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," 1

CHROMATOGRAPHIC AND ELECTROPHORETIC SEPARATIONS

OF CHLORPHENIRAMINE AND ITS METABOLITES

by

Evelyn Chun·Yin Soo

Department ofChemistry

McGill University

Montreal t Canada

1unet 1998

A thesis submined to Ùle Faculty ofGraduate Studies and Research

in partial fulfillment of the requirements of the degree

of

Masters of Science

Copyright © Evelyn Chun-Yin SOOt 1998.

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ABSTRACT

Chlorpheniramine, a reversible competitive inhibitor at the Hl-receptor that has

demonstrated potent, long-lasting antihistaminic activity with only mild side-effects, has

been a popuIar choice for the treatment of allergie conditions and is a common component

of coldlcough preparations. A number of groups have studied the phannacokinetics of

chlorpheniramine since its development, but results have been conflicting. Moreover, most

of the pharmacokinetic studies had only involved analysis of the parent compound and

excluded phannacologically active metabolites.

As part of a new phannacokinetic study of chlorpheniramine, an enantiomeric method to

resoive chlorpheniramine from its N-demethylated metabolites and chlorpheniramine N­oxide was required. The use of high performance liquid chromatography (HPLC) with the

amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (AD-CSP) failed to

resolve the enantiamers of chlorpheniramine and its Metabolites. Capillary electrophoresis

was used ta screen a number of chiral selectors including hydraxypropyl Jk:yclodextrin,

sulfated ~-cyclodextrin and carbaxymethyl ~-cyclodextrin. The resolution of the

enantiomers of chlorpheniramine and its Metabolites was achieved using carboxymethyl ~

cyclodextrin obtained from Wacker Chemie, Munich, Germany. Detection limits of

chlorpheniramine down ta 200 ng/mL were achieved by concentrating samples and usingsample stacking methods.

i

RESUME

La chlorphéniramine, un inhibiteur compétitif réversible des récepteurs Hl de part sa

puissante activité anti-histaminique de longue dureé associée à des effets secondaires

modérés, est un traitement de choix pour diverses allergies et rentre aussi communement

dans la composition de préparations contre le rhume et la toux. De nombreuse équipes ont

conduit des études de pharmacocinétique sur la chlorphéniramine depuis sa développemen~

mais les résultats restent contradictoires. De plus la plupan des études pharmacocinétiques

sur la chlorphéniramine ont porté uniquement sur l'analyse de la molécule mère et ont exclu

les métabolites phannacologiquement actifs.

Dans une nouvelle étude pharmacocinétique sur la chlorphéniramine, on est recours à une

méthode basée sur le principle des énantiomères pour séparer la chlorphéniramine et de ses

métabolites: Les métabolites N-desméthylés et N-oxydés. La chromatographie liquide à

haute performance (HPLC) fut utilisée et la AD-CSP fut évaluée pour le développement de

la méthode, mais l'établissement de la méthode d'une HPLC basée sur la principle des

énantiomères pour séparer la chlorphéniramine et ses métabolites, fut un échee. Des

électrophoréses par capillarité furent utilisées et de nombreuses sélecteurs chiraux testés

incluant l'hydroxypropyl (H:yclodextrine, le sulfate de P-cyclodextrine et la carboxyméthyl

~yclodextrine. Une méthode à partir d'énantiomères pour séparer la chlorphéniramine et

ses métabolites fut trouvée en utilisant le carr-vxyméthyl ~-cyclodextrine obtenu par

Wacker Chemie, Munich, Allemange. Le limite de détection de 200 ng/mL

chlorphéniramine furent atteintes en augmentant les concentrations de échantillons et en

utilisant les méthodes de compilations dtéchantillons.

ü

To

JEFFREY THOMAS PICARD

for

his continued love and support which will a/ways he remembered.

ni

ACKNOWLEDGEMœNTS

1wish ta thank my supervisor Dr 1W Wainer and co-supervisor Dr R Kazlauskas; NSERC

for the fmancial support 1 received for my studies; and Wacker Chemie for their kind

donation of the carboxymethyl ~-cyclodextrins. Many thanks are due to Professor W

Purdy and Dr B Lennox for their invaluable help with the preparation of my thesis, and

aIso ta Dave Uoyd and Georg Schulte for their assistance with the CE troubleshooting.

~Iy collegues Connna, Daphne, Maria and Marion are aIso remembered for all their

assistance, and 1 am truly grateful for the continued support of my special friends Patsy,

Fabrice, Dirk and the Dublin-gang.

Above all, 1wish ta thank my parents and sisters for their constant love and encouragement

during my time in Montréal.

iv

TABLE OF CONTENTS

ABSTRACT

RESUME

DEDICATION

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

GLOSSARY OFTERMS AND ABBREVIATIONS

INTRODUCTION

Ü

ili

iv

v

vü

1

1

2

3

ALLERGY AND ANAPHYLAXIS

1.1 Histamine and Allergic Diseases

1.2 Antihistamines

History and Development of Antihistamines

Phannacology of Antihistamines

Chemical properties of chlorpheniramine

HIGH PERFORMANCE UQUID CHROMATOGRAPHY

2.1 Enantiomeric derivatisation with chiral reagents

2.2 Chiral mobile phases

2.3 HPLC Chiral stationary phases

Type 1

Type 2

Type 3

Type 4

Type 5

CAPll..LARy ELECTROPHORESIS

3.1 General Instrumentation

The capillary

Sample loading techniques

Detection Systems

3.2 Fondamental Principles of CE

3.3 Modes of CE

Capillary zone eleetrophoresis

Isoelectric focusing

v

2

2

3

3

9

10

Il

Il

13

14

15

16

17

19

19

21

22

22

232324

2526

26

Isotachophoresis 27

Capillary gel electrophoresis 27

Micellar electrokinetic capillary chromatography 27

3.4 Applications oCCE 29

Biomolecules 29

Phannaceutical applications 30

Chiral separations 31

RESEARCH OBJECTIVES 33

MATERIALS AND METHOnS 36

RESULTS AND DISCUSSION 39

HPLC STUDIES 40

Amylose tris(3,5-dimethylphenylcarbamate) CSP 40

CE STUDIES 42

Hydroxypropyl (3-cyclodextrin 42

Sulfated JH:yclodextrin 45

Carboxymethyl ~yclodextrin 50

CONCLUSIONS 66

REFERENCES 69

vi

GLOSSARY OF TERMS AND ABBREVIATIONS

AD Amylose tris(3,5-dimethylphenylcarbamate)

AGP al-acid glycoprotein

AS Amylose (S)-a-methylbenzylcarbamate

BSA Bovine serum albumin

CD Cyclodextrin

CE Capillary electrophoresis

CGE Capillary gel electrophoresis

CMC Critical micelle concentration

CNS Central nervous system

CR Crownether

CSP Chiral stationary phase

crA-l Cellulose triacetyl l

CZE Capillary zone electrophoresis

EOF Electroosmotic Dow

FSCE Free solution capillary electrophoresis

HPLC High performance Iiquid chromatography

HSA Homan serum albumin

YÜ

IEF Isoelectric focusing

IgE Immunoglobulin E

IgG Immunoglobulin G

ITP Isotachophoresis

MEKC Micellar electrokinetic chromatography

OB Cellulose 2 nibenzoate

OC Cellulose 2 triphenylcarbamaœ

CD Cellulose 2 tris(3,4-dimethylphenylcarbamate)

01 Cellulose 2 tris(4-methyl-benzoate)

OVM Ovomucoid

SDS Sodium dodecyl sulfate

SRS-A Slow reacting substance of anaphylaxis

TBC Tribenzoyl cellulose

viii

INTRODUCTION

1

Chlorpheniramine is a widely used antihistamine that was developed by Sehering in 1951.

Prepared as a maleate salt, chlorpbeniramine is used for the treatment of various allergie

conditions, and it is a common component of cold/cough preparations [1-5). Since the

development of chlorpheniramine, a number of research groups have studied its

pharmacokinetics but the results have been eontlicting and there are still no reHable

pharmacokinetie data available [1-4].

ReeentIy, a group headed by Dr S. Yasuda at Georgetown University, Washington, OC,

commenced a new pharmaeokinetie study of chlorpheniramine [5]. For part of this on­

going study, a method of separaùng the enantiomers of chlorpheniramine and its

Metabolites is required. High Performance Liquid Chromalography (HPLC) is one of the

Most eSlablished separation techniques for drug analysis, but the use of capillary

electrophoresis (CE) over the decade has become increasingly popular [6,7]. Application

of the two separation techniques to the development of the enantiomeric separation meÙlod

is assessed in part of this research projecl

1 ALLERGY AND ANAPHYLAXIS

Pollen, house dust, animal hair, various foods and drugs can trigger allergie reactions in

susceptible individuals [8,9]. When an antigen (allergen) binds to the specifie IgE

(Immunoglobulin E) antibodies fixed on the surface of Mast cells, the Mast cells

degranulate and release chemical Mediators that induce the symptoms of allergy. Histamine

is probably the MOSt significant Mediator of allergy, but slow reacting substance of

anaphylaxis (SRS-A), bradykinin, prostaglandin, eicosanoids and neuropeptides aIso have

imponant raIes in mediating allergie diseases [8-12].

1. 1 Histamine and Allergie Diseases

Histamine is widely distributed in humans and is mainly round in Mast eeUs and basophils

as stored histamine [9-11]. Although histamine is usually associated with the clinical

symptoms of allergie diseases such as rhinitis, urticaria and anaphylaxis, a number of

normal physiological responses are aIso mediated by histamine. These include gastrie

secretion, smooth muscle contraction in the respiratory and gastrointestinal tracts, and

2

vasodilation [12,13]. Histamine is aIso believed to be a mediator of pain, and to stimulate

the salivary and exocrine glands [15].

Rhinitis is an allergic condition caused by the inhalation of airbome allergens such aspollen, mold spores, house dust and animal haire The release of histamine from Mast cells

in nasal tissues following the inhalation of airborne allergens, causes an increase in the

permeability of capillaries and vasodilation. The leakage of intravascular fluid inIO nasal

tissues, as well as swelling of the nasal mucosa and edema, result from the vascular

changes and give rise ta symptoms such as nasal congestion and mucus discharge.

Histamine aIso stimulates sensory nerves causing pruritus and sneezing [8,12-13].

Urticaria, a disease characterised by intensely itchy, edematous wheals that are surrounded

by a red tIare, and the associated condition angiodema, are both mediated by histamine.

Pruriùs, vasodilation and increased vascular permeability observed in urticaria and

angiodema are the typical pathological responses to histamine. However, prostaglandin,

platelet-activating factor and bradykinin May aIso be important vasoacùve Mediators [12].

The exaet pathogenesis of chronie urtiearia is unknown but studies have implicated IgG

(Immunoglobulin G) autoantibodies, which interaet with and cross-link the alpha subunit

of the IgE receptor, to be the cause of histamine release from mast cells in the skin

[10,11].

Anaphylaxis is a life-threatening allergie disease that targets a number of organs

simultaneously. The skin, cardiovascular system, gastrointestinal tract and respiratory tract

May all be affected, and common allergens of anaphylaxis include foods such as nuts and

shellfish, drugs such as penicillin, and inseet stings. Histamine and eicosanoids are

believed to be Mediators of anaphylaxis and the various symptoms inelude pruritus,

urticaria, angioedema, flushing, tachycardia, hypotension, vomiùng, diarrhea, rhinorrhea,

congestion, wheezing and asphyxiation [9,12-13].

1.2 Antihistamines

Histoty and Development of AotibistamÎQes

The world-wide development of antihistamines was sparked by Staub and Bovet, who in

1937t developed compound 929F that demonstrated potent antihistaminic properties. The

high taxicity of929F ta humans deemed it of no clinical value, but the discovery initiated

3

the research that led to the development of a number of classes of antiallergenic agents,

which are now sorne of the most-widely used drugs in the world [15,16].

929F (~-(5-isopropyl-2-methyl-phenoxyethyl)diethylamine).

Ethylenediamine derivatives

Many antihistamines today are of the ethylenediamine-type and their development began in

1937 when Staub showed that one of Fourneau's compounds, 1571F (N-phenyl-N,N',N'­

triethylethylenediamine), was a potent antihistamine [16].

1571F (N-phenyl-N,N',N'-triethylethylenediamine).

From her study of a further series of ethylenediamines, Staub proposed two backbone

structures for the search of clinically useful antihistaminic compounds:

i) RR'NCH2CH2NR"2

ü) ROCH2CH2NR"2

where R= phenyl or substituted phenyl

R'= aIkyl or araIkyl

Rn= small aIkyl.

4

Subsequent research into ethylenediamines eventually led to useful developments: in

1942, Halpern and Mosnier synthesized phenbenzamine, a patent antihistaminic compound

that became a useful model for the study of other ethylenediamines.

Phenbenzamine (N-benzyl-N',N'-dimethyl-N-phenylethylenediamine).

By 1950, a number of ethylenediamine derivatives (mepyramine, methaphenilene,tripelennamine and chloropyramine), which demonstrated goad antihistaminic activity and

ooly minor side-effects, were developed. Two patent long-lasting antihistamines, zolamine

and tbonzylamine, were aIso synthesized and round application in the treatment ofanapbylaxis.

RO~CH,N(CH,~

U1

M:pyramine: R=CH30 Zolamine. R= QTnpelclU1amme. R=H ~N

Chloropyramine. R=Cl Tbonzylamine. R= V

Ethylenediamine-type antihistamines.

Aminoalkyl ether derivatives

While research into ethylenediamine-type antihistamines was taking place, aminoalkyl

ethers were aIso being studied [16]. In 1946, Rieveschl and Huber introduced

diphenhydramine, the ficst widely used drug in the series, which showed a significant

increase in activity compared to the ethylenediamines but fewer side-effects. Other

aminoalkyl ethers which showed even fewer side-effects include bromodiphenhydramine,

chlorodiphenhydramine, medrylamine, doxylamine, and phenyltoloxamine which helped

prevent the histaminic induction ofasthma in the guinea pig [16].

5

Diphenhydramine. R=HBromodipbenhydramine. R=BrChlorodipbenhydramine. R=ClMedrylamine. R=CH p

Aminoalkyl ether derivatives.

Derivatives ofcyclic basic chains

Early investigations into nitrogen-heteracyclic ring systems led ta a number of important

discoveries: antazoline, anon-initating agent that could be fannulated for local application

to the eye, and the piperazines, which are sorne of the most patent antihistamines exhibiting

aslow onset and prolonged duration of activity [14].

Col\-Qi2~CH:!-<) Antazolinc

C.Hs H

rHs 1\ Cyclizine. R=Cti HsRCH- \..JNCH3 Cblorcyc:lizine. R=4-CIC~4

aOCC~ )-RMeç1izinc. R=CH]. R'="Buclzine. R=H, R'=C(CH])3

Antazoline and the piperazines.

6

Derivatives ofmonoaminopropyl groups

In 1951, two monoaminopropyl compaunds, pheniramine and chlorpheniramine which

showed patent antihistaminic activities, were developed followed by brompheniramine the

next year [16]. Chlorpheniramine is a highly active, long-lasting antihistamine with

minimal side-effects that offers good protection at low doses. Another important

development was mebhydroline, which alleviates the symptoms af allergy without

praducing sedative side-effects cammonly abserved with the use of antihistamines.

Mebbydroline. R=HChlorpbeniramine. R=ClBrompbeniramine. R=Br

Manoamînopropyl-based antihistamines.

Tricyclic derivatives

Derivatives of tricyetic systems have aIso given rise ta a number of useful antihistamines.

Promethazine is an extremely patent agent which, in animal studies, showed the ability ta

counteract doses of histamine much higher than the nannallethal dase. One of its analogs,

diethazine, is even mare patent and has found application in the treatment of Parkinson's

Disease [16].

(X))1CHtM~)N(CH :d2

Promethazine

Diethazinc

Tricycüc antihistamines.

7

The sedative side-effects of these aIder or 'first-generation' antihistamines led to the

deveIapment of the 'second-generation' antihistaminic compounds in the 1980s, such as

terfenadine and astemizoIe, which belong ta a class called the piperidines [13,16].

Terfenadine

r-O-fH 2

CrNH-oCH2CH2 ( )-OCH 3

The piperidines.

8

Astemizole

PharmacQIQiY Qf antihistamines

There are three knawn types Qf histaminic receptQrs ta which histamine ar antihistaminesbind tQ exert their biaIQgicaI effects, and antagonists that bind selectively at each of the

receptars have been develaped. Antihistamines develaped for the treatment Qf allergic

conditiQns are selective antagonists at the Ht-receptor. By cQmpeting with histamine fQr

the Ht-receptor, Ht-antagonists inhibit histamine activity to relieve the symptams of allergy

[13,15].

Although the structures Qf histamine and antihistamines are similar, Hl-antagonists cannot

occupy Ht-receptors directIy. Instead, association with the receptor takes place via

interactions between the amine grQup of the Ht-antagonist and the aniQnie site of thereeeptor. Interactions between the rest af the malecule and regiQns adjacent ta the binding

site are aISQ believed ta take place. Furthennore, the stereoselectivity of the Hl-receptar ta

chiral antihistamines such as chlorpheniramine, is believed ta occur at a region adjacent tothe binding site [16,17].

The frrst-generatian Hl-antagonists are highly effective at relieving the symptoms of

rhinitis and urticaria, but tbey aIsa bind ta Hl-receptors in the central nervolls system

CCNS), causing sedation and diminished alenness. Depression of the CNS becamesparticularly traublesome when it interferes with a patient's daytime activities, especially if

Hl-antagonists are used long-term for the management of allergy [8,10, 12, 15-16]. The

structure of the second-generation Ht-antagonists is believed to prevent them fram crassingthe blood-brain barrier and binding ta Hl-receptars in the CNS. Furthermore, they are less

toxic since they do not show anticholinergic or muscarinic activity and do not stimulate a­

adrenergic and serotonin receptars. The more selective, second-generation Hl-antagonistshave therefore been good alternatives to the classic Ht-antagonists [13,15].

Terfenadine had been a popular alternative to Most of the aider antihistamines because of its

non-sedative properties. However, sorne cases of severe coronary complications have

been associated with its use, so once a popular over-the-counter drug, terfenadine is nowooly available on prescriptian in Nonh America and Europe.

9

Chemical properties Qf cblQrpheniramine

Chlarpheniramine, which belongs ta the class of manoaminQpropyls, shows specific H1­

antagonistic activity. It is a tertiary alkylamine with pKa values af9.2 at the amine, and 4.0

at the pyridine ring. Another important feature of chlorpheniramine is that il has a

stereogenic centre and is Most active as the R-(+)-isomer.

A number of metabQlic studies have cQnfmned that chlQrpheniramine is primarily

eliminated from the body as its monQdesmethyl- and didesmethyl- Metabolites, and as the

parent compound [1-4,18]. Other Metabolites of chlorpheniramine have also been reported

including chlorpheniramine N-oxide, 3-(p-chlorophenyl)-3-(2-pyridyl)-propanol, 3-(p­

chlorophenyl)-3-(2-pyridyl)-N-acetylaminopropane and 3-(p-ehlorophenyl)-3-(2-pyridyl)­

propionic acid [19,20].

a

Chlorphernramine, with * denoting the stereogenic centre.

10

HIGR PERFORMANCE LIQUID CHROMATOGRAPRY (BPLC)

The thalidomide tragedy led pharmaceutical companies and drug regulatory authorities aIl

around the world to acknowledge the need to study the therapeutic and toxicological effects

of drug enantiomers [21]. HPLC has become a widely used chromatographic technique for

the separation and determination of enantiomers, mainly because ofsignificant advances in

HPLC technolagy since the late 1960s but aIso due ta greater understanding of the chiral

recognition mechanism [22].

There are three main approaches for performing an enantiameric separation by HPLC

[23,24]. First, is the reaction of the enantiomers with a chiral reagent to fonn

diastereomeric derivatives thatcan then be separated on an achiral chromatographie column.

Second, is the formation of diastereomers through the use of a chiral reagent dissolved in

the mobile phase. The third approach is direct separation of the enantiamers on chiral

stationary phases (CSPs).

2. 1 Enantiomeric derivatisation with chiral reagents

The use homochiral derivatising reagents to convert enantiomers into diastereomers with

different retention properties, was the classical method for the resolution of enantiomers.

The Most common chiral solutes that have been derivatised with chiral reagents, and

resolved as diastereomers on achiral HPLC columns include amines. carboxylic acids and

alcohols. There are Many different chiral reagents used ta convert enantiomers iota

diastereomeric derivatives but the principal types are acylating agents, chloroformates,

isocyanates, isothiocyanates. and reagents based on o-phthalaldehyde and chiral thioIs[23,25-26].

Acylating reagents are prepared from carboxylic acids and generally used as chlorides,

anhydrides, and N-succinimides [23,25]. Examples of some phannaceutical applications

of acylating reagents include: (+)-6-methoxy-a-methyl-2-naphthaleneacetyl chloride in theenantiomeric resolution of an anticancer compound, cyclophosamide [27],

tluroenylmethyloxycarbonyl-L-proline for the derivatisation ofamphetamine [28], and (S)­

(+)-tlunoxaprofen chloride for the derivatisation oftranylcypromine and other amines [29],and propanolol [30].

Il

The enantiomers of promethazine, a tricyclic HI·antagonist, were resolved as carbamate

diastereomers on an achiral column by HPLC following its derivatisation with (-)-menthyl

chloroformate [31], and aIso aiter fonning urea derivatives with (R)-a-methylbenzyl

isocyanate (32]. Chlorofonnates are prepared by reacting alcohols with phosgene, while

isocyanates generally result from the reaction of amines with phosgene. Sorne recent

applications of isocyanates as chiral reageots in HPLe include (S)-(-)-a-methylbenzyl

isocyanate and (+)-(S)-naphthylethyl isocyanate for the derivatisation of salbutamol [33]

and mefloquine (34], respectively.

Isothiocyanates have mainly been used for peptide sequencing and the analysis of amino

acids. 2,3,4,5-Tetra-O-acetyl-p-D-glucopyranosyl isothiocyanate is one the most widely

used isothiocyanate reagents and has been successfully applied to the enantiomeric

resolution of ~-blockers [35,36], amine acids [37], and epinephrine [38]. 2,3,4,5-Tetra­

O-benzoyl-~-D-glucopyranosyl isothiocyanate has aIso been employed for the HPLC

resolution of ~-blockers as weIl as amine acids [39]. In addition, amino acids are

commonly derivatised with reagents based on o-phthalaldehyde and chiral thiols [40,41].

Diastereomeric derivatives of carboxylic acids are usually formed with chiral amines,

following activation of the carboxyl group. An example is the resolution of the

enanùomers of a non-steroidal anti-inflammatory drug, ibuprofen, and its metabolites after

activation with Ll-carbonyldümidazole and derivatisation with the chiral reagent (S)-(-)-a­

methylbenzylamine [42].

The availability of different chiral derivatising reagents and the wide variety of chiral

solutes that can be resolved are just some of the advantages of using this approach to

perform enantiomeric separations. Although the main purpose of using chiral derivatising

reagents is to conven enantiomers into their diastereomerie derivatives, a chiral reagent may

aIso possess chromophoric or fluorophoric groups which become introduced iota the

solutes during the derivaùsation, to improve the detectability of the enantiomers.

Furthennore, the elution order can be reversed by using the other reagent enantiomer or by

changing the chromatographie conditions from normal-phase to reversed-phase, or vice

versa. However, there are limitations to using chiral derivatising reagents for the resolution

of enantiomers. An optically pure reagent is essential for accurate determinations of

enantiomeric composition and elution arder. Chiral stability of the reagent is also

important, as are the absence ofkinetic resolution and racemisation [23,25].

12

2.2 Chiral Mobile Phases

The limitations of using chiral derivatisng reagents for the resolution enantiomeric

compounds led ta developments in direct HPLC methods for performing

enantioseparations [22]. The separation principle behind the use of chiral mobile phases is

the dynamic formation of stable diastereomers, ion-pairs, Metal complexes or inclusion

complexes with different retention properties between the chiral additive and the

enantiomers, for separation on an achiral column [23].

The use of chiral mobile phases for the resolution ofenantiomers on achiral HPLC columnshas been applied successfully to many chiral drugs. The formation of ion pairs for a

number of ~-blockers (alprenolol, metoprolol and propranolol) with (+)-10­

camphorsulfonic acid in the chiral mobile phase enabled their separation on a diol column

[42]. The enantiomers of propanolol have aiso been separated using N­

benzoxycarbonyIglycyl-L-proline as the chiral selector in the mobile phase [43], and ion­

pairing with quinine as the chiral counterion enabled the separation of 2-phenoxypropionic

acid derivatives [44). The separation of amino acid derivatives has been achieved using N­

acetyl-L-valine tert-butylamide as a chiral mobile phase additive [45], and N,N'­

düsopropyl-tanramide has been reported ta be a useful chiral mobile phase additive for the

enantiomeric separation of hydroxycarboxylic acid, amino acid and 1,2-diol derivatives

[46] as weil as hydroxy ketone, amine alcohol and hydraxy ketoxime derivatives [47].

The use of a chiral copper complex as a mobile phase additive has been the general

approach ta separating ex-amino acids by HPLC. For example, copper(ll) acetate and N,N­

di-n-propyl-L-alanine were added ta the mobile phase for the separation of an ex-amino acid

from its corresponding acid amide [48]. Herbicides (fluazifop and other phenoxyprapionic

acids) have been resolved using L-propyl-n-octylaminide-Ni(ll)-L-proline in the mobile

phase [49], while the enantiomers of a-substituted ornithine and lysine analogs have been

separated with a mobile phase consisting L-praline and copper [50].

~-cycladextrins have been usefuI chiral mobile additives for the resolution of a wide range

of enantiomeric compounds in reversed-phase HPLC, including a number of important

chiral pharmaceuticals [51,52]. The development of cbarged ~cyclodextrins has increased

the scope of their application mainly due ta their increased water solubility and selectivity.

For example, carboxymethyl-(Xyclodextrin has recently been used for the resolution af

doxazosin enantiomers [53], while sulfobutyl-ether p-cyclodextrin was applied to the

13

enantiomeric resolution oC racemic amlodipine [54], and sulfated (i-cyclodextrin to theseparation oC pentazocine enantiomers [55]. Apart from water solubility and the ability to

Conn selective and reversible inclusion complexes, cyclodextrins have other importantproperties. For instance, cyclodextrins are stable over large pH ranges and their lack ofUV absorbance at various ranges of wavelengths generally enable the detection of analytes

at their maximum wavelengths of absorbance. In addition, cyclodextrins are safe, non­

toxic, and can be used to separate nonpolar and polar solutes simultaneously [51].

2.3 HPLC Chiral Stationary Phases

The use of chiral derivatising reagents and chiral mobile phases still remain popularapproaches for resolving drug enantiomers but the application of HPLC chiral stationary

phases (HPLC-CSPs) Cor evaluating the stereoisomeric composition of chiral drugs and

studying the therapeutic and toxicological effects of the enantiomers has virtually becomeroutine [52].

Much of the early progress in the development of HPLC-CSPs was the result of Pirkle's

insights into the chiral recognition mechanism [22,56]. Essentially, chiral discriminationinvolves the formation of temporary diastereomeric complexes of differing stabilities

between the immobilized chiral selector and each enantiomer. From a study on theresolution of a wide range of chiral solutes on a trifluoroanthrylethanol CSP [53], Pirkle

observed that there had to be at least three simultaneous interactions between the CSP andone solute enantiomer, and that at least one of those interactions had ta he stereochemicallydependent for chiral recognition to take place. Pirkle showed the importance of the 1t-1t

interactions between the anthryl 1t-base substituent and 7t-acid group on the solute

enantiomer, as weIl as dipole-dipole interactions and hydrogen bonding between the

stationary phase and solute enantiomer, in the chiral recognition process. Pirkle'sunderstanding of the interactions between the enantiomers and the CSP enabled him topredict the elution order of individual enantiomers based upon the three-point interactionmodel, as illustrated in the following diagram.

14

1.......~--~·~ i/C"IIIE " .. Xill'/C..........

B 0 .. .v z

i i/C"IIIE ....411o---~~ XiII/oC..........

A 0..: .. y Z

Three points of interaction betweenenantiomer and chiral selector;stronger interactions and elutessecond.

Qnly two points of interactionbetween mirror image of enantiomerand chiral selector; weakerinteractions and elutes first

The three-point interaction model and prediction ofelution order.

Although the three-point interaction model has been widely accepted as the basis of chiraldiscrimination, a two..point interaction model for chiral recognition bas a1so been proposed

[54] and the basis of chiral recognition continues ta be a topic of debate. There are Manydifferent types of commercially available HPLC..CSPs and the chiral selectors that bavebeen used are eitber naturally occurring Macromolecules or synthetic polymers [55,56].

The different HPLC-CSPs have been classified into five groups by Wainer, according tothe types of interactions that occur between the solute and CSP in the chiral recognitionprocess. Wainer's classification of the HPLC-CSPs mainly serves to simplify the process

ofchoosing a CSP for performing a chiral separation [52].

l)u 1 CSPs

In the 'type l'or 'Pirkle-type' CSPs, the chiral selectors are mainly amino acid derivatives,

which are attached 10 the silica suppon via achiral spacers [23,56]. The functional groupson the amine acid derivatives include the naphthalene group and 3,S-dinitrobenzene, an

aromatic 1t-base and an aromatic 1t-acid, respectively, which interact with the solute via Jt-1t

interactions in the chiral recognition process. Funhermore, amide, urea or ester groupsmayaIso be present and provide hydrogen bonding and/or dipole interaction sites [26,52].

Solutes that can be resolved on type 1 CSPs must already have interaction sites

complementary to those on the CSPs, or he derivatised ta add the complementary sites.

15

Many chiral solutes have been resolved directIy on type 1 CSPs, such as cyclic imides

[57], benzodiazepenone enantiomers [58] and sulfoxides [59], but most chiral solutes have

required derivatisation. Amines are commonly converted into amides with acid chlorides[60,61], or are derivatised with aryl chloroformates and isocyanates ta fonn the carbamatesand ureas, respectively [62,63]. Alcohols are commonly derivatised into carbamates [64],

while chiral acids are commonly resolved as amide derivatives [65]. Type 1 CSPs areusually used in the normal-phase mode, employing non-polar mobile phases composed ofhexane and an alcoholic polar modifier such as isopropanol [52,56]. Despite the

development and popularity of other commercially available CSPs that generally do not

require a preliminary derivatisation stePt Pirkle-type CSPs are still useful in drug analysis.A recent example is the use of an a-Burke 1column for the separation of a number of ~­

blockers [66].

Ixpe 2 CSPs

Cellulose

Cellulose is a chiral polymer consîsting chains of D-p-glucose subunits and exists as twomajor crystalline forms: the native fonn (cellulose 1) where aIl the chains of D-~-glucosesubunits point in one direction, and the regenerated fonn (cellulose 2) wher allt.•~t1ating

chains of the D-~-glucose subunits point in opposite directions [52]. For use as a HPLC­CSP, bath fonns of cellulose have ta be derivatised to provide the mechanical strength

cellulose requires to withstand the pressures associated with HPLC. The chiralenvironments in cellulose are essentially the cavities between adjacent glucose units, whereinclusion of a solute can occur, and the channels between the polysaccharide chains whereattractive interactions between the solute and D-IJ-glucose subunits take place [52,67].

Cellulose 1 has been derivatised to Conn its triaeetyl (CTA-l) and tribenzoyl (TBC)derivatives. The CTA-l CSP bas found most application with Molecules containing aphenyl moiety, whieh becomes included in the chiral cavity during the formation of thesolute/CSP complex. Examples oC chiral solutes resolved on the CTA-l includes cyelicamides and imides, esters, ketonest ô- and y-Iactones and alkyl-substituted indenes. Chiralcompounds that have been resolved with the TBC-CSP include a1cohols and diols afterderivatisation to fonn their aeetates, severa! p-substituted d-phenyl-d-valerolactones,phenylvinylsulfoxide and trans-stilbene oxide [52].

16

Although triacetate derivatives of cellulose 2 have been used as a CSP, those based on its

ester and carbamate derivatives have shawn broader applicability. A large number of

HPLC-CSPs based on cellulose 2 have been prepared such as the tribenzoate, triphenyl

carbamate, tris(3,4-dimethylphenyl carbamate) and the tris(4-methyl-benzoate) derivatives

which are commonly referred to as the OB, OC, 00 and 01 HPLC-CSPs, respectively

[52]. Examples of their application include the resolution of propanolol on the OO-CSP

[68], acyclic enantiomeric amides [69] and aromatic alcohols [70] on the OB-CSP, and

warfarin on the OC-CSP [71].

Amylose

Carbamate derivatives of amylose, a polymer of O-a-glucose that possess much helicity,

have aIso been prepared and are known as the amylose tris(3,5-dimethylphenylcarbamate)

or AD-CSP, and the amylose (S)-a-methylbenzylcarbamate CSP (AS-CSP). Chiral

recognition is believed to be the result of differential inclusion of the solute itself, or the

aromatic group of a solute, into the chiral cavity within the solute/CSP complex.

Hydrophobie groups are included into the cavities while polar groups give rise to

hydrogen-bonding, dipole and 7t-1t interactions. Examples of phannaceutical applications

include verapamil [72], and ~..amino alcohols [73].

The mobile phases normaIly employed with the type 2 CSPs are hexane-based and

modified with isopropanol, or other polar modifiers such as ethanol, ta manipuIate

retention and selectivity. The CTA-l CSP however, is generally operated with mobile

phases consisting anhydrous ethanol, or ethanol modified with up to 5% water and the use

of mobile phases based on Methanol and isopropanol have offered changes in retention andstereoselectivity [52].

Type 3 CSPs

Cyelodextrins (CDs), polymethacrylate and erown ethers are the different chiral selectors

that have been successfully anached to silica and used as chiral stationary phases (52].

Chiral discrimination is achieved when the solute enters the chiral cavities within the CSP

as a result of hydrophobie or electrostatic interactions, and forms diastereomeric complexes

ofdifferent energies via secondary attractive or sterie interactions.

17

CDs are naturally oecurring cyelic oligosaccbarides composed of D-a-glucose units. CDs

exist as single isomers, and thus have the ability to discriminate between enantiomersthrough the formation of diastereomeric complexes of differing thermodynamic stability.

(X-, ~- and y-cyclodextrin are the three mast cammon forms of native cyclodextrins, and

contain six, seven and eight D-a-glucose units, respectively, linked through the 1,4­

position. CDs possess a relatively hydrophobie interior cavity and chiral recognitionresults from the fonnation of inclusion complexes when the aromatic moiety of a solute

enters the hydrophobie cavity, together with interactions between other functional groups

around the chiral centre and the mouth of the CD cavity [52,74,75].

The solutes that are most successfully resolved by the cyclodextrin CSP usually contain anaromatic group at or next to the stereogenic centre, plus a hydrogen-bonding group or

additional1t system at the stereogenic centre. Sînce the aromatic group becomes includedinto the chiral cavity, the size of the aromatic groups often determines the choice of CD. In

general, the a-CD are for solutes with a single aromatic ring, while solutes with naphthylrings or a greater aromatic system would require the ~- and y-CD, respectively [52,74].

Water-based mobile phases with an organic modifier such as methanol, ethanol or

acetonitrile are commonly employed with the CD-CSPs but a 0.5-1% triethylammoniumacetate or phosphate buffer may help improve efficiency and reduce the retention of chargedspecies [52,74]. Many important chiral pharmaceuticals have been resolved on the p-CD

CSP such as P-blockers, calcium channel blockers, antihistamines and anticonvulsants(76], and amine acid derivatives [77]. Derivatised fooos of cyclodextrins such as the

acetylated, hydroxypropyl ether, carbamate and sulfated forros have increased the scope oftheir application in chiral chromatography [78,79].

Methacrylate monomers are caused ta polymerise for the formation of optically activehelices that are faund in polymethacrylate HPLC-CSPs. As with the CD-CSPs, solutes

enter the chiral cavilies ta fonn a diastereomeric complex and one structural requirement of

the solutes is an aromatic group at or adjacent ta the stereogenic centre. Alcohols, amides,

esters, ethers and ketones with the appropriate aromatic moiety have been resolvedsuccessfully with polymethacrylate CSPs, but chiral aliphatic compounds requirederivatisation before they can he resolved. Comman mobile phases include Methanol

modified with isopropanol, or mixtures of toluene and dioxane [52].

Chiral crown ethers (CR CSPs) are synthetic macrocyclie polyethers which contain chiral

substituents that prevent its rotation around an axis of dissymetery. Solutes, containing a

18

primary amine moiety near or at the stereogenic centre, fonn enantioselective complexes

with chiral crown ethers and can be successfully resolved with the CR CSP. The 18­

crown-6 ether ammonium complex has been immobilized in the commercially available

crown ether CSPs and is used with mobile phases consisting of perchloric acid at a

concentration of around 10 mM and small amounts oforganic modifiers such as alcohoIs or

acetonitrile. The pH of the mobile phase and temperature may be manipulated to obtain the

required retention and enantioselectivity [52]. Crown ether CSPs are usually used for

amino acid separations, such as the recent separation of aspartic acid, leucine, lysine,

phenylalanine and valine [80].

Type 4CSPs

Chiral ligand-exchange chromatography involves the formation of a diastereomeric

complex consisting of a transition Metal ion (M) such as Cu2+, a ligand which is a single

enantiomer of a chiral Molecule (L) such as an amine acid, and either one of the

enantiomers of a racemic solute. Enantioselectivity arises from the difference in stability of

the complexes formed between the R and S enantiomers with the Metal and ligand Le. L­

M-R vs. L-M-S. The commercially available type 4 CSPs immobilise the ligand on a

chromatographie suppon for improved efficiency and reproducibility (53].

Chiral ligand-exchange chromatography is only applicable to solutes that can fonn

complexes with transition metal ions thus are limited ta a-amino acids and mono- and

dicarboxylic acids with an a-hydroxy group. Aqueous mobile phases containing from

0.05 up to 20 mM copper(ll) sulfate are employed, and retention can be manipulated by

changing the pH of the mobile phase, adding modifiers such as Methanol or sodiuIll

chloride, and by changing the ternperatwe at which the chromatography is conducted [53].

Type 5 CSPs

Prateins are campased of L-amina acids, and their ability to bind stereoselectively and

reversibly with small molecules has led ta their immobilisation as type 5 CSPs. The two

main drug-binding serum proteins ofdrug compounds in humans are ul-acid glycoprotein

(AGP), which binds with basic drugs, and human serum albumin (HSA) which interacts

with both neutral and acidie drug compounds. The AGP-CSP and bovine serum albumin

CSP (BSA-CSP) were two of the earliest commercially available type 5 CSPs. Both chiral

calionie and anionic molecuIes can be resolved on the AGP-CSP, but the BSA-CSPs do

not enable the chiral resolution of cationic drugs [52,82,83]. Other proteins such as

19

(HSA), ovomucoid (OVM), a-chymotrypsin, and hen egg yolk ribof1avin have since been

immobilised as CSPs [82,84] and the compatibility of the buffered mobile phases

employed with type 5 CSPs, has rendered them particuIarly useful for toxicological and

pharmacokinetic studies [85].

The AGP-CSP is one of the most established CSPs for the direct separation of drug

enantiomers [85]. The retention and stereoselectivity of a solute on the AGP-CSP are

generally controlled through the use of charged or uncharged mobile-phase modifiers

which enable hydrogen bonding, hydrophobic and electrostatic interactions between the

protein and solute, and by manipulating pH of the mobile phase [52,82]. Examples of the

use of the AGP-CSP include the chiral resolution of ~-adrenolyticand antihistaminic drugs

[83], verapamil and gallopamil [85], salbutaInol [86], sotalol and other ~-blockers [87],

and dihydro-pyranoimidazopyridines [88].

HSA and BSA essentially have similar selectivity, mobile-phase effects and

chromatographic propenies. The mobile phases employed consist of phosphate buffers

and are most commonly modified with I-propanol. Retention and stereoselectivity of

solutes on the serum albumin CSPs are generally regulated by changing the buffer

concentration or pH. Anionic and neutral solutes, usually with aromatic and polar

moieties, are the ideal candidates for enantiomeric separation. Examples of chiral solutes

resolved on the BSA-CSP include leucovorin [89] and warfarin [90], while examples of

thase resolved on the HSA-CSP include dansyI amino acids [64] and D- and L- tryptophan

[91].

20

3 CAPILLARY ELECTROPHORESIS

Capillary electrophoresis (CE) is an established family of related techniques that employ

narrow-bore capillaries and high voltages to separate molecules of varying size and charge.

Over the last decade, the routine use of CE has mainly been for the separation small

Molecules and pharmaceuticals. However, application of CE to biomolecules such asproteins, peptides, nucieic acids and nucleotides has aIso been successful, and is an

imponant development with the growth of the biotechnological industry [92].

CE borrows much from the well-established methodologies developed for conventional

electrophoresis, the main separation technique that had been empIoyed for biomalecules.

Although the concept of CE dates back ta 1953, when Edstrom employed fme sille fibres

(15 JUIl Ld. X 11-12 mm) for the determinatian of RNA in single cells [93], the interest inCE among analytical chemists was limited until the work of Jorgenson and Lukacs in 1983

[94]. By using capillaries with internai diameters of less than 100 ~m, Jorgenson andLukacs separated mixtures of dansyl and fluorescamine derivatives of amino acids,

dipeptides and simple amines with extraordinarily high efficiency. Moreover, the

instrumentation they used was simple, as was the detection technology which aIso praved

ta be sensitive. Sînce then, much work has been carried out on CE and its applications,

and interest in this field continues to grow [95,96].

CE, which is now an established and powerful analytical tool, has severa! unique features

as it:

• employs capillary tubing and high electrical field strengths which enable rapid andhighly efficient electrophoretic separations;

• requires minute amounts of sample, and consumes limited quantities of reagents;

• is easily automated for easy operation and precise quantitative analysis;

• uses modern detector technology such that electropherograms resemble chromatogramsand can be interpreted easily;

• employs a variety of modes which enables its application to wider selection of analytescompared to other separation techniques.

21

3. 1 General Instrumentation

Nearly all of the work in CE prior to 1988 was earried out on homemade deviees but naw

there are Many eammercially instruments. A CE instrument requires a high voltage power

supply, that ean deliver up to 35 kV, connected to twa platinum electrodes which are

dipped into buffer solution contained in two separate buffer vials. A fused-silica capillary,

in which the electrophoretic separations occor, is aIso dipped into the buifer vials and part

of its polYimide coating is bumt at the appropriate position ta ereate an optical viewing

window for on-lïne detection [97].

......-....... DATA ACQUISITION

ELECTROLYTE.......-....~~__ BUFFER

BUFFERRESERVOIR

HV

BUFFERRESERVOIR

Schematic configuration of a CE system

The Capillary

The eapillaries most commonly employed in CE are bare, fused-siliea capillaries with

internai diameters ranging from 20 ta 200~. Better separation is observed with smaller

capillary internai diameters but poorer limits of detection result, due to reduced detector

path length and sample loadability. The polYimide coating on the outside of the capillary

affers it greater strength and flexibility, and May he bumt with a gentle flame to ereate the

optical window, or by electric heating, a method which prevents damage to the eapillary

wall [97]. The temperature in the capillary is kept constant [98], as an increase in

temperature decreases the viscosity of aqueous solutions and increases electrophoretic

mobility, which in turn causes the analyte to migrate faster than normal [99].

22

Bonded-phase capillaries contain an internaI coating and are employed in CE to suppressthe electroasmotic tlow far direct electrophoretic separations, and to reduce sample

adsorption on capillary walls. Examples of bonded phase capillaries include those coated

intemally with cellulose or dextran [100], polyethylenimine [101], and long-chain

hydrocarbons [102,103].

Sample Loadini Techniques

Modem CE instruments are Cully automated, with computer-controlled operations to

inrroduce the sampIe into the capillary. Precise and repeatable sample injection is essential

for reproducible results, and greatly influences the efficiency of the separation process.

There are three basic modes for the introduction of an analyte into the capillary:

hydrodynamic, gravity and electrokinetic. In hydrodynamic injection, the capillary is

placed in the sample solution and a volume of sample solution is introduced into thecapillary by applying a positive pressure or a vacuum. In gravity injection, the inlet end of

the capillary is dipped into the sample vial and raised to a certain height for a given time, to

generate a hydrostatic pressure that introduces the sample ioto the capillary. For bath thesemethods, the amount of sampie injected is dependent upon its viscosity thus the viscosities

of samples and standards have to be matched, and the temperature in the capillary kept

constant. Electrokinetic injection involves placing the capillary end electrode in the sample

solution and applying high voltage, to cause the sample to enter the capillary. The

disadvantage of this method is sampIe discrimination since the sample with highest mobility

will enter the capillary fllSt [97,104].

Detection Systems

AlI commercial CE instruments have UV absorbance detectors and generally provide

wavelength selection from 190 to 700 nm. Other detection methods such as diode array,

fluorescence, laser-excited tluorescence and mass spectrometry have also been used,

mainly ta achieve greater sensitivity and lower detection limits. Sensitivity is still the mainproblem with CE, and concentrated sample solutions are often required since only smallvolumes of sampIe are introduced into the capillary [97].

23

3.2 Fondamental Principles of CE

Separation in capillary electrophoresis is based upon the migration of charged molecules in

an applied field. The migration rate or mobility (JJ.) of a charged molecule is dependent

upon its size and charge, and the actual velocity (v) at which an ion moves is influenced by

its mobility and the magnitude of the applied voltage (E). The relationship between the

mobility of a charged Molecule, its velocity and the electric field strength (E) is given by:

J.L = :t.E

The frictional force that opposes the migration of an ion depends mainly on the viscosity

(11) of the buffer [97].

Fused-silica capillaries have acidic, ionisable silanol groups on the wall of the capillary

which may become ionised when in contact with the buifer contained within the capillary.

The surface charge on the capillary wall attracts positively charged ions from the buffer

solution to form an electrical double layer, and when a voltage is applied across the

capillary the cations in the diffuse part of the layer migrate towards the cathode carrying

water with them. This produces the electroosmotic flow (EDF) which causes a net flow of

solution along the capillary. The extent to which the silanol groups on the capillary areionised is dependent upon the pH of the buffer. At high pH, most of the silanol groups on

the capillary wall are ionised thus a large electroosmotic flow is generated and a migration

order of cations followed by the neutrals and anions is observed. At low pH, ionisation

and hence the EOf is significantly reduced [97,105-106].

..... +- • +.

~ ++ ++

\

+ +

- - ---\

+

\...

\...

\...

\

+EOF -

Effeet of high pH on the electI'oosmotic flow

There is little band broadening observed in capillary electrophoresis compared to other

separation techniques such as liquid chromatography. This is because unIike Iaminar flow

24

in liquid chramatagraphy, the driving force of electroasmotic flow is distributed in a

unifonn manner along the entire length of the capillary, allawing a uniform velocity across

the entire tubing diameter ta be achieved, except where the flow is close ta the wall when

flow velocity approaches zero [97,105].

The efficiency of separatians is influenced by the diffusion coefficient (Dm) of the solute

but a solute with a high diffusion coefficient, which include Macromolecules such as

proteins, May be separated with high efficiency despite their high Dm. The efficiency, N,

of a separation May he expressed by the following equation:

where V is the electric field strength

The efficiency of a separatian May be increased by increasing the voltage. However joule

heating, caused by the resistance of the buifer to the flow of current, results and the heat

generated causes temperature gradients across the capillary. Moreover, ineffective heat

dissipation will aIso lead ta temperature changes with time and drifts in migration times.

Thus narrower capillaries as weIl as efficient cooling systems are employed ta overcome

the problem. However, optimal resolution can be readily achieved through selecting the

appropriate butTer system and mode of capillary electrophoresis [97].

3 •3 Modes of Capillary Electrophoresis

Over the past ten years, five different principal modes of capillary electraphoresis have

been developed which have quite different operative and separative techniques. Thesemodes of capillary electrophoresis are:

• Capillary zone electrophoresis

• Isoelectric focusing

• Iso~chophoresis

• Capillary gel electrophoresis

• Mice1Iar electrokinetic chromatography

25

Capillary zone electrQphoresis

Capillary zone electrophoresis (CZE), also known as Cree solution capillary electrophoresis

(FSCE), is the simplest and most widely used mode of CE where separation is based upon

differences in charge-to-mass ratios. When a sample mixture is injected and the voltage

applied, the components of the mixture separate inta discrete zones depending on their

electrophoretic mobility. Molecules with small differences between their charge-to-mass

ratios can be separated by CZE, but neutraIs remain unresolved. The pH of the buffer isimponant as it influences the net charge of a molecule. At high pH, the EOF is substantial

and the observed migration order will be the cations, neutraIs and anions towards thecathode. At low pH, however, the EOF is significantly reduced thus to measwe the cations

and anions together in a single n1D, the polarity of the electrodes has to be reversed in order

to achieve the appropriate electrical configuration [97,105].

Various buffer additives may be employed in CZE to change the selectivity of the

separation. Examples of buffer additives include: inorganic salts which cause protein

confonnational changes; organic solvents which may act as a solubilizer and modifyelectroosmotic flow; urea which may solubilize and denature proteins and oligonucleotides;

and cellulose derivatives, often used to reduce the electroosmotic flow and to provide a

sieving medium (105).

Isoelectric focusini

The basis of isoelectric focusing (IEF) is the migration of on!y the charged molecules in anelectric field. A series of zwinerionic chemicals known as carrier ampholytes are used [0

generate a pH gradient, 10w at the anode and of high pH at the cathode. When a voltage is

applied, the positively charged ampholytes migrate towards the cathode, which results in an

increase in pH at the cathodic section, while the negatively charged ampholytes migratetowards the anode causing a decrease in pH. As the isoelectric point of each ampholyte isreached, the molecules become neutral, cease to migrate and becomes separated. The

requirements of IEF are the use of an anodic buffer that has a pH lower than the pl of the

Most acidic ampholyte, and a cathodic buffer that bas a pH higher than the most basic

ampholyte. This ensures that migration into the analyte does not occur. Furthermore,

suppression of the electroosmotic flow and other convective forces are necessary, and

bonded-phase capillaries coated with methylcellulose or polyacrylamide are commonlyemployed [97,105].

26

IsQtaehQpbQresis

In iSQtachophoresis (ITP), a leading electrQlyte with a higher mability than any of the

components in the sample mixture is used to fùl the capillary. The sampie is then loaded

and the capillary filled with a tenninating electrQlyte that has a mobility less than any of the

sample camponents. Separatian of the companents occurs, in the gap between the two

electralytes, as fQcused bands in the order Qf their mobilites and migrate at the same

velacity provided that a constant current is applied. One main advantage of ITP is the

cancentrating effect of samples which enables the injection Qf larger volumes of dilute

samples. HQwever, the selectiQn and optimisation of buffers emplQyed are less

straightfQrward in ITP, and simuItaneous detennination Qf bath cations and anions cannat

be carried out in the same run. Moreover, if two focused bands are shorter than the

detector cell length, the adjacent bands remain in contact and unresolved unless spacers

(solutes that are non-absorbing at the operating detection wavelength and have a mobility

value falling in between the mobilities of the two unresalved bands) are employed. This

enables the resolution of the two adjacent bands by migrating between them [105,107].

Capillacy ~l e1ectmphQresis

Capillary gel electrophoresis (CGE) is generally the mode of choice for the separation of

biomolecules such as DNA, carbohydrates and oligonucleotides [108]. Gels, which act asmolecular sieves, are used ta fùl the capillaries for the performance of size separations and

help suppress both the electroosmotic flow and convection [105]. Two main classes of

gels are commonly employed in CGE: the physical or noncrosslinked gels, and the

chemical or crosslinked gels [109-111]. HydroxypropylmethylcelluIose, which consists of

polymers that are entangled to fonn a parous structure, is an example of the physicai gels

used in CGE and is relatively rugged ta changes in the environmenl Chemical gels such aspolyacrylamide are more porous; they consist ofcovalently attached polymers and are legs

rugged. Most commercially available capillaries used in CGE consist of cross-linked

polyacrylamide, the concentration of which may he manipulated ta oplimise resolutioD.

MiceUar electrokinetic cbrornatQ&1Jpby

Until the development of micellar electrokinetic chromatography (MEKC) by Terabe in

1984 [112], neuttal compounds were not resolvable by CE; this was a serioos limitation of

the technique. The separation principle behind MEKC is the use of micelle-fonning

27

surfactants for differential solute distribution (micellar solubilization) and differentiaI

migration of the micelle [113]. Surfactants are long chain molecules that possess a longhydrophobie taU and a hydrophilic head, and aggregate to fonn micelles when above a

certain concentration known as the critical micelle concentration (CMC). Hydrophobie and

electrostatic interactions enable micelles ta organise bath charged and neutral analytes at

their molecular IeveI, and provide anaIytes with the ability to partition into and out of the

micelles for separation to take place.

The analytes have an overall decrease in migration velocity when associated with a micelle,

and migrate towards the cathode at a lower velocity than the bulle Iiquid, after the

endoosmotic flow. The order of migration depends on the relative hydrophobicities of the

analytes, which in tum determines the extent of their association with the micelles. Neutral

Molecules bear no charge and have zero mobility, but the hydrophobie core of micelles is

strongly solubilizing and dissolves the neutral Molecules. By providing a pseudostationary

phase with which neutraI molecules interact, dilferenùal partitioning of the neutralMolecules between the separation electrolyte and the micellar pseudostationary phase occurs

which enables the separation of the neutral species [105,113].

ELECTROOSMOTIC~

FLOW

ELECTROPHORETIC..

-••

"-0 '-0"-0"-0"-0'-0 ••4)

"'-0 "-0'-0'-0•"-0 +-.0"- +-0""- 0""-0

VELOCITY

+

g NEGATIVEly CHARGED ANALYTE

• POSrnVELY CHARGED ANALYTE_ NEUTRALLYCHARGEDANALYTE

o MICELLEeg. SOS

Schematic illustration of the separation principle of MEKC

28

There are four major classes of surfactants: anionic, cationic, zwitterionic and nonionic.

Sodium dodecyl sulfate (SDS) is one of the most commonly used synthetic, amonic

surfactants employed in MEKC. However, naturally occurring compounds such as bile

salts have aIso been shawn to be useful, panicularly for the separation of neutral, lipophilic

compounds. The type of surfactant used in the separation of neutral or non-ionic Molecules

is important in manipulating selectivity. The size, aggregation, and geometry of the

micelles influence the type of surfactant selected for each separation. Organic modifiers

such as Methanol and acetonitrile May be used in MEKC to increase the migration velocity

of hydrophobic species sa that they spend more time in the bulle phase, and enable

separations to be more easily achieved. However, the amount of arganic modifier that can

be used is limited to concentrations of 5-25% as aggregatian and micellar ionisation

numbers are affected [105,113].

3.4 Applications or Capillary Electrophoresis

8ioIDolecules

The separation of amino acid derivaûves was one of the earliest applications of CE [114].

Amino acids exist as cations at law pH and can be readily separated by CE but because

most amine acids do not have good UV-absorbing properties, their analysis has generally

required derivatisation [115,116] or the use of indirect detection methods [117]. CE has

recentIy become more widely used for the analysis of peptides and proteins, such as

peptide mapping ta characterize recombinant proteins [118-121], and the purity control of

pepùdes [122,123]. CZE is usually the mode of choice, and the probIem of protein

adsorption onto the walls of the fused-silica capillary can be overcome by empIoying Iow

pH buffers to minimise the negative charge on the capillary walls and prevent protein

adsorption [124]. An alternative method is the use of buffers al high pH, as bath the

capillary surface and protein would be negatively charged and repulsion would take place

[114]. The main advantages of using CE for peptide analysis are shorter anaIysis times

and the requiremenl of only small sample amounts which is particularly important for the

analysis of expensive or rare peptide preparations [125].

The analysis of bases, nucleosides, nucleotides and oligonucleotides is an important

application of CE. Several important antiviral agents, such as 3'-azido-3'-deoxythymidine

(azT) used for the treatment of AlOS, are nucleosides and nucleotides, and clinical studies

for therapeutic drug monitoring as weIl as metabolism have olten required their

29

quantification [126]. Furthennore, the purification of oligonucleotides used for example in

gene cloning, DNA fmgerprinting, and as primers for DNA sequencing [126], and the

detection of base damage within DNA [127] have all required high-resolution analytical

techniques. The separation of bases, nucleosides and nucleotides can be achieved with

bath CZE and MEKC but the analysis of oligonucleotides generally requires the use MEKC

or CGE. Examples of CZE separations include the resolution of a number of cyclic

nucleotides involved with various brain functions [128]; the detennination ofcytosine-(3-D­

arabinoside, an antileukemic agent, in human serum for a therapeutic drug monitoring

study [129] with bare fused-silica capillaries; and the separation of ribonucleotides using a

polyacrylamide-coated capillary [130]. In MEKC separations, SDS is commonly

employed as the surfactant as in the separation of several dideoxynucleosides used for the

treatment of AlOS [131], and in the separation of adducts of DNA that had been exposed to

alkylating agents [132]. CGE separations of larger oligonucleotides are usually with

polyacrylamide gel-filled capillaries [133,134].

PhaonaceutieW applications

The pharmaceutieal applications of CE can be separated into five main aceas: chiral

separations, identity confmnation, assay, determination of impurities, and determination of

drug counterions [135]. CE is often used in eombination with HPLC for purity testing as il

can offer an alternative selectivity that can provide complementary information. An

example is the additional impurities that were identified for the antibiotics tetracycline and

gentamicin sulfate using CE as a complementary separation technique to HPLC [135].

There is aIso growing use of CE for the determination of active ingredients in

phannaceutical formulations and good precision as weil as detector linearity is often

achieved. To ensure reliable results, consistent sample volumes have to be introduced inta

the capillaryt thus the manix ofsample solutions is made to match exactly those ofstandard

solutions. Good agreement between label claim and results from quantitative assays of

formulations by CE have been observed [135].

Ta improve water solubility, many pharmaceuticals are prepared as saIts. Manufacturing

processes are optimised to give a predetennined ratio or stoichiometry of drug ta caunter­

ion, thus the level of counter-ions must he quantüied in phannaceutical formulations. The

application of CEt using indirect UV detection, for the quantification of counter-ions in

stoichiometric determinations has been shown to be more advantageous than the other

techniques that are used such as titration or ion-exchange chromatography. This is because

30

CE methods are simple, rapid and more cost-effective using cheaper consumables and

having lower waste disposai costs [6,135].

Chiral Separations

Quantitative enantiomeric purity determinations, as well as low levels of undesirableenantiomers, can be detennined by CE methods due ta the high separation efficiencies that

can be readily achieved. Cyelodextrins have been used extensively as chiral selectors in

CE, and with much success, since the pioneering work of Tanali and Terabe in 1989

[136,137]. Although native cyclodextrins have found mueh application in CE, theenantiaselectivity observed is often rather low and their use in the enantioseparation of

neutral solutes has been unsuecessful [78,138]. The law enantiaselectivity may he due ta

the symmetry of the native cycladextrins, and the fact that the achiral portian of a solute isincluded into the hydrophobic cavity, decreasing the interaction between the chiral centresof the cyclodextrin and the solute. With regard to their application being restricted to

charged species, native eyclodextrins cannot resolve chiral neutrals as they lack the

electrophoretic mobility required in free-solution CE. These limitations led ta thedeveloprncnt of modified cyclodextrins, which are obtained by derivatising the hydroxy

groups on the cyclodextrin rim with ionic substituents directIy, or via a short alkyl chain[78,79].

Recently, the use of modified cyclodextrins for chiral resolution in CE has received

incredible attention as they provide a mean far separating neutral compounds and because

enantioselectivity is high. The solutelcyclodextrin complexes formed with the modified

CDs are more stable, and additional electrostatic interactions between Ûle cyclodextrin andsolute contribute ta higher chiral recognition. A number of modified cyclodextrins havebeen developed. First, there are the cationic cyclodextrin derivatives that contain amino andalkylamino groups, or other positively charged nitrogen-, phosphorus-, and sulfur-groups

[79]. Their use as chiral selectors in CE however, has not been explored to any greatextent On the other band, anioDie cyclodextrins including carboxymethyl-, sulfobutyl

ether-, and sulfoethyl ether- p-cycladextrins have found wide application in CE and arecommerciallyavailable [138-143].

Although cyclodextrins have found much application in chiral separations, other chiral

selectors have also been employed in CE with much success. Heparin, a naturally occuring

glycosaminoglycan, was used as a chiral selector for the separation of a number of

31

antimalarial compounds and antihistamines [144], and enabled excellent resolution ofoxamniquine (145]. Separation ofwarfarin and tryptophan enantiomers were achieved by

CE with the use of human serum albumin as a chiral selector [146], and macrocyclic

antibiotics such as vancomycin and ristocetin A have been employed for chiral separations

of a wide range of solutes [147,148].

When compared ta HPLC, the most commonly used separative technique in phannaceutical

analysis, CE offers rapid and efficient separations with limited consumption of reagents.Preconditioning of CE capillaries is unnecessary and the rugged nature of the capillary

enables rinsing to be carried out after each analysis as a means of removing any potentialinterferences. With the increased pressures exerted on the pharmaceutical industry to

produce safety data on new pharmaceuticals, CE offers not anly rapid analysis times butaIso rapid method development, thus its use as a complementary technique to HPLC isincreasing. In addition, much research is ongoing in arder to show the reliability ofCE as

the sole analytical tool [92, 137]. However, there are disadvantages to CE such as the

limited preparative options that are offered, the influence of the sample matrix, and the

lower sensitivity compared to HPLC. However, with commercial instruments that alIow

fluorescence detection and interfacing with mass spectrometers, improvements in

sensitivity have been observed funher showing the usefulness of CE as a rapid and

efficient separative technique [135].

32

RESEARCH OBJECTIVES

33

Chlorpheniramine has been a popular over-the-counter drug for the treatment of allergie

conditions because it is a potent, long-lasting antihistaminic compound with mild side­

effects. Severa! research groups have studied the phannacokinetics of chlorpheniramine

since its development, but the resuIts show considerable discrepancies. For example, there

is much variability in the reported in-vivo half-llfe of chlorpheniramine, ranging from 14

hours up to 40 hours, and there are aIso differences in its reported duration of action [1-4].

Some of these earlier pharmacokinetic studies involved the detennination of the separate

enantiomers of chlopheniramine but excluded the pharmacologically active Metabolite

enantiomers [1,3-51. Where studies had included the determination of bath the parent and

phannacologically active Metabolites of chlorpheniramine, only achiral methods of analysis

were used [1,18,20]. The detennination of the enantiomers of chlorpheniramine as weIl as

its pharmacologically active Metabolites May therefore be a more accurate method of

studying the phannacokinetics of chlorpheniramine.

Studies have shown that chlorpheniramine only accounts for a small amount of the urinary

products in humans. The excretion of two Metabolites, monodesmethyl and didesmethyl

chlorpheniramine, has been confmned in a number of studies but the two N-demethylated

compounds account for less than 10 % of the urinary Metabolites [1,2]. More polar

Metabolites that constitute a significant part of the urinary Metabolites were isolated in early

studies. However, attempts ta deduce their structures were unsuccessful [1]. Subsequent

studies have shown that chlarpheniramine N-oxide is important in animaIs [36,149] and

humans [19,20]. Two other polar metabolites have aIso been isolated in dog studies, and

identifed as the alcohol resulting from oxidative deamination of chlorpheniramine and an

acid which may be conjugated to an amine acid [149]. Other metabolites that have been

reponed in human studies include 3-(p-chloraphenyl)-3-(2-pyridy1)-propanol, 3-(p­

chlorophenyl)-3-(2-pyridyl)-N-acetylaminopropane and 3-(p-chlorophenyl)-3-(2-pyridyl)­

propanic acid [19].

As pan of a recent, new pharmacokinetic study of chlorpheniramine, headed by Dr S.

Yasuda at Georgetown University, Washington, OC, a method to separate the enantiomers

of chlorpheniramine, its N-demethylated Metabolites and chlorpheniramine N-oxide is

required. The separation of the enantiomers of chlorpheniramine bas been achieved by

HPLC using various CSPs including the p-cycladexUin [76], Cll-acid glycoprotein [150]

and ovomucoid [151]. There are aIso existing methads ta separate the enantiomers of

chlorpheniramine by capillary electrophoresis, using neutral [142] and modified

cyclodextrins [143, 152-154]. The aim of this study is to reprJduce the results af same af

34

the existing methods and to apply the separation conditions to resolve the enantiomers ofchlorpheniramine and its pharmacologically active N-demethylated and N-oxideMetabolites.

35

MATERIALS AND METHOnS

36

HPLC Instrumentation

The chromatographie system used in this study was composed of a Spectroflow400 binary

pump set at a flow rate of 1.0 mUmin, and a UV variable wavelength detector (Applied

Biosystems Ine., Toronto, Ontario, Canada) used at a detection wavelength of 254 nm. A

manuaJ injector (Rheodyne, Cotati, CA, USA) equipped with a 20J,lL loop(ThermoSeparation Products, Toronto, Ontario, Canada) was employed for the analyses

and a Spectra-Physies Datajet integrator (ThermoSeparation Products, Toronto, Ontario,

Canada) was used for data collection. Separations were performed on a Chiralpak AD

column (250 mm X 4.6 mm, Chiral Technologies Inc., Exton, PA, USA) and the nitrile-,

amino- and silica-guard columns that were used, were purchased from Regis Chemical

Company, Morton Grove, IL, USA.

CE Instrumentation

A SpectraPHORESIS 1000 CE instrument (ThennoSeparation Products, Mississauga,

Ontario, Canada) was employed for the analyses, and separations were perfonned in

fused-silica capillaries 80 cm long (72.5 cm ta detector) X 50 J.lIIl I.D. and in capillaries 80

cm long (72.5 cm ta detector) X 75 J.lm LD. (Polymicro Technologies, Phoenix, AZ,

USA) at a constant temperature of 20 oC. The voltages used in the study ranged from 10

to 30 kV, and hydrodynamic injections between 2 and 20 seconds were employed. UV

absorbance detection at wavelengths ranging from 200 ta 225 Dm were used for the

analyses.

At the stan of each day, the capillary was rinsed with sodium hydroxide (0.1 M, for 10

minutes), followed by distilled water and the running buffer, each for a further 10 minutes.

Two blanle injections were made prior to the analysis of the chiral solutes to stabilise the

electrophoretic system. Sodium hydroxide (0.1 M, for 5 minutes) and the running buffer

(5 minutes) were used to rinse the capillary between sample injections.

Materials

The hexane, 2-propanol, ethanol and methanol used to prepare the HPLC mobile phases

were ofHPLC grade and obtained from J. T. Baker (phillipsburg, NIt USA), as were the

mobile phase modifiers, diethylamine and trifluoroacetic acid, and the sodium

monohydrogen phosphate and sodium dihydrogen phosphate used to prepare the

37

electrolytes for the CE studies. Sodium dodecyl sulfate, sodium chalate, sodium

deoxycholate, sodium taurocholate and triethanolamine were purchases from Sigma (St.

Louis, MO, USA); and hexanesulfanic acid was abtained from Aldrich (Milwaukee, WIS,

USA). The distilled water used ta prepare the buffers were of Millipore quality from a

Milli-Q Water system.

A number of charged cycladextrins were employed as chiral selectors in the CE studies

including hydroxypropyl ~-cyelodextrin and sulfated ~yclodextrin both from Aldrich

(Milwaukee, WIS, USA), sulfated and carboxymethyl ~-cyclodextrin from Astee Ine.,

(Whippany, NI, USA), native J)-<:yclodextrin purchased from Sigma Chemical Company(St. Louis, MO, USA), and carboxymethyl ~-cyclodextrins from Cyelolab (Budapest,

Hungary) and WackerChemie (Munich, Germany).

Analytes

Racemic chlorpheniramine maleate and its separate enantiomers, monodesmethyl

chlorpheniramine, didesmethyl chlorpheniramine and chlorpheniramine N-oxide were gifts

from Dr S Yasuda, Georgetown University, Washington OC, USA.

Monodesmcthyl chlorpbcniramine Didesmcthyl cb10rpbeninunine

CbJorpheniramine N-oxide

Metabolites ofchlarpheniramine.

38

RESULTS AND DISCUSSION

39

HPLC STUDIES

Amylose tris(3,5.dimethylphenylcarbamate) CSP [AD-CSP]

Excellent resolution of the enantiomers of chlorpheniramine on the AD-CSP was reported

[155] using a mobile phase that consisted of hexane-2-propanol (98:2 v/v) + 0.1 %

diethylamine pumped at a tlow rate of 1.0 mUmin and with UV detection at 254 om.

Injection of a sample solution of racemie chlorpheniramine, dissolved in mobile phase at a

concentration of 100 lJ,g/mL, using the same mobile phase composition and separation

conditions as reported by the authours, was carried out. An enantioseleetivity of 1.3 was

observed for the separation of the enantiomers of chlorpheniramine (Figure 1). Injection of

the separate enantiomers showed that the (R)-isomer eluted frrst at 14 minutes followed by

the (S)-isomer at 18 minutes.

Injection of separate solutions of the two N-demethylated Metabolites and chlorpheniramine

N-oxide, dissolved in mobile phase at concentrations of 100 IJ,g1mL, was also carried out

using the same chromatographie conditions but the Metabolites did not elute from the AD

column. The effect of increasing the polarity of the mobile phase ta elute the Metabolites

was studied (Table 1). The use of greater amounts of 2-propanol in the mobile phase did

not elute the Metabolites from the AD column, and the compounds remained relained when

ethanol or Methanol replaeed 2-propanoI.

Mobile Phase Hexane-alcohol + 02% dietbvlamine Alcohol

1 95:5 2-propanol

2 95:5 etbanol

3 95:5 methanol

4 90:10 2-propanol

5 80:20 2·propanol

6 70:30 2-propanol

7 60:40 2-propanol

8 60:40 ethanol

9 50:50 2-propanol

10 50:50 ethanol

Table 1. Mobile phase modifications studied to elute the Metabolites ofchlorpheniramine from the AD column.

40

PEAK RE'IENltON 1'IMElMiDuta) IDENmY

1 14.17 (R)..chlomheninmine

2 17.93 (Sl-chloroheninmine

1

2

Retention time (minutes)

Figure 1. HPLC separation of (R)- and (S)- chlorpheniramine. Column,Chiralpak AD, 250 mm X 4.6 mm I.D.; mobile phase. hexane-2..propanol(98:2) +0.1 % diethylamine; tlow rate. 1mUmin; deteetion. UV 254 Dm.

41

Booth and Wainer [156] recentIy separated a series of chiral aromatic acids and amides on

the AO-CSP using a hexane-2-propanol mobile phase modified with trifluoroacetic acid,

and proposed that ion-pairing was the basis of retention. Raeemic chlorpheniramine,

dissolved in mobile phase at a concentration of 100 J.LglmL, was injected onto the AD

column. The mabile phase consisted hexane-2-propanol (95:5 v/v) + 0.3 % trifluoraacetic

acid and was pumped at a flow rate of 1.0 mUmin. UV detectian of the analytes was

carried out at 254 nm and partial resolution of the chlorpheniramine enantiomers was

achieved (Figure 2), but the enantiomers of the N-demethylated metabolites and

chlorpheniramine N-oxide were unresolved with the same chromatographie conditions.

Increasing the amount of trifluaroacetic acid ta 1 % enabled partial separation of the

enantiomers of all three Metabolites to be achieved (Figure 3). Hawever, the enantiomeric

separation of the parent compound was lost, and injection of a mixture of the three

Metabolites resulted in co-elution of the anaIytes. The elution orders of the enantiomers of

the Metabolites were not deduced as separate isomers of the Metabolites were not available.

Attempts to resolve the enantiomers of the Metabolites by placing nitrile-, amino- or silica­

guard columns in front of, or behind, the AD column were unsuccessful.

CE STUDIES

Hydroxypropyl·~·cyclode"trin

Good resolution of the enantiomers of chlarpheniramine (Figure 4) was achieved using the

separation conditions employed by St. Pierre and Sentell [153], with hydroxypropyl-~­

cyclodextrin as the chiral selector. Baseline resolution of the enantiomers of didesmethyl

ehlorpheniramine was aIso possible with the same separation conditions, but the

chlorpheniramine N-oxide enantiomers could not be separated (Figure 5). The effect of

increasing the concentration of hydroxypropyl-p-cyclodextrin on the separation of the

enantiomers of chlorpheniramine N-oxide was studied. The enantiomers of

chlorpheniramine and its didesmethyl Metabolite remained separated when the concentration

of bydroxypropyl-~-èyclodextrinwas increased to 2S mM, but the enantiomers of the N­

oxide metabolite were unresolved. Sodium dodecyl sulfate (SDS) was added to the

separation butfer, a technique known as CD-MEKC that olten enhances enantioselectivity

by creating a pseudostationary phase with which chiral solutes ean interaet [152], with the

aim of resolving the enantiomers of chlorpheniramine N-oxide. However, no peaks were

observed in the electropherograms of chlorpheniramine N-oxide samples and peaks were

also absent in th~ electropherograms for the parent drug wbich suggested that the strongly

42

PEAK RETEN"l10N 11ME CMinuta} J1)EN'IIlY

1 16.57 (Ill-e:hloroheniraminc

1 17.48 (Sl-e:blollJbeniraminc

21

Retention lime (minutes)

Figure 2. HPLC separation of (R)- and (S)- chlorpheniramine. Column,Chiralpak AD, 250 mm X 4.6 mm I.D.; mobile phase, hexane-2-propanol(95:5) + 0.3 % trifluoroacetic acid; flow rate, 1mUmin; detectiont UV 254nm.

43

(i)

i

(ili)

i

Retention lime (minutes)

(ü)

Retention time (minutes)

~-e 'l:i-c::•'"~..fIi~as

~

Retention lime (DÙDuteS)

Figure 3. Chromatographie separations of Ci) monodesmethylchlorpheniramine (ii) didesmethyl chlorpheniramine, and (Hi)ehlorpheniramine N...oxide. Column, Chiralpak AD, 250 mm X 4.6 mmI.D.; mobile phase, hexane-2-propanol (95:5) + 1 % trifluoroacetic acid;flow rate, 1 mUmin; deteetion, UV 254 Ml.

44

solubilizing nature of the SOS micelles had completely solubilized the solutes. Analysis of

monodesmethyl chlorpheniramine was not carried out as the limited supply of the

metabolite had been exhausted in the HPLC studies.

The analysis of a mixture of chlorpheniramine and its didesmethylated metabolite showed

that the enantiomers were only partially resolved from each other. The use of a smaller

diameter capillary (50 Ilm LD.) and a 40 mM Na2HP04 buffer did not improve theresolutian, but migration times were decreased without a loss in resalution (Figure 6). The

use of SOS at concentrations down to 10 mM was still too solubilizing, and the addition of

hexane sulfonic acid (1 mM) ta the system as an ion-pairing agent ta decrease the

interaction between the SOS micelles and solutes, was unsuccessful.

Bile salts are chiral compounds that fonn aggregates less solubilizing than SDS micelles,

and offer an alternative surfactant system for the separation of lipophilic compoundsincluding benzothiazepin analogues and corticosteroids [157 t 158]. The bile salts wereinsoluble in phosphate buffer al pH 4.0, thus a 40 mM phosphate buffer at pH 7.0 wasemployed. Sodium chelate alone did not result in the enantiemeric separation of

chlorpheniramine or its didesmethyl Metabolite. When added to the separation buffercontaining 20 mM hydroxypropyl-~-cyclodextrin, sorne resolution of the two solutes wasobserved but there was virtually no enantioselectivity (Figure 7). The required resolutionand enantioselectivity also cauld nat be achieved with the other bile salts, sodium

deoxycholate and sodium taurocholate. Furthermore, the addition of native or sulfated ~­

cyclodextrins to the (WO phosphate buffers containing 20 mM hydroxypropyl-~­

cyclodextrin failed ta resolve the enantiomers of chlorpheniramine and its didesmethylmetabolite.

Sulfated ~.cyclodextrin

Sulfated JH;yclodextrin was reponed ta separate the enantiomers of chlorpheniramine [154]

but the initial attempts to reproduce the separation failed. Upon discussion with thecorresponding authour, the source of the problem was identified ta be the poor quality ofthe sulfated I3-cyclodextrin (Astec Inc., Whippany, NI, USA). The cyclodextrins had not

dissolved completely in the buffer, and the amount that did dissolve formed a yellow

solution. The use of a sample of sulfated p-cyclodextrins (Aldrich, Milwaukee, WIS,

USA) from Stalcup enabled the separation of the chlorpheniramine enantiomers (Figure 8).

45

PEAK MIGRAnON TIME lMinutcsl IDENlTlY

l 29.85 Racemic

2 30.44 chloruheniramine

1 2Ec

0-N-cuui.ccu

Il~,Il

r---- JMigration tilDe (minutes)

Figure 4. Electropherogram showing the separation of racemicchlorpheniramine (LO f.lglmL in 50 mM Na2HP04, pH 4.0). The migrationarder of the separate isomers was not detennined. Separation conditions:capillary, fused-silica, 80 cm X 7S (.lm I.D.; butfer. Na2HP04'(SO mM, pH4.0) containing 18 mM hydroxypropyl-~-cyclodextrin, temperature. 200c;injection, hydrodynamic, 2 seconds; voltage, 10 kV; detection, UV al 210nm.

46

(i)

ec0-M~ t"J t""-

ao

en <: OC ~

r--: f""-~ ('t')• ~

~

Migration lime (minutes)

(ü)

Migration time (minutes)

Figure 5. Electropherograms showing (i) the enantiomeric separation ofdidesmethyl chlorpheniramine (l.0 J,lglmL in 50 mM Na2HP04, pH 4.0),and (ü) chlorpheniramine N-oxide (l.O J.lglmL in sa mM Na2HP04. pH4.0). Separation conditions: capillary, fused-silica, 80 cm X 75 J1II1 I.D.;buffer, Na2HP04 (50 mM, pH 4.0) containing 18 mM hydroxypropyl-l3­cyclodextrin, temperature, 200C; injection, hydrodynamic, 2 seconds;voltage, 10 kV; detection, UV at 210 Dm.

47

PEAK MIGRAlION TIME (Minutes) IDENmY

l 20.98 Didesmethvl cblorobeniramine

2 21.48 Chloll'heniramine

3 21.74 Didemetbvl chlorpheniramine

4 22.24 Cblorobeniramine

Migration lime (minutes)

Figure 6. Enantiomeric separation of chlorpheniramine and didesmethylchlorpheniramine. The migration orders of the separate enantiomers werenot detennined. Separation conditions: capillaryt fused-silica, 80 cm X 50Jl.m 1.0.; butfer, Na2HP04 (50 mM, pH 4.0) containing 18 mMhydroxypropyl-p-cyclodextrin, temperature, 20°C; injection,hydrodynamic, 2 seconds; voltage, 10 kV; deteCtiOD, UV at 210 Dm.

48

PEAK MIGRATION11ME (Minutes) IDENInY

1 32.06 Racemic cblombeDiramine

2 32.8S Didesmethvl chlorobeDiramine

l

2

Migration time (minutes)

Figure 7. Electropherogram showing the resolution of chlorpheniramineand didesmethyl chlorpheniramine with sodiwn cholate (0.1 M) added to theseparation buffer. Separation conditions: capillary, fused-silica, 80 cm XSO J.Lm LD.; buffer, NaH2P04 (50 mM, pH 7.0) containing 20 mMhydroxypropyl-p-cyclodextrin; voltage, 10 kV; temperature, 200C;injection, hydrodynamic, 2 seconds; detectioD, UV at 210 Dm.

49

Unfortunately, more sulfated Ii-cyclodextrins was not availab1e from the supplier andcontinuation of the work to separate the enantiomers of the metabolites could not be carried

out.

Carboxymethyl Ii-cyclodextrin

Another charged ~-cyclodextrin that has received much recent interest is the carboxymethyl

~-cyclodextrin [79,143]. Through persona! communication, a small sample of

carboxymethy11i-cyclodextrin (Wacker Chemie, Munich, Gennany) was obtained fromBlaschke's group, Munster University, Gennany, for investigation into its application forthe enantiomeric separation of chlorpheniramine and its metabolites. By this time, sorne

monodesmethyl chlorpheniramine was available for the study.

Although long migration limes were observed for the analytes, good resolution of the

enantiomers ofchlorpheniramine, its N·demethylated Metabolites and chlorpheniramine N­oxide was achieved using the carboxymethyl ~-cyclodextrin as the chiral selector (Figure9). The downward slope of the baseline observed in the electropherogram is because theelectrophoretic system had not yet completely settled when injections of the sampIe solutionwere made. Analysis of separate sample solutions showed that the N-oxide enantiomers

migrated flfSt, followed by chlorpheniramine, monodesmethyl chlorpheniramine and thendidesmethyl chlorpheniramine.

CarboXYJDethyl-6-eyclodexUins (Cyclolabl

The carboxymethyl ~-cyclodextrin obtained from Blaschke's group was not commercially

available, thus carboxymethyl p-cyelodextrins were purchased from Cyclolab (Budapest,Hungary) and Astee Ine., (Whippany, NI, USA) for continuation of the work. Goodresolution of the enantiomers of ehlorpheniramine and its metabolites was observed with

the carboxymethyl-~-eyclodextrins obtained from Blaschke's group. However, thecyclodextrins from Cylolab showed a different selectivity since application of the previousseparation conditions with the cyclodextrins from Cyclolab resulted in a 10ss ofenantioselectivity for the N-oxide and co-migration of two Metabolite enantiomers (Figure

10).

50

PEAK MIGRATION TIME lMinuleS) IDEN"I1'IY

1 25.13 (S).cblorpheDiramine

2 26.17 CR).cblorpheniramine

1 ·2

Ec::~-~

Migration lime (minutes)

Figure 8. Electropherogram of the enantiomeric separation of racemicchlorpheniramine using sulfated p-cyclodextriDs. Separation conditions:capillary, fused-silica, 80 cm X SO J1IIlI.D.; buffer, Na2HP04 (10 mM, pH3.8) containing 2 % sulfated p-cyclodextrins, temperature, 200c; injection,hydrodynamic, 2 seconds; voltage 15 kV; deteelio~ UV al 214 DD1.

SI

PEAK MlGRAlION 11ME (Minuta) IDEN1IIY

1 30.25 Chlorpheninmine N-oxide

2 32.46

3 51.04 (Sl-chlorpheninmine

4 53.07 (Rl-chlorgheniramine

5 54.99 Monodesmethyl chlorpheninmine

6 56.30

7 56.83 Didesmethyl chlorpheniramine

8 58.06

l 2

5 61

8

Migration time (minutes)

Figure 9. CE resolution of the enantiomers of chlorpheniramine and itsmetaboütes using carboxymethyl IJ-cyclodextrlns (Wacker Chemie).Separation conditions: capillary, fused-silica, 80 l1JIl X SO lIJIl ID.; buffer,NaH2P04 (40 mM, pH 6.0) CODtaining 20 mM carboxymethyl ~­cyclodextrins, temperature, 200c; injection, hydrodynamic, 2 seconds;voltage 20 kV; deteetion, UV al 225 Dm.

52

The enantiomeric separation ofchlorpheniramine had been reported with carboxymethyl ~­

cyclodextrins from Cyelolab at a concentration of 5 mM [143]. Decreasing the

concentration of the cyclodextrins reduced the migration times of the analytes, but

resolution was aIso reduced and the enantiomers of the N-oxide were still not separated

(Figures Il and 12).

A slight improvement in the resolution of the enantiomers of chlorpheniramine and its N­

demethylated Metabolites was achieved when the voltage applied ta the capillary was

decreased ta 12 kV, but broad peaks were abserved for the analytes. Optimisation of

separation conditions by increasing the concentration of the phosphate buffer appeared ta

have enabled the enantiomeric resolution of chlorpheniramine and its N-demethylated

Metabolites but the peak shapes were poor, and the enantiomeric separation of the N-oxide

could not be achieved (Figure 13).

The effeet of buffer pH on the separation of the chiral solutes was aIso studied. Phosphate

buffers at pH 6.5, 7.0 or 7.5, and a phosphate-borate buffer (pH 9.0) were used ta prepare

separation buffers cantaining the carbaxymethyl ~-cyclodextrin for the analysis of

chlorpheniramine and its metabolites, but no improvement in enantiomeric resalution of the

analytes was observed with the various buffers. The use of a butIer prepared framphospharic acid (100 mM) adjusted ta pH 3.0 with triethanolamine containing

carboxymethyl ~-cyclodextrins from Cyclolab enabled excellent resoluùon of the

chlorpheniramine enantiomers (Figure 14), but application of the buffer system for the

enantiomeric resolution of the Metabolites was unsuccessful.

The use of potassium phosphate ta prepare the separatian buffer was alsa studied. Six

peaks were observed in the electropherogram for the separation of chlorpheniramine and its

N-demethylated Metabolites (Figure 15) but anempts ta imprave the resolution of the

analytes were unsucessful and the enantiomers of the N-oxide were unresalved. The use

ofcarbaxymethyl ~cyclodextrins (Astee loe.) aIsa failed to give the required results.

53

PEAK M10RAnONTIME (Minuta) IDENl1"IY

1 29.43 ChloIDheairamiDe N-onde

2 50.62 (S).ch1ol"Dheniramine

3 S4.48 (R).cblol"Dbeniramine

4 S7.30 Monodesmethvl ch10mbeniramine

S 60.12 Monodesmethyl + didesmethyl chlol"Dheniramine

6 63.28 Didesmetbvl chloll)beniramine

15

4

2 3

6 .

Migration lime (minutes)

Figure 10. Electropberogram of the CE separation of cblorpbeniramine andits metabolites using carboxymetbyl IKyclodextrins (Cyclolab). Separationconditions: capillary, fused-silica, 80 cm X SO JUIllD.; butfer, NaH2P04(40 mM, pH 6.0) containing 20 mM carboxymetbyl ~-cyclodextrins,

temperature, 200c; injection, hydrodynamic, 2 seconds; voltage 20 kV;detection, UV at 225 nm

S4

PEAK MIGRATION TIME (Minuta) IDENInY

1 28.46 ChlombeDiramine N-oxide

2 48.19 (S)-cb1orobeDiramine

3 51.92 (R)-cb1oft)beniramine

4 54.64 MODodesmethvl c:blorobeDiramine

5 57.42 MonodesmethYl + didesmethyl c:h1orobeDiramine

6 60.46 Didesmethvl c:b1oroheniramine

1

e 5c::

'" 4MM

~ 2 3ui..clU

ê;

Migration lime (minutes)

Figure Il. Electropherograms of the CE separation of cblorpheniramineand its Metabolites using 10 mM carboxymethyl P-cyclodextrins (Cyclolab)..Separation conditions: capillary, fused-silica, 80 cm X SO J1IIl ID..; buffer,NaH2P04 (40 mM, pH 6.0) containing carboxymethyl p-cyclodextrins,temperature, 200c; injection, hydrodynamic. 2 seconds; voltage 20 kV;deteetion, UV al 225 nID..

ss

PEAK MIGRAnONlIME lMinutes) IDENIrlY

1 14.84 Chlol"Dbeniramine N-oxide

2 21.59 (S).cblomheniramine

3 22.20 (Il).chlofl)heniramine

4 22.64 Monodesmetbvl cblorobeDiramine

S 23.24 Monodesmetbyl + didesmethyl cblo",heniramine

6 23.85 Didesmethvl cblorobeniramine

e 4c:

2 5'" IlM 3M..•ut

.Dte

~

Migration lime (minutes)

Figure 12. E1ectropherogram of the CE separation of chlorpheniramine andits metabolites using S mM carboxymethyl p-cyclodextrins (Cyclolab).Separation conditions: capillary, fused-silica, 80 cm X SO JUIl ID.; buffer,NaH2P04 (40 mM, pH 6.0) containing carboxymethyl p-cyclodextrins.temperature, 200c; injection, bydrodynamic, 2 seconds; voltage 20 kV;deteetion, UV al 225 Dm.

56

PEAK MIORAlION TIME lMinutal IDEN'IIIY

1 21.40 Chlorpheniramine N-oxide

2 28.90 (S)-chloroheniramine

3 31.24 (R)-chlorpbeniramine

4 32.55 MODodesmethyl chIorpbeninmine

5 32.23

6 34.84 Didesmethyl chlorpbeniramine

7 35.49

ec:: 1'"MM

·N• ~ 2 3vi-i~

Migration time (minutes)

Figure 13. E1ectropherogram of the CE separation ofchlorpheniramine andits metabolites using carboxymethylJkyclodextrins (Cyclolab). Separationconditions: capillary, fused-silica, 80 cm X SO lJ.Dl ID.; buffer, NaH2P04(65 mM, pH 6.0) containing 2 mM carboxymethyl ~-cyclodextrins,

temperature, 200c; injection, hydrodynamic, 2 seconds; voltage 12 kV;detection, UV al 22S Dm.

57

PEAK MIGRAnON1'IME lMinuta) IDENmY

1 26.90 (S)-cbloroheniramine

2 32.10 (R)-c:blorobeniramine

1

2

Migration lime (minutes)

Figure 14. Enantiomeric separation of cblorpheniramine by CE with apbosphoric acid butter (100 mM, pH 3.0) containing carboxymethyl ~­

cyclodextrins (Cyclolab, 1.25 mM). Separation conditions: capillary,fused-silica, 80 cm X 50 ~m I.D.; temperature, 20°C; injection,hydrodynamic, 2 seconds; voltage 30 kV; deteetion, UV at 225 Dm.

58

Carboxymetbyl ~-eyclodextrin lWacker Chernie)

The carboxymethyl ~cyclodextrin obtained from Cyclolab offered good stereoselectivity at

low concentrations but its use for the enantiomeric resolution of chlopheniramine and its

Metabolites was not successful. Carboxymethyl ~-cyclodextrin from Wacker Chemie is not

commercially available, but some of the cyclodextrins was kindly donated by Wacker

Chemie for continuation of this study. Unfonuantely by this time, the supply of

monodesmethyl chlarpheniramine was exhausted, and this research continued with the

analysis of only the didesmethyl and N-oxide Metabolites. Application of the initial

separation conditions used with the carboxymethyl ~-cyclodextrins obtained from

Blaschke's group gave excellen~ reproducible baseline resolutions of chlorpheniramine andits didesmethyl and N-oxide Metabolites (Figure 16). To obtain reproducible migration

times, the capillary was rinsed at the stan of each day with sodium hydroxide (0.1 M for 10

minutes), distilled water and the separation buffer, each for 10 minutes. A couple of blank

runs were also required prior ta the analysis of sample solutions ta stabilise theelectraphoretic system.

Application of the enantiomeric separation method ta the pharmacoldnetic study of

chlorpheniramine requires the detection of chlorpheniramine down ta 2 ng/mL.

Hydrodynamic injections of 9 seconds enabled chlorpheniramine concentrations of 2

J,lglmL ta be detected (Figure 17) but lower levels couId not be determined due to noise.

The use of UV absorbance at wavelenghts between 190 and 200 nm have been reponed toincrease sensitivity, and this was possible here as the phosphate background buffer has

zero UV absorbance and the carboxymethyl ~-cyclodextrins absorb at UV wavelengthsabove 200 nm. Another method that has lowered detection limits is on-capillary peakconcentration or sample stacking [129,157]. The priniciple employs the use of

discontinuous buffer systems Le., buffers with different ionic strengths or with different

pH values to suppress an analyte from the relatively long sample plug in a short zone thus

concentrating a large injected sample. In isotachophoretic sample stacking, the sample isdissolved in a buffer of lower ionic strength than the running buffer and is loaded byelectrokinetic injection. The electric field strength in the low-eonducting sarnple medium is

greater than in the running buffer sa ions migrate rapidly 10 the interface between the lower

and high conductivity zones, where analytes stack to cause suppression of the sample zone[102,129,157]. The use of sample stacking methods was studied to lower the detectionlimits of the separation method.

59

PEAK MIGRATION TIME lMinutesl IDENmY

1 21.92 (S)..chlol"Dheniramine

2 23.24 CRl..cblotl'heniramine

3 25.00 Monodesmelhvl chlofl)heDiramine

4 25.32 Didesmethvl c:bloroheniramine

5 25.79 Monodelmclhvl chlorobeniramine

6 26.23 Didesmethvl c:hlorpheDiramine

4 6

l2

Migration time (minutes)

Figure 15. Electropherogram of the CE separation of chlorpheniramineusing carboxymethyl p-cyclodextrins (Cyclolab). Separation conditions:capillary, fused-silica, 80 cm X 50 JUIl I.D.; buffer, KH2P04 (50 mM, pH6.0) containing 2.5 mM carboxymethyl ~yclodextrins, temperature, 200c;injection, hydrodynamic, 2 seconds; voltage 20 kV; detection, UV at 225nm.

60

PEAK MIGRAnONTIME (Minuta) IDENmY

1 22.90 Cblorpheniramine N.gxide

2 23.30

3 39.17 (S)- chlorpbeninmine

4 39.70 fR)· chlombeniramine

S 43.66 Didesmethyl cblorpbeDiramine

6 44.72

4

3

ec:

'"MM• 1 2

Migration time (minutes)

Figure 16. Enantiomeric resolution of chlorpheniramine. didesmethylcblorpheniramine and cblorpheniramine N-oxide using carboxymethyl p­cyclodextrins (Wacker Chemie).. Separation conditions: capillary. fused­silica, 80 cm X 50 llDl 1.0..; buffer, NaH2P04 (50 mM, pH 6..0) containing20 mM carboxymethyl p-cyclodextrins, temperature, 200c; injection,hydrodynamic, 2 seconds; voltage 20 kV; deteetion, UV at 225 Dm..

61

A number of different solvents used to dissolve chlorpheniramine for sample stacking

procedures were studied. No sample concentration was achieved when 2 mM phosphoric

acid was used to dissolve chlorpheniramine, and the injection of chlorpheniramine

dissolved in distilled water resulted in low current A dissolving solvent of lower ionic

strength than the separation buffer, prepared by diluting the 50 mM NaH2P04 (pH 6.0)

buffer 1 to 10 with distilled water, along with hydrodynamic injections of 20 seconds,

resulted in significant sample concentration ofchlorpheniramine and allowed concentrations

of chlorpheniramine down ta 250 ng/mL ta he detected (Figure 18). Electrokinetic

injection methods were employed but results were irreproducible.

Although sample stacking and UV absorbance detection at 200 nm improved the sensitivity

of the method, concentrations of chlorpheniramine down to 2 ng/mL still could not be

detected. In the on-goning pharmacokinetic study of chlorpheniramine at Georgetown

University, 3-mL of plasma is obtained from patients at each time point; concentraùng 3

mL of plasma containing 2 ng/mL chlorpheniramine into 25 ~L would concentrate the

samples [0 240 ng/mL, which is detectable by sample stacking. Studies continued with

drying 3 mLs sample solutions containing 2 ng/mL chlorpheniramine with a speedvac, and

reconstituting the dried sample in 25 J,J.L of diluted buffer. Hydrodynamic injection of the

reconstituted samples for 20 seconds enabled detecùon of the chlorpheniramine

enanùomers but a Doisy baseline was observed due to a low attenutation that was required

to obtain results (Figure 19). Nevertheless, sample concentration followed by stacking

was a successful approach to detecting levels ofchlorpheniramine down to 2 ng/mL.

62

PEAK RETENnON 11ME (Minuta) IDEN1t1Y

1 48.59 (Sl-c:blofl)beninmine

2 SO.95 (Rl-c:b1oroheDiramine

l 2

~

Migration time (minutes)

Figure 17. CE enantiomeric separation of 2 JoLg/mL chlorpheniramine usingcarboxymethy1 p-cyclodextrins (Wacker Chemie). Separation conditions:capillary, fused-silica, 80 cm X 50 JUIl LD.; buffer, NaH2P04 (50 mM, pH6.0) containing 20 mM carboxymethyl P-cyclodextrins, temPerature, 200c;injection, hydrodYDamic, 9 seconds; voltage 20 kV; detection, UV at 225nm.

63

PEAK RE1ENlION1IME (Minutes) IDENIlTY

1 29.53 (S).chlorpheDiramine

2 30.36 (R).chloll3beairaminc

2l

Migration time (minutes)

Figure 18. Enantiomeric separation of 250 ng/mL chlorpheniramine withcarboxymethyl p-cyclodextrins (Wacker Chemie) using sample stackingmethods. Separation conditions: capillary, fused-sfiica, 80 cm X 50 l1IJ1I.D.; buffer, NaH2P04 (SO mM, pH 6.0) containing 20 mM carboxymethylp-cyclodextrins, temperature, 200c; injection, hydrodynamic, 20 seconds;voltage 20 kV; deteetiODt UV al 200 Dm.

64

PEAK 1ŒŒN'l10N 11ME lMinuta) IDENIl'lY

1 32.54 (S)-cbloroheniramine

2 33.33 (R)-cblorl'heniramine

1

Migration lime (minutes)

Figure 19. Enantiomeric separation of 2 ng/mL chlorpheniramine withcarboxymethyl ~-cyclodextrins (Wacker Chemie) following sampleconcentration and sample stacking methods. Separation conditions:capillary, fused-silica, 80 cm X SO JlD1lD.; buffer, NaH2P04 (50 mM, pH6.0) containing 20 mM carboxymethyl p-cyclodextrins, temperature, 200c;injection, hydrodynamic, 20 seconds; voltage 20 kV; deteetion, UV al 200 Dm.

65

CONCLUSIONS

66

A CE method to resolve the enantiomers of chlorpheniramine and its metabolites was

developed. The enantiomeric separation of chlorpheniramine and its Metabolites was

achieved on the AO-CSP but the separations required two separate methods, and the

enantiomeric resolution of the Metabolites could not be achieved. The assessment of

various p-cyclodextrins by CE in this project was rapid and the resolution of the

enantiomers of chlorpheniramine and its Metabolites was achieved using carboxymethyl ~­

cyclodextrins obtained from Wacker Chemie, Munich, Gennany.

Summary or Method

Sample concentration and stacking methods were required ta detect chlorpheniramine

concentrations below 2 J.lglmL and the limit of detection for chlarpheniramine was 200

ng/mL. The electI'opharetic separation conditions are summarised belaw:

Capil/ary:

Separation buffer:

Voltage:

Detection:

Temperature:

Fused silica, 80 cm X 50~ 1.D.

NaH2P04 (50 mM, pH 6.0) containing 20 mM

carboxymethyl P-cyclodextrin (Wacker Chemie)

20kV

UV absorbance al 200 nm

20ce

For concentrations of chlorpheniramine below 2 J,lg/mL, drying the sample and

reconstituting in diluted (1 in 10) buffer, followed by hydrodynamic injection for 20

seconds was required.

At the stan of each day, the capillary was rinsed with 0.1 M sodium hydroxide, followed

by distilled water and the separation buffer, each for 10 minutes. Two blank injections

were required prior to analysis to stabilise the electrophoretic system, and the capillary was

rinsed with 0.1 M sodium hydroxide and separation buffer, each for 5 minutes, in between

each analysis.

Future studies

The levels of chlorpbeniramine and its metabolites in biotluids such as plasma and urine

have ta be determined to study the pharmacokinetics of chlorpbeniramine. An extraction

procedure for chlorpbeniramine and its metabolites is therefore required and a number of

67

groups have successfully extracted biological samples containing chlorpheniramine using

hexane [2], 15 % methylene cbloride in hexane [5], benzene [151] and ether [3,4]. The

extracted solutes could then be analysed easily using the separation conditions developed in

this reseach projecl

CE in chiral analysis

CE has beeome a popular analytical tool and has offered many advantages over more

established analytical techniques such as gas ehromatography (GC) and HPLC. Method

development in CE is rapid and simple; a wide range of buffers can easily be screened as

the preconditioning of CE capillaries is not required, and rinsing the eapillary can

effectively remove any potential interferences when changing from buifer to buffer, and

between analyses. CE aIso offers rapid analysis times and employs buffers that are non­

taxie, cheap to dispose of, and compatible with biotluids which is particularly useful forbioanalytical studies. The need for compounds ta be volatile, required in GC, is

unneccessary and the different modes of operation have enabled a wide range of solutes to

be analysed by CE.

The chiral selectors used in CE are generally less expensive than chiral stationary phases

used in GC and HPLC. Cyelodextrins are the most widely used chiral selectors in CE but

heparin and more recently macrocyclic antibiotics, have also found useful application. Thedevelopment of charged cyclodextrins have offered greater enantioselectivity and have

significantly increased the scope of their application in chiral analysis. Moreover, the

availability of derivatised cyclodextrins with different degrees of substiution have provided

a greater range of selectivities. The usefulness of this was shown here when the

carboxymethyl ~-cyclodextrins obtained from Cyclolab, which contain three carboxymethyl

groups per ~-cyclodextrin ring, could not resolve all the enantiomers of chlorpheniramine

and its metabolites while those obtained from Wacker Chemie, which contain an averagedegree of substitution of0.5 carboxymethyl groups per P-cyclodextrin ring, resolved all theenantiomers; this method can DOW be applied to Dr. Yasuda's pharmacoldnetic study of

chlorpheniramine. CE May he less sensitive compared to other analytical techniques andrequire more concentrated samples, but sample stacking and the use of UV absorbancewavelengths between 190 and 200 nm have improved detection limits. Furthermore, CE

instruments with fluorescence detectors or interfaced with mass ·spectrometers, are

becoming commerically available.

68

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