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CHAPTER-1

GENERAL INTRODUCTION TO IMPURITIES AND

METHOD DEVELOPMENT

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1. 1. Introduction to impurities in pharmaceutical

formulations

Drugs are typically developed and manufactured into

pharmaceutical formulations, these formulations require a variety

of tests like Dissolution, Assay, Related substances and stability

testing to assure therapeutic benefit prior to their use in patients.

The impurity profile of a drug product determines its quality in a

significant manner. The testing and establishment of limits for

impurities in formulations have become important in

pharmaceutical industry. ICH has issued guidelines on impurities

in new drug products and drug substances, These guidelines

showed alert focused on thresholds for identification, qualification

identification and reporting of impurities. (1-4)

Pharmaceutical analysis and analytical chemistry has a major

task in handling the changes facing the Pharmaceutical industry

today. Indeed, the demand for analytical data has become a critical

path activity for the selection of molecules for full development.

Working under full compliance of current good manufacturing

practices (cGMP), pharmaceutical analysts are called on to generate

accurate and precise data almost on demand. The pharmaceutical

analyst plays a major role in assuring the quality of a drug product.

Safety and efficacy studies require that drug substance and drug

product meet the established identity and purity as well as

bioavailability requirements.

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1.2. Various terms used to describe impurities

By-product

Degradation product

Interaction product

Intermediate

Penultimate intermediate

Related product

Transformation product

1.2.1. By-products

The unexpected products generated during the reaction are

generally called by-products.

1.2.2. Degradation products

The compounds produced as a result of decay of the API are

often called degradation products or degradants.

1.2.3. Interaction products

These products are more difficult to evaluate than by-products

and degradation products, these are formed due to interactions of

various chemicals implicated in the synthesis of the API

1.2.4. Intermediates

These are the carry forward compounds formed during multi

step synthesis process.

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1.2.5. Penultimate intermediate

These are the previous compounds in the synthetic chain just

preceding the production of the ultimate desired compound.

Sometimes confusion occurs when the desired material is a salt of a

free base or acid. It is not appropriate to label the free acid or base as

the penultimate intermediate if the drug substance is a salt.

1.2.6. Related products

These products are analogous in chemical structures however,

that the structure alone does not provide any surety about biological

activity.

1.2.7. Transformation products

Transformation products are similar to by-products, except

that this term tends to imply that more is known with reference to the

reaction products.

1.3. Sources of impurities

The following are the potential sources of impurities in a drug substance or drug product

Degradation products

Those generated during the stability studies

Manufacturing process related.

Formed during fabrication of dosage forms

1.3.1. Degradation products

Impurities that may be formed due to degradation of drug

substance. Degradation commonly occurs at different stability

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conditions. In most of the cases, the quantity of these degraded

products increase over time. Penicillin's and cephalosporins are

known example of degradation products. For example Amoxicillin5-6

drug substance, Amoxicilloic acid impurity increases from 0.10% in

control sample to 1.0% in 6M/ 400C/75% RH stability sample.

Degraded products can further react with main molecule to form

dimer impurities. Dimer of Amoxicillin acid increases to a level of 0.5%

during stability.

1.3.2. Impurities formed during storage

Storage or shipment of drug product can impart generation of

stress related impurities. For example, in of cefazolin sodium7, an

impurity. Cefazoloic acids had increased to 0.5% during storage

1.3.3. Manufacturing process related impurity

During the manufacture of drug products, parameters like pH

or temperature may play an important role in the completion of

desired process. In this situation, critical monitoring of the conditions

may be required. For example during preparation of Cefazolin sodium

the reaction is to be conducted at 25-35oC, if there is an increase in

the temperature to 45oC, Cefazolin lactone impurity is observed.

In the Amoxicillin preparation, pH, 3.0-5.0 is to be maintained

during reaction. If there is an increase in the pH to 7.0, dimers and

amoxicilloic acids impurities increase.

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1.3.4. Formed during fabrication of dosage forms.

Excipients interaction can contribute to many impurities in a

drug product..The process of fabricating a dosage form itself can

cause undesirable reactions. For example, in the preparation of

cetirizine oral solution8, propylene glycol and glycerol are used as

excipients. Two impurities, propylene glycol ester of cetirizine and

glyceryl ester of cetirizine were detected in cetirizine oral solution.

1.4. Impurities as per United States Pharmacopoeia:

The pharmacopoeia states that our concept about purity is likely to

change with time and that purity is closely related to current

developments in analytical chemistry. The terms used to describe

these impurities are

• Concomitant components

• Foreign substances

• Ordinary impurities

• Organic volatile impurities

• Signal impurities

• Toxic impurities

1.5. Acceptance criteria for impurities:

The acceptance criteria for impurities is well defined in the ICH

guidelines Q3(2). As per these guidelines process impurities are not

included in the drug product total impurities limit. However, if the

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impurity is a part of the degradation product then it has to be

included. The specifications for both drug substance and drug product

should include specific acceptance criteria for total impurities,

individual known impurities (for which structure is known) and

individual unknown impurities (for which structure is not yet known)

1.6. Qualification of impurities

The qualification threshold levels are described in the ICH Q3

guidelines. The summary is given in the table no 1.1 and flow chart.

In case the value exceeds the limits of ICH then additional toxicity

data is required to be provided.(2)

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1.7. Need for Impurity Characterization:

Impurities are generally assumed to be inferior to the drug

substance because they might not have the same level of

pharmacologic effect. The drug substance is deemed to be

compromised in terms of purity even if it contains another material

with superior pharmacologic or toxicological properties. However, on

further thought it will become clear that if we are to ensure that the

correct amount and type of drug substance is being administered to

the patient, we must assess its purity independent of the extraneous

materials. Hence the irrelevant material present in the drug substance

or active ingredient has to be considered an impurity even if it is static

or has greater pharmacologic actions, so that an appropriate

evaluation of its content in the drug product can be made. The control

of low-level impurities is of great importance when a drug is taken in

large quantities. Impurities that can affect the purity of drug

substance or can be harmful to patients, It is necessary to isolate and

characterize.

Degradant which are obtained during stress testing are

recognized. Chromatographic peaks that a reforming above the

identification thresholds according to ICH during formal stability

studies required to be identified. It is not always possible to

unambiguously characterize them with the widely used hyphenated

methods that are frequently the first line of defense. These methods

utilize detectors such as diode array UV detector (DAD), nuclear

magnetic resonance spectrometer (NMR), and mass spectrometer (MS).

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1.10. General methods of Characterization

Mass spectrometry attached to separation techniques as HPLC

or GC is a great tool in the field of structure elucidation.Applications

of LC-MS for identification of impurities in pharmaceutical analysis

are described in literature 12-15. In GC-MS technique, the molecular

weight is obtained by chemical ionization in both +ve and – ve modes.

Electron impact mode (EI) can also be recorded which gives actual

molecular weight and its fragmentation pattern. A disadvantage of this

technique is its inherent limitations due to volatility and thermal

stability of compounds.

Great advantage of LC-MS technique is its general applicability

16. The data obtained from mass spectra are sufficient to propose a

tentative structure of the impurity. Once a HPLC method is developed

using UV-detection for the detection of impurities of interest, the same

is transferable (with some modification) and to be used in LC-MS

detection. However, a number of factors should be kept in mind when

setting up an efficient LC-MS-MS, method 17-18. Volatile buffers such

as Ammonium acetate 19, Acetic acid, Trifluoroacetic acid, Formic

acid, Ammonium formate etc. is preferred for LC-MS analysis. Buffer

salts like phosphates, citrates and borates which are non-volatile

should be avoided in LC-MS methods. The evaluation of impurities

with poor or no chromophores can also be studied by LC-MS. Several

techniques such as LC-MS-MS, infusion MS-MS. High resolution MS

etc. are also used in the impurity profiling of drugs20 Mass

spectrometry is a powerful marker for the presence of halogen atoms,

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CI and Br in the compound. The compounds having these atoms yield

M+ 2 molecular ion peaks along with molecular ion peak, chloro

pattern (3:1) and bromo pattern (1:1)

1.11. Need for method development for impurities

The need for pharmaceutical analysis is driven largely by

regulatory requirements. Good understanding of physicochemical

behavior of pharmaceutical solids ensures a better selection of

formulation. The selection process includes identification of process-

related impurities and products and studying degradation

mechanisms at an early stage. For all these analytical Method

development for stability testing plays very important role. Stability

are aimed at ensuring that the drug product remains within

specifications taken on by analytical research scientist. The legal

requirements are established to ensure its identity, strength, purity.

and product quality up to end of shelf life.

Pharmaceutical analytical documentation reflects the key

functions of analytical research and development, to monitor and

ensure that the drug substance and dosage forms meet all the

requirements as per current regulatory guidelines Analytical method

development data are the foundation and backbone for

pharmaceutical development, leading to approval and production of

new drugs for market. Now it should be quite apparent that

pharmaceutical analysts play a major role in quality of a drug

product.

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In analytical research and development, the emphasis must be

on finding the optimal methodology that monitors all known and

unknown impurities. This is a daunting task because unknown

impurities have to be theorized based on the best knowledge of

reaction and degradation chemistry, combined with sound knowledge

of physical organic chemistry to theorize potential reactions and

interactions of molecules that could be involved in the material (drug

substance, pharmaceutical aids and solvents used for the preparation

of the drug product). Furthermore, the changes that could occur

during storage in various containers or packages and the effect of a

variety of storage environments must be considered.

1.12. Method Development

Numerous methods are required to characterize drug

substances and drug products. Specifications may include

description; identification, assay (of composite sample), tests for

organic synthetic process impurities, inorganic impurities,

degradation products, residual solvents, and container extractable,

tests of various physicochemical properties, chiral purity, water

content, content uniformity, and antioxidant and antimicrobial

preservative content; microbial tests; dissolution/disintegration tests;

hardness/friability tests; and tests for particle size and polymorphic

form. Some of these tests may be precluded, or additional tests may

be added as dictated by the chemistry of the pharmaceutical or the

dosage form.

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Quantitative tests to characterize drug substance and drug

product composition require method development. Methods such as

thin layer chromatography, gas chromatography, HPLC, supercritical

fluid chromatography, and capillary electrophoresis are used for

pharmaceutical analysis.

1.13. Preformulation Studies

The primary objective of a Preformulation study is to provide

data and information with regard to a drug substance and

manufacturing technology prior to initiating plans for formulation

development activities and product design for a drug product.

Preformulation studies culminate with the preparation of a report

based on these studies that assists the formulators in their

development efforts. With the data and information thus provided, the

finished product can be developed based on sound principles and

technical practices, with due consideration of analytical profiles,

chemical/physical properties, QA/QC practice, modern

manufacturing procedures, stability, and biopharmaceutical

properties.

1.14. Forced Degradation Studies

Forced degradation studies are performed in order to get the

degradants which cannot be made available in real time. This typically

involves subjecting the sample to stress conditions and analyzing for

specific impurities developed. This technique is typically used to

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develop stability indication methods. These studies are typically

protocol driven by best guidance (21) and with some additional

publications (22-24). The general conditions for stress testing are given

in Table 1.2. In case of combination products addressed in literature

(25-26), the drug to drug interaction needs to be addresses even though

the individual drugs separately submitted for FD (27) impurity profile is

completely characterized. A typical flow chart for stability indicating

method development process is given in Fig 1.2.

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b. ICH guideline for appropriate light exposure: Fluorescent=1.2

million lx hours, UV=200 W h/m2, timing depends on chamber

setting.

c. AIBN has poor solubility in water, typically a 1 mg/mL API solution

is prepared in acetonitrile: water (80/20) premixed with 5 mmol of

AIBN. However, ACVA is water soluble.

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1.15. Physico-chemical Properties of the Drugs

The physic chemical properties of the drug substance and its functional groups give a large amount of information on the potential stability problems which may crop us. This is summarized in the

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1.16. Method Development Approach

1.16.1. Stability-Indicating Chromatography Conditions

The quality of stability indicating method (SIM) developed for

impurities was established by using long-term stability samples,

aged samples degraded samples that mostly used for enrichment of

degradation products. (28) Some accepted clear steps for SIM

developing are generally available in literature (29-31). First step in

developing a SIM is what is the objective and intended use of our

method. Then we have to see HPLC or UV method which is

appropriate for our compound.

To select the method it is better to collect the already available

method information in the literature. During method development

other valuable information like functional groups present in our

drug, pKa value, pH, and solubility must be taken into consideration.

Even for new compound browsing the literature for similar

compounds which are used in development will give lot of

understanding (32-33) and literature about the drugs having common

side groups undergoes similar reactions like oxidation as given in one

publication(34).

For method development in chromatography not only the

column, mobile phase, diluent cannot be valuable foundation(35-37).

Selection of starting chromatographic conditions for the method

development for a new drug.

Finally the following chromatographic parameters were selected and

optimized during SIM method development:

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(1) .Diluents

(2) .Columns

(3) .pH of mobile phase

(4) . Mode of pump

(5) .Detector setting

Diluents we will select as one: one organic solvent and water as

it increases the solubility, disintegration of all dosage forms.

Mobile phase pH was selected more or less 1.5 units than pKa.

Mostly pump mode gradient for peak separation of early eluting

compounds. Since impurities present in the drug shows

different wavelength maxima (38) detection of wave length is

critical. So the wavelength maxima at which all the impurities

and main drug shows sufficient response that will be optimized

as detection wavelength for the final method.

1.17. DIFFERENT ANALYTICAL PROCEDURES TO BE VALIDATED:

The parameters which are validated for an analytical method are

listed in the table1.4 below

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1.18. CHALLENGES OF MODERN PHARMACEUTICAL ANALYSIS

The major challenge which exists for commercialization of a

product is its shelf life. In order to assign an acceptable shelf life,

emphasis is given to chemical stability, physical stability, Appearance,

microbial growth and photochemical degradation. The conclusions

regarding the shelf life of a drug substance or drug product depends

entirely on the quality of the analytical data generated. Hence it is

absolutely essential to develop and validate the correct stability

indicating analytical method. So development of SIM is challenging

task in pharmaceutical analysis.

In conclusion identification, isolation and characterization of

impurities in drug products is major requirement in pharmaceutical

industry.

1.19. Scope of present work:

In the present work drug products of different chemical classes

are selected for impurity study and development and aim of the work

given in table 1.5.

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