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Resolution of Enantiomersof Tropicamide by Reversed-Phase High PerformanceLiquid Chromatography UsingHydroxypropyl-β-cyclodextrinas Chiral Mobile Phase AdditiveMohamed M. Hefnawy a & James T. Stewart aa Department of Medicinal Chemistry College ofPharmacy , The University of Georgia , Athens, GA,30602-2352, USAPublished online: 22 Aug 2006.

To cite this article: Mohamed M. Hefnawy & James T. Stewart (1998) Resolutionof Enantiomers of Tropicamide by Reversed-Phase High Performance LiquidChromatography Using Hydroxypropyl-β-cyclodextrin as Chiral Mobile Phase Additive,Analytical Letters, 31:4, 659-667, DOI: 10.1080/00032719808001869

To link to this article: http://dx.doi.org/10.1080/00032719808001869

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ANALYTICAL LETTERS, 31(4), 659-667 (1998)

RESOLUTION OF ENANTIOMERS OF TROPICAMIDE BY REVERSED-

PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING

HYDROXYPROPYL- P-CYCLODEXTRIN AS CHIRAL MOBILE PHASE

ADDITIVE

Key Words: Tropicamide, HPLC, hydroxypropyl-P-cyclodextrin, chiral phase

Mohamed M. Hefnawy and James T. Stewart*

Department of Medicinal Chemistry

College of Pharmacy, The University of Georgia, Athens, GA 30602-2352 USA

ABSTRACT

Hydroxypropyl-P-cyclodextrin (HP-P-CD) was used as an effective chiral

mobile phase additive for the HPLC resolution of the enantiomers of tropicamide. The

HP- P-CD is more soluble in water and hydro-organic solvents than the native-p-

cyclodextrin. The separation of the tropicamide enantiomers was influenced by a

combination of hydrophobic and hydrogen bonding interactions. A baseline separation

(W1.5 ) was achieved for the enantiomers under isocratic conditions on a cyanopropyl

column with 10:90 vlv acetonitrile-aqueous 0.1% triethylammonium acetate buffer pH

4.0 containing 10 mM HP-P-CD. The flow rate was 1.0 mL/min with detection at 255

~~ ~

*Corresponding Author

659

Copyright 0 1998 by Marcel Dekker, Inc

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660 HEFNAWY AND STEWART

nm. The effect of mobile phase composition, concentration of HP-P-CD, and the pH

and type of buffer on peak shape, resolution and retention factors of the enantiomers

were investigated.

INTRODUC TION

In recent years, the major focus of chiral analysis has centered on high-

performance liquid chromatography (HPLC). Of the three types of chiral HPLC

methods, two are based on conventional reversed-phase column materials using either

chiral selectors added in the mobile phase or derivatization of enantiomers to form

diastereomers’.2. Procedures using chiral stationary phases (CSP) are the third type

and have been used by pharmaceutical analysts engaged in the determination of

enantiomeric ratios for quality control and related activities3. The CSP often have

drawbacks, including cost, flow rate, pH, mobile phase restrictions and stationaq

phase stability“.

Some of the most widely used chiral mobile phase additives are cyclodextrins

(CD)je9. They are based on a defined number of glucopyranose sugars linked at the

alpha-1,4-position to give between six and twelve glucose units. Only the a-, p- and

y-CD containing six, seven and eight glucose units, respectively, are commercially

available with p-CD being the most commonly used. The outer surface of the CD

molecule is hydrophilic due to the presence of primaty and secondary hydroxyl groups,

whereas the inner cavity is hydrophobic“). Enantiomers are resolved by differential

inclusion of so-called “host” and “guest” relationships with the guest molecule sitting

more or less tightly in the cavity of the host lo. To obtain a chual separation with a CD,

different interactions must occur between each enantiomer and the CD. These

interactions include dipole-dipole interactions, hydrogen bonding, inductive and

hydrophobic (Van der Waals). If at least one of these interactions is stereochemically

dependent, chiral separation of an enantiomeric solute is possible’’. In the case of a

chemically modified CD. the hydroxyl groups on the rim of the cavity are replaced

with methyl, hydroxypropyl, sulfate or acetyl groups to increase the hydrophobic

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ENANTIOMERS OF TROPICAMIDE 66 1

character of the CD cavity relative to the hydrophilic exterior. These differences

change the inclusive complex strength which can lead to greater selectivity12.

Tropicamide is an anticholinergic agent clinically used in ~phthalmology'~. A

review of the literature revealed that racemic tropicamide has been determined by non-

aqueous titrimetry14, ~olorimetry'~ and differential spectrophotomed6. A method

was reported for the analysis of the racemic mixture in eye drops by HPLC ". The

enantiomers of tropicamide have been separated by capillary electrophoresis using

mobile phases of P-CD in 5% aqueous urea'*, phosphate b ~ f f e f ~ . ~ ' and a-CD in

phosphate buffeg'. However, no chiral HPLC methods have been reported for the

resolution of tropicamide enantiomers using a chiral mobile phase additive. This paper

reports the HPLC separation of (+) and (-) tropicamide on a cyanopropyl column with

10:90 v/v acetonitrile- aqueous 0.1% triethylammonium acetate buffer pH 4.0

containing 10 mM HP-P-CD.

EXPERIMENTAL

Reacents and Chemicals

Racemic tropicamide [N-ethyl-a-(hydroxymethyl)-N-(4-

pyridinylmethyl)benzeneacetamide] was obtained from Sigma (St. Louis, MO, USA).

Hydroxypropyl-0-cyclodextrin(HP-P-CD-degee of substitution 4.3) was kindly

supplied by American Maize Company (Hammond, IN, USA). Acetonitrile and

absolute methanol were purchased from J.T. Baker (Phillipsburg, NJ, USA).

Triethylamine (TEA) was obtained from Fisher Scientific Co. (Orsigeburg, NY, USA)

and tnfluoroacetic acid (TFA) was purchased from Aldrich Chemical Co. (Milwaukee,

WI, USA). All solvents were HPLC grade and mobile phases were filtered through a

0.45 pm filter (Alltech Associates, Deerfield, IL, USA).

Chromatographic Co nditions

Chromatography was performed on an isocratic HPLC system consisting of a

Beckman Model 110A solvent delivery module (Beckman, San Ramon, CA, USA) and

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662 HEFNAWY AND STEWART

Spectroflow 757 absorbance detector (Kratos Analykal, Ramsey, NJ, USA) set at 255

nm. Each chromatogram and peak area response were recorded on a HP Model 3290

integrator (Hewlett Packard. Avondale, PA, USA). The stationary phase was a 250 x

4.6 mm i.d. An apex cyanopropyl RP 5 p column (Jones Chromatography, Lakewood,

CO. US.4) operated at ambient temperature (23’C). The mobile phase consisted of 10

mM HP-P-CD dissolved in 10:90 v/v acetonitrile-0. 1% triethylammonium acetate

buffer pH 4.0 (adjusted with trifluoroacetic acid) delivered at a flow rate of 1.0

mlimin.

RESkILTS AND DISCUSSION

The chemical structure of tropicamide is shown in Fig 1. A reversed phase

cyanopropyl column had previously shown success for the separation of selected c h a l

compounds with 0-cyclodextrin added in the mobile phase22. In this study, an HPLC

separation was achieved for the tropicamide enantiomers with HP-P-CD added in the

mobile phase. Chemical modification of the P-CD will “stretch” the cavity mouth and

change the hydrophobicity of the cyclodextrin and the stereoselectivity of the inclusion

process. In contrast to the secondary hydroxyl groups which are locked into position

on the native p-CD, the hydroxyl moiety of the derivatized hydroxypropyl group is free

to rotate. This flexibility may allow for a closer approach between the hydroxyl groups

and any hydrogen bonding moiety present in the analyte leading to stronger or more

stereospecific interactions than are possible with a native CD.

Tropicamide enantiomers were resolved with HP-P-CD, but not with 9-CD

added in the mobile phase. The resolution probably occurred because, in addition to

the aromatic ring structures for inclusion in the cavity of the CD, tropicamide has a

hydroxyl and a hydrogen at the chiral center which are available for specific hydrogen

bonding interactions with the rim hydroxypropyl groups of HP-P-CD. Table 1 shows

the effect of HP-0-CD concentration in the mobile phase on the separation of the

tropicamide enantiomers. Enantioselectivity generally improved with increasing HP-P-

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ENANTIOMERS OF TROPICAMIDE 663

TROPICAMIDE

Fig. 1 - Chemical structure of tropicamide.

Table 1 Effect of HP-P-CD Concentration on Resolution of Tropicamide Enantiomers on Cyanopropyl Stationary Phasea.

HP-P-CD Concn. added(mM)

Retention Factors

Rs kl k2

5 10 15 20

0.63 0.77 0.92 1.53 1.83 2.17 1.23 1.43 1.87 0.71 0.9 1 1.09

a Mobile phase consisted of 90: 10 v/v aqueous 0.1% triethylammonium acetate buffer pH 4.0 (adjusted with TFA)- acetonitrile containing the appropriate concentration of HP-0-CD at a flow rate of 1 .O mL/min.

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664 HEFNAWY AND STEWART

CD concentrations in the mobile phase. It was thought that each enantiomer formed

an inclusion complex with at least a 5 mM HP-P-CD concentration in the mobile phase

and that the two inclusion complexes had different capacity factors. When the HP-0-

CD concenmtion was < 5 mM, the formation of the inclusion complex was incomplete

and there was either partial or no resolution of the enantiomers.

Increasing the HP-0-CD concentration in the mobile phase resulted in an

increase in resolution of the enantiomers indicating the formation of relatively strong

inclusion complexes. However, the resolution of the enantiomers reached a plateau

region around 20 mh4 HP-P-CD at which resolution decreased. This may be due to the

increased bulk of the hydroxypropyl groups which would be expected to lead to

interactions with the seconday hydroxyl groups of HP-P-CD and result in a reduction

of enantiomeric resolution.

Table 2 shows the effect of organic modifier concentration in the mobile phase

on the resolution of tropicamide enantiomers. With CD-analyte complexes. it is

assumed that the hydrophobic portion of the analyte sits inside the hydrophobic cavity

of the CD and the addition of organic modifier reduces the affinity of analyte for the

HP-P-CD. The organic modifier competes with the analytes for preferred locations in

the hydrophobic cavity resulting in various degrees of interactions with HP-P-CD.

lncreasing the organic content of the mobile phase weakens the strength of the

inclusion complex23 . The retention profiles of tropicamide followed the typical

reverse phase model where the retention factors decrease with increasing organic

modifier concentration 22 . Enantioselectivity was lost when the acetonitrile

concentration was more than 20%. Other organic modifiers such as methanol and

ethanol were investigated, but acetonitrile gave the best resolution. A typical

chromatogram of the enantiomeric separation is shown in Fig. 2 .

CONCLUSION

In conclusion, a cyanopropyl column was successfully employed for the

enantiomeric separation of tropicamide enantiomers with HP-P-CD added in the mobile

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Table 2 Effect of Mobile Phase Composition on Resolution of Tropicamide Enantiomers on Cyanopropyl Stationary Phase

Mobile Phase Composition (v/v)”

Retention Factors

A B Rs kl k2

95 5 0.78 0.93 1.19 90 10 1.53 1.83 2.17 85 15 1.03 1.37 1.87 80 20 0.62 0.8 1 0.92

b 75 25 --- b --- b _ _ _ a Aqueous 0.1% triethylammonium acetate buffer pH 4.0 (adjusted with TFA)- acetonitnle contahhg 10 mh4 HP-PCD. A = Aqueous 0.1% triethylammonium acetate buffer pH 4.0 (adjusted with TFA) B = Acetonitrile

No resolution was achieved.

E, In In

RETENTION TIME, min

Fig.2 - Typical enantiomeric separation of tropicamide on a cyanopropyl

column. Mobile phase: 90:lO v/v aqueous 0.1% triethylammonium

acetate buffer pH 4.0 (adjusted with TFA) -acetonitrile containing 10

mM HP-P-CD at a flow rate of 1.0 mllmin with detection at 255 nm.

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666 HEFNAWY AND STEWART

phase. The c h i d mobile phase additive offered a wide variety of advantages in terms

of the ease of formation of inclusion complexes and the additional number of

interactions possible with various functional groups present in tropicamide.

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