A Sulfated Cyclodextrin Chiral Stationary Phase for High-Performance

Anal. Chem. 1996, 68, 1369-1374

A Sulfated Cyclodextrin Chiral Stationary Phase for High-Performance Liquid Chromatography Apryll M. Stalcup* and Kyung H. Gahm

Department of Chemistry, 2545 The Mall, University of HawaiisManoa, Honolulu, Hawaii 96822 A new sulfated β-cyclodextrin (degree of substitution ≈ 13-14/cyclodextrin) bonded chiral stationary phase (CSP) for high-performance liquid chromatography is introduced. The novel CSP was used to resolve a number of enantiomeric pairs, including antihistamines, antidepressants and phenylhydantoins, using HPLC under a variety of mobile phase conditions. Among the 33 analytes successfully enantioresolved, all but six had some amine functionality. Generally, the best separations were obtained for analytes in which the stereogenic center was either incorporated in a ring system or positioned between two aromatic rings. Retention seemed to arise from either inclusion complexation or electrostatic interactions. Ionic strength and pH were found to influence chiral recognition. Cyclodextrins have been used extensively for enantiomeric separations by high-performance liquid chromatography (HPLC),1,2 thin-layer chromatography (TLC),3 and capillary zone electrophoresis (CZE).4 While the number of analytes enantioresolvable by HPLC using native cyclodextrin-based chiral stationary phases (CSPs) is quite extensive,5 Stalcup et al.6,7 have demonstrated the usefulness of functionalizing the cyclodextrin to expand the range of compounds enantioresolvable by cyclodextrins immobilized on HPLC supports. The hydroxypropyl-β-cyclodextrin CSP was one of the first of the functionalized cyclodextrin-derived CSPs6 shown to enantioresolve a number of compounds that were unresolvable by the native cyclodextrin. The enhanced chiral recognition of the hydroxypropyl-β-cyclodextrin was attributed to the extension of the hydrogen-bonding capabilities at the mouth of the cavity with a more flexible group and decreasing the cavity size with the hydroxypropyl group at the 2 position. In some cases, the configuration of the hydroxypropyl group also seemed to play a role, particularly for compounds in which the stereogenic center might be some distance from the mouth of the cyclodextrin cavity in the included complex. * Current address: Department of Chemistry, P.O. Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172. (1) Armstrong, D. W.; Han, S. M.; Han, Y. I. Anal. Biochem. 1987, 167, 261264. (2) Armstrong, D. W.; Ward, T. J.; Armstrong, R. D.; Beesley, T. E. Science 1986, 232, 1132-1135. (3) Armstrong, D. W.; He, F.-Y.; Han, S. M. J. Chromatogr. 1988, 448, 345354. (4) Ward, T. J. Anal. Chem. 1994, 66, 632A. (5) Han, S. M.; Armstrong, D. W. In Chiral Separations by HPLC; Krstulovı´c, A. M., Ed.; Ellis Horwood Limited: Chichester, England, 1989; pp 208284. (6) Stalcup, A. M.; Chang, S. C.; Armstrong, D. W.; Pitha, J. J. Chromatogr. 1990, 513, 181-194. (7) Stalcup, A. M.; Chang, S. C.; Armstrong, D. W. J. Chromatogr. 1991, 540, 113-128. 0003-2700/96/0368-1369$12.00/0

© 1996 American Chemical Society

Figure 1. Effect of pH on retention ([, 9) and enantioselectivity (+) for DL-glutethimide. Mobile phase, 20% acetonitrile/buffer (100 mM ammonium acetate).

Some of the earliest chiral separations by HPLC under nonaqueous mobile phase conditions on a functionalized cyclodextrin-derived CSP were obtained on the naphthylethylcarbamoylated-β-cyclodextrin (NEC-CD) bonded phase.7 In this case, the addition of the substituent also introduced an additional stereogenic center. Indeed, chromatographic studies using the (R-), (S-), and racemic NEC-β-CD CSP in the normal phase mode revealed that, in many but not all cases, chiral recognition had contributions from both the chirality of the cyclodextrin and the chirality of the carbamate substituent.7 Armstrong et al.8 advanced the “multimodal” character of these NEC-β-CD CSPs because of their ability to exhibit unique enantioselectivity under very diverse mobile phase conditions. In CZE, cyclodextrins are generally added to the background electrolyte as chiral additives. The low solubility of the native β-cyclodextrin, the most widely applicable cyclodextrin in liquid chromatography, has brought about the common use of functionalized cyclodextrins, such as methylated9,10 and hydroxypropylated cyclodextrin.11 However, because neutral cyclodextrins cannot enantioresolve neutral analytes and enantioselectivity is enhanced (8) Armstrong, D. W.; Chang, C. D.; Lee, S. H. J. Chromatogr. 1991, 539, 8390. (9) Sepaniak, M. J.; Cole, R. O.; Clark, B. K. J. Liq. Chromatogr. 1992, 15, 1023-1040. (10) Soini, H.; Riekkola, M. L.; Novotny, M. V. J. Chromatogr. 1992, 608, 265274. (11) Rawjee, Y. Y.; Vigh, G. Anal. Chem. 1994, 66, 619-627.

Analytical Chemistry, Vol. 68, No. 8, April 15, 1996 1369

Table 1. Chromatographic Data Obtained Using a Sulfated Cyclodextrin Chiral Stationary Phase for Various Analytes in Which the Stereogenic Center Is Incorporated in a Ring compound

structure

ketamine

O

k′ a

R

mobile phaseb

12.97

1.08

20% MeCN/buffer (100 mM)

5.02

1.13


5.73 3.22 1.30

1.20 1.18 1.09

50% MeCN/buffer (50 mM) 70% MeCN/buffer (50 mM) 70% MeCN/buffer (167 mM)

2.26

1.12


6.63

1.06


2.35

1.67


1.93

2.03


4.16

1.22


1.17

1.93


0.89

2.01


1.04

1.40


2.28

1.31


1.85

1.41


NHCH3

Cl

tetramisole N

S N

piperoxan O

N

CH2

O

idazoxan

N

NH

O O

tranylcypromine

NH2 C6H5

lorazepam

H

O

N

H OH

N

Cl

Cl

oxazepam

H

O

N N

Cl

nefopam

H OH

C6H5 O

N CH3

aminoglutethimide

O

NH

O C2H5

NH2

glutethimide

O

NH

O C2H5

mephenytoin

CH3 O

N

O

HN

5-(4-hydroxyphenyl)-5phenylhydantoin

C2H5

O HN

NH OH

O

5-cyclobutyl-5phenylhydantoin

O HN

NH

O

1370 Analytical Chemistry, Vol. 68, No. 8, April 15, 1996

Table 1. (Continued) compound

structure

phensuximide

CH3 O

N

k′ a

R

mobile phaseb

0.98

1.16


4.25 2.39

1.09 1.07

50% MeCN/buffer (50 mM) 70% MeCN/buffer (167 mM)

1.82

1.12


O

C6H5

1-aminoindane NH2

5-(4-methylphenyl)-5phenylhydantoin

O HN

NH CH3

O

a

Capacity factor of the first-eluting enantiomer. b MeCN, acetonitrile; MeOH, methanol; buffer, ammonium acetate, pH 6.

if the electrophoretic mobilities of the analyte and the chiral additive are in opposite directions, sulfated cyclodextrins have also been used.12-14 Interestingly, sulfated cyclodextrin is the only known functionalized cyclodextrin investigated as an independent therapeutic agent.15 Recently, we reported16,17 the application of mixtures of randomly substituted sulfated cyclodextrins (degree of substitution ≈ 7-10) as chiral additives for the enantioseparation of over 70 compounds of pharmaceutical interest, including anesthetics, antiarrhythmics, antidepressants, anticonvulsants, antihistamines, antihypertensives, antimalarials, catecholamines, relaxants, and bronchodilators. The compounds enantioresolved by these sulfated cyclodextrins included not only neutral, cationic, and anionic analytes but atropisomers as well. The CZE results suggested that the association between the analytes and the cyclodextrin could arise from several potential interations, including inclusion complexation and electrostatic or ion-pairing interactions. The diversity of structural features present in solutes successfully enantioresolved in these earlier studies suggested that an analogous CSP might prove equally advantageous. The current report describes the results of an investigation of the chiral recognition of a sulfated cyclodextrin bonded CSP. A variety of mobile phase conditions and analytes were used. Mobile phase parameters investigated included type and concentration of organic modifier, ionic strength, and pH. Although ionpairing18,19 has been used effectively for chiral separations by LC, we believe that this is one of the first reports of a CSP offering chiral ion-exchange capability. EXPERIMENTAL SECTION Materials. The analytes were obtained from either Sigma Chemical Co. (St. Louis, MO) or the US Pharmacopoeia (Be(12) Tait, R. J.; Thompson, D. O.; Stobaugh, J. F. Anal. Chem. 1994, 66, 40134018. (13) Wu, W.; Stalcup, A. M. J. Liq. Chromatogr. 1995, 18, 1289-1315. (14) Lurie, I. S.; Klein, R. F. X.; Dal Cason, T. A.; LeBelle, M. J.; Brenneisen, R.; Weinberger, R. E. Anal. Chem. 1994, 66, 4019-4026. (15) Folkman, J.; Weisz, P. B.; Joullie´, M. M.; Li, W. W.; Ewing, W. R. Science 1989, 243, 1490-1493. (16) Stalcup, A. M.; Gahm, K. H. Anal. Chem., in press. (17) Gahm, G. H.; Stalcup, A. M. Chirality, in press. (18) Pettersson, C.; Schill, G. J. Liq. Chromatogr. 1986, 9, 269-290. (19) Duncan, J. D.; Armstrong, D. W.; Stalcup, A. M. J. Liq. Chromatogr. 1990, 13, 1091-1103.

thesda, MD). Ammonium acetate, HPLC grade water, acetonitrile, methanol, acetic acid, and triethylamine were all obtained from Fisher Scientific. Chromatographic Bonded Phase. The bonded sorbent was prepared using a modification of a method developed by Stalcup et al.7 Elemental analysis was performed by Galbraith Laboratories, Inc. (Knoxville, TN). The bonded sorbent was slurry-packed into a 250 mm × 4.6 mm stainless steel column. Confirmation of derivatization reproducibility was carried out by Advanced Separation Technologies (Whippany, NJ). Equipment. The HPLC system used for these experiments consisted of a Shimadzu LC-600 and a SPD-6A UV detector interfaced to a Chromatopac CR-501 data station. The flow rate was typically 0.5 mL/min. Supporting evidence for chiral separation was supplied by repeating the separation with detection accomplished at different wavelengths. Mobile phases were prepared volume/volume, and the various mobile phase compositions are given in the tables. RESULTS AND DISCUSSION Bonding Results. According to the elemental analysis data, the amount of sulfur present in the bonded phase (1.89%) corresponded20 to an average substitution of 13-14 sulfate groups/ cyclodextrin. Chromatographic Results. Chromatographic results for a variety of structurally diverse compounds are included in Tables 1-3. It should be noted that the separations for the compounds included in the tables are not optimized for each of the compounds. Rather, the data presented in the tables were selected to demonstrate the versatility of this new CSP as well as the variety of conditions under which enantiomeric separations can be obtained. For instance, chiral separations were obtained under predominantly aqueous conditions (e.g., 10% acetonitrile/buffer for oxazepam, lorazepam, and assorted hydantoins, Table 1) as well as under “magic mobile phase” 21 conditions (e.g., 88.4% acetonitrile/8.9% methanol/1.8% acetic acid/0.9% triethylamine for pindolol and propranolol, Table 3). (20) Berendsen, G. E.; Pikaart, K. A.; de Galan, L. J. Liq. Chromatogr. 1978, 3, 561. (21) Armstrong, D. W.; Chen, S.; Chang, C.; Chang, S. J. Liq. Chromatogr. 1992, 15, 545-556

Analytical Chemistry, Vol. 68, No. 8, April 15, 1996

1371

Figure 3. Chromatogram of the enantioseparations obtained for hydroxychloroquine (1,1′) and chloroquine (2,2′). Mobile phase, 50% acetonitrile/methanol (50 mM ammonium acetate).

Figure 2. Chromatograms illustrating the effect of pH on the chiral separations of DL-glutethimide (1,1′) and DL-aminoglutethimide (2,2′) at (a) pH 7.0 and (b) pH 4.2. Mobile phase, 30% acetonitrile/buffer (50 mM ammonium acetate).

In general, the overall highest selectivities were observed for analytes in which the stereogenic center was incorporated in a ring (Table 1). Compounds in which the stereogenic center was substituted by two aromatic rings had slightly less overall selectivity (Table 2), while the compounds in which the chiral carbon was more distant from the aromatic moieties had the lowest overall selectivity (Table 3). For those analytes for which enantioseparation was obtained under predominantly aqueous conditions, this may support an inclusion complexation type of mechanism and be consistent with results obtained on a native β-cyclodextrin bonded phase.22,23 However, as noted previously, conditions were not optimized for each analyte. Effect of pH. The presence of the sulfate substituents on the cyclodextrin allows ion-exchange or electrostatic interactions in addition to the more traditional inclusion complexation usually invoked when considering stereospecific interactions with cyclodextrins. Figure 1 illustrates the effect of pH on the retention and enantioselectivity for aminoglutethimide. The pKa for an aromatic amine is expected to be 4-5. Thus, it is reasonable that the retention of aminoglutethimide should decrease rapidly between pH 4 and 6 as the protonated form begins to lose ascendancy. However, as can be seen from Figure 1, as retention increases, the selectivity decreases. Evidently, the subtle differences in the association of the two enantiomers with the cyclodextrin become overwhelmed as the Coulombic or ionic interactions begin to dominate. Figure 2, which further shows the effect of pH on the retention and the enantioseparations of glutethimide and aminoglutethimide, illustrates how pH may be exploited to optimize a given separation. (22) Han, S. M.; Han, Y. I.; Armstrong, D. W. J. Chromatogr. 1988, 441, 376381. (23) Armstrong, D. W.; Han, Y. I.; Han, S. M. Anal. Chim. Acta 1988, 208, 275281.


Figure 4. Effect of the concentration of ammonium acetate on retention (solid lines) and enantioresolution (broken lines) for hydroxychloroquine (b) and chloroquine ([). Mobile phase, 50% acetonitrile/methanol.

As can be seen from Figure 2, lowering the pH from 7.0 to 4.2 decreases the selectivity for both compounds and decreases the retention for glutethimide while increasing the retention for aminoglutethimide. Given the pH/retention behavior for glutethimide shown in Figure 1, it is not too surprising that the retention of aminoglutethimide is more dramatically affected by lowering the pH than that of glutethimide. The concurrent decreased retention for glutethimide as the pH is lowered may be attributable to competition from the acetic acid for the cyclodextrin or the cyclodextrin cavity. It is important to note that aminoglutethimide required gradient elution to effect enantioseparation (R ) 1.03 using acetonitrile/buffer) on the native β-cyclodextrin column.5 Previously, glutethimide was reportedly enantioresolved on a 2,6-dimethylphenyl isocyanate-derivatized β-cyclodextrin column (R ) 1.10 using normal phase conditions)8 and on an (S)-NEC-β-cyclodextrin column (R ) 1.10 using an acetonitrile/buffer mobile phase).24 In either case, the sulfated cyclodextrin column offers superior enantioselectivity (R ) 1.93 for aminoglutethimide and 2.01 for glutethimide; Table 1). Effect of Ionic Strength. Chloroquine and hydroxychloroquine were virtually unenantioresolvable on a native β-cyclodextrin (24) Armstrong, D. W.; Stalcup, A. M.; Hilton, M. L.; Duncan, J. D.; Faulkner, J. R., Jr.; Chang, S. C. Anal. Chem. 1990, 62, 1610-1615.

Table 2. Chromatographic Data Obtained Using a Sulfated Cyclodextrin Chiral Stationary Phase for Various Analytes in Which the Stereogenic Center Is Positioned between Two Ring Systems compound

structure

pheniramine

CH2CH2N(CH3)2

k′ a

R

mobile phaseb

6.08

1.06


5.74

1.10


4.98

1.06


4.48

1.12


3.88

1.00


2.38

1.14


3.96

1.13


N CH

brompheniramine

CH2CH2N(CH3)2 N CH

chlorpheniramine

Br

CH2CH2N(CH3)2 N CH

carbinoxamine

Cl

OCH2CH2N(CH3)2 N CH

doxylamine

Cl

OCH2CH2N(CH3)2 N C CH3

orphenadine

OCH2CH2N(CH3)2 CH CH3

chlorcyclizine

N CH

Cl

N N CH3 a

Capacity factor of the first-eluting enantiomer. b MeCN, acetonitrile; MeOH, methanol; buffer, ammonium acetate, pH 6.

Figure 5. Effect of the molar concentration of ammonium acetate on retention for DL-aminoglutethimide (+) and clenbuterol (b, 9). Separate symbols are used for the individual clenbuterol enantiomers because of the proximal data points on the graph. Mobile phase, 15% acetonitrile/buffer (pH 6.5).

CSP (data not shown) but are easily enantionresolvable on the sulfated β-cyclodextrin column, as can be seen in Figure 3. Figure 4 depicts the relationship between the ammonium acetate concentration and retention or resolution for chloroquine and hy-

droxychloroquine. It is important to note that both the chromatogram depicted in Figure 3 and the data shown in Figure 4 were obtained under nonaqueous reversed-phase conditions. As can be seen from Figure 4, retention for both analytes decreased as the ammonium acetate concentration increased. Competition from the mobile phase ammonium ions shifts the equilibrium between the free analyte and the analyte associated with the CSP cationexchange sites to more free analyte. Hence, the reduction in retention with increasing ionic strength shown in Figure 4 seems to be consistent with an ion-exchange type of mechanism. Alternatively, increased ammonium acetate concentration in the mobile phase may offer a more congenial environment for the chloroquine and hydroxychloroquine, thus resulting in decreased retention. As can be seen from Figure 4, whether the reduction in retention is attributable to enhanced mobile phase affinity or competition from the ammonium for the stationary phase ionophores, decreased retention produced decreased resolution for both analytes. It should also be noted that the relationship between ionic strength and retention depends on whether the analyte interacts with the cyclodextrin predominantly through electrostatic interactions or inclusion complexation. This is nicely illustrated in Figure 5, in which the retention for clenbuterol and aminoglutethimide, at pH 6, is plotted versus the inverse of the ionic strength. At pH 6, aminoglutethimide is not significantly protonated. Thus, its retention should be predominantly based on inclusion complexation. Indeed, as can be seen from Figure 5, the retention for aminoglutethimide increases as the ionic strength increases Analytical Chemistry, Vol. 68, No. 8, April 15, 1996

1373

Table 3. Chromatographic Data Obtained Using a Sulfated Cyclodextrin Chiral Stationary Phase for Miscellaneous Analytes compound benzoin

O

OH

C

C

H OH OH

hydrobenzoin

bupropion

k′ a

structure

C

C

H

H

Cl

R

mobile phaseb

6.81

1.00


1.65

1.40


9.39

1.04


5.25

1.04


5.27

1.05


3.04

1.09


7.71

1.06


34.36

1.05

88.4% MeCN/8.9% MeOH/1.8% acetic acid/0.9% triethylamine

6.16 23.14

1.07 1.05

30% MeCN/buffer (50 mM) 88.4% MeCN/8.9% MeOH/1.8% acetic acid/0.9% triethylamine

12.31

1.05


2.65

1.10


0.54 (+)c

1.21


COCH(CH3)NHC(CH3)3

clenbuterol

Cl H2N

CH(OH)CH2NHC(CH3)3 Cl OCH3

methoxyphenamine

CH2CH(CH3)NHCH3

midodrine

OCH3 CH(OH)CH2NHCCH2(O)NH2 CH3O

normetanephrine

H3CO HO

CH(OH)CH2NH2

pindolol

OH OCH2CHCH2NHCH(CH3)2 HN

propranolol OCH2CH(OH)CH2NHCH(CH3)2

tolperisone CH3

O

H

C

C

CH2

N

CH3

trimipramine N CH2CH(CH3)CH2N(CH3)2

Troger’s base

N

CH3

CH3 N

a Capacity factor of the first-eluting enantiomer. b MeCN, acetonitrile; MeOH, methanol; buffer, ammonium acetate, pH 6. c Configuration of the first-eluting enantiomer.

because the increased ionic strength presents a more hostile mobile phase environment for the hydrophobic analyte. In contrast, the retention for clenbuterol increases as the ionic strength decreases. This behavior is consistent with the previously noted behavior for chloroquine and hydroxychloroquine and suggests an ion-exchange mode of interaction. CONCLUSIONS The results presented here demonstrate the utility and novel chiral recognition of a sulfated cyclodextrin bonded phase. The unique ability of this functionalized cyclodextrin chiral stationary phase to offer not only inclusion complexation but also potential ionophoric moieties for electrostatic interaction enlarges the possible optimization parameters (e.g., pH, ionic strength) for


chiral separation method development. As evidenced by the glutethimide/aminoglutethimide pair, the introduction of electrostatic interactions through the sulfate moiety allows greater differentiation with analytes containing amines and other hydrogenbonding groups. ACKNOWLEDGMENT The authors gratefully acknowledge the generous support of the National Institutes of Health (1R29 GM48180-03) and helpful discussions with T. Beesley of Advanced Separation Technologies. Received for review December 12, 1995. February 6, 1996.X

Accepted

AC951199E X

Abstract published in Advance ACS Abstracts, March 15, 1996.

A Sulfated Cyclodextrin Chiral Stationary Phase for High-Performance

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