1

Chapter- I

Identification, separation, isolation and

characterization of major impurities in some

anticancer and antipsychotic drugs

1.1.0 Introduction

Cancer is the uncontrolled growth and spread cells. It can affect almost any

part of the body. The growths often invade surrounding tissue and can metastasize to

distant sites. In addition, a significant proportion of cancers can be cured by surgery,

radiotherapy or chemotherapy, especially if they are detected early.

Other terms used are malignant tumors and neoplasm. One defining feature of

cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries,

and which can then invade adjoining parts of the body and spread to other organs.

This process is referred to as metastasis. Metastasis, a hallmark of malignancy, is an

extremely complex process resulting from dissemination of tumor cells from the

primary tumor through the vascular and lymphatic system and growth selectively in

distant tissues and organs. Metastases are the major cause of death from cancer.

In general, there are two kinds of anticancer drugs which are available in the

market. One is the plant derived anticancer drugs and the other one is laboratory

synthesized anticancer drugs. FDA (Food and Drug Administration) approved

marketed anticancer drugs: Vincristine, irinotecan, etoposide and paclitaxel are classic

examples of plant derived anticancer drugs where as gemcitabine, anastrozole,

letrozole, capecitabine, etc., are the FDA approved synthetic anticancer drugs.

2

Antipsychotics are a group of psychoactive drugs commonly but not

exclusively used to treat psychosis, which is typified by schizophrenia. Antipsychotics

are also referred to as neuroleptic drugs. The word neuroleptic is derived from Greek:

meaning ‘taking hold of one’s nerves’. This term reflects the drugs’ ability to make

movement more difficult and sluggish. Over a period of time, a wide range of

antipsychotic drugs have been developed. A first generation of antipschotics, known

as typical antipsychotics, was discovered in the 1950s. Most of the drugs in the

second generation, known as atypical antipsychotics, have more recently been

developed. These drugs are commonly used to treat schizophrenia, mania and

delusional disorder. They might be used to counter psychosis associated with a wide

range of other diagnoses, such as psychotic depression.

Impurities present in some of the synthetic anticancer drugs and antipsychotic

drugs were identified, separated, isolated and characterized by using advanced

analytical techniques. The details of these investigations are presented in the present

thesis.

Active pharmaceutical ingredients (API), widely known as healthcare

products, are used for therapeutic effects in pharmaceutical formulations. These are

biologically active chemical substances produced in large quantities using different

manufacturing procedures and commonly called as bulk drugs. The bulk drugs are

used to make individual dosage formulations to cure the diseases of mankind.

The quality of any bulk drug substance depends not only on the technology

adopted but also on the quality of materials used in the manufacturing process. It is

extremely necessary that the purity and safety of the bulk drug substance be ensured

thoroughly before using them in different formulations. Good manufacturing practices

(GMP) are quite useful and provide valuable guidelines for the selection of

3

manufacturing process [1]. There is an ever increasing interest in impurities present

in bulk drug substances. Recently, not only purity profile but also impurity profile has

become essential as per various regulatory requirements. Impurity profile is the

description of identified and unidentified impurities present in new drug substances.

Establishment of impurity profiles, selection of raw materials and specifications for

finished products are some important steps to be carried out during the manufacture of

bulk drugs.

In the pharmaceutical world, an impurity is considered as any other organic

material, besides the drug substance, or ingredients, arise out of synthesis or unwanted

chemicals that remains with API’s. The impurity may be developed either during

formulation, or upon aging of both API’s and formulated API’s in medicines. A good

illustration of this definition may be identification of impurity in API’s like 1-

(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-4[-1-hydroxy-1-methyl-ethyl)-furan-2-

sulphonylurea using multidisciplinary approach [2]. The presence of these unwanted

chemicals, even in small amount, may influence the efficacy and safety of the

pharmaceutical products. Impurity profiling (i.e., the identity as well as the quantity of

impurity in the pharmaceuticals), is now gaining critical attention from regulatory

authorities. The different pharmacopeias, such as the British Pharmacopeia (BP),

United States Pharmacopeia (USP), European Pharmacopeia (EP), Japan

Pharmacopeia (JP), and Indian Pharmacopeia (IP) are recognized standards for

potency and purity of drugs.

The International Conference on Harmonization of Technical Requirements

for Registration of Pharmaceuticals for Human Use (ICH) which took place in

Yokhamma, Japan in1995 released new guidelines on impurities in new drug products

[3]. The ICH has also published guidelines for validation of methods for analyzing

4

impurities in new drug substances, products, residual solvents and microbiological

impurities [4-6]. The main focus of these guidelines deals with the quantification,

reporting, identification and qualification of impurities in new drug substances and

new drug products.

A number of articles [7-9] have stated guidelines and designed approaches for

isolation and identification of process-related impurities and degradation products,

using Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR) Spectroscopy,

High Performance Liquid Chromatography (HPLC), Fourier Transform Ion Cyclotron

Resonance Mass Spectrometry (FTICR-MS), and Tandem Mass Spectrometry for

pharmaceutical substances.

Present work reveals different novel impurities found in some of the

anticancer and antipshychotic category drug substances.

1.2.0 Classification of impurities as per ICH (International Conference on

Harmonization) guidelines

According to ICH guidelines, impurities in the drug substance produced by

chemical synthesis can broadly be classified into following three categories.

- Organic impurities (process and drug related)

- Inorganic impurities

- Residual solvents or Organic volatile impurities

1.2.1 Organic impurities

Organic impurities are generally related to the synthesis. These impurities may

arise during the manufacturing process and or storage of the drug substance, may be

identified or unidentified, volatile or non-volatile, and may include;

5

- Starting materials or intermediates

- By-products of synthesis

- Degradation products

- And materials used in the synthesis such as reagents, ligands and catalysts.

Impurities are found in API’s unless, a proper care is taken in every step

involved throughout the multi-step synthesis. For example, in paracetamol bulk, there

is a limit test for p-aminophenol, which could be a starting material for one

manufacturer or be an intermediate for the others.

In synthetic organic chemistry, getting a single end product with 100% yield is

very rare; there is always a chance of having by-products [10]. In the case of

paracetamol bulk, diacetylated paracetamol may be formed as a by-product.

Impurities can also be formed by degradation of the end product during

manufacturing of the bulk drugs. The degradation of penicillin and cephalosporins are

well-known examples of degradation products. The presence of a β-lactam ring as

well as that of an a-amino group in the C6/C7 side chain plays a critical role in their

degradation. Another example that may be quoted is, the degradation of ibuprofen to

2-(4-formylphenyl)propionic acid, 2-(4-isobutylphenyl) propionic acid, 2-(4-

methylphenyl) propionic acid, 1-(4-ethylphenyl) propionic acid, 4-

isobutylacetophenone, 2-(4-n-propylphenyl) propionic acid and 2-(4-n-butylphenyl)

propionic acid, which are reported to be well known impurities in Ibuprofen [11]. The

degradation products of diclofenac sodium and clotrimazole [12], paclitaxel [13] have

also been reported.

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1.2.2 Inorganic impurities

Generally related to the drug substance synthesis and may include reagents,

ligands, catalysts, heavy metals, inorganic salts, and processing materials used in the

synthesis such as filter aids or charcoal that remain in the final drug substance.

The inorganic salts like NaCl, LiCl, Na2SO4, etc., and other inorganic

impurities are generally quantified by residue on ignition test or sulphated ash test.

This test utilizes a procedure to measure the amount of residual substance not

volatilized from a sample when the sample is ignited at elevated temperature in the

presence of sulfuric acid. This test is usually used for determining the content of

inorganic impurities in an organic substance. The sulfuric acid moistened sample in a

platinum or silica crucible heated at low temperature till the white fumes get

exhausted and then ignited at 600°C ±50°C. The residue remained is quantified

gravimetrically. The pharmacopiea limit for the residue remaining after ignition, in

any active pharmaceutical ingredient, is not more than 0.1% w/w [14].

The heavy metals test is performed as the color comparison test mentioned in

USP w.r.t the known lead standard solution (the general pharmacopea limit is not

more than 20 ppm).

The heavy metals test is used to demonstrate the content of metallic impurities

that are coloured by sulfide ion present in the test substance, under specified test

conditions, are compared visually with the known lead ion standard solution. The

inorganic substances that typically respond to this test are lead, mercury, bismuth,

arsenic, antimony, tin, cadmium, silver, copper and molybdenum [15].

The free halides like Cl-, Br-, will be checked w.r.t the argentometric titration.

The alkali metals can be quantified using AAS and some other metals like Ni, Hg in

ppb level will be checked using ICPMS.

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1.2.3 Residual solvents or organic volatile impurities (OVI)

Organic solvents which are used in the manufacturing process or generated

during the production and remain in the final drug substance. Residual solvents are

also referred as organic volatile impurities (OVI). Some solvents that are known to

cause toxicity should be avoided in the production of bulk drugs. Depending on the

possible risk to human health, residual solvents are divided into three classes [16].

Especially, solvents in Class I, viz benzene (2 ppm limit), carbon tetrachloride (4 ppm

limit), should be avoided in pharmaceutical manufacturing process. In class II, viz,

N,N-dimethylformamide (880 ppm), acetonitrile (410 ppm)methylene chloride (600

ppm), methanol (3000ppm), pyridine (200 ppm), tolune (890 ppm), should be limited.

In Class III solvents (low toxic), viz acetic acid, ethanol, acetone have permitted daily

exposure of 50 mg or less per day, as per the ICH guidelings. A selective gas

chromatography (GC) method has been developed to determine the purity of acetone,

dichloromethane, methanol, and toluene. Using this method, the main contaminants of

each organic solvent can be quantified. Morever, the developed method allows the

simultaneous determination of ethanol, isopropanol, chloroform, benzene, acetone,

dichloromethane, methanol and toluene with propionitrile as the internal standard

[16].

1.2.4 ICH limits for impurities

According to ICH guidelines on impurities in new drug products,

identification of impurities below 0.1% level is not considered to be necessary, unless

potential impurities are expected to be unusually potent or toxic. According to ICH,

the maximum daily dose quantification threshold to be considered is as follows;

≤2g/day 0.1% or 1mg per day intake (whichever is lower) ≥2g/day 0.05%.

8

Nevertheless, as per ICH guidelines, any impurity which is ≥ 0.05% threshold

should be identified, characterized and quantified [17].

In summary, the new drug substance specifications should include, limits for-

ii) Organic Impurities

Each specific identified impurity

- Each specific unidentified impurity at or above 0.1%

- Any unspecific impurity, with limit of not more than 0.1%

- Total impurities

iii) Residual solvents

iv) Inorganic impurities

- Heavy metals ≤ 20 ppm (USP)

- Residue on ignition< 0.1% (USP).

In the present study organic impurities present in some of the anticancer and

anti phsychotic drugs are studied in detail.

1.3.0 Organic impurities in drugs

Organic impurities in drugs are mostly synthesis-related or process-related

which originate from various sources and various stages of synthesis of bulk drugs.

Some by products can also be formed during synthesis. Majority of these organic

impurities are characteristic of the synthetic route used in the process to manufacture

the drug.

The origin of the impurities in drugs is classified as follows:

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1.3.1 Last intermediate of synthesis

Impurities which fall into this category are often called most probable or

expected impurities. For example, the last step in the synthesis of paracetamol (I) is

the acetylation of 4-aminophenol (II). II is a probable impurity in the bulk drug of I.

NH CH3

O

NH2

OH

(I) (II)

Paracetamol 4- aminophenol

1.3.2 Products of incomplete reaction during synthesis

If the intermediate has two functional groups and the final step involves the

same reaction in both, there is always a possibility that only one of them reacts and a

partially reacted impurity appears. Impurities which originate from this kind of

reactions also fall into the category of probable impurities. For example, during the

synthesis of ethynodiol diacetate (III), the final step is the diacetylation of ethynodiol

(IV) in which the monoacetylated product (V) could be an impurity.

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CH

OO

CH3

O

O

CH3

CH3

CH3

(III)

Ethynodiol diacetate

C H

C H 3 OO

C H 3

OH

C H 3C H

C H 3O H

OH

C H 3

(IV) (V)

Ethynodiol Ethynodiol monoacetate

1.3.3 Products of over reaction

In many cases, if the reaction of the final step is not selective enough then the

reagent attacks the last intermediate in addition to the desired site. Over reaction could

take place not only in the final step but also during the previous steps of the synthesis.

The example of over reaction is the chlorination step in the synthesis of pyridinol

carbamate (VI). The reaction product of the photocyclized chlorination of 2,6-lutidine

(VII) is the bis-chloromethyl derivative (VIII). This is converted to the final product

in two steps. Due to additional chlorination of 2,6-lutidine, the tri-chloro

derivative[18] (IX) is formed which in turn leads hydroxyl impurity of pyridinol

carbamate (X).

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NCH3 CH3N

Cl

ClN

ONCH3

O NCH3

OO

HH

NCl Cl

Cl

NON

CH3O N

CH3

OO

HH

OH

Cl2, light 2 steps

2 steps

(VII) (VIII) (VI)

(IX) (X)

1.3.4 Impuriites arising from impurities in the starting material

Impurities present in the starting materials of the drug synthesis can also be

sources of impurities in the drug. In these cases, the impurity undergoes the same

reaction as the main component leading to mainly isomeric impurities. For example

during the synthesis of celecoxib (XI) where the starting material is 4-methyl

acetophenone, the presence of 2-methyl and 3-methyl acetophenone in the starting

material leads to the corresponding isomers of celecoxib[19] (XII & XIII).

N NH

CH3

S

OO

NH2

F

F

F

(XI)

N NH

S

OO

NH2

F

F

F

CH3

(XII)

Celecoxib Celecoxib isomer-I

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N NH

S

OO

NH2

F

F

F

CH3(XIII)

Celecoxib isomer-II

1.3.5 Impurities from the solvent of the reaction

In some cases the solvent used in the reaction or an impurity present in the

solvent is also transformed during synthesis leading to an impurity. For example,

during the synthesis of Ramipril (XIV), the first step of synthesis is a Friedel Crafts

reaction between benzene and acetic maleric anhydride to form 1-phenyl-2-oxobut-2-

ene-4-oic acid. If benzene is used in this case as a solvent of this reaction, traces of

toluene in it leads to a 4-methyl derivative of the above intermediate and this can be a

source of an analogous impurity in the final product [20] (XV).

N

NH

O

CH3

OO

CH3

N

NH

O

CH3

OO

CH3

CH3

(XIV) (XV)

Ramipril Analogous impurity of Ramipril

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1.3.6 Products of side reaction

In majority of the cases side reactions are inevitable, along with the main

reaction, even though the reaction conditions are carefully optimized. For example,

during the synthesis of propranolol (XVI), a typical side reaction occurs where a

dimeric derivative (XVII) is formed as an impurity [21].

O

OH

NH

CH3

CH3

(XVI)

Propranolol

(XVII)

OO

OH

N

OHCH3CH3

Dimeric derivative of propranolol

1.3.7 Degradation products as impurities

Degradation of the final product of the drug can take place in the reaction

mixture of the final step or during the isolation, drying etc. For this reason,

degradation products form a group of impurities in drugs. For example, during the

course of reaction leading to oxipropone (XVIII), piperidine can split off from the

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drug material to form 1-(4-methyl (phenyl)-prop-2-ene-1-one (XIV). The quantity of

this impurity increases during the storage condition.

CH3

O

CH3

N

CH3

O

CH3

CH2

(XVIII) (XIX)

Oxipropone 1-(4-methyl (phenyl)-prop-2-ene-1-one

1.3.8 Crystallization-related impurities

Based on the realization that the nature of structure adopted by a given

compound upon crystallization could exert a profound effect on the solid-state

properties of that system, the pharmaceutical industry is required to take a strong

interest in polymorphism and solvatomorphism as per the regulations laid down by

the regulatory authorities.

Polymorphism is the term used to indicate crystal system where substances

can exist in different crystal packing arrangements, all of which have the same

elemental composition. Whereas, when the substance exists in different crystal

packing arrangements, with a different elemental composition; the phenomenon is

known as Solvatomorphism [22]. For example, Donepezil exerts different polymorphs

in which polymorph form 1 and polymorph form 3 are stable. If any other

polymorphic forms are present in the required form it is considered as polymorphic

impurity.

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1.3.9 Stereochemistry-related impurities

It is of paramount importance to look for stereochemistry related compounds;

that is, those compoumds can be considered as impurities in the API’s. Chiral

molecules are frequently called enantiomers. The single enantiomeric form of chiral

drug is now considered as an improved chemical entity that may offer a better

pharmacological profile and an increased therapeutic index with a more favourable

adverse reaction profile. However, the pharmacokinetic profile of levofloxacin (S-

isomeric form) and ofloxacin (R-isomeric form) are comparable, suggesting the lack

of advantages of single isomer in this regard [23]. The prominent single isomer drugs,

which are being marketed, include levofloxacin (S-ofloxacin), lavalbuterol (R-

albuterol), esomeprazole (S-omeprazole) and escitalopram (S-citalopram).

Carboprost is a prostaglandin analogue belongs to the class of prostaglandins

which are potent stimulants of human uterine contractility and have been used in the

past in various stages of pregnancy. Carboprost is available only through restricted

prescription to major hospitals at more than US$ 100 per 250mcg injection. The author

has developed enantiomeric separation method for the carboprost a prostaglandin drug

#. In which the S-isomeric form of carboprost is producing desired therapeutic activity

and the other enantiomeric form is not producing desired therapeutic effect. A simple

and efficient chiral HPLC method was developed and validated for the separation of S-

carboprost (XX) & R-carboprost (XXI) with commercially available chiral stationary

phase ( chiralpak AD-H).

# The part of this work has been published in CHROMATOGRAPHIA, 68 (2008), 501-505.

16

COOH

HO

HOHO CH3

COOH

HO

HOH3C OH

(XX) (XXI)

S-Carboprost R-Carboprost

1.3.10 Formulation-related impurities

Many impurities in a drug product can originate from excipients used to

formulate a drug substance. In addition, a drug substance is subjected to a variety of

conditions in the process of formulation that can cause its degradation or have other

undesirable reactions. If the source is from an excipient, variability from lot to lot

may make a marginal product, unacceptable for reliability. Solutions and suspensions

are inherently prone to degradation due to hydrolysis or solvolysis[24]. Fluocinonide

Topical Solution USP, 0.05%, in 60-mL bottles, was recalled in the United States

because of degradation/impurities leading to sub-potency [25]. In general, liquid

dosage forms are susceptible to both degradation and microbiological contamination.

In this regard, water content, pH of the solution/suspension, compatibility of anions

and cations, mutual interactions of ingredients, and the primary container are critical

factors.

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Microbilogical growth resulting from the growth of bacteria, fungi, and yeast

in a humid and warm environment may result in unsuitability of an oral liquid product

for safe human consumption. Microbial contamination may occur during the shelf life

and subsequent consumer-use of a multiple-dose product, either due to inappropriate

use of certain preservatives in the preparations, or because of the demi-permeable

nature of primary containers [26].

1.3.11 Impurities arising during storage

A number of impurities can originate during storage or shipment of drug

products. It is essential to carry out stability studies to predict, evaluate, and ensure

drug product safety [22].

1.3.12 Method related impurity

A known impurity, 1-(2,6-dichlorophenyl) indolin-2-one (XXIII), is formed in

the production of a parenteral dosage form of diclofenac sodium (XXII), if it is

terminally sterilized by autoclave[27]. The conditions of the autoclave method (i.e.,

123±2°C) enforce the intramolecular cyclic reaction of diclofenac sodium forming an

indolinone derivative and sodium hydroxide. The formation of this impurity has been

found to depend on initial pH of the formulation.

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NHO

O

Na

Cl Cl

N

O

Cl

Cl

(XXII) (XXIII)

Diclofenac sodium 1-(2,6-dichlorophenyl) indolin-2-one

1.3.13 Mutual interaction amongst ingredients

Most vitamins are very labile and on aging they create a problem of instability

in different dosage forms, especially in liquid dosage forms. Degradation of vitamins

does not give toxic impurities; however, potency of active ingredients drops below

Pharmacopoeal specifications.

Because of mutual interaction, the presence of nicotinamide in a formulation

containing four vitamins (nicotinamide, pyridoxine, riboflavin, and thiamine) can

cause the degradation of thiamine to a sub-standard level within a one year shelf life

of vitamin B-complex injections [28]. The marketed samples of vitamin B-complex

injections were found to have a pH range of 2.8- 4.0. A custom-made formulation

with simple distilled-water and a typical formulated vehicle including disodium

edentate and benzyl alcohol were investigated, and similar mutual interactions

causing degradation were observed.

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1.3.14 Functional group-related typical degradation

Ester hydrolysis can be explained with a few drugs, viz aspirin, benzocaine,

cefotaxime, ethyl paraben [28], and cefpodoxime proxetil [29].

Hydrolysis is the common phenomenon for ester type of drugs, especially in

liquid dosage forms, viz benzylpenicillin, oxazepam and lincomycin. Aspirin (XXIV)

hydrolyses in the presence of water to form salicylic acid (XXV).

O

OH

O

O

CH3

O

OH

OH

(XXIV) (XXV)

Aspirin Salicylic acid

Oxidative degradation of drugs like hydrocortisone, methotrexate, hydroxyl

group directly bonded to an aromatic ring (viz phenol derivatives such as

catecholamines and morphine), conjugated dienes (viz vitamin A unsaturated free

fatty acids), heterocyclic aromatic rings, nitroso and nitrite derivatives, and aldehydes

(especially flavorings) are all susceptible to oxidative degradation.

In mazipredone, the hydrolytic and oxidative degradation pathway in 0.1 mol

L-1 hydrochloric acid and sodium hydroxide at 80°C were studied [30].

Photolytic cleavage includes example of pharmaceutical products that are

exposed to light while being manufactured as solid or solution, packaged, or when

being stored in pharmacy shops or hospitals for use by consumers.

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Ergometrine [31], nifedipine [32], nitroprusside, riboflavin and phenothiazines

are very liable to photo-oxidation. In susceptible compounds, photochemical energy

creates free radical intermediates, which can perpetuate chain reactions. Most

compounds will degrade as solutions when exposed to high-energy UV radiations.

Fluroquinolone antibiotics are also found to be susceptible to photolytic cleavage

[33].

In ciprofloxacin eye drop preparation (0.3%), sunlight induces photocleavage

reaction producing ethylenediamine analog (XXVII) of ciprofloxacin (XXVI) [34].

NN

NH

O

OHO

F

NNH

NH2

O

OHO

F

(XXVI) (XXVII)

Ciprofloxacin Ethylenediamine analog

Decarboxylation of some dissolved carboxylic acids, such as p-aminosalycylic

acid; shows the loss of carbon dioxide from the carboxyl group when heated. An

example of decarboxylation is the photoreaction of rufloxacin [35].

As seen earlier, impurities in drug products can come from the drug or from

excipients or can be brought into the system through an inprocess step by contact with

the packaging material.

21

For most drugs, the reactive species consists of;

• Water – that can hydrolyze some drugs or affect the dosage form performance

• Small electrophiles – like aldehydes and carboxylic acid derivatives

• Peroxides – that can oxidize some drugs

• Metals – which can catalyze oxidation of drugs and the degradation pathway

• Leachable or Extractables – can come from glass, rubber stoppers, and plastic

packaging materials. Metal oxides such as NaO2, SiO2, CaO, MgO are the

major components leached/extracted from glass [36]. Generally most synthetic

materials contain leachable oligomers/monomers, vulcanizing agents,

accelerators, plasticizers, and antioxidants [37]. Some examples of

leachable/extractables from synthetic materials include styrene from

polystyrene, [38] diethylhexylphthalate (DEHP, plasticizer in PVC), [39]

dioctyltin isooctylmercaptoacetate (stabilizer for PVC), [40] zinc stearate

(stabilizer in PVC and polypropylene), [41] 2- mercaptobenzothiazole

(accelerator in rubber stopper), [42] and furfural from rayon [43].

These impurities are needed to be analyzed by suing different

analytical methods.

1.4.0 Impurity profiling

1.4.1 Impurity profiling in new drug discovery

During new drug discovery research for biological screening, the analytical

research plays a vital role mainly in two directions. One is structural elucidation and

the other is estimation of purity profiles for the reaction products by spectroscopic and

chromatographic techniques, respectively. It would be rather difficult to state the

22

point at which the real impurity profile begins since the requirements for the purity of

the samples may be different in various research departments. It sometimes requires

the identification and structural elucidation of the final product which is selected for

biological screening. The estimation of the impurity profile of a drug material also

includes the identification of the main impurities in the intermediates of their

synthesis. In the case of synthesis-related impurities, their mechanism and the source

of their formation should also be presented. Impurity profile also includes the

quantitative determination of residual solvents and inorganic impurities.

1.4.2 Impurity profiling in the bulk drug production

The analytical activities related to the estimation of impurity profiles do not

come to an end after the Research and Development (R & D) phase of the

introduction of a new drug. It is very essential to ensure that no new impurities appear

in the course of scaling up procedure and also that the quantity of impurities in the

bulk drug material which were identified during the synthetic research phase remain

below the specification limits. In this situation detection of impurities by

chromatographic methods and structural elucidation using spectroscopic techniques is

an important task in order to know the amount of impurities and take necessary steps

to control the reaction conditions there by controlling the formation or at least

reducing the quantity of impurities [44-45].

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1.4.3 Impurity profiling in drug formulations

The identification, structure elucidation and quantitative determination of

impurities and degradation products are of prime importance in the course of all the

phases of research, development and production of drug dosage forms. A stability

indicating analytical method is to be used in the course of development of drug

formulation. These studies indicate which of the impurities in the bulk drug are of the

degradation type. During the purity studies, the content of these degradation products

increases while the synthesis-related impurities are likely to remain constant.

1.4.4 Impurity profile for drug registration

The comparison of impurity profiles of several batches from the same

manufacturer provides a good indication for the consistency of manufacturing

process. The comparison of samples originating from different manufacturers can

give a clear picture about the differences between their purity and the level of

manufacturing procedures. The comparison between the impurity profiles of drug

samples from different manufacturers furnish as the information about the synthetic

route used by different companies. Certain impurities can be considered to be

indicators of certain synthetic pathways (often called synthetic markers) even if they

are detected at a much lower level than required by the drug authorities. One of the

rare published studies where the results of the comparison of the impurity profile of a

drug originating from different sources is described in the recent article published by

Lehr et al [46].

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1.4.5 Strategies in impurity profiling

The schematic strategy shown in scheme 1.1 explains the systematic use in the

methods for impurity profiling of drugs.

Scheme 1.1: Schematic diagram depicting the various steps involved

in the impurity profiling of bulk drugs.

25

It is difficult to generalize the strategy regarding impurity profiling in different

industries. Although the same techniques are used in all the laboratories, the manner

of the use of these methods in the individual laboratories can be quite different. The

instrumentation used in the impurity profiling is very rapidly increasing. This can be

summarized/emphathized by the complexity of schemes for the impurity profiling

published in the literature [47-48]. The introduction of hyphenated techniques like

LC-MS-MS, LC-NMR etc. in the late 1990s created an entirely new situation in

pharmaceutical research and analysis and also in the field of impurity profiling. The

hyphenation of chromatographic methods with the spectroscopic techniques really

enables even minor impurities to be detected, identified and characterized within a

short time with more certainty.

1.4.6 Selection of samples for impurity profiling

It is very important to select proper samples for carrying out impurity

profiling. It is important to select right starting materials if isolation of impurities is

necessary. Moreover, it is essential to ensure that the impurities under study are

present in both the main drug and the finished products.

1.5.0 Application of chromatographic, spectroscopic and hyphenated

techniques for identification of impurities

1.5.1 HPLC and related techniques in impurity profiling of drugs

Chromatography in general and High Performance Liquid Chromatography

(HPLC) in particular play an important role in the separation/detection techniques. It

is widely used for separation, identification and estimation of both simple and

26

complex components present in the raw materials, intermediates, bulk drugs and their

formulations in pharmaceutical industry [49]. Thin layer chromatography (TLC) is a

powerful technique for rapid screening of unknown materials in the bulk drugs [50].

Gas liquid chromatography (GLC) has a significant role in the analysis of

pharmaceutical products [51].

HPLC which was introduced in 1960s is the most common of the several

chromatographic employed in the purity control of pharmaceuticals. Impurities in

bulk drug substances at levels of 0.1% or even less can be detected by HPLC.

Gradient elution, temperature programming and wavelength programming techniques

provides valuable information regarding the undetected components of a given drug.

In the case of a UV detection where the impurity components differ in their

absorption spectra/pattern a multiple wavelength scanning program is capable of

monitoring several wavelengths simultaneously. Photo diode-array detectors (DAD)

are generally used not only to see the components through out the entire UV range

but also to record spectra and chromatograms of all the components in a drug. Grady

et al. have outlined different practices towards the establishment of impurity profiles

of synthetic drugs [52-54]. These research articles involve the prediction of likely

impurities with the synthetic process, their isolation and identification by suitable

analytical techniques. However these studies are used only for materials synthesized

by specific routes. Several approaches for the identification of impurities in a drug

substance using HPLC have been reported [55-59].

Most of the literature available on the impurity profiling of drugs, it is very

reasonable instrument that HPLC has proven to be a powerful and effective tool in the

detection of impurities in bulk drugs and their formulations.

27

1.5.2 Analytical method development to separate the impurities from the

bulk drug using Liquid Chromatography (LC)

Before developing any analytical method development, it is preferable to have

the following chemical/physical information of the compound or mixture of

compounds; this involves, chemical structure, molecular weights, solubility,

dissociation constant, presence or absence of uv- chromophores, presence of

functional groups eg. acidic or basic groups, concentration of the sample.

Comprehensive literature search of the chemical and physical property of analyte is

essential. If the analytical method to be developed for a known sample, then check

with Merck index or any other literature which provides the chemical information.

Select appropriate liquid chromatography column for separation. Choice of

column depends on the nature of the sample to be separated. The coloumn selection

details in brief are given below:

• Neutral compounds -Reversed-phase columns like C18, C8, C4, -NH2 etc.,

• Acidic Compounds - Reversed-phase coloumns like C18, C8, C4, -NH2 etc.

or Normal phase coloumns like silica, cyano, phenyl, etc

• Basic compounds -Reversed-phase columns like C18, C8, -NH2 etc., and

wide pH range (pH 2.0 to 12.0) .

• Inorganic ions -Ion-exchange columns (Anion exchange and Cation

exchange coloumns)

The column performance should be carried out before sample analysis

with appropriate column specifications provided by the manufacturer.

Pore size: Select appropriate stationary phase column packing with small pore size

(80-120Å) if the solute molecular weight is less than about 2000 Daltons. Otherwise,

use a column packing with 200-300Å pore size.

28

Particle Size: The standard particle size is 5 µm. If high-speed (faster than 5

minutes/run) analyses are required, packing with 3.0 or 3.5 µm particles in shorter

columns can produce high-resolution separations in less time.

Column Configuration: The column configuration most often recommended for

analytical method development is 4.6 x 150 mm. If more resolution is needed, use a

longer column, 4.6 x 250 mm. After method development, choose the column internal

diameter (e.g., 2.1, 3.0 mm) to accommodate additional application objectives (e.g.,

sensitivity, solvent usage).

Establishment of a starting mobile phase and conditions for Reverse Phase

Chromatography: The reversed-phase chromatography is strongly dependent on pH

and buffer strength (viscosity of moving phase). The initial trial on mobile phases will

reflect this on the chromatogram. In general, if the sample is neutral, start with a

mobile phase comprising of water:acetonitrile in the ratio (60:40). If the sample is

basic (having the basic functional groups like amide, primary amine, secondary

amine,etc.,) start with 5- 50 mM aqueous buffer (pH of 6 to 7) : acetonitrile (60:40).

And for acidic samples, start the initial mobile phase with pH (2-5) aqueous buffer:

acetonitrile (60:40).

Selection of coloumn temperature: Start the method development experiment with

ambient temperature i.e., room temperature, depending upon nature of the compound,

adjust the required coloumn temperature. If the coloumn temperature is increased,

chromatographic peak shape will be improved so that better peak separation, better

theoretical plates and less peak tailing. However, some of the compounds are very

sensitive towards temperature and show degradation at higher coloumn temperature.

In this case the analytical method is to be developed at lower coloumn temperature.

29

Selection of flow rate: In general, start the mobile phase flow rate with 1 ml/ minute

for the conventional HPLC coloumn with particle size of 5.0 µm. The flow rate

depends directly on the particle size of stationary phase used in the coloumn. Lower

particle size (3.0 µm, 2.1µm, etc) means lower flow rate should be used like 0.5

ml/min, 0.2 ml/min, etc.,

Selection of wavelength: Set detector (UV) initially at 254 nm and optimize it later by

considering the intersecting point in the overlaid UV-Visible spectra of all the

impurities and the main peak. In general set a lower wavelengths for samples with

weak chromophores. If the detectors like refractive index (RI) are used, the sample

concentration and injection volume may be increased to obtain acceptable detector

signals.

Sample preparation: While preparing the samples, the following points should be

considered:

• Sample should be free of contamination

• Column should not get damaged because of the diluent used.

• Sample diluent and mobile phase should be miscible and ensure that the

diluent used in the sample preparation is compatible with the mobile phase.

• Sample concentration is adjusted in such a way that all the components

present in the sample should be detected.

• The pH and composition of the diluent (organic and aqueous phase) is

adjusted in such a way that the sample remains stable for a longer time.

Sample Pre-treatment: Some of the sample pre-treatment techniques mentioned

below can be adopted before injecting the sample to the liquid chromatography.

30

• Sample extraction for liquid samples eg. Supercritical fluid

extraction/ultrasonication/solid-liquid extraction, etc.

• Derivatization/concentration for better detection

• Filtration/ Centrifugation/solid phase extraction to remove any

particulates matter present in the sample.

• Dilution, buffering, addition of an internal standard etc.

Mobile phase optimization: The percentage of organic phase to be used should be

optimized after having the information such as nature of sample, pre-treatment of the

sample, selection of the column, detector, etc., the next most important aspect in the

analytical method development will be determining the percentage of organic phase in

the mobile phase i.e., mobile phase optimization.

First of all, use isocratic mobile phase of average solvent strength (aqueous

buffer: organic phase::50:50). Then, the percentage composition of orgainic phase

may be altered so as to increase the solvent strength or decrease the solvent strength

by reducing percentage of organic phase.

Secondly, use the gradient elution to determine the best solvent strength at

which optimum separation is possible between the closely eluting peaks.

Change solvents if required to improve the separation, e.g., instead of acetonitrile,

methanol or combination of different solvents can be used to get the resolution

between the closely eluting peaks atleast 2.0, purity angle should be less than the

purity threshold (provision available in waters empower software) and this indicates

that there is no merging impurities in the chromatogram; theoretical plate count

31

should be more than atleast 2000; peak area should be reproducible and the

chromatographic run time should be minimum.

If the sample is neutral, to increase the retention time and to improve

resolution between closely eluting peaks, decrease the percentage of organic phase

(eg.acetonitrile) and vice versa in order to decrease the retention time of the

compound.

If the sample is acidic, to increase the retention time decrease the organic

phase in the mobile phase and change the pH of the mobile phase, change the buffer

strength, decrease the coloumn temperature, etc., and vice versa for decreasing the

retention time.

If the sample is basic, to increase the retention time decrease organic phase

concentration, change buffer system, decrease temperature and vice versa for

decreasing the retention time. To improve the chromatographic peak shape of some

basic compounds, use the base deactivated silica column.

Triethylamine (TEA) for acid samples or Diethylamine (DEA) for basic

samples can be added to improve the chromatographic peak shape to achieve better

system suitability. It is used as a last option because it complicates the mobile phase,

reduces the retention and most importantly modifies the chemistry of column

stationary phase. TFA and DEA modifying effects may remain even after

discontinuing use, because of their strong affinity for the stationary phase. Because of

this, it is advisable to dedicate certain columns to be used with specific modifiers,

and other columns to be used without modifiers.

32

1.5.2.1 Analytical method development for Chiral separation

Column selection: The original Diacel chiral stationary phases were formed by

coating the chiral polymer from the derivatization of the amylase and cellulose on 10-

micron or 5-micron diameter silica. Because these chiral stationary phases are made

by coating the polymer on the silica, any solvent that will dissolve the polymer will

remove the coating and damage the chiral stationary phase. For this reason there are

strict limits on the mobile phases that may be used.

A wide range of chiral stationary phases available from Diacel. Diacel is

specialized in manufacturing and supplying different category chiral coloumns to the

worldwide pharmaceutical research laboratories. The different Diacell chiral

coloumns are mentioned here to select the right coloumn to separate the enantiomers

of organic compounds having different functional groups.

Organic compunds Diacell Chiral Column

Aliphatic, but not

cycloaliphatic

Chiralcel® AD, AD-H, AS, OD

Cycloaliphatic, but not a

cycloalkanone or lactone

Chiralcel®AD, AS, OD

Cycloaliphatic, and either a

cycloalkanone or lactone

Chiralcel®OB-H,OA, AS, AD

Aromatic only, with no other

functional groups

Chiralcel®OD, OD-H

Aromatic ester Chiralcel®OJ, OB-H, OA

33

Aromatic with other

functional groups

Chiralcel® OD, AD, OG, OF, AS

Low molecular weight

(<100 Daltons)

Chiralcel® AD, AS, OB-H

The chiral stationay phases of branded Diacel chiral coloumns mentioned in the above

table are given below:

AMYLOSE-O-R:

Where, R= NH

O

R=

NH

O

CHIRALCEL® AD-H CHIRALCEL® AS-H

CHIRALCEL® AD CHIRALCEL® AS

CELLULOSE-O-R:

Where, R= NH

O

R= O

CHIRALCEL® OD-H CHIRALCEL® OB-H

CHIRALCEL® OD CHIRALCEL® OB

R= O R= O

CHIRALCEL® OA CHIRALCEL® OJ-H

CHIRALCEL® OJ

34

R= NH

O

R= NH

OCl

CHIRALCEL® OG CHIRALCEL® OF

Mobile phase selection: Some of a sample's functional groups will have a greater

affinity for the stationary phase than others, and will require a stronger solvent to

elute. For samples having multiple functional groups, use the mobile phase

corresponding to the functionality with the greatest affinity for the stationary phase.

In general, if the sample contains oxygen, use mobile phase such as

hexane:isopropyl alcohol (90:10) (v/v ) and use 0.1% trifluroacetic acid modifier to

elute an acid. If the sample contains, nitrogen use hexane: isopropyl alcohol (80/20)

(v/v) and use 0.1% diethyl amine modifier to elute a base. And for the sample having

nitrogen and sulphur atoms, use hexane: isopropyl alcohol (70/30) (v/v). The selection

of coloumn temperature, detector, flow rate, etc., are mentioned in the section 5.2.

Mobile phase optimization: To increase the retention time and improve the resolution

between the closely eluting peaks [(-)R-isomer and (+) S-isomer], reduce the solvent

strength (first change the solvent concentration and then the type of solvent.),

amylose-based columns often show an exaggerated difference in separation factor

when the alcohol component is changed from isopropyl alcohol to ethanol. In this

case, evaluate the effect of the alcohol type before changing the alcohol concentration.

The retention of the compound in the coloumn can be increased by lowering coloumn

temperature within coloumn tolerance limit and also by decreasing the flow rate.

35

To decrease the retention time (this may compromise on resolution between

the closely eluting chromatographic peaks), increase the solvent strength within

column tolerances. Solvent strength should be changed by first changing the solvent's

concentration in the mobile phase, and then by changing to a more polar solvent

(ethanol, isopropyl alcohol, etc.,). Increase the temperature within column tolerances

(this may improve resolution.). Increase the flow rate within column pressure drop

tolerances.

Chromatographic peak optimization: Add appropriate modifiers to the mobile phase.

For acidic samples, trifluoroacitic acid or acetic acid is necessary to use as a modifier

otherwise the sample will fail to elute from the coloumn. For basic compounds

diethylamine or triethylamine is used as a mobile phase modifier. In general, it should

be between 0.1- 0.5% (v/v). For neutral samples, a small quantity of polar solvent,

typically 1-3% methanol, may function as a modifier provided column tolerances

permit it. Polar solvent may be combined with diethylamine or trifluroacetic acid as a

mixed modifier also. Some modifiers may suppress ionization and it can be evidenced

by peak tailing. The diethylamine and trifluroacetic acid modifying effects may

remain even after discontinuing the coloumn use, because of their strong affinity for

the stationary phase. For this reason, it is advisable to dedicate certain columns to be

used with specific modifiers, and other columns to be used without modifiers.

The chiral separation chromatographic system is suitable for indented use

provided the following chromatographic parameters are within the acceptable range:

retention time, precision between the injections (%RSD), resolution between the

peaks, tailing factor, theoretical plates. It is advisable to run a system suitability

solution to check all the above parameters before running the actual samples. The

36

obtained results for the above parameters should be compared with USP

specifications. If it matches with that then, the method is good, if not, the method

should be modified to match the desired specification.

1.5.2.2 Analytical method development using gas chromatography.

Study the nature of the compound or mixture of compounds; this involves

chemical structure, molecular weights, solubility, presence of functional groups,

compound volatility, thermal stability and boiling point of the compound.

Selection of the stationary phase: Stationary phases can be selected based on the

volatility and polarity of the sample components.

• Hydrocarbons and Non-polar compounds : Non polar stationary phase

• Chlorinated hydrocarbons and Solvents : Medium polar stationary phase

• Amino acid, Fatty acid and polar compound : Polar stationary phase

• Optical and positional isomer : Chiral stationary phase

Select the proper stationary film thickness for better capacity factor, retention and

resolution. Capillary columns are preferred over packed column for inertness and

absolute number of theoretical plates.

The selection of inlet or operating conditions will affect the separation of

the compounds. Generally, use the most moderate conditions to minimize sample

degration, inlet overload, contamination, ghosting and column degradation. For high

concentrated sample, split the injection and for trace level sample analysis use split

less injection mode.

Selection of detector: Sensitivity, selectivity, universality and reproducibility should

be considered when selecting a gas chromatography detector. Flame ionization

detector (FID) and thermal conductivity detector (TCD) are considered as universal

detectors because of ease of use, universal response, low cost of operation and

37

reproducibility. For selective response and to achieve lower detection limits, electron

capture detector (ECD) and nitrogen phosphorous detector (NPD) are selected.

Selection of carrier gas: Carrier gas selected should be inert. The optimum flow rate

for gases depends on viscosity and diffusion rates .The optimum flow rates will be in

the order of H2> He>> N2 > Ar. Always use the high pure gas. Use the high capacity

mixture and oxygen traps combined with an oxygen/moisture indicating trap. This

will allow you to maintain an overall improved performance. Optimize the runtime by

performing the temperature program and at the same time maintain the flow rate/

pressure constant.

The most common use of temperature program is to shorten the time of

analysis. Temperature optimization consists only of adjusting the program rate to

yield the fastest analysis while meeting the goals of resolution, peak shape and

reproducibility.

To evaluate the separation’s sensitivity to temperature changes, a new

analysis should be done using a different temperature program rate with the same

flow rate or a different flow rate at the same temperature program rate.

Once a column stationary phase, inlet, and detector are selected and optimized

approximately, then the process of chromatographic peak separation optimization can

be initiated. The first step is by using fast analysis condition, flow rate and

temperature program from low to high at the rate of 25-35°C / min. The low and high

temperature are selected based on the nature of the sample, solvent boiling point,

injection considerations and allowed temperature limits of the stationary phase used.

38

If the chromatographic peak shapes are poor, first correct any reasons for sample or

solvent overload and rerun the screen. If the peaks are still unsatisfactory, switch

immediately to a different stationary phase of very different polarity.

1.5.3 Isolation of impurities by preparative HPLC

Application of this technique comes into picture when the identification of

impurity must be carried out with reliability by means of applying simple analytical

(chromatographic, spectroscopic and hyphenated) techniques. In this situation,

preparative HPLC isolation followed by spectroscopic (NMR, MS) investigation

provides a lot of information to carry out structure elucidation of an unknown

impurity. The quantity of isolated impurity should be sufficient for subsequent

spectroscopic studies. In order to carry out effective isolation by preparative HPLC, it

is advisable to perform the following steps:

In order to carry out successful isolation of impurity of interest an analytical

chromatographic method (HPLC) has to be chosen for its detection and the impurities

to be isolated must be targeted. The course of research, development, analysis, nature

of the drug, solubility, stability, UV spectroscopic data etc. of the drug should also to

be known. The information on the available analytical chromatography (HPLC, TLC)

methods is of great help in the preparative HPLC method development.

Selection of starting materials for isolation of impurities is also very

important. If this material contains only small amounts of impurities (in some cases

below 0.1%) it is always advisable to select crude products or mother liquors which

39

are obtained from crystallization process as starting materials, where-in the impurities

targeted are likely to be high in concentration.

Before beginning the isolation work, it is extremely essential to check the

presence of the impurity of interest in the bulk drug and the starting material chosen

for isolation of impurities. The isolation strategy by preparative HPLC involves

enrichment and purification steps.

The first step is, the isolated impurity sample must not contain other

compounds arising from HPLC separation (mobile phase additives), because these

compounds will hamper structure elucidation work or sometimes even make it

impossible. For this reason it is preferable to select an HPLC method which does not

contain additives in the mobile phase. If this is not possible, the additive used in the

isolation process should be easily removable.

The second step is that after carrying out the isolation of impurities, the

enriched or purified impurity has to be recovered from the mobile phase fractions

without degradation. The simplest way of recovering the impurities is by the

evaporation of solvents used for isolation under vacuum. If buffer salts are used in the

isolation process, liquid-liquid extraction followed by evaporation of the organic layer

is the commonly used method. To avoid contamination from solvents it is necessary

to use HPLC grade solvents.

After the impurity of interest is isolated in the pure form, it is always advisable

to check the purity and identity of the isolated material prior to spectroscopic

investigations. Identity checking can be done by retention matching, peak purity test

and spectral match using photo diode-array detection using HPLC.

All in all, it is quite reasonable to state that preparative HPLC technique is

very much useful in the impurity profiling of drug materials.

40

1.5.4 Mass spectrometry in impurity profiling

Mass spectrometry, with its reproducibility, specificity, selectivity and

sensitivity is an indispensable analytical tool in the field of structure elucidation and

impurity profiling of pharmaceutical compounds [60].

1.5.5 Application of MS after chromatographic separation

In majority of cases, the data obtained from mass spectra (with other chemical

information) are sufficient to propose a tentative structure of the impurity. By the

application of mass spectrometry after chromatographic separation, the isolated

compounds of identical structures can also be analyzed because this technique makes

it possible to differentiate between steroisomers and positional isomers which have

different fragmentation pathways.

For example, the structure elucidation of impurities in allylestrenol (XXVIII), after

preparative HPLC isolation was carried out by electron impact (EI) high–resolution

mass spectral measurements [62]. Both the unknown impurities in the compound

obtained by preparative HPLC have molecular weights of 298 which are two mass

units less than the molecular weight of allylestrenol indicating an additional bond in

both the impurities (XXIX, XXX). This assumption was confirmed by fragmentation

patterns relating to the two different structures.

OH

(XXVIII)

Allylestrenol

41

OH OH

(XXIX) (XXX)

Allylestrenol impurity-I Allylestrenol impurity-II

1.5.6 Application of MS without chromatographic separation

Integrated techniques such as GC-MS and LC-MS are widely used for

characterization of complex mixtures.

The strategies for drug impurity profiling have been presented in the literature

[62]. Once HPLC method is developed using UV-detection for the detection of

impurities of drug, the same is transferable (with some modifications) to be used in

LC-MS system. However, a number of factors should be kept in mind while setting up

an LC-MS method for regular operation [63-64] . The evaluation of drug impurities

having no chromophores, the usage of LC-MS is the most suitable method to study

impurity profiles. Buffer salts like phosphates, citrates and borates which are non-

volatile should be avoided in LC-MS methods. The most suitable buffer salt for LC-

MS analysis is ammonium acetate [65].

Some other techniques such as LC-MS-MS, Infusion MS-MS, High

Resolution MS etc are also used in the impurity profiling studies of drugs. In some

compounds where halogen atoms are present, particularly Cl- and Br-, the presence of

these two atoms act as powerful markers in mass spectrometry. These two atoms yield

M+2 molecular ion peaks.

42

1.5.7 NMR Spectroscopy in Impurity Profiling

NMR is the most widely used technique for structural elucidation of

synthesized organic molecules. NMR plays an important role in identifying even low

level impurities in bulk drug materials with or without preparative chromatographic

isolation. For identification and characterization of drug impurities, modern NMR

offers various ranges of experiments [66].

1.5.8 Application of NMR after chromatographic separation

Structural elucidation of impurities in drug material mostly involves 1H NMR

and 13C NMR experiments; the information obtained from these experiments is

sufficient to ascertain the structure of an unknown impurity in the drug material. In

some cases, particularly 19F NMR and 31P NMR can also be powerful markers [67]

apart from 1H NMR and 13C NMR; the other two dimensional experiments such as

correlation spectroscopy (COSY) [68], heteronuclear multiple bond correlation

(HETCOR) [69], etc. are also very useful for further information with regards to the

problem of resolving the structure of an unknown molecule.

1.5.9 Application of NMR without chromatographic separation

The introduction of NMR probes especially for on-line coupling to HPLC [70-

71] greatly implies the need for preparative isolation of impurities. Stop-flow [72] and

on-flow [73] techniques are used to detect the analytes of interest. HPLC analysis is

carried out in reversed-phase mode using D2O/Buffer – acetonitrile based eluents with

an injection volume of 50-100 µL. The major problem in the use of LC/NMR for the

characterization of impurities of interest is the lack of sensitivity. Sensitivity is not a

major issue if the impurity of interest is present in large amounts (5-10%).

43

Applications describing the use of LC-NMR have been reported in literature in the

field of pharmaceuticals, natural products, environmental samples, drug metabolites

etc.[74-77].

1.5.10 Other techniques

The use of UV-VIS spectroscopy for the identification and determination of

impurities in drug substances without chromatographic separation is of very little

importance. Nevertheless, for determining certain impurities in bulk drugs UV-VIS

spectroscopic method is recommended in pharmacopeias. IR spectroscopy is

generally used to ascertain the functional groups present in the impurities of interest

after chromatographic separation. Capillary electrophoresis technique has its ability to

provide a different sensitivity to characterize the impurity content and profiles in drug

substances [78]. Future techniques for studying impurity profiling of drugs may be

coupling of LC-NMR-MS, CE-NMR, SFC-NMR etc.

In conclusion, each technique has its own unique identity and importance in

the impurity profiling of drug materials.

1.6.0 Objective of present work

In the present study, the impurity profiling (identification, separation, isolation

and characterization of impurities present in the drug substance) is carried out on

some of the anticancer and antipsychotic drugs. After the literature survey, the

following drugs were selected since no impurity profiling is reported for these drugs.

The drugs selected for the impurity profiling study are:

1. Gemcitabine – anticancer drug

2. Anastrozole – anticancer drug

44

3. Capecitabine – anticancer drug

4. Letrozole – anticancer drug

5. Olanzapine – antpsychotic drug

1.7.0 Conclusion

Impurity profiling, in other words, identification, separation, isolation and

structure elucidation of the impurities present in the active pharmaceutical ingredients

is an important analytical scientific research in the process of developing a life saving

drug molecule to cure different kinds of diseases of mankind. The methodology

involved in impurity profiling is discussed in detail. In the present work, the author

has tried to identify, isolate and characterize the novel impurities present in some of

the anticancer and antipsychotic drug substances.

45

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46

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47

[28] Roy, J., Mahmud, M., Sobhan, A., Akhteruzzaman, M., Al-Faooque, M.,

& Ali, E., Drug Dev. Ind. Pharm., 20, 2157 (1994).

[29] Hoerle, S.L., Evans, K.D., & Snider, B.G., Eastern Analytical

Symposium, November 16-20, Somerset, New Jersy 12, (1992).

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