INTRODUCTION

CHAPTER 1

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1.1 Drugs

A drug may be defined as a substance meant for diagnosis, cure, mitigation and

prevention, treatment of diseases in human beings or animals, for altering in structure or

function of the body of human beings or animals.[1] Pharmaceutical chemistry[2-6] is a

science that makes use of general laws of chemistry to study drugs i.e. their preparation,

chemical nature, composition, structure, influence on an organism, the methods of quality

control and the conditions of their storage etc. The family of drugs may be broadly

classified as Pharmacodynamic agents and Chemotherapeutic agents. Pharmacodynamic

agents refer to a group of drugs, which stimulate or depress various functions of body so

as to provide some relief to the body in case of body abnormalities, without curing the

disease. They are mainly used in case of noninfectious diseases so as to correct the

abnormal body functions. Non-selective central nervous system modifiers (depressants or

stimulants), adrenergic stimulants and blocking agents, cholinergic and cholinergic

blocking agents, cardiovascular agents, diuretics, antihistaminic agents and

anticoagulating agents are some examples of this group. These agents have no action on

infective organisms, which cause various diseases.

Chemotherapeutic agents are agents, which are selectively more toxic to the

invading organisms without harmful effect to the host. Some of the examples of this

group are organometallic agents, antimalarials, antibacterials, antiprotozoals, antifungal

agents, antihelmentics, antiseptics, antitubercular agents, antineoplastics, etc.

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1.2 Cardiovascular drugs

Heart diseases or cardiovascular diseases (CVD) are the class of diseases that

involve the heart or blood vessels.[7] While the term technically refers to any disease that

affects the cardiovascular system, it is usually used to refer to those related

to atherosclerosis (arterial disease). Heart attacks and strokes are usually acute events and

are mainly caused by a blockage that prevents blood from flowing to the heart or brain.

The most common reason for this is a build-up of fatty deposits on the inner walls of the

blood vessels that supply the heart or brain. Strokes can also be caused by bleeding from

a blood vessel in the brain or from blood clots.

Every year, 17.1 million lives are claimed by the global burden of heart disease

and stroke 82% of which are in the developing world. The number of deaths especially in

low and middle-income countries (LMICs) is alarming and saddening. These deaths could

be prevented through steps such as eating a healthy diet, regular physical activity and

avoiding tobacco. By the year of 2030, almost 23.6 million people will die from CVD,

mainly due to heart diseases and stroke. These are projected to remain the single leading

causes of death. The largest percentage increase will occur in the Eastern Mediterranean

Region. The largest increase in number of deaths will occur in the South-East Asia

Region.

Each year, heart disease kills more Americans than cancer. In recent years,

cardiovascular risk in women has been increasing and has killed more women than breast

cancer.[8] By the time, the heart problems are detected, the underlying cause

(atherosclerosis) is usually quite advanced, having progressed for decades.

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Therefore increased emphasis on preventing atherosclerosis by modifying risk factors,

such as healthy eating, exercise and avoidance of smoking.

Most countries face high and increasing rates of cardiovascular disease.

Cardiovascular medications are used as a means to control or to prevent certain forms

of heart disease. Many people with advanced heart disease may take several of

these drugs, and drug treatment may change if the disease advances or improves. The

reason people may require several types of drugs are because they may have numerous

symptoms or conditions that need control at the same time. Understanding the various

categories of these medications can be helpful.

Types of cardiovascular drugs may be broken into groups depending upon their

action or what they treat. Treatment categories are more difficult to describe since many

of these medications may address several symptoms of heart disease and have more

than one use. Categories that might describe drug actions include the following:

statins, diuretics, anticoagulants, anti-platelet, beta-blockers, digitalis drugs, vasodilators,

calcium channel blockers, and ACE inhibitors.

Cardiovascular diseases (CVD) are a group of disorders of the heart and blood

vessels and include:

Coronary heart disease – disease of the blood vessels supplying the heart muscle.

Cerebrovascular disease - disease of the blood vessels supplying the brain.

Peripheral arterial disease – disease of blood vessels supplying the arms and legs.

Rheumatic heart disease – damage to the heart muscle and heart valves from

rheumatic fever, caused by streptococcal bacteria.

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Congenital heart disease - malformations of heart structure existing at birth.

Deep vein thrombosis and pulmonary embolism – blood clots in the leg veins,

which can dislodge and move to the heart and lungs.

In practice, cardiovascular disease is treated by cardiologists, thoracic

surgeons, vascular surgeons, neurologists, and interventional radiologists, depending on

the organ system that is being treated. There is considerable overlap in the specialties, and

it is common for certain procedures to be performed by different types of specialists in

the same hospital.

Yet it would be hard to keep track of every single drug intended to assist in heart

disease because of the plethora that exist, and the intense research existing in this area,

which results in frequent development of new drugs.

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Fig. 1.1 Anatomy of Heart

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1.3 Impurity profiling

Worldwide, impurity profiling (identification and characterization of impurities

associated with drugs or drug products) is increasingly viewed as a valuable and essential

part of quality requirements. Various regulatory authorities like United States Food and

Drug Authority[9] (USFDA), European Directorate of Quality Medicine[10] (EDQM),

Therapeutic Goods Administration,[11] World Health Organization (WHO)[12] and other

health agencies[13-16] are emphasizing on the purity requirements and the identification of

impurities in drug substance and products. A key component of the overall quality of a

pharmaceutical is control of impurities, as their presence, even in small amounts, may

affect drug safety and efficacy. International Conference on Harmonization of Technical

Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines[17-23]

are developed with the joint efforts of regulators and industry representative from the

European Union, Japan and the United States. The guidelines helped to ensure that the

different regions have consistent requirements for the data that should be submitted to the

various regulatory agencies. As per ICH Q3A(R)[18] and ICH Q3B(R)[19] guidelines,

unknown impurities associated with bulk drug and dosage form, greater than the

identification threshold should be identified.

Impurity profiles for pharmaceutical products require a basis for well reasoned

and rational argument that seeks to limit both the number and amount of impurities on

safety grounds. The efficacy, safety and the dosage of drug product will determine the

level of impurities to be present in drug compound. The dose of active substance

administered will vary considerably between products and it is obvious, but frequently

ignored, that this will influence the amount of impurity administered when impurities are

controlled on a percentage or parts per unit basis.[24]

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The toxicological assessor in a regulatory agency cannot necessarily be reassured

on safety, simply by the limitation of an impurity to a low percentage level. Some drugs

are administered by mouth at doses of >30 g/day, so that even 0.01% of an

uncharacterized impurity gives a potential patient exposure of >3 mg daily. Conversely,

the analyst may not need to struggle to achieve a low (e.g. 0.1%) limit for detection,

quantification, validation or reporting, if the daily dose to humans will only be in the

microgram or low milligram range. As a rough guide, the limitation to 1 mg daily oral

intake of an uncharacterized or poorly characterized impurity will probably satisfy a

safety assessment for regulatory purposes.[18, 19]

It is therefore necessary from the safety point of view, to know enough about the

impurity profile (both qualitative and quantitative) to allow a judgment that the impurity

will not pose any concern over safety or will be an acceptable risk factor for treating

serious diseases where there is no other therapy suitable for a particular patient.

Impurity profiling of any pharmaceutical substance is a crucial part of process

development. Hence, it was felt necessary to develop the modern chromatographic and

mass spectrometric methods for qualitative and quantitative determination of impurities in

pharmaceutical dosage forms.

1.3.1 Sources of impurities

Various sources of impurities were reported.[25, 26] They are crystallization-related

impurities, stereochemistry-related impurities, process impurities, residual solvents,

inorganic impurities and impurities arising during storage.

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Degradation of the drug substance is one of the main sources of impurities in both

bulk drug and formulated product. Degradation of the drug substance is caused by

chemical instability of the drug substance under the conditions (e.g., heat, oxidation,

humidity, solvent, pH, light, etc.) of manufacturing, isolation, purification, drying,

storage, transportation, and interactions with other chemical entities in the formulation

(e.g., excipients and coating materials). Chemical stability is an inherent property of a

drug substance and is a reflection of the chemical properties of all functional groups in

the drug molecule.

1.3.2 Identification of Impurities

Identification and characterization of impurities is an analytical activity aiming to

elucidate the chemical structures and the possible mechanisms of formation of unknown

impurities. Because of the complexity and diversity of the impurities in both their origins

and properties, the identification strategies are determined by the specific situations.

A general strategy can be set for the identification and characterization of the

impurity of bulk drug substances by the rational use of analytical techniques. The

schematic use of the methods[27] for impurity profiling of drug substances is shown in

figure 1.2. The impurity profile of a drug substance also includes the identification of the

key impurities in the intermediates and key starting materials of their synthesis. In case

of synthesis related impurities, their mechanism and source of their formation can also be

presented. Impurity profiling also includes identification and determination of residual

solvents and inorganic impurities in the drug substances.

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Fig. 1.2 Scheme for impurity profile study

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Detection of an unknown impurity is the first step in impurity profiling. Typically,

unknown impurities are observed during analysis of intermediates or drug substances for

process control or at release. Once the decision for identification is made, the nature and

origin of the impurity can be assessed, based on when, where, and how the unknown is

initially observed. Depending on the structure of the drug substance, the synthetic

scheme, impurities in the starting material, known process related impurities, formulation

ingredients, and the analytical method that is used for the initial detection of the unknown

impurities; it is possible to evaluate the impurities. The impurities can be identified

predominantly by different methods like reference standard method, separation methods

and isolation methods. The characterization of impurities can be carried out using

different spectroscopic and its hyphenated techniques.

Reference standard method can be adapted by making use of the available

standard of the impurity. If the initial analysis indicates that the observed impurity falls

into this category, the impurity identification turns into practice. This can be achieved by

three experiments. First, analysis of sample is followed by analysis of standard and then

spiked sample with standard by applying any of the chromatographic or spectroscopic

techniques.

1.3.3 Quantitation of Impurities

The following techniques are being regularly used for the quantitation of

impurities and degradation products: High Performance Liquid Chromatography

(HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), High

Performance Thin Layer Chromatography (HPTLC), Capillary Electrophoresis (CE),

Supercritical Fluid Chromatography (SFC), and Gel Permeation Chromatography (GPC).

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Recently Ultra Performance Liquid Chromatography (UPLC) is emerging as a fast

separation liquid chromatographic technique. Since, we have used only HPLC and UPLC

in our present work, we have described these techniques in detail as below.

1.3.3.1 High Performance Liquid Chromatography (HPLC)

HPLC is a chemistry based tool for separation, quantification and analysis of

mixtures of chemical compounds. As a consequence of the enormous development of the

analytical technology in the last two decades entirely new possibilities have been created

for the determination of the purity of drug materials. Nearly all organic impurities are

determined by chromatographic or related methods of which HPLC has been the most

important for over a decade.

HPLC is regarded as the most important analytical method in pharmaceutical

analysis as it provides a number of highly selective variants to resolve almost every type

of separation problem.[28] Derivatization of the drugs prior to analysis is normally not

required. HPLC can be operated in both modes i.e. reverse phase and normal phase mode.

Reverse phase analysis involves use of polar mobile phase (e.g. water, methanol,

acetonitrile, etc.) along with stationary phase like C8, C18, phenyl etc. Normal phase

analysis involves use of non polar solvents (e.g., hexane, dichloromethane, ethyl acetate

etc.) along with silica as a stationary phase. Reverse phase analysis is useful for polar

compounds (e.g., amines alcohols, acids etc.) while normal phase provides separation of

non polar compounds.

In reverse phase liquid chromatography, increasing the molecular size increases

the hydrophobicity of solutes and results in a greater retention volume. This indicates that

the van der Waals volume is an important property in optimization. Increasing the number

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of substituent with n-electrons and hydrogen bonding increases the solubility in water,

i.e., they increase the polarity of the solutes. This indicates that dipole-dipole and

hydrogen-bonding interactions contribute to hydrophobicity. Therefore, these properties

are important in controlling the retention volume in reverse-phase liquid chromatography.

However, the n-electrons of stationary phase materials such as polystyrene gel and the

hydrogen bonding of non-end capped bonded silica gels also contribute to the retention.

Many compounds can be analysed by both the methods. For a preparative scale

separation, normal phase chromatography is suitable due to the easy removal of solvent.

Gradient elution (change in mobile phase composition with respect to time), temperature

and wavelength programming techniques provide valuable information regarding the

undetected components of a given drug. UV absorbing components are easily detected, if

present in sufficient quantity. Multiple wavelength UV detection program is capable of

monitoring several wavelengths simultaneously.

Photo Diode Array (PDA) detectors are used to record spectro-chromatograms

simultaneously. PDA allows simultaneous collection of chromatograms at different

wavelengths during a single run. Following the run, a chromatogram at any desired

wavelength can be displayed. The UV spectrum of each separated peak is also obtained as

an important tool for selecting an optimum wavelength for HPLC analysis. By examining

UV spectrum for a peak from beginning to end, peak purity can be evaluated.

Fluorescence (FL) detectors are exquisitely sensitive and selective, making it ideal

for trace analysis. The detection is based on analyte fluorescence by applying

monochromatic light of desired wavelength for excitation of sample molecule.

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Refractive Index (RI) detectors is considered as a universal detector since the RI is a

physical property of all compounds, any compound can be detected at moderate levels.

Electrochemical (EC) detectors commonly used in HPLC can be classified

according to their operation:

(i) Direct- Current Amperometry (DCA) and

(ii) Conductivity.

If the analyte is EC active, a DCA detector is usually preferred because sample

derivatization and related problems are usually avoided. EC detection can be performed

in either oxidative reductive mode depending on the nature of analyte. Conductivity

detectors are mainly used in ion chromatography.[29]

1.3.3.2 Ultra Performance Liquid Chromatography (UPLC)

UPLC takes advantage of technological strides made in particle chemistry

performance, system optimization, detector design, and data processing and control.

Using submicron (about 2 micron) particles and mobile phases at high linear velocities,

dramatic increase in resolution, sensitivity, and speed of analysis can be obtained. This

new category of analytical separation science retains the practicality and principles of

HPLC while creating a step function improvement in chromatographic performance.

As illustrated in figure 1.3, as the particle size decreases to less than 2.5 µm there

is not only a significant gain in efficiency, but also the efficiency does not diminish at

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

capacity (number of peaks resolved per unit time) can be extended to new limits, termed

as Ultra Performance Liquid Chromatography or UPLC.

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The promises of the Van Deemter equation cannot be fulfilled without smaller

particles than those traditionally used in HPLC. The design and development of

submicron particles is a significant challenge, and researchers have been active in this

area for some time to capitalize on their advantages.[30-32] Although high efficiency,

non porous 1.5 µm particles are commercially available, they suffer from poor loading

capacity and retention due to low surface area. To maintain retention and capacity similar

to HPLC, UPLC must use novel porous particles that can withstand high pressures. Silica

based particles have good mechanical strength, but can suffer from a number of

disadvantages, which include a limited pH range and tailing of basic analytes. Polymeric

columns can overcome pH limitations, but they have their own issues, including low

efficiencies and limited capacities.

In 2000, a first generation hybrid chemistry that took advantage of the best of both

the silica and polymeric column worlds was introduced, [33, 34] a classical sol-gel synthesis

that incorporates carbon in the form of methyl groups, these columns are mechanically

strong, with high efficiency, and operate over an extended pH range. But, in order to

provide the kind of enhanced mechanical stability required for UPLC, a second

generation bridged ethane hybrid (BEH) technology was developed [35]. These 1.7 µm

particles derive their enhanced mechanical stability by bridging the methyl groups in the

silica matrix.

Packing 1.7 µm particles into reproducible and rugged columns was also a

challenge that needed to be overcome. Requirements include a smoother interior surface

of the column hardware, and redesigning the end frits to retain the small particles and

resist clogging. Packed bed uniformity is also critical, especially if shorter columns are to

maintain resolution while accomplishing the goal of faster separations.

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Fig. 1.3 Van Deemter plot, illustrating the evolution of particle sizes over the years

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In addition, at high backpressure, frictional heating of the mobile phase can be

quite significant and must be considered[36] with column diameters typically used in

HPLC (3.0 to 4.6 mm). A consequence of frictional heating is the loss of performance due

to temperature induced non uniform flow. To minimize the effects of frictional heating,

smaller diameter columns (1–2.1 mm) are typically used for UPLC. [37, 38]

Faster separations can lead to higher throughput and time savings when running

multiple samples. But, a significant amount of time can also be consumed in developing

the method in the first place. Faster, higher resolution UPLC separations can cut method

development time from days, to hours, or even minutes.

Mass spectrometry (MS) has gained widespread acceptance as an analytical tool

for the qualitative and quantitative analysis of many types of compounds. MS detection is

significantly enhanced by UPLC, increased peak concentrations with reduced

chromatographic dispersion at lower flow rates (no flow splitting) promotes increased

source ionization efficiencies. Jorgenson et al. have shown that higher chromatographic

efficiency, resulting from the use of UPLC, translates into better resolution and higher

peak capacity, which is particularly important for the analysis of peptides and proteins.[39]

The increased resolving power made the resulting data easier to interpret, since

more of the MS peaks consisted of a single compound, and up to a 20 fold improvement

in the quality of the spectral information due to Nano electrospray was obtained.

Lee et al.,[40] used MS detection for the analysis of low molecular weight compounds

similar to those that might comprise a combinatorial library.

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It was demonstrated that, in order to address the very narrow peaks produced by

UPLC, it is necessary to use a very high data capture rate MS such as a TOF or

quadrupole with fast scan rates. Lee et al., also pointed out that, in some instances, related

compounds of the same molecular weight and similar structures could not be

differentiated by MS, necessitating chromatographic resolution on the UPLC time scale.

Plumb et al. have investigated the use of UPLC/MS for the analysis of metabolites[41, 42]

and as a tool for differential metabolic pathway profiling in functional genomics

studies.[43] Their data illustrate the benefit obtained from the extra resolution of UPLC,

both in terms of specificity and spectral quality, revealing new information and reducing

the risk of not detecting potentially important metabolites.

1.3.4 Isolation techniques

It is often necessary to isolate impurities for their structural elucidation and

qualification. Generally chromatographic and non chromatographic techniques are used

for isolation of impurities prior to characterization.[44] A list of techniques that can be

used for isolation of impurities is given below.

(i) Solid phase extraction (ii) Liquid-liquid extraction (iii) Accelerated Solvent

extraction (iv) Supercritical fluid extraction (v) Column chromatography (vi) Preparative

HPLC (vii) Preparative TLC and (viii) Flash chromatography

Since preparative HPLC was used in the present investigation, this technique

alone is described in detail as under

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Preparative HPLC is the most powerful and commonly used technique for

isolation and purification tasks in the pharmaceutical industry.[45] Application of this

technique comes into picture when the identification of impurity cannot be carried with

acceptable certainty by use of simple analytical (chromatographic, spectroscopic and

hyphenated) techniques. In this case, preparative HPLC isolation followed by

spectroscopic (NMR, MS and IR) investigation provides an appropriate solution for

structural elucidation of any unknown impurity.

In order to carry out successful isolation of targeted impurity, an appropriate

analytical LC method needs to be developed for its detection. The HPLC and TLC

information is very important for development preparative HPLC.

Selection of input sample is also important for isolation of impurities. If the

material contains only small amounts of impurities, it is advisable to select crude samples

or mother liquors which are obtained from recrystallization process, as an input sample

wherein the targeted impurities are likely to be high in concentration. Extraction or

desalting procedures are to be adopted to remove any buffers from the collected fraction.

Rotary evaporator or freeze drying technique can be implemented for concentration of

aqueous/organic fractions during work up process to get solid impurities.

1.3.5 Characterization techniques (Spectroscopic and Hyphenated Techniques)

Structural characterization of unknown impurities is a challenging task in

Pharmaceutical Analysis. Ultraviolet (UV), Fourier Transform Infrared (FT-IR), Mass

Spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (NMR) are used for

the characterization of synthesized, isolated and degradation impurities.

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FT-IR spectroscopy gives information about the functional groups present in the

molecule and used as a valuable tool in the structure elucidation in combination with

other spectral techniques. UV absorption spectroscopy can characterize those types of

compounds which absorbs UV radiation. Identification is done by comparing the

absorption spectrum with the spectra of known compounds and generally used for

characterizing aromatic compounds and aromatic olefins. It gives useful information

about the presence or absence of unsaturation and the presence of hetero atoms.[46]

Liquid Chromatography-Mass Spectrometry[47] (LC-MS) is an analytical

technique that couples high resolution chromatographic separation with sensitive and

specific mass spectrometric detection. This includes high performance liquid

chromatography (HPLC)-MS, Capillary Electrophoresis (CE)-MS and more recently

Capillary Electro Chromatography (CEC)-MS. The technique is still fast developing,

particularly in the mass spectrometry area, with vastly improved sensitivity and

resolution. It is probably the most powerful technique currently available for

pharmaceutical analysis.

Since impurities and degradants are usually present in relatively small quantities

compared to the drug, an analytical technique capable of separating a mixture containing

highly varied concentrations of analytes with sensitive and specific detection is required.

LC-MS is therefore widely used for this purpose. LC-MS has proved to be an extremely

sensitive and specific technique for the analysis of pharmaceuticals. It plays important

roles in the studies of drug metabolism,[48] discovery of new drug candidates and the

analysis, identification and characterization of impurities and degradants in drug

substances and products. Technical advances in MS continue, with improvements in

sensitivity and resolution. The trend is towards the further development of hybrid

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instruments such as Q-TOF, FT-ICR will become more prominent as developments are

made and instruments become less complex and more available. The likely importance of

proteomics in pharmaceutical development will have implications for MS, leading to the

further requirements for high resolution sequencing. Coupling high-throughput sample

preparation techniques with multiplexed LC-MS-MS will lead to even faster analysis and

the potential of interfacing LC-NMR with MS to give an LC-NMR-MS system will allow

the unequivocal identification of drugs and metabolites. .[49,50] LC-MS-MS in turn gives

very useful information about the structure of the molecules by means of selective

fragmentation. Advance instrumentation techniques like Ion Trap will help in structural

identification by the fragmenting molecule up to MS8. The technical difficulties in linking

micro and capillary separation techniques with nanospray MS are being solved and

advances in this front can be expected. The use of microfluidic systems offers prospects

for miniaturized chip separations and even the possibility of miniaturized mass

spectrometers in the rather more distant future.

NMR is the most widely used technique for structural elucidation of synthesized

and isolated organic molecules. For identification and characterization of drug impurities

modern NMR offers various ranges of experiments[51] like 1H, 13C, DEPT, NOE, COSY,

Homo and Hetero Nuclear Irradiation and LRC (Long Range Correlation experiments).

Structural elucidation of impurities in drug materials mostly involve 1H, D2O Exchange

1H and 13C experiments, the information obtained from these experiments is sufficient to

ascertain the structure of the unknown impurity in the drug material along with the other

advanced 2D NMR techniques such as Correlation spectroscopy (1H-1H COSY and

1H-13C HSQC) etc.,[52] The 15N, 19F and 31P in special cases are powerful tools for the

molecules containing the respective elements.

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The introduction of advanced NMR probes especially for on-line coupling to

HPLC greatly reduces the need for preparative isolation of impurities. Stop-flow and

on-flow techniques are used to detect the analytes of interest.[53] HPLC analysis is carried

out in the reverse phase mode using D2O/buffer, acetonitrile based eluents with a higher

injection volume (50-100 µl).

1.4 Analytical methods

Pharmaceutical analysis[54] deals not only with medicaments (drugs and their

formulations) but also with their precursors i.e. with the raw material on which degree of

purity and the quality of medicament depend. The quality of a drug is determined after

establishing its authenticity by testing its purity and the quality of pure substance in the

drug and its formulations. Quality[55] is important in every product or service but it is

vital in medicine as it involves life. Unlike ordinary consumer goods there can be no

“second quality” in drugs. Quality control is a concept, which strives to produce a perfect

product by series of measures designed to prevent and eliminate errors at different stages

of production.

The ability to provide timely, accurate, and reliable data is central to the role of

analytical chemists and is especially true in the discovery, development, and manufacture

of pharmaceuticals. Analytical data are used to screen potential drug candidates, aid in the

development of drug synthesis, support formulation studies, monitors the stability of bulk

pharmaceuticals and formulated products, and test final products for release. The quality

of analytical data is a key factor in the success of a drug development program.

The process of method development and validation has a direct impact on the quality of

these data.

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Analytical method is a specific application of a technique to solve an analytical

problem. The use of instrumentation is an exciting and fascinating part of chemical

analysis that interacts with all areas of chemistry and with many other areas of pure and

applied science. Analytical instrumentation plays an important role in the production and

evaluation of new products and in the protection provides the lower detection limits

required to assure safe foods, drugs, water and air. Instrument or physicochemical

methods are based on the theory of relation between the content and the corresponding

physicochemical and physical properties of the chemical system being analyzed.

Physicochemical methods [56, 57] are used to study the physical phenomena that occur as a

result of chemical reactions. Changes in the system properties are either detected or

recorded through the measurement of current, electrode potential, electrical conductivity,

optical density, refractive index etc., with suitable and sensitive instruments. In

instrument analysis physical property of a substance is measured to determine its

chemical composition. The instrument is only one compound of the total analysis. Often

it is necessary to use several instrumental techniques to obtain the information required to

solve the analytical problem. Instrumental methods may be used by analytical chemists to

save time, to avoid chemical separations or to obtain increased accuracy. The time saving

feature can be realized in routine analysis, or where a considerable number of

determinations are to be made. Most important techniques fit into one of the three

principal areas: spectroscopy, electrochemistry and chromatography.

The analytical method plays a vital role in the dossier submission and it becomes

inevitable to comply with the regulatory requirements. The intricacy of analytical

methods such as assay and related substances remains challenging to the scientists.

Regulatory authority states that the analytical method which is employed for the assay of

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drug substance and drug product should be stability indicating. The stability indicating

assay method may have the advantage of evaluation of degraded components in the

presence of active pharmaceutical substance. The analytical method involved in the

related substances should be sensitive to estimate the synthetic or degraded impurities in

the level of 0.03% and any potential impurities less than 0.03%. The analytical method

for the related substances paves the way for the impurity profiling, elucidation of

degradation pathway, mass balance etc. The dosage forms including the presence of

various excipients pose challenge during the development of assay procedure. Standard

analytical procedures for the estimation of new drug substance for assay, related

substances, dissolution may not be available in Pharmacopoeia; hence it becomes

essential to develop newer analytical methods in such a situation. The estimation of

degraded and synthetic impurities in the presence of the analytes can be analyzed by

HPLC[58], HPTLC, LC-MS, LC-MS/MS, GC[59], GC-MS, CE and CE-MS techniques.

HPLC method is used because of several advantages like rapidity, specificity, accuracy,

precision and ease of automation has the advantage over the other methods.

The chromatographic methods are characterized by high sensitivity, selectivity

and economical consumptions of chemicals. Some of the advantages of HPLC methods

are speed (analysis can be accomplished in 20 minutes or less), greater sensitivity

(various detectors can be employed), improved resolution (wide variety of stationary

phases), reusable columns (expensive columns but can be used for many samples), ideal

for the substances of low volatility, easy sample recovery, handling and maintenance,

instrumentation lends itself to automation and quantitation (less time and less labor),

precise and reproducible, calculations are done by software itself and suitable for

preparative liquid chromatography on a much larger scale.

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Specific stability indicating assay method can be defined as a method that is, able

to measure unequivocally the drug in the presence of all degradation products, excipients

and additives, expected to be present in the formulation. The in vitro evaluation of any

drug substance(s) and drug product(s) requires sensitive analytical methods. Dissolution

testing is an important parameter to assess the drug product for its quality control and in

vivo behaviour. The goal of dissolution testing is to ensure the pharmaceutical quality of

product(s). It would therefore, be desirable to develop dissolution test that can assess the

ability of dosage form to release the drug completely and to simultaneously indicate how

a product will perform in vivo. Dissolution testing is a vital parameter in the stability

studies. Stability conditions (accelerated, intermediate and long term stability studies) can

vary the drug content and its release pattern. Stability studies are carried out to predict

the shelf life, container closure system and expiry of the drug product(s).

1.4.1 Method Development

The development of any new or improved method for the analysis of an analyte

usually depends on tailoring the existing analytical approaches and instrumentation.

Method development [60, 61] usually involves selecting the method requirements and on the

type of instrumentation. Today the development of a method for analysis is usually based

on prior art or existing literature, using the same or quite similar instrumentation. It is rare

today that an HPLC based method is developed that does not in some way relate or

compare to existing, literature based approaches. The development of any new or

improved method usually tailors existing approaches and instrumentation to the current

analyte, as well as to the final needs or requirements of the method. Method development

usually requires selecting the method requirements and deciding on what type of

instrumentation to utilize and why. In the development stage, decisions regarding choice

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of column, mobile phase, detectors, and method of quantitation must be addressed. In

this way, development considers all the parameters pertaining to any method. There are

several valid reasons for developing new method of analysis:

a) There may not be a suitable method for a particular analyte in the specific sample

matrix.

b) Existing method may be inaccurate, contamination prone, or they may be

unreliable (have poor accuracy or precision).

c) Existing methods may be too expensive, time consuming or energy intensive, or

they may not be easily automated.

d) Existing methods may not provide adequate sensitivity or analyte selectivity in

samples of interest.

e) Newer instrumentation and techniques may have evolved that provide

opportunities for improved methods, including improved analyte identification or

detection limits, greater accuracy or precision, or better return on investment.

f) There may be a need for an alternative method to confirm analytical data

originally obtained by existing methods for legal or scientific reasons.

Once the instrumentation has been selected, based on the criteria suggested above,

it is important to determine, “analyte parameters” of interest. To develop a method it is

necessary to consider the properties of the analytes of interest that may be advantageous

to establish optimal ranges of analyte parameter values. It is important that method

development is to be performed using only analytical standards that have been identified

and characterized well, and whose purity is already known. Such precautions will prevent

problems in the future and will remove variables when one is trying to optimize or

improve initial conditions during method development.

Chapter 1

26

Before starting the any method development one has to have knowledge about the

information of the nature of sample, define separation goals, number of compounds

present, chemical structures, molecular weights, pKa values, solubility and UV spectrum

of the compounds. Perhaps maximum method development involves the trial and error

procedures. The most difficult problem usually is where to start, what type of column is

worth trying with what kind of mobile phase. While there are a number of HPLC methods

available to the development chemist, perhaps the most commonly applied method is

reverse phase chromatography method. A typical pharmaceutical compound is considered

to be an active pharmaceutical ingredient (API) of less than 1000 Daltons, either soluble

in water or in an organic solvent.

The water soluble drug is further differentiated as ionic or nonionic which can be

separated by reverse phase. Similarly, the organic soluble drugs can be classed as polar

and non polar and equally separated by reverse phase. In some cases the non polar API

may have to be separated using adsorption or normal phase HPLC, in which mobile phase

would be non polar organic solvent. The other chromatographic modes may need to be

necessary for separation. These include ion exchange, chiral and size exclusion

chromatography. In case of samples like proteins, peptides nuclic acids and synthetic

polymers analysed by using the some special columns or ion pair reagents.

1.4.1.1. General conditions to initiate HPLC method development

Either isocratic or gradient mode may be used to determine the initial conditions

of the separation. In general, one begins with reverse phase chromatography, when the

compounds are hydrophilic in nature with many polar groups and are water soluble. The

organic phase concentration required for the mobile phase can be estimated by gradient

Chapter 1

27

elution method. For aqueous sample mixtures, the best way to start is with gradient

reverse phase chromatography. Gradient can be started with 5-10% organic phase in the

mobile phase and the organic phase concentration can be increased up to 100%

within 20-30 minutes. separation can then be optimized by changing the initial mobile

phase composition and the slope of gradient according to the chromatogram obtained

from preliminary run. The initial mobile phase composition can be estimated on the basis

of where the compounds of interest were eluted, namely at what mobile phase

composition. Changing the polarity of a mobile phase can alter elution of drug molecules.

The elution strength of a mobile phase depends upon its polarity, the stronger the polarity,

higher is the elution. Ionic samples (acidic and basic) can be separated, if they are present

in the undissociated form. Dissociation of ionic samples may be suppressed by proper

selection of pH. The buffer selected for a particular separation should be used to control

pH over the range of ≈ pKa ± 2.0. The buffer should transmit light at or below 200 nm so

as to allow low UV detection and pH of the buffer should be adjusted before adding

organic. Optimization can be started only after a reasonable has been obtained. A

reasonable chromatogram means that more or less symmetrical peaks on the

chromatogram with detection of all the compounds. The optimized chromatogram is the

one in which all the peaks are symmetrical and are well separated in less run time.

The peak resolution can be increased by using a more efficient column (column

with higher theoretical plate number), which can be achieved by using a column of

smaller particle size, or a longer column in length. These factors, however, will increase

the analysis time. Flow rate does not influence resolution, but it has a strong effect on the

analysis time.The parameter that are affected by the changes in chromatographic

Chapter 1

28

conditions are Capacity factor (K’), Selectivity (α), Column efficiency (N) and Peak

asymmetry factor or Tailing factor (As)

1.4.1.2. Selection of mobile phase

The selection of the mobile phase mainly based on the solubility and polarity of

the compound. Usually, in RP-HPLC method water and organic solvents are used as the

mobile phase. In NP-HPLC method non polar solvents like Hexane and THF were used.

If the sample contains ionic or ionizable compounds, then use of a buffered mobile phase

to ensure the reproducible results. Under unfavorable circumstances, pH changes as little

as 0.1 pH units can have a significant effect on the separation. On the other hand properly

used buffer allows controlling the pH easily. Buffer works best at the pKa values of its

acid. At this pH, the concentration of the acidic form and the basic form of the buffering

species are equal, and the buffering capacity is maximum. Phosphate has three pKa

values in the range of interest for silica based chromatography at pH 2, pH 7 and

pH 12.32. The pKa of acidic buffer is 4.75. Citrate has three pKa values 3.08, 4.77

and 6.40. Between citrate and phosphate buffers, the entire pH range useful for silica

chromatography can be covered.

In many cases, a silanophilic interaction causes tailing, mainly for the basic

compounds due to ion-exchange interaction. This can usually be reduced or suppressed

by the use of mobile phases modifiers (0.1% v/v triethylamine for basic analyte or 1% v/v

glacial acetic acid for the acidic analyte), or a combination thereof. Whenever buffers or

other mobile phase activities are used, check the solubility in mobile phase. This is

especially true for gradient applications. acetonitrile is the preferred organic modifier in

reverse phase chromatograpy. acetonitrile based mobile phases can give up to two fold

Chapter 1

29

lower pressure drop that can methanol based mobile phases at equal flow rate. This means

that column efficiency is higher. The elution strength increases in the order methanol,

acetonitrile and tetrahydrofuran. The retention changes by roughly 10% for every 1%

change in the concentration of organic modifier.

1.4.1.3. Role of pH

pH is another factor in the resolution that will affect the selectivity of the

separation in reverse-phase HPLC. In reverse-phase chromatography sample retention

(K’) increases when the analyte is more hydrophobic (nonpolar). Sample retention (K’)

decreases when the analyte is more hydrophilic (polar). Thus when an acid or base is

undergoes ionization it becomes more hydrophilic and less interacting with column

binding sites. When the pH value of the mobile phase equal to the pKa value of the

analyte, it is said to be half ionized, i.e. the concentration of the ionized and unionized

species are equal. As mostly all of the pH caused changes in the retention occur

within ± 2.0 pH unit of the pKa value, it is best to adjust the mobile phase to pH value

atleast ± 2.0 pH unit above or below the pKa to ensure practically 100% unionization of

analyte for retention purpose. Generally at low pH peak tailing is minimized and method

ruggedness is maximized. On the other hand, operating in the intermediate pH offers an

advantage in increased analyte retention and selectivity.

1.4.1.4. Role of buffer

In reverse phase mobile phase pH values are usually between 2.0 and 7.5. Buffers

are needed when an analyte is ionizable under reverse phase conditions or the sample

solution is outside this pH range. Analyte ionisable under reverse phase conditions often

have amine or acid functional group with pKa between 1.0 and 11.0. A correctly chosen

Chapter 1

30

buffer pH will ensure that the ionisable functional group is in a single form, whether ionic

or neutral. If the sample solution is at pH damaging to the column, the buffer will quickly

bring the pH of the injected solution to a less harmful pH.

If the analyte contain only amine fuctional group buffer selection is easier. Most

amine will be in cationic form at pH value less than 9.0, so any buffer effective at pH 7.0

or lower will work. Buffer at pH 7.0 are used, even though pH of water is 7.0, because

amine retention and peak shapes are pH dependent. As pH is lowered amine retention

time shortens and peak shaps sharpens as the buffer protonates the acidic silanols on silica

surface. Any buffer with pKa less than 7.0 is suitable, but we have found potassium

phosphate at pH 3.0 in the best for amines. In both condition (acidic and alkaline)

potassium phosphate buffer pH 3.0 works well in general is an excellent buffer for

analyte that contain acidic and amine functional groups. The potassium salt works better

than the sodium salt for amines.

1.4.1.5. Selection of column

The HPLC column is the heart of the method, critical performing the separation.

The column must posses the selectivity, efficiency and reproducibility to provide good

separation. Commonly used reverse phases are C18 (octadecyl silane, USP L1), C8

(octylsilane, USP L7), phenyl (USP L11) and cyno (USP L18). They are chemically

different bounded phases and demonstrate significant changes in the selectivity using the

same mobile phase.

During method development selection of column can be streamlined by starting

with shorter column (150, 100 or even 50 mm long). By selecting a shorter column with

an appropriate phase run time can be minimized so that an elution order and an optimum

Chapter 1

31

mobile phase can be quickly determined. It can also advantageous to consider the column

internal diameter, many laboratories use 4.6 mm i.d. as standard. But it is worth

considering use of 4.0 mm i.d. column as an alternative. This requires only 75% of the

solvent flow that a 4.6 mm column used. Selecting an appropriate stationary phase can

also help to improve the efficiency of the method development. For example, a C8 phase

(reverse phase) can provide a further time saving over a C18 as it doesn’t retain analyte as

strongly as the C18 phase.

1.4.1.6. Role of temperature

Temperature variation over the course of a day has quite significant effect on

HPLC separations. This can even occur in air conditioned rooms. While temperature is a

variable that can affect the selectivity, its effect is relatively small.

1.4.1.7. Role of flow rate

Flow rate, more for isocratic than gradient separation, can sometimes be useful and

readily utilized to increase the resolution, although its effect is very modest. The slower

flow rate will also decrease the column back pressure.

1.4.2 Validation of Analytical Methods

Validation of analytical method is an integral part of product development. The

developed analytical methods are validated in order to demonstrate that it is suitable for

its intended purpose. The analytical methods were validated for the following parameters

in accordance with ICH Harmonized Tripartite Guidelines.[22, 62, 63]

Chapter 1

32

1.4.2.1 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an accepted

reference value and the value found. This is sometimes termed as trueness of the

analytical method. Accuracy of the analytical method is proved by application of the

analytical procedure to synthetic mixtures of the drug product components to which

known quantities of the drug substance to be analysed have been added.

In cases where it is impossible to obtain samples of all drug product components,

it may be acceptable either to add known quantities of the analyte to the drug product or

to compare the results obtained from a second, well characterized procedure, the accuracy

of which is stated and/or defined. Accuracy may be inferred once precision, linearity and

specificity have been established.

Accuracy should be assessed using a minimum of 9 determinations over minimum

of 3 concentration levels covering the specified range (e.g., 3 concentrations / 3 replicates

each of the total analytical procedure). Accuracy should be reported as percent recovery

by the assay of known added amount of analyte in the sample or as the difference

between the mean and the accepted true value together with the confidence intervals.

1.4.2.2 Precision

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple sampling of

the same homogeneous sample under the prescribed conditions. Precision may be

considered at three levels like repeatability, intermediate precision and reproducibility.

Chapter 1

33

Precision should be investigated using homogeneous and authentic samples. However, if

it is not possible to obtain a homogeneous sample it may be investigated using artificially

prepared samples or a sample solution. The precision of an analytical procedure is usually

expressed as the variance, standard deviation or coefficient of variation of a series of

measurements.

1.4.2.3 Repeatability

Repeatability expresses the precision under the same operating conditions over a

short interval of time. Repeatability is also termed as intra-assay precision. Repeatability

should be assessed using a minimum of 9 determinations covering the specified range for

the procedure (e.g., 3 concentrations /3 replicates each) or a minimum of 6 determinations

at 100% of the test concentration.

1.4.2.4 Intermediate precision

The extent to which intermediate precision should be established depends on the

circumstances under which the procedure is intended to be used. The applicant should

establish the effects of random events on the precision of the analytical procedure.

Typical variations to be studied include days, analysts, equipment, etc., Intermediate

precision expresses within-laboratories variations such as different days, different

analysts, different equipment, etc.

1.4.2.5 Reproducibility

Reproducibility expresses the precision between laboratories (collaborative

studies, usually applied to standardization of methodology).

Chapter 1

34

1.4.2.6 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. The procedures used to demonstrate specificity will

depend on the intended objective of the analytical procedure. It is not always possible to

demonstrate that an analytical procedure is specific for a particular analyte. In this case a

combination of two or more analytical procedures is recommended to achieve the

necessary level of discrimination.

1.4.2.7 Detection Limit

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.

1.4.2.7.1 Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be

used with instrumental methods. The detection limit is determined by the analysis of

samples with known concentrations of analyte and by establishing the minimum level at

which the analyte can be reliably detected.

Chapter 1

35

1.4.2.7.2 Based on Signal-to-Noise

This approach can only be applied to analytical procedures which exhibit baseline

noise. Determination of the signal-to-noise ratio is performed by comparing measured

signals from samples with known low concentrations of analyte with those of blank

samples and establishing the minimum concentration at which the analyte can be reliably

detected. A signal-to-noise ratio of 3 or 2:1 is generally considered acceptable for

estimating the detection limit.

1.4.2.7.3 Based on the Standard Deviation of the Response and the Slope

The detection limit (DL) may be expressed as

DL =3.3 σ

S

Where σ = the standard deviation of the response

S = the slope of the calibration curve

The slope(S) may be estimated from the calibration curve of the analyte. The

estimation of slope may be carried out in a variety of ways, for example:

1.4.2.7.4 Based on the Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by

analyzing an appropriate number of blank samples and calculating the standard deviation

of these responses.

Chapter 1

36

1.4.2.7.5 Based on the Calibration Curve

A specific calibration curve should be studied using samples containing an analyte

in the range of DL. The residual standard deviation of a regression line or the standard

deviation of y-intercepts of regression lines may be used as the standard deviation.

1.4.2.8 Quantitation Limit

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.

1.4.2.8.1 Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be used

with instrumental methods. The quantitation limit is generally determined by the analysis

of samples with known concentrations of analyte and by establishing the minimum level

at which the analyte can be quantified with acceptable accuracy and precision.

1.4.2.8.2 Based on Signal-to-Noise Approach

This approach can only be applied to analytical procedures that exhibit baseline

noise. Determination of the signal-to-noise ratio is performed by comparing measured

signals from samples with known low concentrations of analyte with those of blank

Chapter 1

37

samples and by establishing the minimum concentration at which the analyte can be

reliably quantified. A typical signal-to-noise ratio is 10:1.

1.4.2.8.3 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

The slope(S) may be estimated from the calibration curve of the analyte. The estimation

of slope may be carried out in a variety of ways for example

1.4.2.8.4 Based on Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by

analyzing an appropriate number of blank samples and calculating the standard deviation

of these responses.

1.4.2.8.5 Based on the Calibration Curve

A specific calibration curve should be studied using samples, containing an

analyte in the range of QL. The residual standard deviation of a regression line or the

standard deviation of y-intercepts of regression lines may be used as the standard

deviation.

Chapter 1

38

1.4.2.9 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.

1.4.2.10 Range

The range of an analytical procedure is the interval between the upper and lower

concentration 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.

1.4.2.11 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.

Chapter 1

39

1.4.2.12 Stress degradation methods

The purpose of stability testing is to provide evidence on how the quality of a drug

substance or drug product varies with time under the influence of a variety of

environmental factors such as temperature, humidity, and light, and enables

recommended storage conditions, retest periods, and shelf lives to be established.

Stress testing helps to determine the intrinsic stability of the molecule by

establishing degradation pathways in order to identify the likely degradation products and

to validate the stability indicating power of the analytical procedures used. In principle,

the influence of each factor is first individually explored, then cross-influences are

evaluated. The duration of the study will depend on the studied factor and the sensitivity

of the product to that factor.

1.4.2.12.1 Influence of Temperature

The behaviour of the drug substance under extreme temperatures such as 50° C or

even 70° C should be investigated. The ICH guidelines on stability, recommend a 10°C

increment above the accelerated conditions (40°C).Also sensitivity of drug substance

towards relative humidity (RH) from a dry atmosphere up to a water-saturated

atmosphere (from 10 to 90 percent RH) was investigated. Temperature and humidity will

influence the physical stability.

1.4.2.12.2 Influence of Light

Forced degradation testing evaluates the overall photosensitivity of the material

for method development purposes and/or degradation pathway elucidation. The intensity

Chapter 1

40

of light and duration of exposure will vary depending on the photosensitivity.

These results will serve as basis to define conditions such as protect drug substance from

light during analysis, handling or storage.

1.4.2.12.3 Influence of pH

The study of the influence of pH will show the susceptibility of hydrolysis of the

drug substance in acidic or alkaline media. As a first approach, the extreme conditions

could be 1 N hydrochloric acid and 1 N sodium hydroxide. Then, when it is evident that

the product is sensitive to pH variations, a step-by-step approach will start to allow the

degradation in relation to pH. The results of these studies will serve to explain or to better

select some conditions for drug substance and drug product manufacture, as well as for

preformulation studies.

1.4.2.12.4 Influence of Oxygen

Oxygen can be a very critical parameter, since it is not always easy to protect the

drug substance or product against oxidation. The stability of the drug substance as a bulk

or in solution under oxygen, air or nitrogen atmospheres, with high contact between the

drug and the atmosphere, can be studied. The results could be aimed at recommending

protections and preliminary limits for residual oxygen.

Chapter 1

41

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