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CHAPTER-I General Introduction

Study of Impurities and Importance of Analytical Method

Development and Method Validation in pharmaceutical

Research and Development

Chapter-I General Introduction

General Introduction

1.1 Study of Impurities

1.1.1 Introduction

Drugs play a vital role in the progress of human civilization by curing diseases. The word

drug is derived from the French word drogue, which means a dry herb. In general, a drug may

be defined as a substance used in the prevention, diagnosis, treatment or cure of diseases in

man or other animals. According to world health organization (WHO), a drug may be defined

as any substance or product which is used or intended to be used for modifying or exploring

physiological systems or pathological states for the benefit (physical, mental as well as

economical) of the recipient.

The quality of a drug plays an important role in ensuring the safety and efficacy of the

drugs. Quality assurance and control of pharmaceutical and chemical formulations is essential

for ensuring the availability of safe and effective drug formulations to consumers. Hence

analysis of pure drug substances and their pharmaceutical dosage forms occupies a pivotal

role in assessing the suitability to use in patients. The quality of the analytical data depends

on the quality of the methods employed in generation of the data. Hence, development of

rugged and robust analytical methods is very important for statutory certification of drugs and

their formulations with the regulatory authorities.

The quality and safety of a drug is generally assured by monitoring and controlling

the assay and impurities effectively. While assay determines the potency of the drug and

impurities will determine the safety aspect of the drug. Thus, the analytical activities

concerning impurities in drugs are among the most important issues in modern

pharmaceutical analysis. Assay of pharmaceutical products plays an important role in

efficacy of the drug in patients. The impurity profile of pharmaceuticals is of increasing

importance as drug safety receives more and more attention from the public and from the

media.

Quality is the most important attribute of any pharmaceutical product. Over the last

few decades much attention is paid towards the quality of pharmaceuticals that enter the

market. The major challenge for both bulk drug industries and pharmaceutical industries is to

produce quality products. It is necessary to conduct vigorous quality control checks in order

to maintain the quality and purity of output from each industry. The bulk drug industry forms

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base of all pharmaceutical industries as it is the source of active pharmaceutical ingredients

(APIs) of specific quality. The quality of a pharmaceutical product was determined by the

content of active ingredient using appropriate techniques when advanced techniques were not

available. In the past half a century, analytical techniques have undergone a revolutionary

evolution which has enabled the detection of smaller and smaller quantities of any kind of

analyte. These advancements have also resulted in far reaching changes in the regulatory

aspects related to the development of pharmaceuticals.

In the changed perspectives, not only the content of active ingredient is essential but

also a complete detailing of the impurities present or likely to appear during the course of

usage has become mandatory. During the manufacturing process, whether by chemical

synthesis, extraction, cell culture/fermentation, recovery from natural sources, or any

combination of these processes, impurities may arise. Purity of active pharmaceutical

ingredient depends on several factors such as raw materials, their method of manufacture and

the type of crystallization and purification process. Concept about purity changes with time

and it is inseparable from the developments in analytical chemistry. The pharmacopoeias

specify not only purity but also puts limits which can be very stringent on levels of various

impurities.

Safety and efficacy of pharmaceuticals are two fundamental issues of importance in

drug therapy. The safety of a drug is determined by its pharmacological-toxicological profile

as well as the adverse effects caused by the impurities in bulk and dosage forms. The

impurities in drugs often possess unwanted pharmacological or toxicological effects by which

any benefit from their administration may be outweighed. Therefore, it is quite obvious that

the products intended for human consumption must be characterized as completely as

possible. The quality and safety of a drug is generally assured by monitoring and controlling

the impurities effectively. Thus, the analytical activities concerning impurities in drugs are

among the most important issues in modern pharmaceutical analysis. Control is more

important today than ever. Until the beginning of the 20th century, drug products were

produced and sold having no imposed control. Quality was generally not verified. Many

products were patent medicines of dubious value. Some were harmful and addictive. In 1937,

ethylene glycol was used as a vehicle for an elixir of sulfanilamide, which caused more than

100 deaths. Thereupon the food, drug and cosmetic act was revised requiring advance proof

of safety and various other controls for new drugs. The impurities to be considered for new

drugs are listed in regulatory documents of the FDA, ICH and United States Pharmacopoeia.

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1.1.2 Importance of impurity profile evaluation in pharmaceutical industry

Impurities are extraneous compounds that are not the drug substance (also known as active

pharmaceutical ingredient), but arise during the synthesis, extraction, purification, or storage

of the drug. Understanding the origin, control, and measurement of impurities is critical to the

production of high quality drug substances. In addition to guidance from the local authorities

of many countries, a series of guidelines developed in recent years by expert working group

of the international conference on harmonization of technical requirements for registration of

pharmaceuticals for human use have been increasingly accepted by the pharmaceutical

community. The expert working group ICH has defined an impurity as “any compound of the

medicinal product which is not the chemical entity defined as the active substance or as an

excipient in the product”. Similarly impurity profile has been defined as “a description of the

identified and unidentified impurities present in the medicinal product” [1]. Chiral drugs

constitute about 56% of the drugs currently in use and about 88% of these chiral synthetic

drugs are used therapeutically as racemates. The racemates can exhibit variable metabolic

pathways and pharmacologic activity and under some special circumstances, enantiomers as

well as polymorphs are also considered as impurities [2].

The identification of impurities and/or degradants in pharmaceuticals is critically

important for reasons of both product efficacy and patient safety. The impurities and /or

degradants may evoke any form of adverse response, either pharmacologic or toxicological in

patients undergoing medication. Modern separation methods clearly play a dominant role in

scientific research today because these methods simultaneously separate and quantify the

components. Hence making the separation and characterization of impurities easier [3]. To

ensure patient safety, impurity profiling which can be defined as a group of analytical

activities aimed at the detection, identification or structure elucidation and quantitative

determination of organic and inorganic impurities as well as residual solvents in bulk drugs

and pharmaceutical formulations is very essential.

Impurities in pharmaceuticals are unwanted chemicals that remain with the APIs or

develop during formulation or develop upon ageing of both APIs and formulated APIs to

medicines [4-7]. The presence of these unwanted chemicals even in small amounts may

influence the efficacy and safety of the pharmaceutical products. Different pharmacopoeias

such as british pharmacopoeia (BP) and the united states pharmacopoeia (USP) are

incorporating limits to allowable levels of impurities present in the APIs or formulations. The

ICH has published guidelines on impurities in new drug substances, products and residual

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solvents [8-10]. Impurity profile is description of the identified and unidentified impurities

present in a typical batch of API produced by a specific controlled production process [11].

The main reasons for the increasing interest of drug manufacturers and drug

registration authorities in the impurity profiles of bulk drug substances are as follows. (i) In

the course of the development of a new drug or a new technology for manufacturing an

existing drug it is essential to know the structures of the impurities: by possessing the

information that the synthetic organic chemists are often able to change the reaction

conditions in such a way that the formation of the impurity can be avoided or its quantity

reduced to an acceptable level. (ii) Having suggested structures for the impurities, they can be

synthesized and thus provide final evidence for their structures previously determined by

spectroscopic methods. (iii) The material synthesized can be used as an ‘impurity standard’

during development of a selective method for the quantitative determination of the impurity

and the use of this method as part of the quality control testing of every batch. (iv) In case of

major impurities the synthesized or isolated material can be subjected to toxicological studies

thus greatly contributing to the safety of drug therapy. (v) For drug authorities the impurity

profile of a drug substance is a good fingerprint to indicate the level and constancy of the

manufacturing process of the bulk drug substance.

Impurities present in excess of 0.1% should be identified and quantified by selective

methods. The suggested structures of the impurities can be synthesized and will provide the

final evidence for their structures, previously determined by spectroscopic methods.

Therefore it is essential to know the structure of these impurities in the bulk drug in order to

alter the reaction condition and to reduce the quantity of impurity to an acceptable level.

Isolation, identification and quantification of impurities help us in various ways, to obtain a

pure substance with less toxicity and, safety in drug therapy. Regulatory authorities such as

USFDA (United States Food and Drug Administration), cGMP (Current Good Manufacturing

Practices), TGA (Therapeutic Goods Administration), and MCA (Medicines Control Agency)

insist on the impurity profiling of drugs. In a chemical synthesis, the unwanted compounds

that are not removed during the synthetic or purification steps will become impurities. In a

similar fashion, the extraction, purification, and later synthetic steps for natural, fermentation,

or recombinant products may also give rise to such impurities. Biotechnological processes

may give rise to impurities such as media components and host cell proteins. For

biotechnology and classical fermentation products, the process begins with the cell bank,

master and working cell banks, as appropriate. The further source step is from the final end

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product, the less likely the presence of an impurity in the drug substance. Understanding the

source of the impurity will make it easier to devise a means of eliminating the impurity, thus

resulting in a drug substance of improved quality.

1.1.3 Classification of impurities

Impurities are classified into various types such as identified impurity (an impurity for which

a structural characterization has been achieved), unidentified impurity (an impurity for which

a structural characterization has not been achieved and that is defined solely by qualitative

analytical properties), specified impurity (an impurity that is individually listed and limited

with a specific acceptance criterion in the specification) and unspecified impurity (an

impurity that is limited by a general acceptance criterion but not individually listed with its

own specific acceptance criterion in the specification).

Organic impurities: Organic impurities may be unwanted by-products of a chemical

synthesis. These may arise by many different routes. These can arise during the

manufacturing process and/or storage of the new drug substance.

Inorganic impurities: Inorganic impurities include water, salts from buffers, reagents,

ligands, catalysts, heavy metals, or other residual metals, and inorganic compounds used in

processing, such as filter aids and charcoal. Inorganic impurities can also arise by leaching

from equipment as a result of the unit manufacturing process. These chemicals are less

commonly found in APIs. However, in some cases they may pose a problem as impurities.

Chemical reagents, ligands, and catalysts used in the synthesis of a drug substance can be

carried over to the final products as trace level impurities. Many chemical reactions are

promoted by metal based catalysts. Some of the catalysts used for reduction reactions,

oxidation and de-protection of functional groups those reagents and catalysts are Pd/C

(palladium/carbon), zinc dust and nickel etc.

Residual solvents: Residual solvents are considered a subset of organic impurities. Residual

solvents in pharmaceuticals are defined here as organic volatile chemicals that are used or

produced in the manufacture of drug substances or excipients, or in the preparation of drug

products. Solvents used to create a solution or suspension during the manufacturing process

may not be completely eliminated in the course of manufacture. Appropriate selection of the

solvent for the synthesis of drug substance may enhance the yield, or determine

characteristics such as crystal form, purity, and solubility. Therefore, the solvent may

sometimes be a critical parameter in the synthetic process.

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Contamination impurities: Contamination impurities are unexpected adulterating

compounds found in the drug substance. Current manufacturing technology has reduced

many of the contaminant impurities observed in drugs prepared decades ago.

1.1.4 Formation of impurities

Process impurities arise during the manufacturing of the drug substance, degradation

impurities arise during the storage of the drug substance and contaminant impurities are not

drug related but are inadvertently introduced during processing or storage, and are not part of

the synthesis, extraction, or fermentation process. Impurities that cause the greatest concern

are those that are toxic, defined by the USP as impurities that have significant undesirable

biological activity [12] and host cell contaminants in biopharmaceuticals that have potential

risks of allergic reaction or other immune pathological effects [13].

Impurities in the starting materials and intermediates: Possibility of impurities in the

starting materials may follow the same reaction pathways as the starting material itself, and

the reaction products could carry over to the final product as process impurities.

Understanding of the impurities in starting materials helps to identify related impurities in the

final product, and to understand the formation mechanisms of related process impurities.

Starting materials or intermediates are the most common impurities found in every API

unless a proper care is taken in every step involved in throughout the multi-step synthesis.

Although the end products are always washed with solvents, there are chances of remaining

residual unreacted starting materials unless the manufacturers are very careful about the

impurities.

Stereochemistry-related impurities: It is of paramount importance to look for stereo

chemistry related compounds, that is, those compounds that have similar chemical structure

but different spatial orientation, these compounds can be considered as impurities in the

API’s. Chiral molecules are frequently called enantiomers. Since the discovery of difference

between thalidomide enantiomers in pharmacological and toxicological actions,

discrimination of optical isomers has been one of the major subjects in the field of pharmacy,

because optical purities of substrates with asymmetries are critical for the evaluation of their

biological activities [14]. Enantiomers of racemic drugs often show different behaviors in

pharmacological action and metabolic process. Often one enantiomer is active and other can

be non-active, poorly active or toxic. The pharmaceutical industry has raised its emphasis on

the generation of enantiomerically pure compounds before under taking pharma kinetic,

metabolic, physiological and toxicological evaluation in the search for drugs with greater

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therapeutic benefits and low toxicity [15,16]. In recent years, stringent regulations for

marketing for marketing enantiomeric drugs have been implemented by the regulatory

agencies of all the major countries (pharmaceutical consumers) of the world [17-19].

Formation of impurities from product side chains: Some of the intermediates and API’s

undergo reaction with the side chains in presence of some of the solvents like methanol,

acetone and other organic solvents resulting in the formation of impurities in final

compounds.

Impurities originated from reaction solvents: Some solvents which are the part of the

reaction act as a source of impurities.

Impurities originating from stability studies and degradants: Impurities can also be

formed by storage conditions of the final product during stability of API’s. However, stability

impurities may impact on the quality of API’s. The general stability conditions are

accelerated, intermediate and cold conditions as per ICH guidelines. These stability

conditions may vary from drug to drug.

Impurities originating from dimerization: Many compounds containing thiols (disulfide

formation) olefins, alcohols and carboxylic acids undergo dimerization reactions. Phenols

dimerize under free radical initiated oxidative, usually to ortho phenols.

Formation of impurities from degradation of the API’s: Normally, degradants are

chemical breakdown compounds of the drug substance formed during storage. The definition

of degradation of the end product in the ICH guideline is a molecule resulting from a

chemical change in the substance brought about by overcome or the effect of light,

temperature, acid, base and peroxide. The goal of the stability indicating method is to obtain

baseline resolution of all the resulting products (the API and all the degradation products)

with no co-elutions [20,21]. The degradation products generated in the stressed samples are

termed as “potential” degradation products that may or may not be formed under relevant

storage conditions. The major forced degradation studies are acid, base, oxidative, photolytic

and thermal degradation.

Acid/Base degradation: Impurities can also be formed by stress degradation conditions of

the final product during specificity study of API’s. The general acid/base conditions are 0.1 to

2N hydrochloric acid (HCl), 0.1 to 1N sodium hydroxide (NaOH) at RT or 60-70°C up to 1

to 3 days, these conditions may vary case to case based on formation of % of degradation.

Oxidative degradation: In oxidative (peroxide) stress degradation conditions, impurities can

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also formed in addition to the final product during specificity study of API’s. The general

peroxide conditions are dilute hydrogen peroxide (0.3 to 6.0 %) at room temperature or 60-

70°C up to 1 to 3 days, these conditions may vary case to case based on formation % of

degradation.

Thermal degradation: The general thermal conditions in thermal degradation are at 60-70°C

up to 7 to 10 days, these conditions may vary case to case based on formation % of

degradation and melting point of the respective API. Impurities can also be formed by

thermal stress degradation of the pure drug.

Photolytic degradation: Impurities can also be formed by photo stress degradation condition

of the final product during specificity study of API’s. The general thermal conditions are uv

(ultra-violet) at 254 nm up to 7 to 10 days, these conditions may vary case to case based on

formation % of degradation.

1.1.5 Control of impurities

In theory, all impurities should be eliminated. In practice, it is generally not economically

feasible to totally eliminate all impurities. In most cases, only low level of impurities should

be allowed, but in rare cases, even quite high levels of impurities are tolerated.

Control of drug substance impurities: In some cases, for example, biotechnology derived

products such as macrocyclic antibiotics or extracts of a botanical source such as some

dietary supplements, the drug substance or active component contains multiple compounds,

all of which have biologically activity.

Control of residual solvents: Since there is no therapeutic benefit from residual solvents, all

residual solvents should be removed to the extent possible to meet product specifications,

good manufacturing practices, or other quality-based requirements. Drug products should

contain no higher levels of residual solvents than can be supported by safety data. Various

techniques are used to reduce the amounts of these solvents left in the drug substance, such as

washing with water to remove water soluble solvents, distillation, or drying under reduced

pressure. Modification of the reaction scheme to use more easily removed solvents may also

be a valid approach.

Control of synthetic impurities: Generally, the reaction conditions are adjusted to reduce

the amounts of by-products produced during each step of the reaction. The reaction

conditions are tightly controlled to prevent varying levels of impurities, or even new

impurities, from arising. High quality starting materials may also lead to lower amounts of

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impurities in the final product when starting material impurities are carried through to drug

substance impurities. Similarly, the use of high-quality reagents may help to avoid the

generation of unwanted by-products. Other options to reduce these impurities are the

introduction of additional intermediate or final purification steps.

1.1.6 Polymorphs

The physicochemical properties of active pharmaceutical ingredients are key factors to the

development of appropriate dosage forms. Most organic substances exist in solid state as

polymorphs, pseudo-polymorphs (solvates) or amorphous forms. Since all physico-chemical

properties in solid state are affected mainly in terms of solubility, dissolution, bioavailability,

processability and stability, it is mandatory [22] to investigate the polymorphic behaviour of

active ingredients. Polymorphism is the ability of a substance to exist in more than one

crystalline form. The different crystalline forms are known as polymorphs. Polymorphism is

a consequence of different packing arrangements and/or conformation of the molecules

within the crystal lattice [23-26]. All physicochemical characteristics of the solid state are

involved in the polymorphism and pseudo-polymorphism. The main difference in structural

arrangements leads to different physical properties such as melting point, sublimation

temperatures, heat capacity, conductivity, volume, density, viscosity, crystal hardness, crystal

shape, colour, refractive index, processability, solubilities, dissolution rate, stability,

hygroscopicity, optical, electrical and solid state reactions. In the solid state, the atoms,

molecules or ions may be arranged in one of the fundamental crystal systems: triclinic,

monoclinic, orthorhombic, tetragonal, trigonal, hexagonal or cubic. Polymorphs show the

same properties in the liquid or gaseous state but they behave differently in the solid state.

Amorphous solids are not crystalline because the arrangement of the molecules is disordered.

The name glassy state is given to amorphous products which change from glassy state to

rubber state by undergoing a glass transition [27].

Crystalline modifications of a substance formed by the incorporation of either

stoichiometric or non-stoichiometric amounts of a solvent incorporated within the crystal

structure, known as solvates, are considered as pseudopolymorphs. The effect of

polymorphism on bioavailability or toxicity is the most important consequence for drug

substances if the bioavailability is mediated via dissolution. For the most famous case of

chloroamphenicol palmitate [28], the active polymorph is not the thermodynamically stable

one. The polymorphism of the excipients may also play an important role in bioavailability

[29]. Chemical reactivity in the solid state is correlated with the nature of the crystalline

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modifications. Walkling [30] found that the two crystalline modifications of fenretinide

behave quite differently. After 4 weeks at 25°C, the stable form showed no detectable

degradation, whereas the unstable form showed 8% degradation. In a recent example, the

hydrolysis of an investigational compound led to a toxic degradation product for which one

solid form was much less stable than the other [31]. Typically obtained during lyophilisation,

spray-drying, granulation, grinding or milling, the amorphous form is responsible for the

higher reactivity of some batches. It gives rise to major problems with activity and stability.

Amorphous forms generally tend to crystallize in the presence of moisture (e.g.

indomethacine, lactose [32]). Since processing and storage imply changes of temperature,

pressure and humidity, polymorphic transitions are undesirable phenomena. Different

polymorphic forms may be categorized as impurities in the drug substance. Different

polymorphs may be generated by changes in the manufacturing process, or be seeding effects

if a new polymorph arises. In some cases, the presence of different polymorphic forms in

various batches of drug substance is not important, for example, if the drug product is a

solution and all polymorphs are highly soluble, but in other cases, the polymorphic form of

the drug substance is critical to the performance of the drug product. For example, the

original formulation of the protease inhibitor ritonavir (Norvir) was that of a capsule

containing a hydroalcoholic solution of ritonavir [33]. The appearance of a new and

dramatically less soluble polymorph of ritonavir made it impossible to manufacture this

product and necessitated a change in the formulation.

Pharmaceutical compounds have often a great number of solid phases, even in

metastable state and interpretations are difficult because of kinetic factors. Therefore several

techniques are currently used for the study of polymorphism and pseudopolymorphism.

Thermoanalytical techniques combining differential scanning calorimetry, microcalorimetry

and thermogravimetry with microscopy, spectroscopy, X-ray diffraction or mass

spectrometry are state of the art techniques.

1.2 Analytical Techniques

Organic impurity levels can be measured by a variety of techniques, including those that

compare an analytical response for an impurity to that of an appropriate reference standard or

to the response of the new drug substance itself. In cases where the response factors of the

drug substance and the relevant impurity are not close, this practice can still be appropriate,

provided a correction factor is applied or the impurities are, in fact, being over estimated.

Currently organic impurities are almost exclusively measuring by using of chromatographic

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procedures. Chromatographic procedures should involve a separation mode that allows for

the resolution of impurities from the drug substance and a detection mode that allows for the

accurate measurement of impurities.

In general, non-volatile impurities are analyzed by HPLC techniques, among which

reversed phase HPLC (RPLC), normal phase (NPLC), LC-MS, GC and GC-MS are the most

widely used separation modes [34,35]. Hydrophilic interaction liquid chromatography

(HILIC) seems complementary to RPLC for the retention and separation of small molecule

polar analytes, and has thus gained increasing attention recently. Good retention can be

achieved for more polar analytes, which is not possible on RPLC columns. In the hydrazine

group, the HILIC method was used in addition to the HPLC-UV and LC-MS methods [36].

However, if the analytes are ionized, ion chromatography (IC) may provide better retention

and separation. IC is based on ion-ion interactions between charged analytes and oppositely

charged groups embedded in the stationary phase. GC methods are commonly used for the

analysis of several volatile small molecules. Some examples include the liquid injection

technique and the headspace sampling technique. Liquid injection is prone to contamination

in which injection of a large amount of non-volatile API can accumulate in the injector liner

or on the head of the GC column, which can cause a sudden deterioration in method

performance. Headspace injection, on the other hand, is desirable because it minimizes

potential contamination of the injector or column by avoiding the introduction of a large

quantity of API. Jouyban and Kenndler (2008) [37] reviewed the applicability of capillary

electrophoresis (CE) methods for the analysis of pharmaceutical impurities. FT-IR (fourier

transform infrared spectroscopy), NIR (near-infrared spectroscopy), raman spectroscopy,

DSC (differential scanning calorimetry), TGA (thermogravimetry) and P-XRD (powder X-

ray diffraction) techniques are used for identification and quantification of crystalline phases

and polymorphic impurities.

1.2.1 High-performance liquid chromatography (HPLC)

Introduction:

High-performance liquid chromatography technique is a widely accepted separation

technique for both sample analysis and purification in a variety of areas including the

pharmaceutical, biotechnological, environmental, polymer, and food industries. HPLC was

used for the separation of a wide variety of compounds such as organic, inorganic, biological

compounds, chiral compounds, and analysis of impurities, isolation and purification of

compounds, separation of closely related compounds, ultra trace to preparative and process

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scale separations. The successful use of liquid chromatography for a given problem requires

the right combination of a variety of operating conditions such as the type of column packing

and mobile phase, column length and diameter, mobile phase flow rate, column temperature,

and sample size. The most extensively used technique in pharmaceutical applications for

impurity evaluation is high-performance liquid chromatography (HPLC) among different

types of chromatography techniques [38,39]. A few RP-HPLC methods [40-47] were

developed and validated for assay and impurities of some drugs in dosage forms. Some

stability-indicating HPLC methods [48-51] were also reported in the literature for the study

of degradation under the stressed conditions. Some RP-HPLC methods have been developed

[52-60] for the simultaneous estimation of drugs in alone and combination with other drugs.

Principle:

In HPLC, separations are achieved by partition, adsorption or ion-exchange, depending on

the nature of interactions between the solute and the stationary phase, which may arise from

hydrogen bonding, vanderwall’s forces, electrostatic forces, hydrophobic forces (or) based on

the size of particles (e.g. size exclusion chromatography). The principle of separation in

normal phase and reverse phase HPLC is adsorption. When a mixture of components is

introduced in to a HPLC column, they travel according to their relative affinities towards the

stationary phase. The component which has more affinity towards the adsorbent travels

slower. The component which has less affinity towards the stationary phase travels faster.

Since, no two components have the same affinity towards the stationary phase, the

components are separated.

Instrumentation:

HPLC instrumentation (Fig.1.1) is made up of eight basic components: mobile phase

reservoir, solvent delivery system, sample introduction device, column, detector, waste

reservoir, connective tubing, and a computer, integrator, or recorder.

Mobile phase reservoir:

The mobile phase reservoir can be any clean, inert container such as an empty solvent bottle,

laboratory flask. This should be with small covered opening. This prevents or lessens

evaporation. It usually contains 1-2 litre of solvent, and it should have a cap that allows the

tubing inlet line to pass through. It is important to degas solvents before use, because small

gas bubbles present in the mobile phase can create a problem in other components,

particularly in the pump heads and the detector, and the analysis.

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Fig.1.1: Schematic diagram of an HPLC instrument

Solvent delivery system: The purpose of the pump, or solvent delivery system, is to ensure

the delivery of a precise, reproducible, constant, and pulse-free flow of mobile phase. There

are two classes of HPLC pumps: constant pressure and constant flow pumps, late one is most

common. The most common type of HPLC constant flow pump is the reciprocating piston

pump, in which a piston is driven in and out of a solvent chamber by an eccentric cam or

gear. On the forward stroke, the inlet check valve closes, the outlet check valve opens, and

the mobile phase is pumped to the column. On the reverse stroke, the check valves reverse

and solvent is drawn into the chamber.

Sample introduction devices: A variety of sample introduction devices exist, manual and

automatic that used primarily a valve mechanism. When the valve is in the load position, the

sample loop is filled. For best results, a two to five fold excess of sample should be passed

through the loop to ensure that the previous sample has been thoroughly purged.

Column: The column is heart of the HPLC instrument because the separation occurs here. It

is generally made of 316-grade stainless steel, which is relatively inert to chemical corrosion

and is packaged with the desired stationary phase. Common dimensions for analytical scale

columns are in the range of 10 to 25 cm long and 3 to 9 mm inner diameter.

Detectors: The important role of the HPLC detector is to monitor the solutes as they are

eluted from the column. The detector generates an electrical signal that is proportional to the

level of some property of the mobile phase or solutes. Some characteristics of a good HPLC

detector are sensitivity, linearity, predictability in response, reliability, non-destructiveness,

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ease of use, and low dead volume. Over 70% of all of the HPLC detectors are UV absorbance

detectors. The mobile phase is passed through a small flow cell, where the radiation beam of

a UV/visible photometer or spectrophotometer is located. As a UV-absorbing solute passes

through the flow cell, a signal is generated that is proportional to the solute concentration.

Only UV-absorbing compounds, such as alkenes, aromatics and compounds that have multiple

bonds between C and O, N, or S are detected. The mobile phase components should be

selected carefully so that they absorb little or no radiation. These types of absorbance

detectors are available: fixed-wavelength, variable-wavelength and photodiode array. A fixed

wavelength detector uses a light source that emits maximum light intensity at one or several

discrete wavelengths that are isolated by appropriate filters. A variable-wavelength detector

uses a relatively wide band-pass UV/visible spectrophotometer. It offers an increased number

of UV and visible wavelength, but is more expensive than the fixed wavelength detector. In

order to generate real-time spectra for each solute as it is eluted, a photodiode array is used.

Computer, integrator and recorder: A data collection device such as a computer,

integrator, and recorder is connected to the detector. It takes the electronic signal produced by

the detector and plots it as a chromatogram, which can be evaluated by the user. Recorders

are rarely used because they are unable to integrate the data. Both integrators and computers

can integrate the peaks in the chromatograms, and computers have the further advantage that

they electronically save chromatograms for later evaluation.

1.2.2 Ultra performance liquid chromatography (UPLC)

Introduction: Ultra performance liquid chromatography is a novel technique used in the

analysis of pharmaceutical formulations. HPLC technology doesn’t have the capability to

take full advantages of sub-2 µm particles. Therefore ultra-performance liquid

chromatography (UPLC) regarded as new invention for liquid chromatography which is

applied at high pressure (8,000 to 15, 000 psi). It brings dramatic improvements in sensitivity,

resolution and speed of analysis. It has instrumentation that operates at high pressure than

that used in HPLC & in this system uses fine particles (less than 2.5 µm) & mobile phases at

high linear velocities, decreases the length of column, reduces solvent consumption and save

the time. According to the van deemter equation, as the particle size decreases to less than 2.5

µm, there is a significant gain in efficiency, while the efficiency does not diminish at

increased flow rates or linear velocities. Ultra performance liquid chromatography methods

have been developed for the estimation of drugs in pure and dosage forms and their related

impurities [61-66]. A number of stability indicating UPLC methods [67-71] were reported in

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the literature for the simultaneous determination of drugs and assay in pharmaceutical

formulations.

Principle: The UPLC is based on the principal of use of stationary phase consisting of

particles less than 2.5 μm. The underlying principles of this evolution are governed by the

van deemter equation, which is an empirical formula that describes the relationship between

linear velocity (flow rate) and plate height; H=A+B/v+Cv; Where A, B and C are constant, v

is the linear velocity. The advent of UPLC has demanded the development of a new

instrumental system for liquid chromatography, which can take advantage of the separation

performance (by reducing dead volumes) and consistent with the pressures (about 8000 to

15,000 psi, compared with 2500 to 5000 psi in HPLC). Efficiency is proportional to column

length and inversely proportional to the particle size. In second generation bridged ethane

hybrid (BEH) technology 1.7 µm particles derive their enhanced mechanical stability by

bridging the methyl groups in the silica matrix.

Instrumentation: Instrumental (Fig.1.2) requirements are pumps-solvent delivery system,

mixing unit, gradient controller and solvent degassing, injector-manual or auto injectors,

guard column, analytical columns, detectors and recorders and integrators.

Fig.1.2: Schematic representation of UPLC instrumentation

High pressure pump: Pump is the main part of the solvent delivering system. The mobile

phase must be pumped through the column at a high pressure about 8000 to 15000 psi. There

are different types of pumps available like mechanical pumps and pneumatic pumps.

Mechanical pumps operate with constant flow rate and use a sapphire piston. Pneumatic

pumps operate with constant pressure and use highly compressed gas.

15


Analytical column: Analytical column is the most important part of the UPLC technique

which decides the efficiency of separation. There are several stationary phases available

depending up on the technique or mode of separation used. Column materials are made up of

stainless steel, glass, polyethylene and PEEK (poly ether ether ketone). Most widely used are

stainless steel which can with stand high pressure. Column length, diameter and practical size

vary from 25 to 150 cm, 1.0-4.6 mm and 1.5-2.5 µm respectively. The functional group

present in the stationary phase depends on the type of chromatographic separation. In normal

phase mode it contains silanol groups (hydroxyl group). In reverse phase columns it contains

the following groups: BEH C18, BEH C8, BEH Phenyl, and BEH Shield RP-18.

Detectors: Detectors used depend up on the property of compounds to be separated. The

most commonly used detector in the analysis is UV & PDA detector, later which result a

spectra in 3D (3-Dimension) plot of response vs time vs wavelength. The advantage is that

wavelengths need not to be selected, as the detector detects the responses of all the

compounds.

Recorders and integrators: Recorders are used to record the responses obtained from

detectors after amplification, if necessary. They record the baseline and all the peaks

obtained, with respect to time. Retention time of all peaks can be found out from such

recordings, but the area of individual peaks cannot be known. Integrators are improved

version of recorders with some data processing capabilities. They can record the individual

peaks with retention time, height and width of peaks, peak area and percentage of area etc.

integrators provide more information on peaks than recorders. Now a day’s computers and

printers are used for recording and processing the obtained data and for controlling several

operations.

1.2.3 Liquid chromatography-mass spectrometry (LC-MS)

LC-MS method has been applied to the identification of impurities and degradants in API and

drug product. LC-MS method was utilized for the determination of impurities. These methods

gave enhanced sensitivity compared with the standard LC-UV or stand alone MS methods. In

pharmaceutical industry LC-MS has become method of choice in many stages of drug

development. Recent advances in electro spray, thermo spray, and ion spray ionization

techniques offer unique advantages of high detection sensitivity and specificity.

Principle: Principle of LC-MS is based on the fragmentation of charged ions and detection of

the resulting fragments. There are two common atmospheric pressure ionization (API) LC-

16


MS processes: Electron spray ionization (ESI) and atmospheric pressure chemical ionization

(APCI). Both of these processes are compatible with most chromatographic separations. In

LC-MS analysis, a sample is injected into an LC column after introduction of the sample

from HPLC, the sample is ionized by the ion source. Types of sources include electron

impact ionization (EI), chemical ionization (CI) and atmospheric pressure ionization (API).

In this technique API is mostly used. The vacuum interface ensures the transition of ions

from the API source to the mass analyser, which is kept under a vacuum. From the ion

source, ions are transferred to mass analyzer where there are separated according to their

mass-to-charge (m/z) values. The mass analyser operates under a vacuum to ensure that ions

travel with maximum efficiency. The detector measure the abundance of electrons generated

from the ions for each m/z ratio. Most MS systems use some type of electron multiplier.

Normally pulse counting detector is used.

Instrumentation: LC-MS instrument (Fig.1.3) consist of three main components namely LC

(to resolve a complex mixture of components), an interface (to transport the analyte into the

ion source of a mass spectrometer) and mass spectrometer (to ionize and mass analyze the

individually resolved components) and a detector, which measures the value of an indicator

quantity and thus provides data for calculating the abundances of each ion present.

Fig.1.3: Schematic representation of LC-MS instrument

17


Interface: There are many types of interfaces available such as electro spray ionization

(ESI)-positive & negative electro spray ionization (Fig.1.4), atmospheric pressure chemical

ionization and atmospheric pressure photo ionization (APPI). APCI sources ionize the

samples at atmospheric pressure and then transfer the ions into mass spectrometer. These

techniques are used to ionize thermally labile samples such as peptides, proteins and

polymers directly from the condensed phase. The sample is dissolved in an appropriate

solvent and this solution is introduced into the mass spectrometer, APCI source introduce the

sample through a series of differentially pumped stages. This maintains the large pressure

difference between the ion source and mass spectrometer. In addition a drying gas is used to

break up the clusters that form as the solvent evaporate. Because the analyte molecules have

more momentum that the solvent and air molecules, they travel through the pumping stages

to mass analyzer.

Fig.1.4: Schematic diagram of a direct-EI, LC-MS interface

Mass analyzer: In a mass spectrometer, the ions are separated according to mass charge ratio

(m/z) using a mass analyzer. There are many different mass analyzers that can be used in LC-

MS. Commonly used mass analyzers are quadrupole type (single quadrupole, triple

quadrupole, quadrupole-time of flight), time of flight type (TOF type) and ion trap type. The

quadrupole analyzer is the most widely used analyzer due to its ease of use, mass range

covered, good linearity for quantitative work, resolution and quality of mass spectra all these

for a relatively accessible price. The quadrupole is composed of two pairs of metallic rods.

One set of rod is at a positive electrical potential, and the other one at a negative potential. A

18


combination of DC (direct current) and RF (radio frequency) voltages is applied on each set.

The positive pair of rods will act as a high mass filter, the other pair is acting as a low mass

filter. The resolution depends on the dc value in relation to the RF value. The quads are

operated at constant resolution, which means that the RF/DC ratio is maintained constant.

Detector: A series of dynodes maintained at ever-increasing potentials. Ions strike the

dynode surface, resulting in the emission of electrons. These secondary electrons are then

attracted to the next dynode where more secondary electrons are generated. Ultimately

resulting in a cascade of electrons. The mass spectrometer (MS) is very important HPLC

detector because of its ability to generate structural and molecular weight information about

the eluted solutes. The combination of HPLC and mass spectrometry allows for both

separation and identification in the same step, an advantage none of the other detectors

provide.

1.2.4 Chiral chromatography

Chiral column chromatography is a variant of column chromatography in which the

stationary phase contains a single enantiomer of a chiral compound rather than being achiral.

The two enantiomers of the same analyte compound differ in affinity to the single-enantiomer

stationary phase and therefore they exit the column at different times. The chiral stationary

phase can be prepared by attaching a suitable chiral compound to the surface of an achiral

support such as silica gel, which creates a chiral stationary phase (CSP). Many common

chiral stationary phases are based on oligosaccharides such as cellulose or cyclodextrin (in

particular with β-cyclodextrin, a seven sugar ring molecule). As with all chromatographic

methods, various stationary phases are particularly suited to specific types of analytes. Chiral

stationary phases are much more expensive than comparable achiral stationary phases such

as C18.

Chiral analysis and purification has become increasingly important in research and

development and supercritical fluid chromatography (SFC) technology has unmatched chiral

performance. Nearly 80% of new API's are chiral active compounds. CE (capillary

electrophoresis) and SFC technology, which offers superior speed, resolution and specificity,

is up to five times faster than standard HPLC methods. It allows high purity enantiomeric

material to be analyzed or collected rapidly, plus it increases throughput, reduces run times,

improves analysis and provides higher yield and purity in preparative situations. In addition

the amount of solvent use is dramatically reduced.

19


1.2.5 Powder X-ray diffraction (P-XRD)

Introduction:

X-ray diffraction (XRD) is an analytical technique looking at X-ray scattering from

crystalline materials. Each material produces a unique X-ray "fingerprint" of X-ray intensity

versus scattering angle that is characteristic of it's crystalline atomic structure. Qualitative

analysis is possible by comparing the XRD pattern of an unknown material to a library of

known patterns. About 95% of all solid materials can be described as crystalline. When

X-rays interact with a crystalline substance (phase), one gets a diffraction pattern. The X-ray

diffraction pattern of a pure substance is, therefore, like a fingerprint of the substance. The

powder diffraction method is thus ideally suited for characterization and identification of

polycrystalline phases. Today about 50,000 inorganic and 25,000 organic single component,

crystalline phases, diffraction patterns have been collected and stored on magnetic or optical

media as standards. The main use of powder diffraction is to identify components in a sample

by a search/match procedure. Furthermore, the areas under the peak are related to the amount

of each phase present in the sample. Solid matter can be described as:

Amorphous: The atoms are arranged in a random way similar to the disorder we find in a

liquid. Glasses are amorphous materials.

Crystalline: The atoms are arranged in a regular pattern, and there is as smallest volume

element that by repetition in three dimensions describes the crystal. E.g. we can describe a

brick wall by the shape and orientation of a single brick. This smallest volume element is

called a unit cell. The dimensions of the unit cell are described by three axes: a, b, c and the

angles between them alpha, beta, gama. About 95% of all solids can be described as

crystalline.

Powder X-ray diffraction is the analysis of a powder sample. The typical output is a plot of

intensity versus the diffraction angle (2θ). Such a plot can be considered a fingerprint of the

crystal structure, and is useful for determination of crystallographic sameness of samples by

pattern comparison. A crystalline material will exhibit peaks indicative of reflections from

specific atomic planes. The patterns are representative of the structure, but do not give

positional information about the atoms in the molecule. One peak will be exhibited for all

repeating planes with the same spacing. Large particles or certain particle morphologies, such

as needles or plates, can result in preferred orientation. Preferred orientation is the tendency

of crystals to pack against each other with some degree of order and it can affect relative peak

20


intensities, but not peak positions, in P-XRD patterns. If a powder is packed into a P-XRD

sample holder and the surface is smoothed with a microscope slide or similar device, crystals

at the surface can become aligned so that a non statistical arrangement of crystal faces is

presented to the X-ray beam. The result is that some reflections are artificially intensified and

others are artificially weakened. One way to determine if preferred orientation is causing

relative peak intensity changes is to grind and reanalyze samples. Qualitative analysis of

powder patterns can be used to determine if multiple samples are the same crystal form or if

multiple crystal forms have been produced. Mixtures of samples can also be evaluated. When

mixtures are obtained, P-XRD, DSC, FT-IR, Raman spectroscopy can also be used in a

quantitative mode to calculate the amount of each phase present [72-88].

Principle:

An electron in an alternating electromagnetic field will oscillate with the same frequency as

the field. When an x-ray beam hits an atom, the electrons around the atom start to oscillate

with the same frequency as the incoming beam. In almost all directions we will have

destructive interference, that is, the combining waves are out of phase and there is no

resultant energy leaving the solid sample. However the atoms in a crystal are arranged in a

regular pattern, and in a very few directions we will have constructive interference. The

waves will be in phase and there will be well defined x-ray beams leaving the sample at

various directions. Hence, a diffracted beam may be described as a beam composed of a large

number of scattered rays mutually reinforcing one another.

Fig.1.5: Schematic diagram of X-Ray diffraction

21


This model is complex to handle mathematically, and in day to day work we talk about x-ray

reflections from a series of parallel planes inside the crystal. The orientation and inter planar

spacing of these planes are defined by the three integers h, k, l called indices. A given set of

planes with indices h, k, l cut the a-axis of the unit cell in h sections, the b axis in k sections

and the c axis in l sections. A zero indicates that the planes are parallel to the corresponding

axis. The schematic powder x-ray diffraction is represented in Fig.1.5. The x-ray is focused

on the sample at angle θ, while the detector opposite the source reads the intensity of the X-

ray it receives at 2θ away from the source path. The incident angle is than increased over time

while the detector angle always remains 2θ above the source path. Diffractometers can

typically be operated in either reflection or transmission configurations. Reflection is by far

the more common and also referred to as Bragg-Brentano geometry (Fig.1.6). In reflection

measurements, incoming x-rays are “reflected” off the sample surface and focused by the

instrument optics onto the detector. X-ray diffraction techniques used for characterizing

pharmaceutical solids include the analysis of single crystals and powders. The two parallel

incident rays 1 and 2 make an angle (THETA) with these planes. A reflected beam of

maximum intensity will result if the waves represented by 1’ and 2’ are in phase. The

difference in path length between 1 to 1’and 2 to 2’ must then be an integral number of

wavelengths, (λ). We can express this relationship mathematically in Bragg’s law.

2d sin θ=n λ

(n=1, 2,3,. . .), where, λ=X-ray wavelength, d=spacing between the diffracting planes,

θ=diffraction angle.

The process of reflection is described here in terms of incident and reflected (or diffracted)

rays, each making an angle THETA with a fixed crystal plane. Reflections occurs from

planes set at angle THETA with respect to the incident beam and generates a reflected beam

at an angle 2-THETA from the incident beam. The possible d-spacing defined by the indices

h, k, l are determined by the shape of the unit cell.

Rewriting Bragg’s law we get: sin θ=λ/2d

Therefore the possible 2-THETA values where we can have reflections are determined by the

unit cell dimensions. However, the intensities of the reflections are determined by the

distribution of the electrons in the unit cell. The highest electron densities are found around

atoms. Therefore, the intensities depend on what kind of atoms we have and where in the unit

cell they are located. Planes going through areas with high electron density will reflect

strongly, planes with low electron density will give weak intensities.

22


Instrumentation: A schematic of the powder x-ray diffractometer given in (Fig.1.7).

Laboratory x-ray diffractometer typically consists of an x-ray source (x-ray tube), sample

stage and detector. X-rays will be diffracted at an angle defined as θ, knowing the diffraction

angle and the x-ray wavelength, the spacing between the planes can be calculated.

Fig.1.6: Bragg-Brentano geometry of powder x-ray diffractometer

Fig.1.7: Schematic diagram of powder x-ray diffractometer

23


X-ray Tubes: The traditional method of producing x-rays in a crystallographic laboratory is

by means of an x-ray tube. The most commonly used x-ray source in laboratory experiments

with organics is Cu (copper), though instruments typically allow different sources to be used

with some configuration. The tube is evacuated and contains a copper block with a metal

target anode, and a tungsten filament cathode with a high voltage between them. The filament

is heated by a separate circuit, and the large potential difference between the cathode and

anode fires electrons at the metal target. The accelerated electrons knock core electrons out of

the metal, and electrons in the outer orbitals drop down to fill the vacancies, emitting x-rays.

The x-rays exit the tube through a beryllium window. Due to massive amounts of heat being

produced in this process, the copper block must usually be water cooled. Slits and optics are

used to focus the incident and diffracted radiation on the sample and detector, respectively.

Specimens usually can be rotated to alleviate some of the intensity artifacts.

X-ray detectors: While older machines used film as a detector, most modern equipment uses

transducers that produce an electrical signal when exposed to radiation. These detectors are

often used as photon counters, so intensities are determined by the number of counts in a

certain amount of time. Detectors can be point, linear or area, with the latter offering

advantages in both speed of acquisition and ability to assess the particle statistics and

preferred orientation in a sample through examination of debye rings.

Gas-filled transducers: A gas-filled transducer consists of a metal chamber filled with an

inert gas, with the walls of the chamber as a cathode and a long anode in the center of the

chamber. As an x-ray enters the chamber, its energy ionizes many molecules of the gas. The

free electrons then migrate towards the anode and the cations towards the cathode, with some

recombining before they reach the electrodes. The electrons that reach the anode cause

current to flow, which can be detected. The sensitivity and dead time (when the transducer

will not respond to radiation) both depend on the voltage the transducer is operated at. At

high voltage, the transducer will be very sensitive but have a long dead time, and at low

voltage the transducer will have a short dead time but low sensitivity.

Scintillation counters: In a scintillation counter, a phosphor or NaI (Sodium iodide) is

placed in front of a photomultiplier tube. When x-rays strike the phosphor, it produces flashes

of light, which are detected by the photomultiplier tube.

Semiconductor transducers: A semiconductor transducer has a gold coated p-type

semiconductor layered on lithium containing semiconductor intrinsic zone, followed by an n-

type semiconductor on the other side of the intrinsic zone. The semiconductor is usually

24


composed of silicon. Germanium is used if the radiation wavelength is very short. The entire

crystal has a voltage applied across it. When an x-ray strikes the crystal, it elevates many

electrons in the semiconductor into the conduction band, which causes a pulse of current.

Transmission mode analysis overcomes many of the limitations of reflection mode when

carefully configured. In transmission measurements, the incident x-rays pass through the

sample and are diffracted not only on the surface but throughout the sample. This type of

analysis is possible for organics due to their relative transparency to x-rays. The sample no

longer needs to be level with the holder but thickness of the sample is important and can

cause peak displacement errors (when the sample is too thick). Transmission configurations

generally allow lower angle measurements than reflection, making them useful for large

molecules. However, it becomes essential that the holder containing the sample is as x-ray

transparent as possible, because its “fingerprint” will be part of the x-ray pattern collected on

each sample.

1.3 Analytical Method Development

1.3.1 Method development in HPLC and UPLC

Analytic method development and validation are key elements of any pharmaceutical

development program. HPLC analysis method is developed to identify, quantity or purifying

compounds of interest. Effective method development ensures that laboratory resources are

optimized, while methods meet the objectives required at each stage of drug development.

To know information concerning of target compound or analyte is worth. Understand

its physical and chemical characteristics allow selecting the most appropriate HPLC method

development from vast literature. Information concerning sample, for example, molecular

mass, structure and functionality, pKa values and UV spectra, solubility of compound(s)

should be compiled. The requirement of removal of insoluble impurities by filtration,

centrifugation, dilution or concentration to control the concentration, extraction (liquid or

solid phase), derivatization for detection etc. should be checked. For pure compound,

determine sample solubility whether it’s organic soluble or water soluble, as this helps to

select the best mobile phase and column to be used in your HPLC Method development.

Analytic methods are intended to establish the identity, purity, physical characteristics

and potency of the drugs that we use. Methods are developed to support drug testing against

specifications during manufacturing and quality release operations, as well as during long

term stability studies. Methods may also support safety and characterization studies or

25


evaluations of drug performance. Once a stability-indicating method is in place, the

formulated drug product can then be subjected to heat and light in order to evaluate potential

degradation of the API in the presence of formulation excipients [89].

The three critical components for a HPLC method are: sample preparation (%

organic, pH, shaking/sonication, sample size, sample age), HPLC analysis conditions

(%organic, pH, flow rate, temperature, wavelength, and column age) and standardization

(integration, wavelength, standard concentration, and response factor correction). During the

preliminary method development stage, all individual components should be investigated

before the final method optimization. This gives the scientist a chance to critically evaluate

the method performance in each component and streamline the final method optimization

[90-93]. The degraded drug samples obtained are subjected to preliminary chromatographic

separation to study the number and types of degradation products formed under various

conditions [94]. Scouting experiments are run and then conditions are chosen for further

optimization [95]. Resolving power, specificity, and speed are key chromatographic method

attributes to keep in mind during method development [96].

Method development in UPLC remains same as of HPLC but few areas of

chromatographic conditions are different e.g. for gradient elution column equilibration time is

very less as compare to HPLC due to lower column volume. Advance technology in column

filled material for HPLC as well as UPLC allows higher pH and temperature stability for

column for wider choice of mobile phase for different applications. The factor responsible for

the development of HPLC/UPLC technique was evolution of packing materials used to effect

the separation. The principles behind this evolution are governed by the van deemter equation

that describes the relationship between linear velocity and plate height. According to van dee

mter equation, decrease in particle size increases the efficiency of separations while on other

hand efficiency diminishes on increased flow rates or linear velocities. At a particle size less

than 2.5 µm, there is a significant gain in efficiency and the efficiency does not diminish at

increased flow rates or linear velocities. By using smaller particles, speed and peak capacity

can be extended to new limits, termed ultra-performance liquid chromatography (UPLC).

Selecting a detector: Various detectors include: UV/visible, photodiode array detector,

fluorescence detector, conductivity detector, refractive index detector, electrochemical

detector, mass spectrometer detector, evaporative light scattering detector. In most of the

HPLC method development UV & PDA detector (200 nm to 400 nm) is generally used,

selection is based on chemical nature of analytes and potential interference. Good analytical

26


results will be obtained only by careful selection of the wavelength used for detection. The

wavelength chosen for UV detection must provide acceptable absorbance. The mobile phase

must transit sufficiently at the wavelength used for detection. Detection affects assay

precision via the signal/noise (S/N) ratio.

Selecting the separation mode: The nature of the sample determines the best approach to

select the mode of separation in method development. Samples are classified as polar or non

polar, large portion of analytes are water soluble. Reversed phase chromatography (RPC) is

the first choices for most the polar compounds. Wide ranges of stable stationary phases and

simple mobile phases are available for many applications in RPC. After selection of the mode

and solubility of the sample the initial separation conditions like column, temperature, flow

rate and mobile phase will come. A temperature of 35 or 400C is usually a good starting

point, however ambient temperature is required if the method will be used for lack of column

thermo stating. In RPC mobile phase is polar, stationary phase is non polar. In RPC major

distinction between analytes is their hydrophobicity and the sample must be soluble in water

or polar organic solvents.

Mobile phase and pH: Mobile phase used for liquid chromatography typically are mixtures

of organic solvents like acetonitrile (ACN), methanol (MeOH) and tetrahydrofuran (THF)

and water or aqueous buffers. Mixture of acetonitrile and water is the best initial choice for

the mobile phase during method development. Isocratic methods are preferable to gradient

methods. Gradient methods will require when the molecules being separated have vastly

different partitioning properties. When gradient elution method is used, care must be taken to

ensure that all solvents are miscible. Retention can be preferably adjusted by changing mobile

phase composition or solvent strength in RPC. A change in organic solvent type is often used

to change peak spacing and improve resolution. Characteristics of mobile phases are; it is

essential to establish that the drug is stable in the mobile phase; excessive salt concentration

should be avoided. High salt concentration can result in precipitation, the mobile phase

should have a pH 2.5 to 7.0 to maximize the lifetime of the column, mobile phase must be

water soluble, low viscosity and uncreative in nature and mobile phase must have low UV

cutoff and minimize the absorbance of buffer.

The selection of solvents for this purpose is guided by solvent properties that are believed to

affect selectivity, acidity, basicity and dipolarity. A retention range of 0.5 to 20 is allowable

for sample to be separated using isocratic condition. pH is the important consideration in

method development. At a pH close to the pKa peak distortion will results.

27


Acidic compounds are retained at low pH while basic compounds are more retained at

higher pH. The neutral compounds remain unaffected. The pH range 4-8 is not generally

employed because slight change in pH in this range would result in a dramatic shift in

retention. However, by operating at pH extremes (2-4 or 8-10), not only is there a 10-30 fold

difference in retention that can be exploited in method development [97,98]. Some acidic or

basic samples undergo a change in absorbance as pH is varied and band spacing will occur.

Method development can proceeds by investigating parameters of chromatographic

separations first at low pH and then at higher pH until optimum results is achieved.

Column and column packing material: The column is the heart of liquid chromatography

separation process. The availability of a stable, high-performance column is essential in

developing a rugged, reproducible method. An HPLC column packed with stationary phase

of C18-bonded silica (C18 Column) and C8-bonded silica (C8 Column) is used in RP-HPLC

separation of a wide range of organic compounds. Different columns can vary in plate

number, band symmetry, retention, band spacing and lifetime. The choice of common

packing material and mobile phases depends on the physical properties of the drug. Reverse

phase columns differ by the carbon chain length, degree of end capping and percent carbon

loading. Most columns for liquid chromatography method development use straight lengths

of stainless steel tubing with highly polished interior walls. Stainless steel is useful with all

organic solvents and most aqueous buffers. The use of silica-based packing is favored in most

of the present HPLC columns due to several physical characteristics. Silica substrates are

available in spherical or irregular shapes and can be prepared with different surface areas,

pore sizes and particle sizes, which make them suitable for most HPLC applications. Totally

porous silica particles with 5 μm diameter provide the desired characteristics for most HPLC

separations. Column having 3 µm particles means fast separation is going on. Column having

1.5 µm means very fast separation will take place. Plate number, peak asymmetry, selectivity,

column back pressure, retention, bonded phase concentration and column stability are the

column specifications. The separation selectivity for certain components vary between the

columns of different manufacturer as well as between column production batches from the

same manufacturer. Separation of many samples can be enhanced by selecting the right

column temperature. Higher column temperature reduces system backpressure by decreasing

mobile phase viscosity, which in turn allows use of longer columns with higher separation

efficiency. However, an overall loss of resolution between mixture components in many

samples occurs by increasing column temperature. The optimum temperature is dependent

28


upon nature of the mixture components. The overall separation can be improved by

simultaneous changes in column temperature and mobile phase composition [99-101].

Recently, normal phase HPLC is back popular with the birth of HILIC technology that

proved to improve reproducibility in separating polar and hydrophilic compounds such as

peptides, carbohydrate, vitamins, polar drugs and metabolites. In order to develop a HPLC

method effectively, most of the effort should be spent in method development and

optimization as that will improve the final method performance [102].

1.3.2 Method development in P-XRD Technique

Qualitative analysis usually involves the identification of a phase or phases in a specimen by

comparison with “standard” patterns (i.e., data collected or calculated by someone else), and

relative estimation of proportions of different phases in multiphase specimens by comparing

peak intensities attributed to the identified phases.

Quantitative analysis of diffraction data usually refers to the determination of amounts

of different phases in multi-phase samples. Quantitative analysis may also be thought of in

terms of the determination of particular characteristics of single phases including precise

determination of crystal structure or crystallite size and shape. In quantitative analysis, an

attempt is made to determine structural characteristics and phase proportions with

quantifiable numerical precision from the experimental data itself. Though “standard”

patterns and structural data are used as a starting point, the most successful quantitative

analysis usually involves modeling the diffraction pattern such that the calculated pattern(s)

duplicates the experimental one.

All quantitative analysis requires precise and accurate determination of the diffraction

pattern for a sample both in terms of peak positions and intensities. While some kinds of

analysis (i.e., particle shape and clay structure) rely on the existence of preferred orientation,

most require a uniformly sized, randomly oriented fine (ideally 1-2 μm) powder specimen to

produce intensities which accurately reflect the structure and composition of the phase(s)

analyzed. As will become evident, the successful application of quantitative methods requires

careful sample preparation, good quality data and a very thorough understanding of the

material you are working with and the possible sources of error in your experiments. Since

diffraction data are generally very dependent on the systematics of your diffractometer and its

data collection system, application of quantitative methods that involve ratios of peak

intensities requires careful calibration with well-known standards before a quantitative

analysis is attempted.

29


The most effective quantitative methods, particularly those involving pattern

modeling, are computationally intensive can only be applied with powerful analytical

software. Commercial versions of this type of software are very expensive. Fortunately there

are several versions of software to do pattern refinements and quantitative analysis available

at no cost to the user. Though they are not as “user friendly” as the commercial versions,

once the learning curve is climbed, they can be very effective analytical tools. The diffraction

pattern includes information about peak positions and intensity. The peak positions are

indicative of the crystal structure and symmetry of the contributing phase. The peak

intensities reflect the total scattering from the each plane in the phase’s crystal structure, and

are directly dependent on the distribution of particular atoms in the structure. Thus intensities

are ultimately related to both the structure and composition of the phase.

Sample preparation issues:

With the possible exception of whole-pattern (rietveld and similar) methods of quantitative

analysis (in which specimen characteristics become another parameter to be modeled),

successful quantitative analysis requires a specimen that presents a very large number of

randomly oriented, uniformly sized crystallites to the X-ray beam. To achieve peak intensity

errors of less than ±1% for a single phase (100% of specimen) requires particles between 0.5

and 1.0 μm in size. This particle size range, in practice is extremely difficult to obtain. The

best methods generally result in a 1-5 μm size range, and the statistics are further degraded by

the fact that every phase in a multi-phase sample will be less than 100% of the whole. All of

this means that a statistical error of ± 5% for major phases in an intensity-related quantitative

analysis should be considered reasonable. Be suspicious of analyses that report lower errors.

Clearly the most successful quantitative analyses will be with materials in which particle size

is uniform, small and well known. Particular caution must be exercised in situations where

crystallite sizes vary widely within a particular sample. A goal of the analyses was to produce

repeatable quantitative determinations of the amounts of different phases in the specimens.

Although techniques were developed using an internal standard that could produce repeatable

results (±5%) in known binary mixtures, the method could not be successfully applied to the

actual rocks and results varied by up to 30% from independent determinations with

petrographic microscope and chemical techniques.

Measurement of line intensities:

All of the methods of quantitative analysis (even whole-pattern methods) require accurate

intensity measurements. The below (from Jenkins and Snyder, p. 356) summarizes the

30


various factors which control absolute and relative intensities in a powder pattern.

Structure sensitive factors:

These factors are mostly included in the K (hkl) α term in the intensity equation. Most of

these factors are intrinsic properties of the phase producing the reflection, but their intensity

can be modified both temperature and the wavelength of the incident radiation. 1) Structure-

sensitive (atomic scattering, structure factor, polarization, multiplicity, temperature) 2)

Instrument-sensitive (a) absolute intensities (source intensity, diffractometer efficiency,

voltage drift, take-off angle of tube, receiving slit width, axial divergence allowed (b) relative

intensities (divergence slit aperture, detector dead time) 3) Sample-sensitive (micro

absorption, crystallite size, degree of crystallanity, residual stress, degree of particle overlap,

particle orientation) 4) Measurement-sensitive (peak area measurement, degree of peak

overlap, background subtraction, Kα2 stripping or not, degree of data smoothing employed).

Instrument-sensitive parameters:

Variation in power supplied to the X-ray tube can cause notable variation in incident beam

intensity over time. Fortunately most modern digital power supplies include very

sophisticated circuitry to virtually eliminate voltage drift. All X-ray tubes will decrease in

intensity as they age, however, and it is important to monitor this over time. Detector dead

time can cause very intense peaks to be measured with lower intensity than would be

warranted by actual amount of the phase present producing the peak. The proper dead time

correction should be applied to correct your data for this.

Sample-sensitive parameters:

These are by far the most important class of factors affecting quantitative work. All of these

factors have the capability of severely compromising the usefulness of diffraction data. Keep

crystallite size at 1 μm for all phases and eliminate preferred orientation in your specimen and

you’ve got a chance of getting usable data.

Measurement-sensitive parameters:

The selection of the 2θ points at which background will be measured is critical to

determination of accurate integrated peak intensities. The choice of where the peak starts and

ends relative to background will have a significant effect on integrated intensity as illustrated

in the figures (from Jenkins and Snyder, 1996) below.

31


Fig.1.8: Trace of the Si (111) peak using Cr radiation

Fig.1.8 shows an experimental trace of the (111) peak of Si using Cr radiation. Note that the

peak has a notable “tail” and the start of the peak could be picked at some point between 41.5

and 42.2° 2θ. The reference intensity ratio (RIR) is the ratio between the integrated intensities

of the peak of interest and that of a known standard (corundum in this case).

Fig.1.9 shows how the RIR varies as a function of where the background is picked

(holding the Al2O3 line constant). Clearly, where the background is picked will have a

significant effect on peak ratios, and thus the amount of the unknown determined. It must be

noted that peak areas for well-defined peaks will be proportional to peak heights, but that this

relationship breaks down in peaks which show significant broadening. Peak broadening,

either by strain or particle size, results in integrated peak intensities which are not

representative of the amounts present, thus some sort of correction for this broadening is

desirable for quantitative analysis.

Fig.1.9: The variation of RIR as a function of scan width of the peak shown in Fig.1.8.

32


Fig.1.10: Types of line measured in quantitative analysis

This effect is shown in the left-most two peaks in (Fig.1.10a and 1.10b). Another

significant problem in the use of integrated intensities is in overlapping peaks of interest and

the difficulty of calculating integrated intensities. This requires one of two approaches:

selection of peaks which do not overlap or decomposition of overlapping peaks into their

components prior to calculating integrated intensities. The peak overlap situation is shown in

Fig.1.10c. Sophisticated digital tools for processing diffraction data make peak

deconstruction or “deconvolution” possible.

Quantitative methods based on intensity ratios:

Numerous methods have been developed to use peak intensities for quantitative analysis of

diffraction data. Many of them are specialized in nature (i.e., require binary mixtures or

involve polymorphs having the same mass absorption coefficients). The methods with the

greatest chance of producing successful results generally involve the addition of a known

amount of an internal standard and calculating ratio of the areas of the standard peaks to those

of the phases being determined.

Method of standard additions:

This method requires a variety of diffraction patterns run on prepared samples in which

varied amounts of a well-known standard, β are added to the unknown mixture containing

phase α, the each mixture is analyzed. This method was developed and still widely used for

elemental analysis by X-ray Fluorescence. Many methods of quantitative P-XRD involve the

addition of a known amount of a well characterized standard. The main difficulties with this

method involve rather laborious sample preparation, the requirement for uniform crystallite

33


size between sample and standard powders, the necessity of a well-mixed random sample

powder, and (in many cases) production of an extensive database of diffraction patterns for

known mixtures of the internal standard and individual standard phases.

Internal standard method:

The internal standard method, or modifications of it, is most widely applied technique for

quantitative P-XRD. This method gets around the (μ/ρ)s problem by dividing two intensity

equations to yield:

where α is the phase to be determined, β is the standard phase and k is the calibration constant

derived from a plot of I (hkl)α/I(hkl)'β vs. Xα / Xβ. Direct application of this method requires

careful preparation of standards to determine the calibration curves, but can produce

quantitative determinations of identified phases that are substantially independent of other

phases in the specimen.

Care must be taken when choosing standards to select materials with reasonably simple

patterns and well-defined peaks that do not overlap peaks in phases of interest. It is also very

important that the crystallite size of the specimen and standard be the same, ideally about 1

μm. Other quantitative methods such as reference intensity ratio methods, generalized RIR

method, normalized RIR method, full-pattern analysis–the rietveld methods is also useful in

quantitative analysis of unknown mixtures, contamination of polymorphic impurities by

powder x-ray diffraction analysis.

1.4 Analytical Method Validation

Analytical method validation is the process to confirm that the analytical procedure employed

for a specific test is suitable for its intended use. Methods need to be validated or revalidated

Validation is required for any new or amended method to ensure that it is capable of giving

reproducible and reliable results, when used by different operators employing the same

equipment in the same or different laboratories. Validation is a procedure having of

documental evidence to demonstrate the procedure (method/process) is able or not to produce

the expected results under the stated experimental conditions. For this a number of definitions

can imply but the final concept of the validation is to assure the fitness of the procedure for

its intended purpose. Assurance of the procedure will be demonstrated by establishing the

34


suitable parameters (nothing but experimental conditions) of validation. These parameters

shall be selected with the consideration of intended application, pharmacopoeial or industrial

guidelines, conditions to be challenged, time, chemistry of the substances. In pharmaceutical

industry the validation exercise is mainly applicable to process validation, analytical method

validation), cleaning validation and instrument validation-DQ (design qualification)/IQ

(installation qualification)/OQ (operation qualification)/PQ (performance qualification)/MQ

(maintenance qualification), personal validation. Typical validation characteristics which

should be considered are listed below.

Specificity: Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expected to be present. Typically these might include impurities,

degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be

compensated by other supporting analytical procedure(s). Specificity can be evaluated with

PDA detector by comparing collected spectrum across a peak. It indicates that homogencity

of peak and also evaluated using modern LC-MS/GC-MS techniques. Specificity is

calculated and documented in a separation by the resolution, measuring of theoretical plates

and tailing factor.

Precision and accuracy: The purpose of carrying out a determination is to obtain a valid

estimate of a 'true' value. When one considers the criteria according to which an analytical

procedure is selected, precision and accuracy are usually the first time to come to mind.

Precision and accuracy together determine the error of an individual determination. They are

among the most important critical for judging analytical procedures by their results.

Precision: Precision refers to the reproducibility of measurement within a set, i.e., to the

scatter of dispersion of a set about its central value. One of the most common statistical terms

employed is the standard deviation of a population of observation. Standard deviation is the

square root of, the sum of squares of deviations of individual results for the mean, divided by

one less than the number of results in the set. The standard deviation S, is given by

∑=

−−

=n

ii xx

nS

1

2)(1

1

Standard deviation has the same units as the property being measured. The square of standard

deviation is called Variance (S2). Relative standard deviation is the standard deviation

expressed as a fraction of the mean, i.e. S/x. It is sometimes multiplied by 100 and expressed

as a percent relative standard deviation. It becomes a more reliable expression of precision.

35


% Relative standard deviation= S X 100/x

Accuracy: Accuracy normally refers to the difference between the mean x, of the set of

results and the true or correct value for the quantity measured. According to IUPAC accuracy

relates to the difference between results (or mean) and the true value. For analytical methods,

there are two possible ways of determining the accuracy, absolute method and comparative

method.

Absolute method: The test for accuracy of the method is carried out by taking varying

amounts of the constituents and proceeding according to specified instructions. The

difference between the means of an adequate number of results and amount of constituent

actually present, usually expressed as parts hundred (%) is termed as % error. The constituent

in question will be determined in the presence of other substances, and it will therefore be

necessary to know the effect of these upon the determination. This will require testing the

influence of a large number of probable compounds in the chosen samples, each varying

amounts. In a few instances, the accuracy of the method controlled by separations (usually

solvent extraction or chromatography technique) involved.

Comparative method: In the analysis of pharmaceutical formulations (or solid laboratory

prepared samples of desired composition), the content of the constituent sought (expressed as

percent recovery) has been determined by two or more (proposed and official or reference)

supposedly "accurate" methods of essentially different character can usually be accepted as

indicating the absence of an appreciable determinate error.

Detection limit (DL): The detection limit of an individual analytical procedure is the lowest

amount of analyte in a sample which can be detected but not necessarily quantitated as an

exact value. Several approaches for determining the detection limit are possible, depending

on whether the procedure is a non-instrumental or instrumental. Approaches other than those

listed below may be acceptable. Signal-to-Noise (S/N) approach can only be applied to

analytical procedures which exhibit baseline noise.

Quantitation limit (QL): The quantitation limit of an individual analytical procedure is the

lowest amount of analyte in a sample which can be quantitatively determined with suitable

precision and accuracy. The quantitation limit is a parameter of quantitative assays for low

levels of compounds in sample matrices, and is used particularly for the determination of

impurities and/or degradation products. Several approaches for determining the quantitation

limit are possible, depending on whether the procedure is a non-instrumental or instrumental.

36


Approaches other than those listed below may be acceptable. Signal-to-noise approach can

only be applied to analytical procedures that exhibit baseline noise. Based on the standard

deviation of the response and the slope, the quantitation limit (QL) may be expressed as

QL=10 σ/S, where σ=the standard deviation of the response, S = the slope of the calibration

curve. For instrumental techniques or other methods that rely upon a calibration curve for

quantitative measurements, the IUPAC approach employs the standard deviation of the

intercept (Sa), which may be related to DL and the slope of calibration curve, b, by

DL= 3Sa / b QL=10Sa / b

Linearity: The linearity of an analytical procedure is its ability (within a given range) to

obtain test results which are directly proportional to the concentration (amount) of analyte in

the sample. The correlation coefficient, y-intercept, slope of the regression line and residual

sum of squares should be submitted. A plot of the data should be included. In addition,

analysis is the deviation of the actual data points from the regression line may also be helpful

for evaluating linearity.

Range: The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for which it

has been demonstrated that the analytical procedure has a suitable level of precision, accuracy

and linearity. For the determination of an impurity, from the reporting level of an impurity to

120% of the specification. For impurities known to be unusually potent or to produce toxic or

unexpected pharmacological effects, the detection/quantitation limit should be commensurate

with the level at which the impurities must be controlled.

Robustness: The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters and provides an

indication of its reliability during normal usage. The evaluation of robustness should be

considered during the development phase and depends on the type of procedure under study.

It should show the reliability of an analysis with respect to deliberate variations in method

parameters. If measurements are susceptible to variations in analytical conditions, the

analytical conditions should be suitably controlled or a precautionary statement should be

included in the procedure. One consequence of the evaluation of robustness should be that a

series of system suitability parameters (e.g., resolution test) is established to ensure that the

validity of the analytical procedure is maintained whenever used. Examples of typical

variations are, influence of variations of pH in a mobile phase, influence of variations in

mobile phase composition, different columns (different lots), temperature, flow rate.

37


Selectivity of the method: Matrix and interference effects may disturb the determination of

an analyte. Some of the excipients, incipients and additives present in pharmaceutical

formulations may sometimes interfere in the assay of drug and in such instances appropriate

separation procedure is to be adopted initially. The selectivity of the method is ascertained by

studying the effect of a wide range of excipients and other additives usually present in the

pharmaceutical formulations to be determined under optimum conditions. Initially,

interference studies are carried out by the determination of fixed concentration of the drug

several times by the optimum procedure in the presence of a suitable (1-100 fold) molar

excess of the foreign compound under investigation and its effect on the absorbance of the

solution is noticed. The foreign compound is considered to be interfering at these

concentrations if it constantly produces an error of less than 3.0% in the absorbance produced

in pure solution.

1.5 Chromatographic Parameters and Statistical Evaluation

1.5.1 Chromatographic parameters

Resolution: Chromatographers measure the quality of separation by resolution R of adjacent

bands.

21

12 )(2WWttR

+−

=

t1 and t2 are retention times of the first and second adjacent bands ;W1 and W2 are base line

band width.

Capacity factor (k): It is the measure of how well the sample molecules are retained by the

column during an isocratic separation. It is affected by the solvent composition, separation,

aging and temperature of separation.

O

OR

ttt

'K−

= Where tR =Band retention time and tO=Column dead volume

Column efficiency (N): It is called as the number of theoretical plates. It measures the band

spreading of a peak. When band spread in smaller, the number of theoretical plates is higher.

It indicates a good column and system performance. Column performance can be defined in

terms of values of N

Column efficiency (N)=16 (tR/W)2

Plate height H=N/L (length)

38


Peak asymmetry/Peak tailing (As): Peak with poor symmetry can result in (i) Inaccurate

plate number and resolution measurement (ii) imprecise quantization (iii) degraded resolution

and undetected minor bands in the peak tail (iv) poor retention reproducibility. Increased

peak asymmetry value, k>1.5 the sign that the column should be changed.

AS = W0.05/2f, where W0.05 is the width of the peak at 5% height and f is the distance from

the peak maximum to the leading edge of the peak, the distance being measured at a point 5%

of the peak height from the baseline.

Selectivity: It measures relative retention of two components. Selectivity is the function of

chromatographic surface (column), melting point and temperature.

01

02'1

'2

VVVV

kk

−−

==α

1.5.2 Statistical evaluation

Calibration: Calibration is one of the most important steps in bioactive compound analysis.

A good precision and accuracy can only be obtained when a good calibration procedure is

used. In instrumental methods, the concentration of a sample cannot be measured directly, but

is determined using another physical measuring quantity 'y’ of a solution. An unambiguous

empirical or theoretical relationship can be shown between this quantity and the

concentration of analyte. The calibration between y=g(x) is directly useful and yields by

inversion of the analytical calculation function. The calibration function can be obtained by

fitting an adequate mathematical model through the experimental data. The most convenient

calibration function is linear, goes through the origin and is applicable over a wide dynamic

range. In practice, however, many deviations from this ideal calibration line may occur. For

the majority of analytical techniques the analyst uses the calibration equation. Y=a+bx

Linear regression-method of least squares:

Least-squares regression analysis can be used to describe the relationship between response

(y) and concentration (x). We adopt the convention that the x values relate to the controlled

on independent variable (e.g. the concentration of a standard) and the y values to the

dependent variable (the response measurements). This means that the x values have no error.

On the condition that the errors made in preparing the standards are significantly smaller than

the measuring error (which is usually the case in analytical problems). The values of the

unknown parameters must be estimated in such a way that the model fits the experimental

data points (xi, yi) as well as possible. The true relationship between x and y is considered to

39


be given by a straight line. The relation between each observation pair (Xi, Yi) can be

represented as Yi =α+βXi +ei. The signal yi is composed of deterministic component

predicted by linear model and a random component ei. One must now find the estimates of a

and b of the two values α and β. This can be done by calculating the values a and b for which

ei2 is minimal. The component ei represent the differences between the observed yi values

and the predicted yi values by the model. The ei are called the residuals, a and b are the

intercept and slope respectively.

2

1 1

2

1 11

∑ ∑

∑ ∑∑

= =

= ==

−

=−

n

i

n

iii

n

i

n

iii

n

iii

xxn

yxyxnb

2

1 1

2

1 11

2

1

∑ ∑

∑ ∑∑∑

= =

= ===

−

−=

n

i

n

iii

n

i

n

iii

n

iii

n

ii

xxn

yxxxya

Standard error on estimation (Se):

The standard error on estimation is a measure of the difference between experimental and

computed values of the dependent variable. It can be represented by the following equation,

)2/()(1

2 −−= ∑=

nyySn

iiie

Yi, and yi, are the observed and predicted values, respectively. Standard deviations on slopes

(Sb) and intercepts (Sa) are quoted less frequently, even though they are used to evaluate

proportional differences between or among methods as well as to compute the independent

variables such as concentration etc. It is important to understand how uncertainties in the

slope are influenced by the controllable properties of the data set such as the number and

range of data points and also how properties of data sets can be designed to optimize the

confidence in such data.

Standard deviation on slope (Sb):

The standard deviation on slope is proportional to standard error of estimate and inversely

proportional to the range and square root of the number of data points.

)2(

)(1

2

−

−=∑=

n

yyS

n

iii

b Where Xi is the arithmetic mean of xi

values

∑=

−n

iii xx

1

2)(

1

40


Standard deviation on intercept (Sa):

Intercept values of least squares fits of data are often to evaluate additive errors between or

among different methods

)2(

)(1

2

−

−=∑=

n

yyS

n

iii

a

Correlation coefficient (r):

To establish whether there is a linear relationship between two variables xi and yi, use

Pearson’s correlation coefficient r. The value of r must lie between +1 and -1; the nearer it to

±1, the greater the probability that a definite linear relationship exists between the variables x

and y, values close to +1 indicate positive correlation and values close to -1 indicate negative

correlation. Value r of that towards zero indicate that x and y are not linearly related.

222 )1/()()(

)1/())((

−

∑ −−

−

∑ −−

=nyyxx

nyyxxr

n

ii

n

ii

1.6 Profile of the Selected Drugs

1.6.1 Solifenacin succinate drug profile

Solifenacin succinate (SFS) is chemically known as (1S)-(3R)-1-azabicyclo [2.2.2] oct-3-yl

3,4-dihydro-1-phenyl-2(1H)-iso-quinolinecarboxylate (1:1) having an empirical formula of

C23H26N2O2.C4H6O4 and molecular weight is 480.55 grams/mole. It appears to be white to

pale-yellowish-white crystal or crystalline powder, freely soluble in water, glacial acetic acid,

dimethyl sulfoxide and methanol. It is available in the market under the brand name of

VESIcare in the form of 5 mg and 10 mg tablets manufactured by Astellas Pharma

Technologies, Inc. Norman, Oklahoma and marketed and distributed by Astellas Pharma US,

Inc. Deerfield, Illinois. It reduces muscle spasms of the bladder and urinary tract.

Solifenacin is used to treat symptoms of overactive bladder, such as frequent or urgent

urination, and incontinence. The chemical structure of SFS is represented in Fig.1.11.

∑=

−n

iii xx

1

2)(

1

n

xn

ii∑

=1

2

41


Fig.1.11: Chemical structure of solifenacin succinate

1.6.2 Fesoterodine fumarate drug profile

Fesoterodine fumarate (FST) is an isobutyric acid 2-((R)-3-diisopropyl ammonium-1phenyl

propyl)-4-(hydroxymethyl) phenyl ester hydrogen fumarate. The empirical formula and its

molecular weight are C30H41NO7 and 527.66 grams respectively. It is a white to off-white

powder, which is freely soluble in water. It is soluble in some polar protic organic solvents

and polar non protic solvents. It is commercially available under the brand name of Toviaz

which contains fesoterodine fumarate and is an extended-release tablet. Each toviaz

extended-release tablet contains either 4 mg or 8 mg of FST. It is genitourinary smooth

muscle relaxants (antispasmodic; an antimuscarinic agent). The chemical structure of

fesoterodine fumarate is shown in Fig.1.12.

Fig.1.12: The chemical structure of fesoterodine fumarate

1.6.3 Darunavir ethanolate drug profile

Darunavir ethanolate (DRE) has the chemical name [(1S,2R)-3-[[(4-aminophenyl) sulfonyl]

(2-methylpropyl)amino)-2-hydroxy-1(phenylmethyl)propyl]carbamicacid (3R,3aS, 6aR)-

hexa hydrofuro[2,3-b]furan-3-yl ester mono ethanolate. Its molecular formula is

C27H37N3O7S .C2H5OH and its molecular weight is 593.73 grams/mole. It is a protease

(PRO-tee-ayz) inhibitor and used an antiviral medication that prevents human

immunodeficiency virus (HIV) cells from multiplying in your body. It is used to treat HIV,

which causes acquired immuno deficiency syndrome (AIDS). It is available in the market

42


under the brand name of prezista. It is a white to off-white powder with a solubility of

approximately 0.15 mg/ml in water at 20°C. It is available in the trade name as prezista100

mg/ml oral suspension, prezista 75, 150, 400 and 600 mg tablets. Darunavir ethanolate has

the following structural formula (Fig.1.13).

Fig.1.13: The structural formula of darunavir ethanolate

1.6.4 Levosimendan drug profile

Levosimendan (LSM) (Fig.1.14) is chemically known as (R)-[[4-(1,4,5,6-tetrahydro-4-

methyl-6-oxo-3-pyridazinyl) phenyl]hydrazono] propanedinitrile belongs to a new class of

drugs, the calcium sensitisers used in the management of acutely decompensated congestive

heart failure. Levosimendan is a moderately lipophilic drug. It is a weak acid with pKa 6.3.

Solubility of levosimendan in distilled water and phosphate buffer (pH 8) is poor (0.04 mg/ml

and 0.9 mg/ml, respectively). Solubility in ethanol is 7.8 mg/ml and therefore levosimendan

in its pharmaceutical composition (levosimendan 2.5 mg/ml infusion concentrate) is diluted

in ethanol. It is marketed under the brand name of simdax. The molecular formula of

levosimendan is C14H12N6O and the molecular weight is 280.28 grams/mole.

Fig.1.14: The chemical structure of levosimendan

43


1.6.5 Sitagliptin phosphate drug profile

Sitagliptin phosphate (SGP) is a new oral hypoglycemic (anti-diabetic drug) chemically

known as 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(tri

fluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate. The empirical

formula and molecular weight of it are C16H15F6N5O.H3PO4.H2O and 523.32 grams/mole

respectively. It is a white to off-white, crystalline, non-hygroscopic powder, soluble in water

and slightly soluble in methanol; very slightly soluble in ethanol, acetone, and acetonitrile;

and insoluble in isopropanol and isopropyl acetate. The chemical structure of Sitagliptin

phosphate was shown in Fig.1.15. Sitagliptin is a new oral hypoglycemic (anti-diabetic drug)

of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. This enzyme-inhibiting

drug is to be used either alone or in combination with metformin or a thiazolidinedione for

control of type 2 diabetes mellitus. Currently it is available in the market under the brand

name of januvia and janumet (25 mg, 50 mg or 100 mg) tablets.

Fig.1.15: Chemical structure of sitagliptin phosphate monohydrate

1.6.6 Raltegravir potassium (RVK) drug profile

Raltegravir potassium, a human immunodeficiency virus integrase strand transfer inhibitor.

The chemical name for raltegravir potassium is N-[(4-fluorophenyl)methyl]-1,6-dihydro5-

hydroxy-1-methyl-2-[1-methyl-1-[[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino]ethyl]-6-

oxo-4pyrimidinecarboxamide monopotassium salt. The empirical formula is C20H20FKN6O5

and the molecular weight is 482.51 grams/mole. Raltegravir potassium is a white to off-white

powder. Currently it is available in the market under the brand name of isentress. It is soluble

in water, slightly soluble in methanol, very slightly soluble in ethanol and acetonitrile and

insoluble in isopropanol. It has no chiral centres although polymorphism is observed.

Isentress available as 25 mg, 100 mg chewable tablets and oral 400 mg tablets. The chemical

structure of raltegravir potassium is shown in Fig.1.16.

44

http://www.rxlist.com/script/main/art.asp?articlekey=6845


Fig.1.16: Chemical structure of raltegravir potassium

1.6.7 Irbesartan (IRB) drug profile

Irbesartan (Fig.1.17) is an angiotensin II receptor antagonist used mainly for the treatment of

hypertension. It is available under the trade names aprovel, karvea, and avapro. Irbesartan

may also delay progression of diabetic nephropathy and is also indicated for the reduction of

renal disease progression in patients with type 2 diabetes, hypertension and micro

albuminuria (>30 mg/24 hours) or proteinuria (>900 mg/24 hours). Irbesartan is also

available in a combination formulation with a low dose thiazide diuretic,

invariably hydrochlorothiazide, to achieve an additive antihypertensive effect. Irbesartan/

hydrochloro thiazide combination preparations are marketed under similar trade names to

irbesartan preparations, including irda, coIrda, coaprovel, karvezide, avalide and avapro

hydrochloro thiazide.

Fig.1.17: Chemical structure of Irbesartan

1.7 Objective of the Present Investigation

The main objective of the present investigation presented in chapter-II is to develop a reverse

phase, gradient liquid validated chromatographic method for the determination of solifenacin

succinate in pure and pharmaceutical formulations, characterization of three impurities of

solifenacin succinate and determination of these impurities by the developed method, study

45


of forced degradation under stress condition and to resolve all known impurities that were

generated during the forced degradation studies and assay of API sample by the proposed

method. The aim and scope of the proposed work in chapter-III is to develop a suitable HPLC

method for the assay study of fesoterodine fumarate and darunavir ethanolate API and tablet

dosage forms independently, study of forced degradation of these drugs under stress

condition, to resolve all known impurities and generated during the force degradation studies.

In chapter-IV (section-A), developed chiral HPLC method for the determination of S-isomer

content in levosimendan, active pharmaceutical ingredients followed by validation as per ICH

guidelines. As per the chiral theory two isomers are possible for this structure. Out of them R-

isomer is the identified and reported as an active pharmaceutical ingredient for the treatment

in the management of acutely decompensated congestive heart failure. Finally S-isomer needs

to be control as per guidelines. Simple and precise analytical normal phase isocratic HPLC

methods for determination of S-isomer was developed and validated. In chapter-IV (section-

B), due to the chiral nature of sitagliptin phosphate it is felt necessary to develop a chiral LC

method for enantiomeric separation and accurate quantification of unwanted enantiomer ((S)-

enantiomer) of sitagliptin phosphate. So far no chiral HPLC methods were reported in the

literature for the enantiomeric separation of sitagliptin phosphate and accurate quantification

of its potential (S)-enantiomer. The main aim of the present work describes the quantitative

determination of S-enantiomer of sitagliptin phosphate in bulk and tablet dosage forms by

using normal phase chromatography. In chapter V, a stability indicating RP-UPLC method

was developed for determination of raltegravir potassium in bulk and dosage forms. The

developed method is validated with respect to specificity, precision, linearity and accuracy. In

chapter VI of this thesis, provided a detailed physical characterization of form-A and form-B

polymorphs of irbesartan and validation of the method used for the analysis of irbesartan

form-B polymorphic impurity content in irbesartan form-A API and tablet dosage forms by

powder x-ray diffraction (P-XRD).

46


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