Post on 28-Mar-2018
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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.
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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
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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
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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;
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- 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%.
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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.
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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.
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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
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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.
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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.,
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[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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