CHIRALITY 4:268-272 (1992)
Resolution of Terfenadine Enantiomers by P-Cyclodextrin Chiral Stationary Phase High-Performance Liquid Chromatography HENRI WEEMS AND KAVEH ZAMANI Llepartmmt of Pharmacology and Division of U h k a l Pharmacology, Uniformed Services University of the Health Sciences, F. Edward He'bert school of Medicine, Bethesda, Maryland
ABSTRACT Enantiomers of terfenadine were resolved by high-performanceliquid chromatography (HF'LC)using a chiral stationary phase (CSP)column packed with p-cyclodextrin (p-CD) covalently bound to silica. Separation was achieved in both the reverse phase and normal phase modes.Resolution of enantiomers was confirmed by ultraviolet-visible absorption, circular dichroism, and mass spectral analysis.
KEY WORDS: HPLC,chiral stationary phase, optical isomers, absoluteconfiguration,circular dichroism, chiral, enantiomers, terfenadine, cyclodextrin, normal phase
INTRODUCTION
Terfenadine (SeldaneB, Merrell Dow Pharmaceuticals Inc.) (Fig. 1)is a selective histamine H1-receptor antagonist used in the treatment of allergic rhinitis. It has been reported to have little affinity to H2-histamine receptors, a- or P-adrenergic receptors, and to have insignificant antiserotoninergic,anticholinergic, and a- or p-adrenergic activity.l Since it lacks CNS depressant activity, terfenadine has been shown to be a clinically effective antihistamine which has a less adverse sedative effect when compared with the classical histamine H1-receptor antagonists such as chlorpheniramine,diphenhydramine, promethazine, and triprolidine. Administered as a racemate, the drug has an asymmetric center at the a carbon (Fig. 1).Biotransformation of terfenadine in humans produces several metabolites2The major metabolite (metaboliteI), an oxidative product, retains the asymmetric carbon and antihistaminic activity (Fig. 1).Recently a stereoselective biotransformation of terfenadine to this major metabolitewas demonstrated by highperformance liquid chromatography (HF'LC). The enantiomeric resolution of terfenadine and the major metabolite were accomplished using a protein-based ovomucoid chiral stationary Phase (CSP). In this report the direct resolution of racemic terfenadine by some nonprotein-based CSPs was investigated. Other drugs possessing a hydroxyl group at the asymmetic a carbon have been resolved utilizing the carbohydrate-based p-cyclodextrin CSP.4 Here the resolution of terfenadine enantiomers is demonstrated in both the normal phase mode as well as in the reverse phase mode utilizing the p-cyclodextrinCSP. MATERIALS AND METHODS Materials Terfenadine was obtained as a racemateDI.[( = 0") from Sigma Chemical Co.(St. Louis, MO). The following chemicals were purchased: triethylamine, Fisher Scientific (Fair Lawn, NJ); glacial acetic acid, Mallinkrodt,Inc. (Paris, KY); and ammonium acetate, Sigma Chemical Co. (St. Louis, MO). Solvents @
1992 Wiley-Liss, Inc.
were HPLC grade [methanol (MeOH) and 2-propanol (IsOH), Baker Chemical, Phillipsburg, NJ; hexane, and acetonitrile (ACN),Mallinkrodt, Paris, KY; ethanol (EtOH), Midwest Grain Co., Perkin, IL]. High-PerformanceLiquid Chromatography
HPLC was performed using a Waters Associates (Milford, MA) Model 510 solvent delivery system, an Autochrom (Milford, MA) Model OPG/S gradient system, a Kratos (Ramsey, NJ) Model 757 variable absorbance detector set at 218-220 nm, a Valco (Houston, TX) Model C6U loop injector, and a HewlettPackard (Palo Alto, CA) Model 3390A integrator. Chiral stationary phase HPLC Resolution of enantiomeric terfenadine was carried out on chiral stationary phases CYCLOBOND I, CYCLOBOND II, or CYCLOBOND I SN columns, 4.6 mm x 250 mm, Advanced SeparationsTechnologies,Inc. (Astec)(Whippany,NJ), packed, respectively, with P-cyclodextrins (p-CD), y-cyclodextrins (yCD), or S-naphthylethyl carbamate derivatized p-cyclodextrins (P-CD-SN)bonded to 5 pm silica gel. Enantiomers were resolved on p-CD CSP columns using ethanol/acetonitrile/hexane (6.6/3.3/90, v:v:v) or ethanol/methanol/hexane (5/5/90, v:v:v) at a flow of 2 ml/min (1,ooO psi) in the normal phase mode. In the reverse phase mode elution was carried out with ammonium acetate (NH4Ac)(50 mM, pH 4,5,6,7, and 8)/methan01 (lo/%, v:v) or triethylamine acetate (TEAA) (0.1'30, pH 7)/methanol(l0/90,v:v) at a flow rate of 1ml/min (1,500 psi). Enantiomeric elution order in the different modes was established by chromatographing unequal mixtures of the two resolved enantiomers. Absolute stereochemistry was assigned from an earlier report.3 Received for publication October 8, 1991; accepted January 13, 1992. Address reprint requests to Henri B. Weems, Department of Pharmacology and Division of Clinical Pharmacology, Uniformed Services University of the Health Sciences, F. Edward H6bert School of Medicine, 4301 Jones Bridge Road, Beth&a, MD 208144799.
269
CYCLODEXTRIN RESOLUTION OF TERFENADINE ENANTIOMERS
TERFENADINE
A
/Oxidation
N-Demethylation
J
\
Ll
,d
METABOLITE I1
METABOLITE I
Fig. 1. Structure and metabolic transformation of terfenadine to two metabolites (modified from 2). Asymmetric a-carbon is indicated with asterisk
Spectral Analysis Mass spectral analysis was performed on a Finnigan 4OOO (San Jose, CA) gas chromatograph-massspectrometer with a Technivent 1050 (Maryland Heights, MO) data system. Samples were introduced by solid probe in the electron impact mode at 70 eV with a 250°C ionizer temperature or by a Vacumetrics DCI (Ventura, CA) desorption probe with a 150°C ionizer. Ultraviolet-visibleabsorption spectra of samples were determined using a 1 cm path length quartz cuvette with either a Varian Cary 11% (Palo Alto, CA) or SLM DW2OOO (Urbana, IL) W-VIS scanning spectrophotometer. Circular dichroism spectra of samples (in hexane) in a quartz cell of 1 cm path length were measured at ambient temperature with a Jasco 5OOA maston, MD) spectropolarimeter equipped with a Model DP500 data processor.
tem, TEAA, yielded a similar selectivity value (u)with a slight decrease in resolution due to broader peak width (Table l).5 Even though terfenadine contains two hydroxyl groups, its retention by reverse phase ODs columns appears quite nonpolar. The long retention time encountered in this mode has been shortened by use of the moderately retentive CN c01umns.~ This nonpolar characteristicmay be due in part to the nonpolar phenyldimethylethyl moiety at one end of the molecule and a nonpolar diphenyl group at the opposite end which may be shielding the smaller hydroxy group which shares the same carbon (Fig. 1).To this extent, terfenadine elutes early under normal phase conditions and may be a mode of choice for some studies. Bonded p-CD phases have been utilized in the normal phase mode of HF'LC for the separationof a variety of nonchiral compounds, yet application to chiral separations to date have shown little enantioselectivity. Recently, newer derivatized RESULTS AND DISCUSSION p-CD bonded phases have been synthesized and applied to CSP HPLC Separation of Enantiomers enantiomericseparation under normal phase conditions. It is The retention times, absolute configurations, and resolution believed that in the normal phase mode, the nonpolar hexane values of terfenadine enantiomers are listed in Table 1. Re- occupies the hydrophobic cavity of cyclodextrin thus blocking solved enantiomerswere confirmed by UV-VISabsorption,cir- the necessary inclusion complex. Use of isopropanol in hexcular dichroism, and mass spectra. Since some chiral why- ane, for example, produces a resolution value (RV) of 0 as seen droxy compounds have been resolved with p-CD, we utilized a from Fig. 2. In this case, as in earlier studies, it is likely that the similar NH4Ac buffered solvent ~ y s t e mAn . ~ investigation into less polar hexane occupies the p-CD cavity thus forcing separathe effect of buffer pH showed that increasing the pH would tions to occur on the outside surface of the cyclodextrin moleincrease the retention time with little increase in resolution cule resulting in no enantiomeric resolution. Ethanol, how(Table 2). Resolution was found to maximize at approximately ever, is known to be an effective solvent for displacing 10 to 20% NH4Ac buffer in methanol. Resolution was also substances from the p-CD cavity.5 In addition, ethanol has found to decrease with increasing flow rate as had previously been used in the normal phase mode for the resolution of been reported (Table 2).5 Use of a recommended solvent sys- enantiomers of some chiral compounds which could not be 6p7
y'7
270
WEEMS AND ZAMANI
TABLE 1. Cyclodextrin CSP-HPLC resolution of enantiomeric terfenadine
Retention time (min)c Solventb
CSPa
Rt,
Rt2
ud
RVe
Reverse Phase Mode (10% of solvent in methanol at 1 ml/min, 1,500 psi)/ p-CD TEAA (PH 7) p-CD NH,Ac (PH 7) NH,Ac (pH 7) 1-CD P-CD-SN NH,AC (pH 7)
8.9 (S) 10.9 (S) 3.5 4.1
9.6 (R) 11.9 (R) 3.5 4.1
1.15 1.16 1.00 1.00
0.62 0.76 0 0
Normal Phase Mode (10% of solvent in hexane at 2 ml/min, 1,OOO PsiY
p-CD p-CD p-CD p-CD p-CD yCD p-CD-SN
ISOH EtOH EtOHrMeOH (211) EtOHlMeOH (1:l) EtOHlACN (2:l) EtOH:ACN (2:l) EtOHlACN (2:l)
8.1 14.6 (S) 11.5 (S) 10.6 (S) 12.9 (S) 21.1 4.0
8.1 15.9 (R) 12.7 (R) 11.7 (R) 14.1 (R) 21.1 4.0
1.00 1.12 1.12 1.13 1.10 1.00 1.00
0 0.37 0.63 0.74 0.76 0 0
A
E
8 CI W
0 Z
s
a 0 r n m a
l C
0
8
16
',*CSPs and solvents are desaibed in Materials and Methods. CEnantiomers1 and 2 are designated by retention times, Rt, and Rt,, respectively. Absolute confimtion is based on ref. 3. du = Rt, - RtJRt, - Rb, where Rt,, is the unretained sample time. "V = resolution value = 2(V, - V,)/(W, + W,), where V is the retention volume and W is the peak width at base. fBackpressure in psi.
TABLE 2. Effect of pH and flow rate on the CSP-HPLC retention and resolution with p-CD
Fig. 2. Effect of differentalcohol mixtures in hexane on enantiomericresolution of terfenadine. (A) 10% isopropanol (RV = 0); (B)10%ethanol (RV= 0.37): (C)10% ethano1:methanol (2:1, v:v) (RV=O.63); @) 10% ethano1:methanol (1:1, v:v) (RV = 0.74). Flow rate in each was 2 ml/min.
Retention timeb
MeOH(%)"
flow
Rt,
Rt,
ciC
RVd
8
75 75 75 75 75
7 7 7
80 90 90
1 1 1 1 1 1 1 1.5
14.7 16.6 23.4 23.9 25.7 17.2 10.9 7.3
16.3 18.3 25.9 26.4 28.4 19.0 11.9 8.0
1.14 1.14 1.13 1.13 1.12 1.14 1.16 1.15
0.65 0.68 0.73 0.74 0.76 0.77 0.76 0.65
pH" 4 5 6 7
"NH4Ac (50 mM) buffer in methanol with a flow rate in ml/min. *Enantiomers1and 2 are designated by retention time, Rt, and Rt,, respectively. (u = RG - Rt,,/Rt, - Rto, where Rt,, is an unretained sample time. dRV = resolution value = 2(Vz - V,)/(W, + W,), where V is the retention volume and W is the peak width at base.
resolved under reverse phase conditions. Even though it is more polar, the increased solvent strength of ethanol in hexane provides an increase in resolution (RV = 0.4) as seen in Figure 2. Increasing amounts of methanol, as an organic modifier, have improved resolution of propranolol when using p-CD in capillary zone electrophoresis.l1 Methanol, however, is too polar and not readily soluble in hexane, yet it is soluble in ethanol which is soluble in hexane. By increasing the amount 9910
RETENTIONTIME (min)
of methanol in ethanol to equal portions, a normal phase resolution with a similar resolution value (RV = 0.74) to that found in the reverse phase mode was achieved (Fig. 2 and Table 1).The elution order was identical to that in the reverse phase mode. Increases in methano1:ethanolratios to 3:1decreased retention time; however, this did not improve resolution beyond that found with a ratio of 1:l.Acetonitrile like methanol is a polar solvent, not readily soluble in hexane, and is commonly used in place of methanol since it has a lower viscosity. Both ethanol and acetonitrile have been reported to have a greater aflinity for the cyclodextrin cavity than methanol.12 Use of a polar solvent system of ethano1:acetonitrilemixtures in a ratio of 2: 1 in hexane, originally developed for application to the Pirkle type chiral stationary phases, yielded a resolution (RV =0.76) similar to that found with the ethanol:methanol mixtures as seen in Table 1.l3 The larger cavity y-CD was tried under these solvent conditions, but gave no resolution in either mode (Table 1).This finding supports the concept that p-CD chiral interactions require a tight fit on the intersurface of the cyclodextrin molecule.8 As with the hydrophobic naphthylene end of propranolol, the nonpolar aromatic moiety, phenyldimethylethyl, of terfenadine is likely inserted into the nonpolar cavity of p-CD
CYCLODEXTRIN RESOLUTION OF TERFENADINE ENANTIOMERS
with potential binding sites likely around the entrance of the cavity close to the chiral center. l4 This is further supported by the fact that derivatization of the surface hydroxyls with (S)-1naphthylethyl isocyanate, p-CD-SN, blocks resolution and retention achieved with the native cyclodextin under identical chromatographicconditions in both the reverse phase and normal phase modes (Table 1). Lowering the organic modifier, EtOH:ACN (2:1), concentration from 10 to 2.5% in hexane increased normal phase retention to 15.8 min but resulted in only a slight indication of resolution (RV=O.l), likely due to some underivatized groups. Previous studies with this p-CDSN CSP have shown that incorporation of a stereogenic substituent often provides insight into chiral recognition mechanisms.l5 In some cases the substituent and p-CD combine synergistically, while in other cases the combination proves antagonistic.l5 In addition, it has been found that compounds which are easily resolved on p-CD, in the reverse phase mode, are generally not resolved on the naphthylethylcarbamatederivatized p-CD also noted in this example.16 Specific chiral interactions of the inclusion complex are often difficult to acertain; however, enantiodifferentation may be clarified through detailed X-ray studies.17 Recent gas phase calculations have shown that cyclodextrins are flexible rather than rigid and that the highly symmetrical truncated cone is really a time-averaged structure of a wide range of shapes.18 As with many complexes,the binding constant is often temperature dependent. Cyclodextrincomplexes are known to bind stronger at lower temperatures which is sometimes useful in increasing resolution in the reverse phase mode.5 A temperature comparison in the normal phase mode with p-CD at ambient temperature (23°C)and in an ice bath (OOC)showed not only an increase in resolution and retention time, but also an increase in back pressure associated with the lower temperature (Table 3). It is interesting to note that the higher backpressure resulting from this increase in viscosity did not exceed that found in the reverse phase mode at ambient temperature and a lower flow rate (Table 1).The increase in separation factor (01) at lower temperatures was greater than the change in resolution (RV) since a wide peak width due to band spreading associated with a longer retention time decreased the peak efficiency. The solvent used in this case was 10% MeOH:EtOH (1:l) in hexane since the same 10% concentration of EtO-
271
H:ACN (2:l)in hexane will separate into phases at lower temperatures. Column sample capacity was tested under several conditions in the normal phase mode utilizing 10% EtOH:ACN (2:1, v:v) in hexane. Using a stock solution of 1 pg/ul the resolution was unchanged up to 15 pg (15 pl). At 20 pg (20 pl) and above the resolution began to decrease until 50 pg (50 pl) where the resolution reached zero. Since the increased injection volume may account for some loss in resolution, a second tial of injections was made using increasing concentrations within a constant 10 pl volume. Resolution remained constant from 0.4 to 25 pg and began to decrease at 50 pg. While there are numerous examples of enantiomeric resolution ulilizing p-CD in the reverse phase mode, only a few chiral applications have been reported to date for the normal phase mode. This is likely the first example of dual mode resolution with this CSP and should be applicable to some other compounds. CONCLUSION
Terfenadine was directly resolved on p-CD CSP by reverse phase at pH 7. Resolution and elution order achieved in the reverse phase mode were duplicated in the normal phase mode after adjusting solvent conditions. With optimal solvation, the enantioselective mechanisms of j3-CD appear identical in both modes. Temperature interactionsreported for reverse phase are similar in the normal phase mode. The application of p-CD in the normal phase mode for other non-polar compounds which have been resolved in the reverse phase mode should be considered in future investigations. ACKNOWLEDGMENTS
The authors thank Drs. Yang, Conner, and Cantilena for helpful discussions. Dr. Leon Moore is acknowledged for technical assistance in computer graphics of HPLC chromatograms. This work was supported by USUHS Protocol R07502 to Dr. Shen K. Yang and FDA Project 224-88-3006to Drs. Dale P. Conner and Louis R. Cantilena, Jr.. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense, the Food and Drug Administration,or the Uniformed Services University of the Health Sciences. NOTE ADDED IN PROOF
TABLE 3. Effect of temperature on p-CD CSP-HPLC resolution Retention time (min). Temp
23'C O'C
Pressure
Flowb
Rt,
Rt,
ud
RV
1,OOO 1,500
2.0 2.0
10.9 22.0
12.1 26.6
1.14 1.22
0.74 0.79
%ackpressure in psi. bSolvent was 10% [methanol:ethanol (l:l,v:v)] in hexane. cEnantiomers 1 and 2 are designated by retention times, Rt, and R$, respectively. du = Rt, - Rt,,/Rt, - Rt,, where Rt,, is the unretained sample time. eRV = resolution value = 2(Vz - V,)/(W, + W,), where V is the retention volume and W is the peak width at base.
Subsequent to submission of this manuscript Chan et. al. reported in a short communication (J. Chromatogr. 57129297,1991)the reverse phase resolution of terfenadine on W D . Near baseline resolution was achieved at 44 min. with methanokO.Ol4M sodium perchlorate (75:25,v/v) at 0.2 ml/min. Attempts to separate the enantiomers of the carboxylic acid metabolite were unsucessful likely due to the inability of the polar acid group to interact with the CD cavity as had the p-tertbutylphenyl moiety of terfenadine. LITERATURE CITED 1. McTavish, D., Goa, K. L., Ferrill, M. Terfenadine:An updated review of its pharmacological properties and therapeutic efficacy. Drugs 39(4):552-574, 1990. 2. Garteiz, D. A., Hook,R. H., Walker, B. J., Okerholm, R. A. Phannacokinetics and bio!mnsformational studies of terfenadine in man. Armeim.-F0rsch.l Drug Res. 32(1I):11&1190,1982.
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WEEMS AND ZAMANl
3. Zamani, K.,Conner, D. P., Weems, H. B., Yang, S. K., Cantilena, L. R., Jr. Enantiomeric analysis of terfenadine in rat plasma by HPLC.Chirality 3467470,1991. 4. Walhagen, A., Edholm, L-E., Kennedy, EM., Xiao,L. C. Determination of terbutaline enantiomers in biological samples using liquid chromatography with coupled columns. Chirality 1:20-26,1989. 5. CYCLOBOND HANDBOOK A Guide to Using Cyclodextrin Bonded Phases.Advanced SeparationsTechnologiesInc., Whippany, NJ,19876 7 . 6. Chang, A. C., Wu, Q., Tan, L. Normal-phase high performance liquid chromatographic separations of positional isomers of substituted benzoic acids with amine and p-cyclodextrin bonded-phasecolumns. J. Chromatogr. 361:19%7,1986. 7. Armstrong, D.W., Stalcup, A.M., Hilton, M. L., Duncan. J. D., Faulkner, J. R., Chang, SC. Derivatized cyclodextrins for normal-phase liquid chromatographic separation of enantiomers.Anal. Chem. 62:161&1615,1990. 8. Armstrong, D.W., DeMond, W., Czech, B. Separation of metallocene enantiomers by liquid chromatography: Chml recognition via cyclodextrin bonded phases. Anal. Chem. 57481484,1985. 9. Macaudiere, P., Caude, M., Reset, R., Tambute, T. A note on use of various commercially available c h d stationary phases in supercritical fluid chromatography. In: Chml Separations ( P r d i n g s of the Chromatographic Society International Symposium on Chml Separations). Stevenson, D., Wilson, I. D. eds. New York Plenum Press, 1988:115120. 10. Nusser, E., Banerjee, A., Gal, J. Excavations in drug chirality: 1. Cyclothiazide. Chirality 3(1):2-13, 1991. 11. Fanali, S.Use of cyclodextrins in capillary zone electrophoresis.Resolution
of terbutaline and propranolol enantiomers. J. Chromatogr. 545437444, 1991. 12. Ward, T. J., Armstrong, D. W. Cyclodextrin-stationary phases. In: ChromatographicChiral Separations (ChromatographicScience, a Series of Monographs, Vol. 40). Zief, M., Crane, L. J., eds. New York Marcel Dekker, 1m 160. 13. Weems, H.B., Yang, S. K. Resolution of optical isomers by chiral highperformance liquid chromatography: Separation of dihydrdiols and tetrahydrodiols of berm$ajpyrene and w a h t h r a c e n e . Anal. Biochem. 125: 156-161.1982. 14. Armstrong,D. W., Ward, T. J., Armstrong, R. D., Beesley, T. E. Separation of drug stereoisomers by the formation of P-cyclodextrin inclusion complexes. Science 2321132-1135,1%6. 15. Stalcup, A. M., Chang, SC., Armstrong, D. W. Effect of the configuration of the substituents of derivatized p-cyclodextrin bonded phases on enantioselectivity in normal-phase liquid chromatography. J. Chromatogr. 540: 115-128.1991. 16. Armstrong, D. W., Chang, CD., Lee, S., H. (R) and (S) Naphthylethylcarbamate-substituted p-cyclodextrin bonded stationary phases for the reversed-phase liquid chromatographic separation of enantiomers. J. Chromatogr. 539KWO.1991. 17. Hamilton, J. A., Chen L. Crystal structure of an inclusion complex of P-cy clodextrin with racemic fenoprofen: Direct evidence for chiral recognition.J. Am. Chem. Soc. llOS33-5841,lW. 18. Lipkowib, K. B. Symmetry breaking in cycyodextrins: A molecular mechanics investigation. J. Org. Chem. 5663574267,1991.
Resolution of Terfenadine Enantiomers by P-Cyclodextrin Chiral Stationary Phase High-Performance Liquid Chromatography HENRI WEEMS AND KAVEH ZAMANI Llepartmmt of Pharmacology and Division of U h k a l Pharmacology, Uniformed Services University of the Health Sciences, F. Edward He'bert school of Medicine, Bethesda, Maryland
ABSTRACT Enantiomers of terfenadine were resolved by high-performanceliquid chromatography (HF'LC)using a chiral stationary phase (CSP)column packed with p-cyclodextrin (p-CD) covalently bound to silica. Separation was achieved in both the reverse phase and normal phase modes.Resolution of enantiomers was confirmed by ultraviolet-visible absorption, circular dichroism, and mass spectral analysis.
KEY WORDS: HPLC,chiral stationary phase, optical isomers, absoluteconfiguration,circular dichroism, chiral, enantiomers, terfenadine, cyclodextrin, normal phase
INTRODUCTION
Terfenadine (SeldaneB, Merrell Dow Pharmaceuticals Inc.) (Fig. 1)is a selective histamine H1-receptor antagonist used in the treatment of allergic rhinitis. It has been reported to have little affinity to H2-histamine receptors, a- or P-adrenergic receptors, and to have insignificant antiserotoninergic,anticholinergic, and a- or p-adrenergic activity.l Since it lacks CNS depressant activity, terfenadine has been shown to be a clinically effective antihistamine which has a less adverse sedative effect when compared with the classical histamine H1-receptor antagonists such as chlorpheniramine,diphenhydramine, promethazine, and triprolidine. Administered as a racemate, the drug has an asymmetric center at the a carbon (Fig. 1).Biotransformation of terfenadine in humans produces several metabolites2The major metabolite (metaboliteI), an oxidative product, retains the asymmetric carbon and antihistaminic activity (Fig. 1).Recently a stereoselective biotransformation of terfenadine to this major metabolitewas demonstrated by highperformance liquid chromatography (HF'LC). The enantiomeric resolution of terfenadine and the major metabolite were accomplished using a protein-based ovomucoid chiral stationary Phase (CSP). In this report the direct resolution of racemic terfenadine by some nonprotein-based CSPs was investigated. Other drugs possessing a hydroxyl group at the asymmetic a carbon have been resolved utilizing the carbohydrate-based p-cyclodextrin CSP.4 Here the resolution of terfenadine enantiomers is demonstrated in both the normal phase mode as well as in the reverse phase mode utilizing the p-cyclodextrinCSP. MATERIALS AND METHODS Materials Terfenadine was obtained as a racemateDI.[( = 0") from Sigma Chemical Co.(St. Louis, MO). The following chemicals were purchased: triethylamine, Fisher Scientific (Fair Lawn, NJ); glacial acetic acid, Mallinkrodt,Inc. (Paris, KY); and ammonium acetate, Sigma Chemical Co. (St. Louis, MO). Solvents @
1992 Wiley-Liss, Inc.
were HPLC grade [methanol (MeOH) and 2-propanol (IsOH), Baker Chemical, Phillipsburg, NJ; hexane, and acetonitrile (ACN),Mallinkrodt, Paris, KY; ethanol (EtOH), Midwest Grain Co., Perkin, IL]. High-PerformanceLiquid Chromatography
HPLC was performed using a Waters Associates (Milford, MA) Model 510 solvent delivery system, an Autochrom (Milford, MA) Model OPG/S gradient system, a Kratos (Ramsey, NJ) Model 757 variable absorbance detector set at 218-220 nm, a Valco (Houston, TX) Model C6U loop injector, and a HewlettPackard (Palo Alto, CA) Model 3390A integrator. Chiral stationary phase HPLC Resolution of enantiomeric terfenadine was carried out on chiral stationary phases CYCLOBOND I, CYCLOBOND II, or CYCLOBOND I SN columns, 4.6 mm x 250 mm, Advanced SeparationsTechnologies,Inc. (Astec)(Whippany,NJ), packed, respectively, with P-cyclodextrins (p-CD), y-cyclodextrins (yCD), or S-naphthylethyl carbamate derivatized p-cyclodextrins (P-CD-SN)bonded to 5 pm silica gel. Enantiomers were resolved on p-CD CSP columns using ethanol/acetonitrile/hexane (6.6/3.3/90, v:v:v) or ethanol/methanol/hexane (5/5/90, v:v:v) at a flow of 2 ml/min (1,ooO psi) in the normal phase mode. In the reverse phase mode elution was carried out with ammonium acetate (NH4Ac)(50 mM, pH 4,5,6,7, and 8)/methan01 (lo/%, v:v) or triethylamine acetate (TEAA) (0.1'30, pH 7)/methanol(l0/90,v:v) at a flow rate of 1ml/min (1,500 psi). Enantiomeric elution order in the different modes was established by chromatographing unequal mixtures of the two resolved enantiomers. Absolute stereochemistry was assigned from an earlier report.3 Received for publication October 8, 1991; accepted January 13, 1992. Address reprint requests to Henri B. Weems, Department of Pharmacology and Division of Clinical Pharmacology, Uniformed Services University of the Health Sciences, F. Edward H6bert School of Medicine, 4301 Jones Bridge Road, Beth&a, MD 208144799.
269
CYCLODEXTRIN RESOLUTION OF TERFENADINE ENANTIOMERS
TERFENADINE
A
/Oxidation
N-Demethylation
J
\
Ll
,d
METABOLITE I1
METABOLITE I
Fig. 1. Structure and metabolic transformation of terfenadine to two metabolites (modified from 2). Asymmetric a-carbon is indicated with asterisk
Spectral Analysis Mass spectral analysis was performed on a Finnigan 4OOO (San Jose, CA) gas chromatograph-massspectrometer with a Technivent 1050 (Maryland Heights, MO) data system. Samples were introduced by solid probe in the electron impact mode at 70 eV with a 250°C ionizer temperature or by a Vacumetrics DCI (Ventura, CA) desorption probe with a 150°C ionizer. Ultraviolet-visibleabsorption spectra of samples were determined using a 1 cm path length quartz cuvette with either a Varian Cary 11% (Palo Alto, CA) or SLM DW2OOO (Urbana, IL) W-VIS scanning spectrophotometer. Circular dichroism spectra of samples (in hexane) in a quartz cell of 1 cm path length were measured at ambient temperature with a Jasco 5OOA maston, MD) spectropolarimeter equipped with a Model DP500 data processor.
tem, TEAA, yielded a similar selectivity value (u)with a slight decrease in resolution due to broader peak width (Table l).5 Even though terfenadine contains two hydroxyl groups, its retention by reverse phase ODs columns appears quite nonpolar. The long retention time encountered in this mode has been shortened by use of the moderately retentive CN c01umns.~ This nonpolar characteristicmay be due in part to the nonpolar phenyldimethylethyl moiety at one end of the molecule and a nonpolar diphenyl group at the opposite end which may be shielding the smaller hydroxy group which shares the same carbon (Fig. 1).To this extent, terfenadine elutes early under normal phase conditions and may be a mode of choice for some studies. Bonded p-CD phases have been utilized in the normal phase mode of HF'LC for the separationof a variety of nonchiral compounds, yet application to chiral separations to date have shown little enantioselectivity. Recently, newer derivatized RESULTS AND DISCUSSION p-CD bonded phases have been synthesized and applied to CSP HPLC Separation of Enantiomers enantiomericseparation under normal phase conditions. It is The retention times, absolute configurations, and resolution believed that in the normal phase mode, the nonpolar hexane values of terfenadine enantiomers are listed in Table 1. Re- occupies the hydrophobic cavity of cyclodextrin thus blocking solved enantiomerswere confirmed by UV-VISabsorption,cir- the necessary inclusion complex. Use of isopropanol in hexcular dichroism, and mass spectra. Since some chiral why- ane, for example, produces a resolution value (RV) of 0 as seen droxy compounds have been resolved with p-CD, we utilized a from Fig. 2. In this case, as in earlier studies, it is likely that the similar NH4Ac buffered solvent ~ y s t e mAn . ~ investigation into less polar hexane occupies the p-CD cavity thus forcing separathe effect of buffer pH showed that increasing the pH would tions to occur on the outside surface of the cyclodextrin moleincrease the retention time with little increase in resolution cule resulting in no enantiomeric resolution. Ethanol, how(Table 2). Resolution was found to maximize at approximately ever, is known to be an effective solvent for displacing 10 to 20% NH4Ac buffer in methanol. Resolution was also substances from the p-CD cavity.5 In addition, ethanol has found to decrease with increasing flow rate as had previously been used in the normal phase mode for the resolution of been reported (Table 2).5 Use of a recommended solvent sys- enantiomers of some chiral compounds which could not be 6p7
y'7
270
WEEMS AND ZAMANI
TABLE 1. Cyclodextrin CSP-HPLC resolution of enantiomeric terfenadine
Retention time (min)c Solventb
CSPa
Rt,
Rt2
ud
RVe
Reverse Phase Mode (10% of solvent in methanol at 1 ml/min, 1,500 psi)/ p-CD TEAA (PH 7) p-CD NH,Ac (PH 7) NH,Ac (pH 7) 1-CD P-CD-SN NH,AC (pH 7)
8.9 (S) 10.9 (S) 3.5 4.1
9.6 (R) 11.9 (R) 3.5 4.1
1.15 1.16 1.00 1.00
0.62 0.76 0 0
Normal Phase Mode (10% of solvent in hexane at 2 ml/min, 1,OOO PsiY
p-CD p-CD p-CD p-CD p-CD yCD p-CD-SN
ISOH EtOH EtOHrMeOH (211) EtOHlMeOH (1:l) EtOHlACN (2:l) EtOH:ACN (2:l) EtOHlACN (2:l)
8.1 14.6 (S) 11.5 (S) 10.6 (S) 12.9 (S) 21.1 4.0
8.1 15.9 (R) 12.7 (R) 11.7 (R) 14.1 (R) 21.1 4.0
1.00 1.12 1.12 1.13 1.10 1.00 1.00
0 0.37 0.63 0.74 0.76 0 0
A
E
8 CI W
0 Z
s
a 0 r n m a
l C
0
8
16
',*CSPs and solvents are desaibed in Materials and Methods. CEnantiomers1 and 2 are designated by retention times, Rt, and Rt,, respectively. Absolute confimtion is based on ref. 3. du = Rt, - RtJRt, - Rb, where Rt,, is the unretained sample time. "V = resolution value = 2(V, - V,)/(W, + W,), where V is the retention volume and W is the peak width at base. fBackpressure in psi.
TABLE 2. Effect of pH and flow rate on the CSP-HPLC retention and resolution with p-CD
Fig. 2. Effect of differentalcohol mixtures in hexane on enantiomericresolution of terfenadine. (A) 10% isopropanol (RV = 0); (B)10%ethanol (RV= 0.37): (C)10% ethano1:methanol (2:1, v:v) (RV=O.63); @) 10% ethano1:methanol (1:1, v:v) (RV = 0.74). Flow rate in each was 2 ml/min.
Retention timeb
MeOH(%)"
flow
Rt,
Rt,
ciC
RVd
8
75 75 75 75 75
7 7 7
80 90 90
1 1 1 1 1 1 1 1.5
14.7 16.6 23.4 23.9 25.7 17.2 10.9 7.3
16.3 18.3 25.9 26.4 28.4 19.0 11.9 8.0
1.14 1.14 1.13 1.13 1.12 1.14 1.16 1.15
0.65 0.68 0.73 0.74 0.76 0.77 0.76 0.65
pH" 4 5 6 7
"NH4Ac (50 mM) buffer in methanol with a flow rate in ml/min. *Enantiomers1and 2 are designated by retention time, Rt, and Rt,, respectively. (u = RG - Rt,,/Rt, - Rto, where Rt,, is an unretained sample time. dRV = resolution value = 2(Vz - V,)/(W, + W,), where V is the retention volume and W is the peak width at base.
resolved under reverse phase conditions. Even though it is more polar, the increased solvent strength of ethanol in hexane provides an increase in resolution (RV = 0.4) as seen in Figure 2. Increasing amounts of methanol, as an organic modifier, have improved resolution of propranolol when using p-CD in capillary zone electrophoresis.l1 Methanol, however, is too polar and not readily soluble in hexane, yet it is soluble in ethanol which is soluble in hexane. By increasing the amount 9910
RETENTIONTIME (min)
of methanol in ethanol to equal portions, a normal phase resolution with a similar resolution value (RV = 0.74) to that found in the reverse phase mode was achieved (Fig. 2 and Table 1).The elution order was identical to that in the reverse phase mode. Increases in methano1:ethanolratios to 3:1decreased retention time; however, this did not improve resolution beyond that found with a ratio of 1:l.Acetonitrile like methanol is a polar solvent, not readily soluble in hexane, and is commonly used in place of methanol since it has a lower viscosity. Both ethanol and acetonitrile have been reported to have a greater aflinity for the cyclodextrin cavity than methanol.12 Use of a polar solvent system of ethano1:acetonitrilemixtures in a ratio of 2: 1 in hexane, originally developed for application to the Pirkle type chiral stationary phases, yielded a resolution (RV =0.76) similar to that found with the ethanol:methanol mixtures as seen in Table 1.l3 The larger cavity y-CD was tried under these solvent conditions, but gave no resolution in either mode (Table 1).This finding supports the concept that p-CD chiral interactions require a tight fit on the intersurface of the cyclodextrin molecule.8 As with the hydrophobic naphthylene end of propranolol, the nonpolar aromatic moiety, phenyldimethylethyl, of terfenadine is likely inserted into the nonpolar cavity of p-CD
CYCLODEXTRIN RESOLUTION OF TERFENADINE ENANTIOMERS
with potential binding sites likely around the entrance of the cavity close to the chiral center. l4 This is further supported by the fact that derivatization of the surface hydroxyls with (S)-1naphthylethyl isocyanate, p-CD-SN, blocks resolution and retention achieved with the native cyclodextin under identical chromatographicconditions in both the reverse phase and normal phase modes (Table 1). Lowering the organic modifier, EtOH:ACN (2:1), concentration from 10 to 2.5% in hexane increased normal phase retention to 15.8 min but resulted in only a slight indication of resolution (RV=O.l), likely due to some underivatized groups. Previous studies with this p-CDSN CSP have shown that incorporation of a stereogenic substituent often provides insight into chiral recognition mechanisms.l5 In some cases the substituent and p-CD combine synergistically, while in other cases the combination proves antagonistic.l5 In addition, it has been found that compounds which are easily resolved on p-CD, in the reverse phase mode, are generally not resolved on the naphthylethylcarbamatederivatized p-CD also noted in this example.16 Specific chiral interactions of the inclusion complex are often difficult to acertain; however, enantiodifferentation may be clarified through detailed X-ray studies.17 Recent gas phase calculations have shown that cyclodextrins are flexible rather than rigid and that the highly symmetrical truncated cone is really a time-averaged structure of a wide range of shapes.18 As with many complexes,the binding constant is often temperature dependent. Cyclodextrincomplexes are known to bind stronger at lower temperatures which is sometimes useful in increasing resolution in the reverse phase mode.5 A temperature comparison in the normal phase mode with p-CD at ambient temperature (23°C)and in an ice bath (OOC)showed not only an increase in resolution and retention time, but also an increase in back pressure associated with the lower temperature (Table 3). It is interesting to note that the higher backpressure resulting from this increase in viscosity did not exceed that found in the reverse phase mode at ambient temperature and a lower flow rate (Table 1).The increase in separation factor (01) at lower temperatures was greater than the change in resolution (RV) since a wide peak width due to band spreading associated with a longer retention time decreased the peak efficiency. The solvent used in this case was 10% MeOH:EtOH (1:l) in hexane since the same 10% concentration of EtO-
271
H:ACN (2:l)in hexane will separate into phases at lower temperatures. Column sample capacity was tested under several conditions in the normal phase mode utilizing 10% EtOH:ACN (2:1, v:v) in hexane. Using a stock solution of 1 pg/ul the resolution was unchanged up to 15 pg (15 pl). At 20 pg (20 pl) and above the resolution began to decrease until 50 pg (50 pl) where the resolution reached zero. Since the increased injection volume may account for some loss in resolution, a second tial of injections was made using increasing concentrations within a constant 10 pl volume. Resolution remained constant from 0.4 to 25 pg and began to decrease at 50 pg. While there are numerous examples of enantiomeric resolution ulilizing p-CD in the reverse phase mode, only a few chiral applications have been reported to date for the normal phase mode. This is likely the first example of dual mode resolution with this CSP and should be applicable to some other compounds. CONCLUSION
Terfenadine was directly resolved on p-CD CSP by reverse phase at pH 7. Resolution and elution order achieved in the reverse phase mode were duplicated in the normal phase mode after adjusting solvent conditions. With optimal solvation, the enantioselective mechanisms of j3-CD appear identical in both modes. Temperature interactionsreported for reverse phase are similar in the normal phase mode. The application of p-CD in the normal phase mode for other non-polar compounds which have been resolved in the reverse phase mode should be considered in future investigations. ACKNOWLEDGMENTS
The authors thank Drs. Yang, Conner, and Cantilena for helpful discussions. Dr. Leon Moore is acknowledged for technical assistance in computer graphics of HPLC chromatograms. This work was supported by USUHS Protocol R07502 to Dr. Shen K. Yang and FDA Project 224-88-3006to Drs. Dale P. Conner and Louis R. Cantilena, Jr.. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense, the Food and Drug Administration,or the Uniformed Services University of the Health Sciences. NOTE ADDED IN PROOF
TABLE 3. Effect of temperature on p-CD CSP-HPLC resolution Retention time (min). Temp
23'C O'C
Pressure
Flowb
Rt,
Rt,
ud
RV
1,OOO 1,500
2.0 2.0
10.9 22.0
12.1 26.6
1.14 1.22
0.74 0.79
%ackpressure in psi. bSolvent was 10% [methanol:ethanol (l:l,v:v)] in hexane. cEnantiomers 1 and 2 are designated by retention times, Rt, and R$, respectively. du = Rt, - Rt,,/Rt, - Rt,, where Rt,, is the unretained sample time. eRV = resolution value = 2(Vz - V,)/(W, + W,), where V is the retention volume and W is the peak width at base.
Subsequent to submission of this manuscript Chan et. al. reported in a short communication (J. Chromatogr. 57129297,1991)the reverse phase resolution of terfenadine on W D . Near baseline resolution was achieved at 44 min. with methanokO.Ol4M sodium perchlorate (75:25,v/v) at 0.2 ml/min. Attempts to separate the enantiomers of the carboxylic acid metabolite were unsucessful likely due to the inability of the polar acid group to interact with the CD cavity as had the p-tertbutylphenyl moiety of terfenadine. LITERATURE CITED 1. McTavish, D., Goa, K. L., Ferrill, M. Terfenadine:An updated review of its pharmacological properties and therapeutic efficacy. Drugs 39(4):552-574, 1990. 2. Garteiz, D. A., Hook,R. H., Walker, B. J., Okerholm, R. A. Phannacokinetics and bio!mnsformational studies of terfenadine in man. Armeim.-F0rsch.l Drug Res. 32(1I):11&1190,1982.
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WEEMS AND ZAMANl
3. Zamani, K.,Conner, D. P., Weems, H. B., Yang, S. K., Cantilena, L. R., Jr. Enantiomeric analysis of terfenadine in rat plasma by HPLC.Chirality 3467470,1991. 4. Walhagen, A., Edholm, L-E., Kennedy, EM., Xiao,L. C. Determination of terbutaline enantiomers in biological samples using liquid chromatography with coupled columns. Chirality 1:20-26,1989. 5. CYCLOBOND HANDBOOK A Guide to Using Cyclodextrin Bonded Phases.Advanced SeparationsTechnologiesInc., Whippany, NJ,19876 7 . 6. Chang, A. C., Wu, Q., Tan, L. Normal-phase high performance liquid chromatographic separations of positional isomers of substituted benzoic acids with amine and p-cyclodextrin bonded-phasecolumns. J. Chromatogr. 361:19%7,1986. 7. Armstrong, D.W., Stalcup, A.M., Hilton, M. L., Duncan. J. D., Faulkner, J. R., Chang, SC. Derivatized cyclodextrins for normal-phase liquid chromatographic separation of enantiomers.Anal. Chem. 62:161&1615,1990. 8. Armstrong, D.W., DeMond, W., Czech, B. Separation of metallocene enantiomers by liquid chromatography: Chml recognition via cyclodextrin bonded phases. Anal. Chem. 57481484,1985. 9. Macaudiere, P., Caude, M., Reset, R., Tambute, T. A note on use of various commercially available c h d stationary phases in supercritical fluid chromatography. In: Chml Separations ( P r d i n g s of the Chromatographic Society International Symposium on Chml Separations). Stevenson, D., Wilson, I. D. eds. New York Plenum Press, 1988:115120. 10. Nusser, E., Banerjee, A., Gal, J. Excavations in drug chirality: 1. Cyclothiazide. Chirality 3(1):2-13, 1991. 11. Fanali, S.Use of cyclodextrins in capillary zone electrophoresis.Resolution
of terbutaline and propranolol enantiomers. J. Chromatogr. 545437444, 1991. 12. Ward, T. J., Armstrong, D. W. Cyclodextrin-stationary phases. In: ChromatographicChiral Separations (ChromatographicScience, a Series of Monographs, Vol. 40). Zief, M., Crane, L. J., eds. New York Marcel Dekker, 1m 160. 13. Weems, H.B., Yang, S. K. Resolution of optical isomers by chiral highperformance liquid chromatography: Separation of dihydrdiols and tetrahydrodiols of berm$ajpyrene and w a h t h r a c e n e . Anal. Biochem. 125: 156-161.1982. 14. Armstrong,D. W., Ward, T. J., Armstrong, R. D., Beesley, T. E. Separation of drug stereoisomers by the formation of P-cyclodextrin inclusion complexes. Science 2321132-1135,1%6. 15. Stalcup, A. M., Chang, SC., Armstrong, D. W. Effect of the configuration of the substituents of derivatized p-cyclodextrin bonded phases on enantioselectivity in normal-phase liquid chromatography. J. Chromatogr. 540: 115-128.1991. 16. Armstrong, D. W., Chang, CD., Lee, S., H. (R) and (S) Naphthylethylcarbamate-substituted p-cyclodextrin bonded stationary phases for the reversed-phase liquid chromatographic separation of enantiomers. J. Chromatogr. 539KWO.1991. 17. Hamilton, J. A., Chen L. Crystal structure of an inclusion complex of P-cy clodextrin with racemic fenoprofen: Direct evidence for chiral recognition.J. Am. Chem. Soc. llOS33-5841,lW. 18. Lipkowib, K. B. Symmetry breaking in cycyodextrins: A molecular mechanics investigation. J. Org. Chem. 5663574267,1991.
