, Biological and Medical Research Department (MBC-03-65), King Faisal Specialist Hospital and Research
`Center, PO Box 3354, Riyadh-11211, Saudi Arabia
`
`Received 8 July 2002; accepted 9 August 2002
`
`ABSTRACT: The chiral resolution of some clinically used drugs namely metoprolol, teratolol, tolamolol, nebivolol (b-adrenergic
`blockers), econazole, miconazole (anti-fungal agents), cromakalim (anti-hypertensive agent) and etodolac (anti-inflammatory agent)
`was achieved on cellulose tris (3,5-dichlorophenylcarbamate) chiral stationary phase. The mobile phase used was 2-propanol at
`0.5 mL/min with detection at 220 nm. The separation factors (a) of these drugs ranged from 1.24 to 3.90 while the resolution factors
`were from 1.05 to 5.0. The chiral recognition mechanisms between the racemates and the chiral selector are discussed. Copyright
`# 2003 John Wiley & Sons, Ltd.
`
`KEYWORDS: econazole; etodolac; cromakalim; metoprolol; nebivolol; miconazole; teratolol; tolamolol
`
`INTRODUCTION
`
`The different pharmacological activities of the enantio-
`mers have created an interest in studying the pharmaco-
`logical and toxicological properties of the enantiomers of
`drugs and agrochemicals (Stevenson and Wilson, 1988;
`Zief and Crane, 1988; Krstulovic, 1989a, 1989b; Allen-
`mark, 1991; Subramanian, 1994; Aboul-Enein and
`Wainer, 1997). Only about 20–25% of the optically
`active pharmaceuticals are sold and administrated as pure
`enantiomers. The US Food and Drug Administration has
`issued certain guidelines to pharmaceutical and agro-
`chemical industries to specify the enantiomeric purity of
`the optically active compounds (FDA Policy, 1992).
`Therefore, the enantiomeric resolution of optically active
`compounds became an urgent need of pharmaceutical,
`agrochemical and other chemical-based industries. Ac-
`cordingly, there is an increasing demand for the direct
`methods of chiral resolution of enantiomers of the
`optically active compounds. HPLC has been used in the
`last 15 years as the method of choice for chiral resolution
`(Zief and Crane, 1988; Krstulovic, 1989a, 1989b; Allen-
`mark, 1991; Subramanian, 1994; Aboul-Enein and
`Wainer, 1997). The development of the chiral stationary
`phases (CSPs) in HPLC has proved to be an effective
`
`*Correspondence to: H. Y. Aboul-Enein, Pharmaceutical Analysis
`Laboratory, Biological and Medical Research Department (MBC-03-
`65), King Faisal Specialist Hospital and Research Center, PO Box
`3354, Riyadh-11211, Saudi Arabia.
`E-mail: enein@kfshrc.edu.sa
`Abbreviations used: CSP, chiral stationary phase; RS, resolution
`factors.
`
`Copyright  2003 John Wiley & Sons, Ltd.
`
`racemic compounds.
`modality in the resolution of
`Various chiral columns have been used for the enantio-
`
`Figure 1. Chemical structure of cellulose Tris (3,5-dichlorol-
`phenylcarbamate) CSP.
`
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`114
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`ORIGINAL RESEARCH
`
`I. Ali and H. Y. Aboul-Enein
`
`Figure 2. Chemical structures of some of the clinically used drugs resolved in this study. The asterisk
`denotes the position of chiral carbon.
`
`racemates
`meric resolution of a wide variety of
`(Krstulovic, 1989a, 1989b; Allenmark, 1991; Subrama-
`nian, 1994; Aboul-Enein and Wainer, 1997). Among
`these, polysaccharide-based derivatives are currently the
`most useful chiral stationary phases in HPLC enantiose-
`partion because of their wide range of applications
`(Shibata et al., 1989; Subramanian, 1994; Aboul-Enein
`and Wainer, 1997; Beesley and Scott, 1998; Okamoto
`and Yashima, 1997). Recently, a new polysaccharide
`CSP namely cellulose Tris (3,5-dichlorophenylcarba-
`mate) (Fig. 1) was developed and coated on silica surface
`(Chankvetadze et al., 2000). The authors have also
`advocated the good chiral resolution capacity of this CSP.
`Furthermore, they explained the good chiral recognition
`ability because of its intact rigid linear and helical
`structure as it is in the coated form on silica gel. In view
`of this, we have tried the chiral resolution of some
`
`clinically used drugs (Fig. 2), namely metoprolol,
`teratolol, tolamolol, nebivolol (b-adrenergic blockers),
`econazole, miconazole (anti-fungal agents), cromakalim
`(anti-hypertensive agent) and etodolac (anti-inflamma-
`tory agent) on this CSP. The results of this study are
`presented herein.
`
`EXPERIMENTAL
`
`Chemicals and reagents. The racemic mixture of teratolol was
`supplied by Les Laboratories Servier, Gidy, France while
`tolamolol was obtained from Pfizer, Groton, CT, USA. The
`racemic [(‡)-RRRS and ()-SSSR] nebivolol
`(product no.
`R67555) was kindly supplied as gifts by Janssen Research
`Foundation, Beerse, Belgium. Racemic mixture [(‡)-3R,4R and
`()-3S,4S] of cromakalim was obtained from SmithKline
`
`Copyright  2003 John Wiley & Sons, Ltd.
`
`Biomed. Chromatogr. 17: 113–117 (2003)
`
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`Enantioseparation of drugs
`
`ORIGINAL RESEARCH
`
`115
`
`Table 1. The chromatographic parameters, retention factor
`(k), separation factor (a) and resolution factor (Rs) for
`enantiomeric resolution of some some clinically used drugs
`on cellulose Tris (3,5-dichlorophenylcarbamte) CSP using
`2-propranol as mobile phase at 0.5 mL/min
`
`Metoprolol
`Nebivolol
`Teratolol
`Tolamolol
`Econazole
`Miconazole
`Cromakalim
`Etodolac
`
`k1
`0.33
`0.46
`0.60
`1.38
`2.78
`4.05
`0.83
`0.11
`
`k2
`0.59
`0.79
`1.46
`1.76
`3.46
`5.00
`2.05
`0.43
`
`For details see Experimental section.
`
`a
`
`1.78
`1.72
`2.43
`1.28
`1.25
`1.24
`2.47
`3.90
`
`Rs
`
`1.10
`1.40
`1.80
`1.05
`1.72
`2.40
`5.00
`1.30
`
`Beecham (Frythe, Welwyn, UK). Econazole, miconazole, meto-
`prolol and metoprolol were purchased from Sigma Chemical Co.
`(St Louis, MO, USA). Etodolac was supplied by Wyeth-Ayerst,
`Maidenhead, Berks, UK. The solutions of the individual drugs
`(0.1 mg/mL) were prepared in ethanol. 2-Propanol of HPLC grade
`was purchased from Fisher Scientific (Fairlawn, NJ, USA). The
`absolute ethanol was obtained from E. Merck (Darmstadt,
`Germany).
`
`Chromatographic conditions. Aliquots of 20 mL of each of the
`solutions were injected onto an HPLC system consisting of Waters
`solvent delivery pump (model 510), Waters injector (model WISP
`710B), Waters tunable absorbance detector (model 484) and
`Waters integrator (model 740). The column used was cellulose Tris
`(3,5-dichlorolphenylcarbamate) (25 cm  0.46 cm; Fig. 1) coated
`on Daisogel SP-2000 (particle size 10 mm) and was kindly donated
`by Professor B. Chankvetadze. The mobile phase used in this study
`was 2-propanol, which was filtered and degassed before use. The
`flow rate of the mobile phase was 0.50 mL/min. The chart speed
`was kept constant at 0.1 cm/min. All the experiments were carried
`out at 23  1°C. The detection was carried out at 220 nm. The
`chromatographic parameters such as retention factor, separation
`factor and resolution factor were calculated.
`
`RESULTS AND DISCUSSION
`
`The chromatographic parameters, retention factor (k),
`separation factor (a) and resolution factor (Rs) for the
`resolved enantiomers of the reported drugs are given in
`Table 1. It may be observed from Table 1 that the best
`resolution was achieved for cromakalim. A typical
`chromatogram of cromakalim enantiomers on this new
`CSP is presented in Fig. 3. A variation in the
`chromatographic parameters was carried out to obtain
`the best resolution. To optimize the chromatographic
`conditions, methanol and ethanol were tried as the mobile
`phases. The mixtures of alcohols and alcohol–water were
`also tested but no good resolution could be achieved.
`
`Figure 3. Chromatogram showing the enantiomeric resolution
`of cromakalim on cellulose Tris (3,5-dichlorophenylcarba-
`mate) column using 2-propanol as the mobile phase at 0.5 mL/
`min flow rate.
`
`After experimentation the best chromatographic condi-
`tions were developed and are reported herein.
`The a values of these drugs ranged from 1.24 to 3.90
`while the Rs values were from 1.05 to 5.0. The resolution
`of these drugs was in the order cromakalim > miconazole
`> teratolol > econazole > nebivolol > etodolac > meto-
`prolol > tolamolol. This sort of
`resolution may be
`explained on the basis of the different magnitudes of
`the different types of bonds between racemates of these
`drugs and the CSP. The chiral recognition mechanism at
`a molecular level on the cellulose-based CSPs is still
`unclear, although it has been reported that the chiral
`resolution by these CSPs is achieved through hydrogen
`bonding, p–p and dipole–dipole induced interactions
`between the chiral stationary phase and the enantiomers
`of the analytes (Wainer and Alembic, 1986; Wainer et al.,
`1987; Yamamoto et al., 1999). The cellulose-based chiral
`stationary phases are the semi synthetic polymers, which
`
`Copyright  2003 John Wiley & Sons, Ltd.
`
`Biomed. Chromatogr. 17: 113–117 (2003)
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`116
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`ORIGINAL RESEARCH
`
`contain the polymeric chains of derivatized D-(‡)
`glucose residues in b-1,4 linkage and these chains lie
`side by side in a linear fashion. The structure of the
`reported drugs (Fig. 2) contains several electronegative
`atoms, namely nitrogen, oxygen, sulfur and chlorine
`along with aromatic rings. Therefore, resolution of the
`enantiomers of these drugs occurred due to the different
`hydrogen bonding and dipole–dipole induced interac-
`tions of different magnitudes between the electronegative
`atoms of the racemates and the reported cellulose chiral
`stationary phase (Fig. 1). It has also been reported
`(Wainer and Alembic, 1986; Wainer et al., 1987) that the
`p–p interactions between the substituted phenyl moieties
`of cellulose-based chiral
`stationary phase and the
`aromatic rings of the analytes play an important role in
`the chiral resolution mechanisms. The steric effect has
`also been observed to play a crucial role in chiral
`resolution (Aboul-Enein and Ali, 2001). Finally,
`the
`enantiomers of
`these drugs fit stereogenically in a
`different fashion into the chiral grooves of the reported
`stationary phase, which is stabilized by the different types
`of bonds mentioned above which result in the resolution
`of enantiomers.
`Basically, the chiral resolution of the racemates is
`controlled by the overall effect of bonds (as discussed
`above), steric effect and the pattern of fitting of the
`enantiomers in the chiral grooves. However, we have
`tried to explain the chiral resolution behavior of these
`drugs by considering the structures of the reported drugs
`and the possible bonds involved. The best resolution of
`cromakalim may be due to its small molecular size which
`experiences less steric force and hence has the maximum
`fitting in the chiral groove of the CSP. The better
`resolution of miconazole in comparison to econazole may
`be attributed to the stronger hydrogen bond in micona-
`zole. The stronger hydrogen bonding in miconazole may
`be due to one additional chlorine atom in miconazole.
`The retention times of the b-blockers were in the order
`tolamolol > teratolol > nebivolol > metoprolol. This or-
`der of retention times could be due to the increase of
`hydrogen and p–p bondings in the same order as the
`number of the electronegative atoms and phenyl rings
`increased in the same order except in nebivolol where the
`steric effect may be dominant.
`Furthermore, to ascertain the mechanisms of the chiral
`resolution, attempts have been made to resolve these
`drugs on the cellulose Tris (3,5-dimethylphenylcarba-
`mate) (Chiralcel OD), cellulose Tris (4-methylphenyl-
`carbamate) (Chiralcel OG), cellulose triphenylcarbamate
`(Chiralcel OC) and cellulose 4-chlorophenylcarbamate
`(Chiralcel OF) CSPs using the same experimental
`chromatographic conditions as described in the experi-
`mental section. No resolution or partial resolution was
`observed on these CSPs. Of course the above-mentioned
`CSPs contain various sites for hydrogen and p–p
`bondings but they showed no or poor chiral capability
`
`I. Ali and H. Y. Aboul-Enein
`
`in comparison to the cellulose Tris (3,5-dichlorol-
`phenylcarbamate) CSP. This could be due to their poor
`bonding capacities in comparison to the cellulose Tris
`(3,5-dichlorophenylcarbamate) column. Cellulose Tris
`(3,5-dichlorophenylcarbamate) CSP contains six chlorine
`atoms per unit of cellulose, and hence provide stronger
`hydrogen bonding. Therefore, it may be concluded that
`the hydrogen bonding is the major contributor for the
`chiral resolution on this polysaccharide-based CSP.
`
`CONCLUSION
`
`This study indicates the good chiral resolution capacity of
`cellulose Tris (3,5-dichlorophenylcarbamate) CSP for
`several chemically used drugs. The baseline chiral
`resolution of metoprolol, teratolol, tolamolol, nebivolol
`(b-adrenergic blockers), econazole, miconazole (anti-
`fungal agents), cromakalim (anti-hypertensive agent) and
`etodolac (anti-inflammatory agent) has been achieved on
`the reported CSP. Taking into consideration the results
`obtained, one can conclude that
`the enantiomeric
`resolution of these drugs on this chiral stationary phase
`is governed by hydrogen bondings. Besides, p–p, dipole–
`dipole induced interactions and steric effect also
`contribute towards chiral resolution. However,
`it
`is
`essential
`to mention here that
`the reported CSP has
`certain limitations as a mixture of hexane and alcohol
`(supposed to be suitable mobile phases in polysacchar-
`ides CSPs) cannot be used due to the solubility of this
`CSP in these mixtures. Therefore,
`to increase the
`application of the reported CSP some modifications are
`still
`required. Finally,
`the developed stereoselective
`HPLC method can be used for the resolution of the
`reported drugs on a semi preparative scale for further
`pharmacological investigations of the individual enan-
`tiomers.
`
`Acknowledgments
`
`The authors (I.A. and H.Y.A-E.) would like to thank the
`King Faisal Specialist Hospital and Research Center,
`Riyadh administration for their support for the Pharma-
`ceutical Analysis Laboratory Research Program. The
`authors are also thankful to Professor B. Chankvetadze,
`Institute of Pharmaceutical Chemistry, Mu¨nster, Ger-
`many for providing the chiral column.
`
`REFERENCES
`
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`Aboul-Enein HY and Wainer IW. The Impact of Stereochemistry on
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`
`Copyright  2003 John Wiley & Sons, Ltd.
`
`Biomed. Chromatogr. 17: 113–117 (2003)
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
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`IPR2016-00379
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`
`Enantioseparation of drugs
`
`ORIGINAL RESEARCH
`
`117
`
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`Copyright  2003 John Wiley & Sons, Ltd.
`
`Biomed. Chromatogr. 17: 113–117 (2003)
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