CHAPTER!

INTRODUCTION

INTRODUCTION

Hypertension is considered multifactorial in origin and is caused by a breakdown

of the control mechanisms, which regulate cardiac output, blood volume, sodium balance

and systemic vascular resistance. It is observed that blood pressure increases with age,

and thus the prevalence of hypertension increases from about 25 % in the middle-aged

population, and to round 50 % in those aged over 75, with risk factor for coronary heart

diseases, cerebrovascular diseases, chronic renal failure and congestive heart failure. It is

therefore, rapidly becoming a foremost problem in developing countries and is

considered as the principal cause of stroke that leads to disease of the coronary arteries

with myocardial infarction and sudden cardiac death. Along with this, it is also a major

contributor to cardiac failure, renal insufficiency and dissecting aneurysm of the aorta;

hence, antihypertensive drugs have been chosen for the present study [ 1]. As far as

treatment is concerned, only a few patients having hypertension, which includes adrenal

disease, renal artery stenosis, unilateral renal parenchymal disease etc., are amenable to

surgical correction. Rest of the patients requires treatment with antihypertensive drugs

and, because this must continue indefinitely, its acceptability in terms of dosage

frequency and side effects must be considered majorly. The choice oftreatment should be

tailored to the individual since side effects are rarely obtrusive and impotence in

particular. In addition, these drugs do not have unfavourable effects on glucose or lipid

metabolism and, but may protect against development of arterial disease in later life.

2

In recent years, most of the drugs available are in the combination of low doses of

two different drugs with an additive effect, which may have fewer side effects than the

higher doses of one agent. Antihypertensive drugs such as atenolol, metoprolol

propranolol, hydrochlorothiazide and nifedipine are the group ofthe drugs that are widely

used in the treatment of edema, hypertension, hypoparathyroidism, hyperthyroidism,

angina pectoris, vascular disease, glaucoma, acute myocardial arrhythmias etc. [2], either

alone or in combination. Hence, in order to ensure quality and stability of the final

product, the analyst must be able to separate these mixtures into individual components

prior to quantitation analysis. These drugs are selected, since, these drugs are preferred as

first line therapy for hypertension; including efficacy, side effect profile and cost. In

addition, they are generally well tolerated, relatively inexpensive and widely given alone

and in combinations [3]. ~-blockers have a useful role in patients with exaggerated

tachycardia or an anxiety component to their hypertension, while for many patients

calcium antagonists are the best tolerated and most effective drugs. Thus, if a single

agent provides inadequate blood pressure control, a second can be added-ideally as a

combined preparation for ease of administration and better compliance, while persistent

hypertension requires the addition of a third agent and, in the most severe cases, four or

more different drugs may be necessary.

Also, drugs when administered, in formulation, are not in pure form but contain

several excipients like binders, fillers, disintegrants, diluents, colours, flavours etc. In

addition, they may contain impurities in the final product, which generally are confined

to those arising from incomplete and side reactions, or due to environmental conditions

3

(i.e. oxidation, reduction, hydrolysis etc.). This has necessitated the chemist for

determining the assay of drug since drug impurities induce toxicity, which may prove to

be fatal. In addition, the comparison of the relative efficacy of the different dosage forms

of the same drug entity requires the analysis of the active ingredient in biological

matrices, such as blood, urine and tissues, which is necessary to study bioavailability or

bioequivalence study. Hence, quality assurance and control have become very important

parameters for the complete assay of drug within the limits of accuracy and precision. In

addition, the impurity check of the drug substance is currently the most important

analysis, thereby giving essentially the fingerprint of the chemical synthesis and of the

stability studies. Literature cites several conventional methods like volumetry, gravimetry

etc., but today they are being fastly replaced by instrumental techniques viz.

spectrophotometry, GC, CE, HPLC etc., which allows even traces of impurities to be

separated and detected and finds numerous applications in the field of science like

medicine, clinical chemistry, pharmaceutical, agricultural, forensic, metallurgy,

oceanography etc.

As drug purity is equated with drug safety, enormous effort is expended by the

pharmaceutical industries to minimize and control the impurity levels in its products.

This effort begins at the initial stages of pharmaceutical development and continues into

quality control of the final products. Impurity assays by liquid chromatography are

developed and validated to support the manufacturing process, for stability study of the

formulations and for release assays of each lot of bulk drugs. Since impurity levels are

often very low in most pharmaceutical bulk products, the choice of the method is very

4

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important. The method must be accurate, sensitive, selective, reproducible and

convenient.

Taking this into view, the literature for the separation of antihypertensive drugs

viz. atenolol, propranolol, metoprolol, hydrochlorothiazide and nifedipine of the recent

five years have been overviewed. Separation of drugs via chromatographic methods is

further discussed by tw<;> methods since some ofthese drugs exist in their racemic forms.

I ACHIRAL METHODS

II CHIRAL METHODS

Separation of the drugs via achiral methods

In chromatography, High Performance Liquid Chromatography (HPLC) has

expanded to include ion chromatography, affinity, immunoaffinity, and chiral

chromatography in addition to the more common modes of adsorption, ion-exchange,

normal phase, reversed phase and size exclusion chromatography. An abundant number

of packing materials have been developed of which some are highly specialized for

specific applications and some have multipurpose use. However, use of reversed phase

packings helped to give high resolution separations that could not be readily

accomplished by ion-exchange, adsorption, or normal phase chromatography. Initially

silica was the only solid support but other supports gradually began to evolve. Organic

5

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resins have comeback after declining its popularity in 1970s and early 1980s. These

supports have advantages, as do polymer-coated silicas, especially carbons also have

great potential as solid supports. They are chemically unreactive, can be used over a

wide pH range, are highly reproducible and stereoselective, can be modified by

adsorption of high molecular weight materials, and can be used for size exclusion.

Different methods have been reported in the literature for the separation of

antihypertensive drugs. Most of these drugs have been separated in combination with

other antihypertensive drugs, while some with other class of drugs in RP-mode using

variety of columns under different conditions with polar mobile phases. As far as drugs

are concerned, most of the drugs are either polar or semipolar in nature and hence

reversed phase chromatography is more preferred compared to normal phase m

pharmaceutical industries. The pharmaceutical industry is facing the challenge of

ensuring quality and purity in drug production. As pressure from regulator increases,

detecting trace impurities in drugs becomes vital. In such cases, separation has been

carried out by different reversed phase columns and polar mobile phases using various

detectors viz. UV detector, Fluorescence detector, Refractive index detector,

Electrochemical detector etc. In the present work, a review of literature of last five years

have only been taken into consideration.

A modified HPLC method with tluorimetric detection has been developed to

determine the pharmacokinetics and comparative bioavailability of atenolol tablets

manufactured by two companies [4]. Chiap et a!. have separated atenolol from human

plasma on C8 column using UV detector [5], while the same group of the authors have

6

also reported the automated liquid chromatographic determination of atenolol in plasma

using dialysis and trace enrichment on a cation exchange precolumn [6]. Quantification

of atenolol in human plasma has been carried out by HPLC [7] and Capillary Zone

Electrophoresis method [8] having linear range 25-800 ng/mL and 30-585 ng/mL,

respectively. Mislanova and Hutta have studied influence of various biological matrices

like plasma, blood microdialysate etc., on chromatographic performance for the

determination of f3-blockers viz. atenolol, pindolol and propranolol using alkyl-diol silica

precolumn [9]. A general method has been developed for the detection of f3-blockers viz.

alprenolol, atenolol, betaxolol, bupranolol, butofilolol, nadolol, oxprenolol, penbutolol

and timolol in human urine by GC-MS-MS-EI with an average sensitivity limit of 20

ng/mL [I 0]. Kriikku and co-workers have carried out analysis of f3-blockers viz.

oxprenolol, atenolol, timolol, propranolol, metoprolol and acebutolol in human urine by

combination of isotachophoresis and zone electrophoresis with poly (methyl

methacrylate) chip system [11]. Also, optimization of the separation of f3-blockers like

atenolol, sotalol, betaxolol and metoprolol have been carried out by Servais et a!. by ion

pair capillary electrophoresis in non-aqueous media using univariate and multivariate

approaches [12]. Simultaneous determination of f3-blockers viz. (atenolol, sotalol,

diacetolol, carteolol, nadolol, pindolol, acebutolol, metoprolol, celiprolol, oxprenolol,

labetalol, propranolol, tertatolol and betaxolol); (alprenolol, atenolol, metoprolol, nadolol,

pindolol, propranolol, sotalol and timolol); and (acebutalol, alprenolol, atenolol,

betaxolol, bisoprolol, varvedilol, celiprolol, esmolol, labetalol, metoprolol, oxprenolol,

pindolol, practolol, propranolol, sotalol and timolol) have been carried out be HPLC

7

methods using UV and fluorescence [ 13]; photodiode-array UV [ 14 ]; and mass

spectrometry [ 15] detectors, respectively. Atenolol in combination with different calcium

channel blockers viz. nifedipine [ 16], amlodipine [ 17, 18] and amlodipine, nifedipine.

nimodipine and nitrendipine [19] have been separated by RP mode. Abdei-Hamid has

separated 13-blockers viz. atenolol, propranolol, pindolol and acebutalol along with

certain antiepileptic drugs using LC-MS method, which proved to be faster. more

sensitive and specific [20]. Also, atenolol alone [21, 22] and in combination with 13-

blockers [23-26] and chlorthalidone [27] have been separated using variety of mobile

phases. Bhatia et al. have separated atenolol in combination with antihypertensive drugs

like hydrochlorothiazide and amiloride on cyanide bonded column using acetonitrile­

phosphate buffer as mobile phase [28]. In a similar way, Hermansson et al. have

separated atenolol, propranolol and ibuprofen with three different columns viz. Zorbax­

CN column, Hypersil Elite C1s column and CT-Sil C1s column, respectively using

acetonitrile and phosphate buffer in different proportions [29]. An HPTLC method has

been reported for atenolol with calcium channel blocker amlodipine [30, 31] and

nitrendipine [32] using different mobile phases. Similarly, Dul'tseva et al. have reported

separation of atenolol but by TLC and GLC method in which, GLC was found to be

suitable for the chemical toxicology analysis [33].

Propranolol has been separated on different columns like 15_.um Shimpack CLC­

ODS column [34], 5-Qm Inertsil ODS 80 A column [35],..Q-Bondapack C18 column [36]

and column containing polystyrene seed particles with ~ I.Qm diameter [37] and in each

case detection has been carried out by fluorescence detector. Further, propranolol has

8

been analysed using various methods like fluorescence optosensor [38], continuous flow

method based on chemiluminescence [39], phosphorescence [40], and direct

chromatographic detection of a-napthoxylactic acid in urine [ 41 ]. Panchagnula and co­

workers have carried out RP-LC method with UV detection for simultaneous quantitation

of indinavir and propranolol from ex-vivo rat intestinal permeability studies [42] with

limit of detection 40 and 30 ng/mL, respectively. Further, propranolol and metoprolol

have been detected by AAS and spectrophotometric method [43], and rapid

electrochemical detection in pharmaceutical preparations using stabilized lipid film [44].

Braza et a!. have developed two liquid chromatographic methods with fluorimetric

detection to quantify atenolol and propranolol in human plasma [45], while Pulgarin and

co-workers have used first derivative non-linear variable-angle synchronous fluorescence

spectrometry for atenolol, propranolol, dipyridamole and amiloride to improve the

• selectivity of fluorescence measurements without loss of sensitivity [46]. El-Saharty has

reported an efficient RP-HPLC method for the determination of furosemide and

propranolol with linear range 0.1-200 and 5-200 !lg/mL, respectively [ 4 7]. Du et a!.

have separated propranolol, caffeine and phenytoin sodium in Kapuben tablets on Inertsil

ODS column with linear range 3-24, 20-160 and 35-280 11g/mL [ 48]. A size exclusion

chromatographic method has been reported for propranolol using Amberlite LA2 as anion

exchangers and methyl isobutylketone and water as mobile phase and its detection has

been done by NMR spectroscopy [ 49]. Jonczyk and Nowakowska have separated

propranolol along with hydrochlorothiazide and triamterene by HPLC and

spectrophotometric methods. lt was concluded that the results from both the methods

9

results from both the methods were comparable and could be successfully applied to

assay in their pharmaceutical preparation [50]. Yamini et al. have studied solubilities of

phenazopyridine, propranolol and methimazole in supercritical carbon dioxide and results

shows good self-consistency of the data obtained using semi empirical model [51].

Alpdogan and Sungar have separated metoprolol on..t.l.:-bondapak C 18 column after

forming Cu (II) - dithiocarbamate complex by precolumn derivatization of secondary

amino group ofmetoprolol with CS2 and CuC12 in presence of ammonia [52]. Metoprolol

and IK-hydroxy metoprolol from urine and plasma samples have been separated on

Novapak C 18 column and CJE column, respectively in RP mode in acidic pH and

fluorimetry detectors have made detection [53, 54]. Metoprolol and alprenolol have been

separated on two different columns viz. 5;Mm Hypercarb column and Lichrosorb column

in which results have indicated that the separation was more robust using Lichrosorb

column than the another one [55]. Metoprolol along with other B-blockers viz.

bisoprolol, carazolol, propranolol and alprenolol have been separated on Lichrocart RP

column using acetonitrile and 0.02 M sodium dihydrogen phosphate (I :3) as mobile

phase [56]. Braza and co-workers have developed two HPLC methods for the individual

determination of bisoprolol and metoprolol in human plasma with tluorimetric detection

having linear range 6.25-200 ng/mL for both [57]. Metoprolol tartarate along with

allopurinol, procaterol hydrochloride, dipyridamole, timepidium bromide and

mequitazine in pharmaceutical preparation have been determined by HPLC method and

this method was found to be rapid and was suitable for QC-analysis of commercial

samples [58].

10

Hydrochlorothiazide has been separated on Hypersil Cis column and Nucleosil

Cis column with acetonitrile- 0.1 %acetic acid as mobile phase in different proportion

having detection limit of 1 ppm and 0.2 ppb, respectively [59, 60]. Razak has carried out

electrochemical study for the determination of hydrochlorothiazide in urine and tablets

[61]. Hydrochlorothiazide in presence ofvarious angiotensin converting enzyme inhibitor

viz. benazepril [62], ramipril [63, 64], captopril [65], cilazapril [66], enalapril [67, 68],

fosinopril [69], quinapril [70], and angiotensin receptor antagonist viz. losartan [71-73],

valsartan [74], and diuretic viz amiloride [75, 76] have been separated by RP-HPLC

method. Manna et al. have done simultaneous determination of benazepril

hydrochloride, fosinopril sodium, ramipril and hydrochlorothiazide in pharmaceutical

formulations by liquid chromatographic ion pairing method [77]. Further, Zecevic and

co-workers have separated hydrochlorothiazide in combination with methyldopa and

amiloride in Alatan tablets [78]. While Ferraro et al. have carried out chemometric

determination of amiloride hydrochloride, atenolol, timolol maleate and

hydrochlorothiazide in synthetic mixtures of pharmaceutical formulations [79]. HPLC

and spectrophotometric method have been reported for the determination and separation

of hydrochlorothiazide and other drugs like benazepril [80, 81], lisinopril [82], captopril

[83], amiloride [84, 85], losartan [86, 87], va1sartan [88, 89] and fosinopril [90]. Further,

HPLC and TLC method for hydrochlorothiazide alone [91] and in presence of benazepril

[92, 93] have been reported. El-Gindy et al. have reported spectrophotometric and

HPTLC determination of hydrochlorothiazide in combination with lisinopril and losartan

[94].

11

Nifedipine has been separated on different RP column viz. Lichrocart RP -18

[95], Pecosphere Cis [96], Hypersil RP Cis [97] and Novapak Cis column [98] with

different mobile phase but best linear range was obtained on Hypersil Cis column which

was 2-200 ng/mL with diazepam as internal standard. Determination of nifedipine from

human plasma by solid-phase extraction have been carried out by RP-HPLC having

linear range 5-400 ng/mL [99], 2-200 ng/mL [100], and 10-200 ng/mL [101] using

different mobile phase with limit of quantification to be 5 ng/mL, 2 ng/mL, and 3 ng/mL,

respectively. Castro et al. have developed first derivative spectrophotometric and liquid

chromatographic methods for determination of nifedipine in oil/water/oil multiple

microemulsions during stability studies [1 02]. Nifedipine in combination with rifampicin

is separated on radiapak Cis column at pH 4 using UV detector [1 03]. GC

spectrophotometry and HPLC method have been used for determining nifedipine with

mefruside [ 104] and acebutolol hydrochloride [ 1 05] using UV detector for HPLC and

flame ionization detector in GC. Results further have revealed that, all these methods

show good linearity, precision and reproducibility. Nifedipine in combination with other

antihypertensive drugs viz. diltiazem, dimethyl diltiazem, deacetyl diltiazem, verapamil,

norverapamil, nitrendipine and dehydronitrendipine have been separated on 4-: m

novapak Cis column using imipramine as internal standard [1 06]. Thus, the achiral

methods reported in the literature for the separation of chosen drugs viz. atenolol,

metoprolol, propranolol, hydrochlorothiazide and nifedipine have been discussed as

above. Separation of chiral drugs viz. atenolol, metoprolol, and propranolol via chiral

chromatographic methods is discussed in detail as follows.

12

Separation of drugs via chiral methods

The majority of the new drugs approved and most often prescribed, have at least

one asymmetric centre, and 75-90% are marketed as racemates. Since, most often only

one of the enantiomer is responsible for a compound's activity and in exceptional case,

both the isomers may be active and in some cases, the correct ratio of enantiomers is

necessary for maximum response. Also, it is well known that stereochemistry of these

compounds have dramatic effects on their properties, especially in a biological

environment and due to this, the enantiomers will differ in absorption, distribution,

protein binding and affinity to the receptor [ 1 07]. Thus, it is required to study the

properties of individual drug enantiomers. Considering this, it is appropriate to assess the

progress of chiral methods in pharmaceutical industries that have traditionally been

carried out by liquid chromatography.

Methods for the resolution of chiral drugs via indirect and direct methods are

described below.

The ideal way to obtain pure drug enantiomers would be enantioselective

synthesis. This is, however, not always easy and usually complicated as well as

expensive. Therefore, the separation of racemic mixtures of intermediate or final

products is often required. The classical methods for the chiral separation that has been

used for the resolution of pharmaceutical intermediates includes crystallization, kinetic

resolutions, reaction resolution combinations, membrane based separations etc.

13

[82,83,84]. The most practically useful technique for the separation of chiral analyte

includes "Separation Methods" based on indirect and direct chromatographic separation.

(A) INDIRECT METHOD

Indirect method includes derivatization of enantiomers via highly pure optically

active reagent, followed by separating them as diastereomeric derivatives, which differ in

their chemical and physical behaviours and therefore can be separated on achiral

stationary phase. However, derivatization represents an additional step that can involve

undesirable side reactions, formation of decomposed products and racemization.

Furthermore, the chiral derivatization reagent has to be of high enantiomeric purity and

the presence of derivatizable groups in the analyte is a prerequisite. Nevertheless, this

approach circumvents the need for expensive columns with chiral stationary phase and is

more flexible.

The most frequently used chiral derivatization reagents are 1-(9-fluorenyl)

ethylchloroformate and o-phthaldialdehyde in combination with chiral thiols [Ill].

Kleidernigg et a!. [ 112] used (0, 0' -R, R)- diacylated tartaric acid anhydrides for the

derivatization of 2-blockers. Same group of authors have introduced a new chiral

derivatization reagent, (I R, 2R) - or (IS, 2S)-N- [(2-isothiocyanato) cyclohexyl]-3, 5

dinitrobenzoylamide (DDITC) for the derivatization of primary and secondary amines

and amino alcohols [ 113]. 1-(6-Methoxy-2-naphthyl)-ethyl isothiocyanate (NAP-IT) and

2-( 6-methoxy-2-naphthyl)-1-propylchloroformate (NAP-C) were used by Buschges et a!.

14

workers have developed RP-HPLC method for atenolol enantiomers m rat hepatic

microsome after chiral derivatization with 2,3,4,6-tetra -0-acetyl- 2-D-glycopyranosyl

isothiocynate having detection limit 0.055 1-lg/mL [ 126]. While Zhou and co-workers

have derivatized propranolol enantiomers in transgenic Chinese hamster CHL using

2,3,4,6-tetra -0-acetyl- 2-D-glycopyranosyl isothiocynate as derivatizing agent [ 127].

Kim et al. have derivatized certain I)-blockers (acebutolol, arotinolol, betaxolol,

bisoprolol, celiprolol, metoprolol and pindolol) with (-) menthyl chloroformate and have

resolved their diastereomeric derivatives on achiral column [128]. Chiral purity test of

metoprolol enantiomers have been studied by Kim and co-workers by direct method

using Chiralcel OD column and by indirect method via derivatization with (-) menthyl

chloroformate and then separating on Inertsil C8 column in RP-mode [ 129]. Direct

enantioselective analysis of metoprolol in plasma was carried out using Chiralpak AD

and Chiralcel OD columns while indirect analysis was done by using S-(-) menthyl

chloroformate as diastereomeric derivatives [130]. Further, various other chiral

derivatizing agents like S(-)-1-(1-napthyl)ethylamine, R( + )-K-methylbenzylamine [ 131-

133], (S)-1-phenylethylamine [134] and amino acid based CDA like (S) - N- (4-

nitrophenoxycarbonyl ) phenyl alanine methoxy ethyl ester [ 135], L- phenylalanine

methyl ester[ 136] and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amine [ 137] have been

used for the separation of different group of drugs.

16

- -------~---

for the derivatization of adrenoreceptor antagonists and antiarrythmic drugs [ 114].

Bruckner and Wachsmann have synthesized N- [4-[(S)-1-carbamoyl-2-methyl­

propylamino ]-6-chloro-[ I ,3,5]triazin-2-yl] -L-phenylalanine starting from cyanuric

chloride and used this reagent for the indirect enantioseparation of amino acids [ 115].

Toyo'oka has given an excellent overview of fluorescent chiral derivatization reagents

[Ill]. A new chiral fluorescent tagging reagent, (IR, 2R)-N-[(2-isothiocyanato)

cyclohexyl]-6-methoxy-4-quinolinylamide was prepared by Kleidernigg and Lindner and

applied to amino acids and amines [ 116]. AI-Kindy et a!. have synthesized a series of

new fluorescent chiral benzoxadiazole-amino acid derivatives for amines [117]. Yasaka

et al. have reported the preparation of (S)-(+)-1-methyl-2- (6,7-dimethoxy-2,3-

naphthyalimido )ethyl trifluoromethansulfonate as chiral fluorescent derivatization

reagent for carboxylic acids [118]. More recently, lnoune et al. [119] have introduced 4-

(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a chiral

fluorescent labeling reagent for prolyl dipeptides. Indirect resolution of 2-blockers viz.

propranolol, oxprenolol, pindolol, metoprolol and atenolol have been carried out by RP

capillary electro chromatographic method using (+)-1-(9-fluorenyl)ethyl chloroformate as

a derivatizing agent and then separating on octadecylsilanized silica gel based stationary

phase [ 120].

Derivatives of isocyanates including (1R, 2R)-1,3-diacetoxy-1- (4-nitrophenyl)-

2-propyl isothiocyanate, R-( + )-1-phenylethylisocyanate, S-(-)-K:-methylbenzyl isocyanate

and S (+)-napthyl ethyl isocyanate have been used for the enantioseparation of series of

9-blockers, racemic amino alcohols and terbutaline enantiomers [121-125]. Li and his co-

15

(B) DIRECT METHOD

Separation of enantiomers through direct method includes two fundamentally

different cases, depending upon whether enantiodifferentiation takes place through a

chiral recognition effect by the stationary phase or by a chiral constituent of the mobile

phase forming a diastereomeric complex in situ during the chromatographic process.

From an experimental point of view, a clear distinction can be made between the

uses of chiral column (i.e a column containing a chiral stationary phase) together with an

achiral mobile phase, and the use of an achiral column together with a chiral mobile

phase. In the later case, however, the actual mode of chiral separation will depend on the

relative affinity of the chiral constituent for the stationary phase and the analyte,

respectively. In one extreme case, the chiral constituent may become strongly adsorbed

on the stationary phase, thereby converting it into a CSP, and the separation process

would then be regarded as a chiral recognition by the CSP thus generated. In the opposite

case, the chiral constituent has a much lower affinity for the stationary phase than for the

analyte. This means that diastereomeric complexes are generated in the mobile phase and

the separation takes place as normal LC separation of diastereomers. Thus, direct

approach, which makes use of columns with chiral stationary phases, is more convenient

and applicable for the separations on preparative scale, but requires a collection of

expensive columns to solve a variety of problems. In case of mobile phase additive,

though it represents a simple and flexible alternative, is not always applicable, since, the

17

mobile phase containing the chiral selector cannot be reused and hence the technique

cannot be applied with expensive reagents.

Chiral sorbents for LC in case of chiral columns may further be classified with

respect to their general structure types. Some are based on synthetic or natural polymers

and are totally and intrinsically chiral. Other consists of chiral selector of low molecular

weight, which is bound to a hard, incompressible matrix, usually silica. There are also

sorbents consisting of polymers anchored to silica in order to give improved column

performance, but the difficulty is that, each new class of enantiomeric compound would

appear to require a different stationary phase.

A rough classification of various modes of chiral LC is given below, which is

mainly based on the general nature of chromatographic sorbent, without regard to the

type of retention process involved, which is often quite complex and difficult to evaluate

in detail.

Natural (polysaccharides and protein derivatives) and Synthetic

intrinsically cltiral polymeric stationary phases

POLYSACCHARIDE DERIVATIVES

The linear polysaccharide cellulose represents the most common organic

compound of all with chemical constitution of a linear poly -13-D-1 ,4-glucoside. Gubitz

and co-workers have observed that the native cellulose showed only weak chiral

18

recognition ability [138]. Considering this, Hesse and Hagel have discovered that on

acetylating cellulose which yielded microcrystalline cellulose triacetate (CTA-1) produces

a tertiary structure upon swelling and forms chiral cavities which are able to include

stereoselectivity with aromatic residues and gave better resolution compared to that of

natural cellulose [139]. Thus, with the advent of time, number of papers and review

articles has been reported for the separation of drugs using polysaccharide phase. Most

of them have been excluded and only relevant references have been quoted over here.

The chromatographic resolution of enantiomers of numerous pharmaceutical

intermediates and final products with the chiral discrimination mechanism regarding

polysaccharide phases have been discussed by Miller and co-workers [ 140] and Aboul­

Enein [141]. Several acidic racemic drugs [142] and 2-blockers [143] have been

separated on cellulose derivatives. Similarly, various other chiral drugs like metipranolol

and desacentylmetipranolol [144], and propranolol analogues [145] have been separated

via. Chiralcel OD column using different mobile phases. Ferdam and Modier have

separated enantiomers and diastereomers of propranolol derivatives on cellulose tris (3.5

dimethyl phenyl carbamate) [146] while Rumiantsev and lvanova have determined

propranolol content by achiral NP-HPLC and their enantiomeric ratio· by chiral HPLC

using silica bonded cellulose tris(3,5 dimethyl phenyl carbamates) [147]. Santoro and co­

workers have described the separation and quantitative determination of atenolol isomers

using Chiralcel OD column and hexane-ethanol-diethylamine as mobile phase [148].

Singh et a/. have developed a simple, rapid, precise and accurate method for separating

the enantiomers of atenolol and metoprolol in tablet preparation on a Chiralcel OD

19

column with hexane-ethanol-diethylamine-acetic acid as mobile phase in different

proportions [149]. In a similar way, Svensson et al. have separated metoprolol

analogues along with several racemic amino alcohols on a Chiralcel OD column using

chemometrics [ 150]. Direct stereoselective separation of four stereoi somers of a­

hydroxymetoprolol in human plasma and urine has been carried out on Chiralpak AD

column [151]; and Chiralcel OD-R and Chiralcel OD-H column [152]. Kim et al. have

determined metoprolol enantiomers in human urine by coupled achiral-chiral column,

quantifying metoprolol and internal standard first on silica column and then separating

enantiomers on Chiralcel OD CSP [153] with detection limit 25ng/mL for each

enantiomers. ~-adrenergic blocking drugs viz. propranolol and atenolol were resolved by

HPLC using Chiralcel OD-H and Chiralpak AD as CSP with separation factor in the

range 1.34-4.55 and resolution 1.50-10.65 [ 154]. Yang et a!. have derivatized

propranolol, ateriolol and metoprolol with a fluorogenic reagent 4-(N­

chloroformylmethyl-N-methyl)amino-7 -N ,N-dimethylaminosulfonyl-2, I J­

benzoxadiazole (DBD-COCl). Further, propranolol has been separated on Chiralcel OD­

R and the other two (i.e. atenolol and metoprolol) on Chiralcel OJ-R column [155].

Schmid et a!. have done comparative study of chiral resolution of several ~-blockers on

cellulose tris (3, 5-dimethyl phenyl carbamate) phase's viz. (Chiralcel OD NP-column,

Chiralcel OD-RH RP column and Chiralcel OD-RH RP narrow bore column). In this

condition, sixteen ~-blockers were resolved under NP conditions and eleven under RP

conditions [156]. Ali and Aboui-Enein have carried out chiral resolution of some clinical

used drugs namely ~-blockers (metoprolol, teratolol, tolamolol, nebivolol), antifunagal

20

agent (miconazole), antihypertensive agent (cromakalin) and anti-inflammatory agent

( etodolac) on cellulose tris (3,5-dichlorophenylcarbamate) CSP [ 157].

0~~ H~o--

OH H

Fig. 1 Cellulose, linear poly [1--- 4-P -D-glucosej

The other widespread polysaccharide which is built from (+) 0-glucose units is

starch. Substitution of cellulose by amylase was found to result in different

enantioselectivity [ 158]. Contrary to cellulose, left-handed 411 he I ical structure is

postulated for amylase [159]. Zhou et al. have synthesized amylopectin -tris (phenyl

carbamate) as a CSP and separated racemic biphenyl diesters of acidic drugs [ 160].

Greiser et al. have described direct, preparative enantioselective chromatography of

propranolol hydrochloride and thioridazine hydrochloride [ 161]. Comparative

enantioseparation of certain drugs on four different polysaccharide type CSPs viz.

Chiralcel OJ, Chiralpak AD, Chiralcel OD and Cellulose tris (3,5- dichlorophenyl

carbamates) have been done with polar organic mobile phases, however the study

21

confirms that the Cellulose tris ( 3,5- dichlorophenyl carbamates) has high potency for

the enantioseparation over other three CSPs [ 162].

H /Q~~OH l-

HO jH 0

0 0 o ...

o--

\

Fig. 2 Amylopectin, 6-branched poly [1-- 4-a-D-g/ucosej

The new crystalline carbohydrates, so-called dextrins form inclusion complexes

with various compounds of the correct size due to its relatively non-polar central cavity

and the stability of the complex is largely dependent on the hydrophobic and steric

character of the guest [163]. Cyclodextrin.s have been used in HPLC both in the form of

chiral mobile phase additives as well as CSPs. Bressolle eta!. have given an overview of

the use of cyclodextrins in HPLC and CE [ 164]. Radulovic and co-workers have

investigated inclusion complex formation between metoprolol tartrate and P-

cyclodextrins, which further indicated the potential applications of precolumn

derivatization for enantiomeric separation of p- blockers [165]. In a similar way, Park

and co-workers have studied inclusion complexes of metoprolol and carboxymethyl P-

22

cyclodextrin, binding studies performed using HPLC, UV-spectrophotometry, and CE

has revealed that a complex with I: I stoichiometry was predominant [ 166].

Duval and co-workers have discussed the use of mono-2-pentenyl f)- cyclodextrin

and mono-6-pentenyl f)- cyclodextrin based chiral stationary phase in HPLC and SFC for

enantioseparation of aminoglutethimide and thalidomide and revealed that hetero atom

present in spacer arm was essential for the chiral recognition mechanism (167]. Schurig

et al. have developed a new chiral polymer column, Chirasil-Dex which found to have a

unified enantioselective chromatographic approach utilizing common methods of GLC,

SFC. LC (open tubular and packed) and CEC for chiral separation (168]. Propranolol

analogues have been separated on sulphated f)-cyclodextrin bonded stationary phase

( 169]. Similarly, an approach has been made to use an anlaytical column packed with

novel perphenylcarbamate f)-cyclodextrin bonded CSP to separate propranolol

enantiomers using triethylammonium acetate buffer and methanol mixture as mobile

phase [ 170]. A chemically bonded f)-cyclodextrin chiral stationary phase for HPLC has

been prepared by Tazerouti et al. and using this chiral stationary phase, various racemic

analytes such as aminoalcohol, adrenergic f)-blockers, benzodiazepine anxiolytics like

arylpropionic acid antiinflammatory agents and herbicides like aryloxy propionic acids

and esters have been separated [ 171 ]. A comparative study has been carried out by Ng et

a!. between commercially available cyclodextrin based column (CYCLOBOND l 2000

SN) and newly synthesized novel perfunctionalized cyclodextrin CSP immobilized onto

aminized silica gel by a single ureido chemical bond. Studies have revealed that ureido

bonded CSP column showed unique properties especially on enantioseparation of f3-

23

adrenergic blockers and amine containing racemic compounds that proved to be

complementary to CYCLOBOND column that can separate acidic compounds more

effectively [172].

0

H

OH

~H ~H OHD \ OH \ ~-~O 0 CHOH

CH20H 2

Fig. 3 f3-cyclodextrin

24

PROTEINS

Proteins are biomolecules that are directly responsible for cellular structure and

functions. The ability of proteins to bind drugs stereoselectively has been utilized for the

chromatographic separation of drug enantiomers. Proteins consist of chiral amino acid

building blocks, which form three-dimensional structure, whereby hydrophobic.

electrostatic interactions and hydrogen bonds are assumed to be the possible interactions

responsible for chiral recognition. During the developments of enantiomeric separations

in HPLC, a variety of protein chiral selectors like bovine serum albumin, a 1 -acid

glycoprotein, conalbumin and cellulose typically immobilized to silica-based carriers

have been used as stationary phase, though they may also be used as mobile phase

components [173]. Haginaka has given comprehensive review reports on the

developments of protein based CSPs and their application [ 174]. Peyrin el a!. have used

Human Serum Albumin column to investigate measurement selectivity data using a new

chiral recognition model [ 175]. With the same column, Haque et al. have also obtained

the baseline resolution of several drug enantiomers [176]. Martinez-Pia and co-workers

have developed fast enantiomeric separation of propranolol by affinity capillary

electrophoresis using human serum albumin as chiral selector to control the application of

propranolol in quality control of pharmaceuticals [ 177]. a-Adreno receptor antagonists

have been separated on Human Serum Albumin and a 1 - Acid Glycoprotein (AGP) using

HPLC methods while, the same have also been separated by Capillary Electrophoresis

using heptakis (2,6-di - o-methyl)-j3-cyclodextrin (DMCO) 1-hydroxy propyl -13-

25

cyclodextrin (HPCD) & ~-Cyclodextrin as chiral selector [ 178]. A different study has

been carried out by Goetmar et al. with respect to adsorption isotherms of enantiomers of

~-blockers namely metoprolol, alprenolol and propranolol on cellobiohydrolase-1

immbolized on silica gel in the concentration range between 0.25 J.!M and 1.7 mM at pH

5.0, 5.5 and 6.0 [ 145], while, Fornstedt and co-workers have separated atenolol in

microdialysis and plasma samples by Chiral CBH column with Lichrospher precolumn

[ 180].

Another human plasma protein called a 1-acid glycoprotein (AGP) or ovomucoid

present in a concentration of 55-140 mg per I 00 mL of plasma has been claimed as the

main cationic binding protein in the humans [18l.J. Five basic hydrophobic drugs

alprenolol, propranolol, promethazine, chlorphenaramine and disopyramide have been

separated on a 1 • AGP column with diamine sparteine as cationic mobile phase modifiers

[ 182), similarly Berthault et al. have separated several ~-blockers by chiral AGP column

[ 183].

CHIRAL SYNTHETIC POLYMERS

Synthetic chiral polymers can be produced either by the use of chiral reagent or

by a chiral catalyst. In the first case, a chiral derivatization of a suitable monomer is

performed and the product is then polymerized to form a polymer network having chiral

substituents. In the second case, the monomer is polymerized under the influence of a

chiral catalyst, which will produce an optically active polymer, since the stereoregulatory

26

influence of the catalyst will yield an isotactic polymeric structure of a certain preferred

helicity. Thus, CSP prepared in this manner are relatively economic and excellent for the

optical resolution of racemates. Nakano has given a comprehensive review on the

synthesis and application of chiral synthetic polymeric CSPs [ 183]. Krause eta!. studied

different separation modes i.e. normal phase and reverse phase nano HPLC and CEC

with and without pressure assistance in fused silica capillaries packed with silica gel

which was modified by covalent attachment of poly-N-acryloyl -1-phenylalanine

ethylester (chiraspher®) or by coating with cellulose tris (3,5-dimethylphenylcarbamate)

[ 185]. Two affinity adsorbents for the vancomycin group antibiotics have been prepared

by immobilizing 0- alanine and D, L-alanine, respectively, onto crosslinked

polyacrylamide resin through Mannich reaction by Yuan et a!. and they further have

studied adsorption ofN- dimethylvancomycin on these adsorbents [ 186].

I ~(Nt+... I "'R*

suspension copolymerization ~ with crosslinker to obtain polymer network n

~ NH/ R*

>==< R

anionic polymerization with chiral initiator

Fig. 4 Synthetic po(vmers

·- ... ~ .... '· ~!·1,· !.. . ..: . f : •• • •

,.

·. 2~

(network)

R R R

-~ n

(helical, stereoregular, linear polymer)

Molecular imprinting technique includes construction of polymer network cavities

by synthetic means to fit only one oftwo enantiomers. The principle rests on an imitation

of an enzyme's binding site, which can usually be regarded as a chiral cavity or cleft in

the protein, often highly specific with respect to binding of substrate enantiomers because

of the precise steric requirements for multiple bond attachment. Since the experimental

technique can be compared with making a plaster cast from an original template, it is also

called "Molecular Imprinting". Hence, the molecules of a particular compound act as

templates around which a rigid polymeric network is cast. Specialized review articles

have been reported in the literature regarding molecular imprinted polymers [ 187 -189].

Amino acid derivatives and peptides have been separated by antibody mimicking

polymers as CSP and chiral separation has showed higher load capacity, increased

selectivity with better resolving capability [190]. Similarly, Liao and co-workers have

prepared polymeric molecule transporters using molecular imprinting techniques with 0-

tryptophan, 0-phenylalanine and 0-histidine as the template and further revealed that

molecular "receptors" prepared using molecular imprinting techniques could potentially

be used for the separation of enantiomers through serial enantioselective transports [ 191].

Owens and co-workers have reviewed the potential of analytical techniques based on

molecular imprinting from view point of bio and pharmaceutical analysis and focussed on

how these benefits have been used for the improvement in the quality of analytical

procedures [ 192]. In this regard, Aboul-Enein and Al-Ouraibi have developed direct

isocratic method for enatioseparation of propranolol using newly developed chirose C1

CSP as chiral polymer [193]. Haginaka and Sakai have prepared uniformed size MIP for

28

(S)-propranolol by a multistep swelling and thermal polymerization method usmg

methacrylic acid and ethylene glycol dimethacrylate as a host functional monomer and

cross linker, respectively. These MIP have proved to have some specific recognition for

(S)-propranolol and moderate recognition for some structurally related J3-adrenergic

antagonists but no recognition for other basic, acidic or neutral compounds [ 194]. Martin

el a!. have investigated three propranolol derived MIPs as selective sorbents for the solid

phase extraction of J3-blockers. Results have revealed that aqueous based elution solvents

gave little or no selectivity for propranolol analogues compared to elution using toluene

based solvents, which showed significant selective extraction [ 195]. Further, same group

of authors have prepared propranolol derived MIP against the drug tamoxifen, which

surprisingly showed considerable selectivity towards tamoxifen, and was indeed much

more selective than the MIP prepared using tamoxifen as the imprint molecule ( 196]. In

addition, Suedee et al. have prepared several MIPs using the enantiomers of either J3-

blockers drugs R-(+)-propranolol, R-(+) or S-(-) atenolol, or the NSAID, S-(+)-naproxen

and S-(+)-ibuprofen as print molecules. They have coated these polymers on glass

supports and have resolved racemates of propranolol and ketoprofen [ 197]. Thus, chiral

phases based on MIPs have high antibody like selectivity, however, suffers from

relatively low efficiency and the restricted range of applicability, since one special phase

shows chiral recognition only for the same molecule used as template or very closely

related compounds.

29

Bonded Synthetic Chiral Selectors

The CSPs described under bonded synthetic chiral selectors are all

characterized by their well-defined molecular structures bonded to some solid support,

usually silica.

CROWN ETHERS

Crown ethers are macrocyclic polyethers, which are known to form host-guest

complexes with alkali- and alkaline earth-metal ions as well as primary ammonium

cations. Cram et a!. have been the first to synthesize optically active crown ethers for

optical resolution purposes [ 198] and this principle was then transferred to an LC

separation technique by the use of the chiral crown ether in the mobile phase or

covalently bound to a silica support [ 199]. They prepared crown ether phases based on

polystyrene or silica for classical LC and demonstrated the applicability of these phases

to the chiral separations. Moreover, substituents of the crown ether are perpendicular to

the plane of the macrocyclic ring, forming a chiral barrier, which divides the space

available for the substituents at the chiral centre of the analyte into two domains. Thus,

two different diastereomeric inclusion complexes are formed.

Several selected amino acid enantiomers have been separated using Crownpak R~

(+)column and enantiomeric impurities as low as 0.001% (IOppm) was determined by

this method [200]. Mach ida et al. have used (+) 18-Crown-6-tetracarboxy1ic acid on 3-

30

amino propyl silanized silica gel to separate thirteen DL-amino acids out of eighteen and

seven racemic amino alcohols [201]. A CSP derived from(+)- (18-crown-6)-2,3,11-12-

tetra carboxylic acid has been used for direct separation of an antibacterial agent.

tluoroquinolines, however the resolution depends not only on the contents, but also on

the type of acidic and organic modifiers in mobile phase and on column temperature

[202]. Nishiaok and co-workers have developed novel CSPs covalently bonded with

chiral pseudo crown ether containing phenyl groups as chiral barrier that has high ability

of discriminating enantiomers in both RP and NP mode for the separation of wide range

of chiral amines, amino alcohols and amino acids especially for the hydrophobic amines

[203].

Fig. 5 Chira/ crown ether

31

METAL COMPLEXES (CHIRAL LIGAND EXCHANGE)

Chiral ligand exchange chromatography has been extensively studied by

Davankov and co-workers, the pioneers, in resolving a-amino acids [204, 205]. Here,

the chiral recognition on chiral stationary phases is based on the formation of ternary

mixed metal complexes between the selector and the analyte ligand.

Mobile Phase (m)

Stationary Phase (s)

A: Analyte

M: Metal

S :Selector

,..__ AMSs

The ability of transition metals to participate in complex formation has been

exploited earlier for the purpose of enantiomeric separation. Since, diastereomeric

complexes are formed from amino acid ligands of opposite configuration, any difference

in stability of these complexes will of course result in different chromatographic

mobilities of the amino acid enantiomers. The original phases prepared by Davankov for

classical column chromatography were based on polystyrene-divinylbenzene polymers

containing amino acid residues complexed with metal ions. These phases showed

remarkable enantioselectivity for amino acids.

32

Kurganov has extensively reported chiral chromatographic separations based on

I igand exchange chromatography [206]. W achsmann and Bruckner have reported the

synthesis of a new chiral ligand exchange chromatography phase by binding L-proline or

L-lysine to aminopropylsilanized silica through a triazine spacer. While the first phase

was found to be suitable to resolve underivatized amino acids and N-(2,4-dinitrophenyl)

amino acids, the second phase resolved dansylamino acids [207]. Wan et a!. have

synthesized chiral selectors derived from L-proline and L-phenylalanine by alkylation

and arylation. The selectors containing C7, C9, C 12- chains or methoxybenzyl,

naphthylmethyl or anthrylmethyl groups were adsorbed onto the surface of porous

graphitic carbon. Using this, authors have resolved thirty-six racemic amino acids [208].

Ma et al. have separated amino acid enantiomers using polyvinyl alcohol with L-proline

pendant as CSP in ligand exchange chromatography [209].

CSP BASED ON CHARGE TRANSFER COMPLEXATION

The chiral selectors have been characterized by the operation of an aromatic n-n

bonding interaction as an essential element of the retention process. This technique that

includes interactions between so called n-acceptor and n-donor molecules has been first

introduced by Pirkle. A selector of this type is designed to have a cleft, consisting of n­

acidic and n-basic aromatic parts directed almost perpendicular to each other, to which

one enantiomer is preferentially bound. The enantioselectivity binding has been shown

by NMR studies to be due to a simultaneous face-to-face and face-to-edge n-n interaction

33

[21 0]. These phases (commercially available as Whelk-0™, Regis Co.) typically operate

under normal phase conditions with hexane containing a retention modifier like propan-

2-ol. Chilmonczyk et al. have enantioseparated several clinically used racemic drugs on

N- (3,5-dinitrobenzoyl) -3-amino-4-phenylazetidin-2one bound to silica known as pirkle-

1 J CSP and they have explained chiral recognition mechanism involved in it by using

molecular modeling semi-empirical AM I method [211].

Vinkovic and co-workers have investigated mechanism of chiral recognition in

the enantioseparation of 2-aryloxypropionic acids on new brush type CSPs comprising N-

3, 5, 6-trichloro-2, 4-dicyanophenyl -L-a-amino acids bound to y-aminopropyl silica

[212], while Lin et al. have used CSP based on a-amino acid and pyrrolidine

disubstituted cyanuric chloride for the separation of amino acids [213]. Petersen et ul.

have investigated eighteen f3- blockers using Pirkle type a-Burke CSP, of which fifteen

have been successfully separated by NP-HPLC within an acceptable analysis time [214].

Eaginger et al. have separated (R)- and (S)- propranolol in human plasma have been

separated on (R, R) dinitrobenzoyl diamino cyclohexane CSP using dichloromethane­

methanol as mobile phase [215]. Cleveland has studied separation of chiral drugs of

certain classes (like antihypertensive, antiarrhythmics, antianginals, diuretics, adrenergic

drugs, anti inflammatory and analgesic compounds, topical anesthetics, antihistamines

and antimalarial) on Pirkle concept CSP [216]. Mach ida et al. have synthesized a tartaric

diamide phase bearing a p-chlorophenyl residue and applied it to the resolution of I, 2-

diols, 2, 2' -dihydroxy-1, I' -binaphthyl and some f3-blockers [217].

34

Enantiomers of N-aryloxazolinones, N-arylthiazolinones & their thia analogs

[218]; as well as aryl propionic acid, non steriodal antiinflammatory drug, aryl epoxides,

sulfoxides, alcohols, am ides and esters [219] on Whelk-0 1 column have been studied and

the results revealed that the separation has been achieved in spite of an element of

uncertainity due to the presence of 2n-basic interaction sites. A number of racemic

thiazide diuretics and their analogues have been resolved on two diastereomeric chiral

stationary phases prepared from (S)- or (R)- a [1-(6,7- dimethyl) -napthyl] - 10-

dodecenyl-amine and (S)-2- phenyl propanoic acid by Hyun and Pirkle and studies

showed former CSP to be better than latter one [220].

0 (J NH"-._

0

---si, I ''OH

_I IIIII II

Fig. 6 Selector based on ~ 7l interaction

35

OTHER TYPES OF SELECTION

Though the bonded selectors are reasonably well understood in terms of their

chiral recognition behaviour, a number of other selectors have been developed which

probably act by more complicated and less well-understood mechanisms.

CSP BASED ON UREA AND AMIDE DERIVATIVES

Very promising CSPs based on amide or urea derivatives have recently been

prepared and studied. The amides have only one H-bonding acceptor/donor group, and

can form only a single bond with a neighboring selector-molecule. The free rotation

along the single bond, and the alternative positions on the L-peptide molecule, where

such a bond can be formed, prevented establishing a predominant selector/select and

associate that woud lead to chiral selectivity. To enhance chiral selectivity of a complex

formed through a single bond, the bonding site of the selector was placed between two

asymmetric centers, as can be seen in the structure of the "L-urea" phase. The C2-

symmetry around the bonding site should maximize the number of the different

associates and maximize the contribution of the complex(es) where the asymmetric

carbons of the selector and selectand are brought to close proximity.

Norephedrine enantiomers have been separated on urea derivative as chiral stationary

phase using hexane, dichloroethane and isopropanol, and studies have been done by

36

altering the organic modifier by substituting ethanol, acetonitrile and tetrahydrofuran for

isopropanol to decrease the retention time [221]. Similarly, Zou and Yun have separated

racemic propranolol using amide derived CSP using hexane- I, 2-dichloroethane-ethanol

as mobile phase system in composition (71-26 -3) [191] while, Zhang et al. have

directly separated atenolol on amide derived CSP via NP-HPLC using n-hexane - 1.2-

dichloroethane - methanol as mobile phase. Further, chiral resolution of atenolol with

propranolol and celiprolol has also been discussed [222]. Zou and Yun have resolved

celiprolol enantiomers by using NP-mode with urea derivative as CSP [223]. In contrast.

formoterol, labetalol, nadolol, indenolol and U-54494A have been resolved by urea type

CSP column of (S)-indoline- 2-carboxylic acid and R-(1) (a-napthyl) ethyl amine by

Aboul-Enein and AI- Duraibi [225].

R = -CH(CH3)2

R= -C02CH(CH3)2

Fig. 7 Derivatized urea phase

37

MACROCYCLIC ANTIBIOTICS

One of the latest and perhaps the most advanced classes of chiral selectors

includes the macrocyclic antibiotics, which may either be immobilized or covalently

bonded to a solid support and also be used as a chiral additive. These chiral selectors are

of a relatively complex structure and the mechanism behind their chiral discrimination

ability is virtually unknown, although it has been suggested that they act by a

combination of hydrogen bonding, 1t-1t complexation, dipole stacking, inclusion and

steric interactions [226]. Vancomycin contains I 8 stereogenic centres, whereas the

others used, rifamycin B, thiostrepton and teicoplanin, contain 9, I 7 and 23, respectively.

These selectors are immobilized via a two to three carbon spacer to the silica surface and

columns packed with these sorbents are now commercially available (such as

Chirobiotic ™, Astec Corp.) and have been used for the resolution of large number of

different racemates. Vancomycin, teicloplanin and rifamycin are the major antibiotics

that have been used in both HPLC and CE to separate wide variety of enantiomers [227].

Rojkovocova et a!. have summarized properties of the macrocyclic antibiotics viz.

vancomycin, teicoplanin and ristocetin A which have been used as stationary phases in

HPLC for the separation of various enantiomeric drugs and their derivatives [228].

Lamprecht et al. carried out an enantioselective analysis of (R)- and (S)- atenolol in urine

samples by HPLC column switching setup using Lichrospher ADS restricted access

column and Chirobiotic T column and effluent were detected by fluorescence detector

[229]. Mistry and co-workers have developed sensitive and stereoselective HPLC assay

38

for the determination of enantiomers of metoprolol and diastereoisomers of a­

hydroxymetoprolol using chirobiotic T bonded phase column with linear range 0.5-100

ng/mL and 1-100 ng/mL, respectively [230]. Mislanova et a!. have reported direct HPLC

determination of (R)- and (S)- propranolol in rat microdialysate using on-line two column

switching procedures viz. RP-18 ADS precolumn coupled with ovomucoid analytical

column and RP-8 ADS precolumn with a teicoplanin analytical column giving limit of

detection 10 ng/mL and 15 ng/mL for (R)- and (S)- propranolol, respectively [231]. A

number of compounds including amino acid and their related compounds, compounds

with a ring containing stereogenic centre, compounds bearing aromatic structures near

their stereogenic centres and alcohols have been tested for enantioseparation on these two

CSPs [232].

Vancomycin Rifamycin

Fig. 8 Antibiotics

39

Technique based on addition of chiral constituents to mobile phase

Modern RP-HPLC sorbents are excellent materials for the achievement of high

column efficiency. However, the principle of using mobile phase additives in RP-LC in

order to regulate the retention behaviour of an analyte is widely used today. By the use of

an optically active counter-ion, diastereomeric pairs have been separated on an achiral

ordinary RP-column. Many of the principles had been already described, which are based

on covalently bound chiral phases, which can also be applied to the technique of adding

the chiral selector to the mobile phase.

Cyclodextrins and their derivatives are currently the most powerful chiral

selectors, which are applicable to wide range of organic compounds. The enantiomeric

separation of metoprolol and its metabolites in human urine was carried out by Capillary

Electrophoresis using carboxymethyl-f3-cyclodextrin as chiral selector [233]. Shuang and

Choi have described retention behaviour of procaine hydrochloride on an Alltima

octadecyl silica C18 column with a mobile phase containing negatively charged

carboxymethyl f3-cyclodextrin as a chiral additive by RP-HPLC and the results show that

proposed method can be successfully applied to real sample analysis [234]. Me Murtrey

et al. have resoluted enantiomers of dihydroxyphenylalanine and selected salsolinol

derivatives using sulfated f3-cyclodextrin as chiral selector with conventional RP-ODS

column, however sulfated f3-cyclodextrin gave very effective resolution of salsolinol

derivatives with little less effective resolution for dihydroxyphenylalanine [235]. Li and

co-workers have performed enantiomeric resolution of epenephrine, isoproterenol and

40

ephedrine by RP-mode using f3-cyclodextrin, DM-f3-cyclodextrin and TM-f3-cyclodextrin

as mobile phase additive and results showed that chromatographic systems with a

dynamically generated stationary phase with methylated f3-cyclodextrin proved to be a

versatile tool for enantiomeric separation [236].

Dolezalova and Tkaczykova have reviewed direct HPLC and CE separation for

enantiomers of interest by using teicoplanin and sulfobutylether-f3-cyclodextrin as chiral

selectors. They have also described ligand exchange liquid chromatography with N,N­

dimethyi-L-phenylalanine[237]. In a similar way, aryloxyphenoxypropanoic acid and an

antitumor agent have been separated by using teicoplanin as the chiral selector in HPLC

and hydroxypropyl cyclodextrins in CE method with the result showing that CE assay is

much less precise and accurate than HPLC [238].

Gotmar et al. have studied the influence of the solute hydrophobicity on the

enantioselective adsorption of f3-blockers on cellulose protein, which has been used as

chiral selector, and results have showed the hydrophobicity of propranolol is greater than

alprenolol and metoprolol [239]. Fornstedt and co-workers have studied the complex

dependence of the selectivity factor on pH of mobile phase for propranolol enantiomers

in which cellulose protein was used as chiral selector [240]. f3-lactoglobulin has been

used as CSP in HPLC while as chiral additive in CE in separation of several chiral acidic.

basic and uncharged drugs [241 ]. Teicoplanin, a macrocyclic antibiotic has also been

used as a CSP and as chiral selector in HPLC for amino acid, peptides, a-OH carboxylic

acid and neutral analytes including cyclic amides and amines [242].

41

An alternative technique in ligand exchange chromatography is the use of chiral

metal complexes as additives to the mobile phase in combination with achiral stationary

phases. In this case, a monodendate (MS) or bidendate selector metal complex (SMS) is

present in the mobile phase and forms a mixed selector-analyte complex (AMS).

Partition between the mobile (m) and the stationary phase (s) takes place according to the

following equilibria:

Mobile Phase

Stationary Phase

A: Analyte

M: Metal

S :Selector

........

Dolezalova and Tlaczykova have determined 0-DOPA in levodopa by chiral

ligand exchange chromatography with an ordinary C 18 column and a chiral mobile phase

containing N, N-dimethyl L-phenyl alanine and Cu (II) acetate by HPLC on a teicoplanin

column. However, HPLC methods have been proved much more sensitive than

polarimetric methods [243]. Using the same approach, Bazylak and Aboui-Enein have

42

separated stereoisomers of labetalol [244], carbuterol [245] and clenbuterol enantiomers

[246] by RP-HPLC on conventional Octadecyl silica packed column and double helical

chelate (-) (M) ( A., !!.. ) - 4, 4' - ( S) -methyl - (2R) - (propyl ethane - diyldiimino ) bis (

pent -3 - en-2-onato) nickel ( II) as chiral mobile phase additive. In addition, Jiang et al.

have determined R (+) isomer and its related substances in levotloxacin HCI on C18

column with 0.008 M L-phenylalanine and methanol as a mobile phase [247].

43

AIM AND SCOPE

The review of the literature work reveals that impurity assays in pharmaceutical

industries by liquid chromatography are developed and validated to support the

manufacturing process, for stability study of the formulations and for release assays of

each lot of bulk drugs. Since, impurity levels are often very low in most pharmaceutical

bulk products, the choice of the method is very important. The method must be

accurate, sensitive, selective, reproducible and convenient. The development of such

reliable analytical methods enables quantitative assessments required to ensure that the

product, which reaches to the public, is fit for its purpose or not. For this, liquid

chromatography is the premier method for stability and impurity assays of

pharmaceuticals, which allows traces of impurities to be separated and detected.

Apart from this, in recent years, the impact of chirality in the design. development

and utilization of drugs has gained widespread recognition. As a result. there has been a

dramatically increasing demand for chiral separations. When an analyst faces the task to

develop a separation method for a given problem, it is important that there exists

sufficient background knowledge on the potentials of the techniques available in relation

to the analyte(s) of interest, so that a rational choice can be made towards the best

possible solution. For the separation of non-chiral analytes, extensive theoretical

knowledge is available. Yet, despite various efforts, when it comes to the resolution of

chiral analytes, knowledge about the basic principles is still fragmentary and most of the

phase systems available are non-predictable with regard to their enantioselectivity and

44

retention properties for a given chiral problem. Keeping this in view, there emerged a

need to synthesize a sorbent that can separate an active ingredient from the given drug.

The present work involves the synthesis of polymeric sorbent and using that resolution of

antihypertensive drugs has been carried out. Further, a SFC method has also been

described for bioequivalence study of atenolol in human plasma.

45

PRESENT INVESTIGATION

The present work describes the synthesis of amide based stationary phases. their

characterization and applications.

A novel method for preparing stationary phase based on amberlite XAD-4 resin is

presented. Functionalized polymers were obtained by reactions which involves Friedal­

Crafts acetylation, oxidation and chloroformoylation of the resin. CSP viz. m-(+)[a­

methyl benzyl carboxamide] XAD-4 was synthesized by reacting R(+)-1-

phenylethylamine with chloroformoyl XAD-4 under weakly alkaline conditions. An

amino acid based stationary phase was synthesized by reacting methyl ester L- histidine

dihydrochloride with chloroformoyl XAD-4 to give m - [2- carbamoyl-3-(4- imidazolyl)

methyl propanoate] XAD-4. These synthesized compounds were characterized by mp.

elemental analysis, FT -IR spectra and SEM analysis.

The newly synthesized m-(+)[a-methyl benzyl carboxamide] XAD-4 chiral resin

was used for the resolution of ~-blockers viz. atenolol, metoprolol and propranolol by

column chromatography. Physico-chemical properties of the resin viz. moisture content

swelling, density, void volume were determined for the synthesized resin. Hydrogen

bonding and n - n interactions are supposed to be the major analyte- chiral stationary

phase interactions. The resin was highly selective for the separation of the selected ~­

blockers with high resolution.

46

Separation and estimation of antihypertensive drugs vtz. propranolol,

hydrochlorothiazide, atenolol and nifedipine has been carried out on m -[2-carbamoyl-3-

( 4-imidazolyl) methyl propanoate] XAD-4 stationary phase. Antihypertensive drugs

were absorbed in methanol media and separated by eluting them with the mixture of

acetonitrile : sodium acetate - acetic acid buffer (3:7, v/v) (pH = 4.1) solution. The

proposed method was further applied to the analysis of tablets containing these drugs.

A new and novel separation technique is developed for atenolol using

Supercritical Fluid Chromatography, which is cost effective, viable and speedy technique

with usage of less toxic solvents and hence, is an ecofriendly method. The method was

applied to determine atenolol in blood plasma for its bioequivalence study. Thus, the

developed method is accurate, sensitive, selective, reproducible and convenient.

47

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