Chapter 1 Introduction

1. INTRODUCTION (1-2)

Analytical techniques and methods are used for the quality control of pharmaceutical
compounds and thereby assure patient safety and efficacy, they have become an essential part
of pharmaceutical Quality by design. The scientific understanding gained during the method
development process can be used to devise method control elements and to manage the risks
identified. This approach ensures a very high likelihood of method success during the product
lifecycle. Thus, the validation which is usually performed after method development will
serve the purpose of confirming method performance as opposed to identifying potential
problem areas1.Pharmaceutical analysis plays a very significant role in quality control of
pharmaceuticals through a rigid check on raw materials used in manufacturing of
formulations and on finished products. It also plays an important role in building up the
quality products through in process quality control. It also plays a major role in isolation and
characterization of impurities2.
1.1 ANALYTICAL CHEMISTRY (3-4)
Analytical Chemistry is a measurement of science consisting of a set of powerful
ideas and methods that are useful in all fields of science and medicine. It seeks ever improved
means of measuring the chemical composition of natural and artificial materials. This branch
of chemistry, which is both theoretical, and a practical science, is practiced in a large number
of laboratories in many diverse ways while analytical method, is a specific application of a
technique to solve an analytical problem. Methods of analysis are routinely developed,
improved, validated, collaboratively studied and applied. The discipline of analytical
chemistry consists of qualitative and quantitative analysis.
Qualitative analysis – Information regarding the presence or absence of one or more
components of the sample.
Quantitative analysis– Information regarding the amount of components of the sample,
however the required information is finally obtained by measuring some physical property
that is characteristically related to the compound of interest.(Nash et al).

1.1.2. Factors Affecting the Choice of Analytical Method

Analytical techniques have different degrees of sophistication, sensitivity and selectivity, as
well as, different cost and time requirements. An important task for the analyst is to select
best procedure for a given determination this will require careful consideration of the
following criteria:

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Chapter 1 Introduction

a) The type of analysis required: elemental or molecular, routine or occasional.

b) Problem arising from the nature of the material to be investigated, e.g. radio-active
substance, corrosive substance, substances affected by water.
c) Possible interference from components of the material other than those of interest.
d) The concentration range to be investigated.
e) The accuracy required.
f) The facilities available, particularly the instrument.
g) The time required to complete the analysis.
h) The number of analysis of similar type which have to be performed.

1.1.3. Classification of Analytical Methods

The various methods of analysis can be broadly classified into two categories; Classical
methods and Instrumental methods: -

1. Classical Methods
a. Volumetric Methods
In volumetric, also called titrimetric procedures the volume or mass of a standard reagent
required to react completely with the analyte was measured.
b. Gravimetric Methods:
In gravimetric measurements, the mass of the analyte or some compound produced from the
analyte was determined. The extent of their general application is, however, decreasing with
the passage of time.
2. Instrumental Methods
These methods are based upon the measurement of some physical properties as conductivity,
electrode potential, light absorption or emission, mass-to-charge ratio and fluorescence of
substance. There are many techniques available for the analysis of analytes.
a) Spectroscopic Analysis
1. Ultraviolet and visible spectrophotometry,
2. Fluorescence and phosphorescence spectrophotometry,
3. Atomic spectrophotometry (emission & absorption),
4. Infra-red spectrophotometry,
5. Raman spectroscopy,
6. X-ray spectroscopy,
7. Radio chemical techniques including activation analysis,
2
Chapter 1 Introduction

8. NMR spectroscopy,
9. ESR spectroscopy.
b) Electrochemical Techniques
1. Potentiometry,
2. Voltametry,
3. Stripping techniques,
4. Amperometric techniques,
5. Coulometry,
6. Electrogravimetry,
7. Conductance techniques.
c) Chromatographic Methods
1. Gas chromatography (GC),
2. High performance liquid chromatography (HPLC),
3. High-performance thin layer chromatography (HPTLC).
d) Miscellaneous Techniques
1. Thermal analysis,
2. Mass spectrometry,
3. Kinetic techniques.
e) Hyphenated Methods
1. GC-MS,
2. ICP-MS,
3. GC-IR,
4. MS-MS.

Amongst all the techniques mentioned above UV-Visible spectrophotometry, High

performance liquid chromatography (HPLC) and High performance thin layer
chromatography (HPTLC) are the most widely used techniques for quantitative analysis of
pharmaceutical substances.

1.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (5)

The term ‘Chromatography’ covers those processes aimed at the separation of the
various species of a mixture on the basis of their distribution characteristics between a
stationary and a mobile phase.

3
Chapter 1 Introduction

1.3 MODES OF CHROMATOGRAPHY (6)

Modes of chromatography are defined essentially according to the nature of the

interactions between the solute and the stationary phase, which may arise from hydrogen
bonding, Vander walls forces, electrostatic forces or hydrophobic forces are based on the size
of the particles (e.g. Size exclusion chromatography)

Different modes of chromatography are as follows -

▪ Normal Phase Chromatography

▪ Reverse Phase Chromatography

▪ Reverse Phase – ion pair Chromatography

▪ Ion Chromatography

▪ Ion-Exchange Chromatography

▪ Affinity Chromatography

▪ Size Exclusion Chromatography

1.3.1 Reverse Phase Chromatography (7)

Methods can be chosen based on solubility and molecular mass. In most of the cases
for non-ionic small molecules (µ < 2000), reversed phase methods are suitable.

Fig.1 Selection of LC Modes

In 1960’s chromatographers started modifying the polar nature of silanol group by

chemically reacting silica with organic silanes. The objective was to make less polar or non
polar so that polar solvents can be used to separate water-soluble polar compounds.

A large number of chemically bonded stationary phases based on silica are available
commercially. Silica based stationary phases are still most popular in reversed phase

4
Chapter 1 Introduction

chromatography however other absorbents based on polymer (styrene-divinyl benzene

copolymer) are slowly gaining ground.

Simple compounds are better retained by the reversed phase surface, the less water-
soluble (i.e. the more non-polar) they are. The retention decreases in the following order:
aliphatic > induced dipoles (i.e. CCl4) > permanent dipoles (e.g.CHCl3) > weak lewis bases
(ethers, aldehydes, ketones) > strong Lewis bases (amines) > weak Lewis acids (alcohols,
phenols) > strong lewis acids (carboxylic acids). Also the retention increases as the number
of carbon atoms increases.

In reverse phase systems the strong attractive forces between water molecules arising
from the 3-dimentional inter molecular hydrogen bonded network, from a structure of water
that must be distorted or disrupted when a solute is dissolved. Only higher polar or ionic
solutes can interact with the water structure. Non- polar solutes are squeezed out of the
mobile phase and are relatively insoluble in it but with the hydrocarbon moieties of the
stationary phase.

Chemically bonded octadecyl silane (ODS) an alkaline with 18 carbon atoms, it is the
most popular stationary phase used in pharmaceutical industry. Since most pharmaceutical
compounds are polar and water soluble, the majority of HPLC methods used for quality
assurance, decomposition studies, quantitative analysis of both bulk drugs and their
formulations use ODS-HPLC columns. The solvent strength in reverse phase
chromatography is reversed from that of adsorption chromatography (silica gel) as stated
earlier. Water interacts strongly highly with silanol groups, so that, adsorption of sample
molecules become highly restricted and they are rapidly eluted as a result. Exactly opposite
applies in reverse phase system; water cannot wet the non-polar (hydrophobic) alkyl groups
such as C18 of ODS phase and therefore does not interact with the bonded moiety. Hence
water is the weakest solvent of all and gives slowest elution rate. The elution time (retention
time) in reverse phase chromatography increases with increasing amount of water in the
mobile phase.

1.3.2 Adsorption Chromatography /Normal Phase Chromatography (8)

In normal phase chromatography, the stationary phase is a polar adsorbent and the
mobile phase is generally a mixture of non-aqueous solvents.

The silica structure is saturated with silanol groups at the end. These OH groups are
statistically disturbed over the whole of the surface. The silanol groups represent the active

5
Chapter 1 Introduction

sites (very polar) in the stationary phase. This forms a weak type of bond with any molecule
in the vicinity when any of the following interactions are present.

▪ Dipole-induced dipole,

▪ Dipole-dipole,

▪ Hydrogen bonding,

▪ π-Complex bonding

These situations arise when the molecule has one or several atoms with lone pair
electron or a double bond. The absorption strengths and hence k’ values (elution series)
increase in the following order. Saturated hydrocarbon < olefins < aromatics < organic
halogen compounds < sulphides < ethers< esters < aldehydes and ketones < amines <
sulphones < amides < carboxylic acids. The strength of interactions depends not only on the
functional groups in the sample molecule but also on steric factors. If a molecule has several
functional groups, then the most polar one determines the reaction properties.

Chemically modified silica, such as the amino propyl, cyan propyl and diol phases is
useful alternatives to silica gel as stationary phase in normal phase chromatography.

The amino propyl and cyan propyl phases provide opportunities for specific
interactions between analyse and the stationary phases and thus offer additional options for
the optimisations of separations. Other advantages of bonded phases lie in their increased
homogeneity of the phase surface.

Resolution with water in weak mobile phase may be most conveniently achieved by
drying the solvents and then adding a constant concentration of water or some very polar
modifier such as acetic acid or triethylamine (TEA) to the mobile phase. The addition of such
polar modifiers serves to deactivate the more polar shape as well as the reproducibility of the
retention times.

Chromatographic methods can be classified most practically according to the

stationary and mobile phases, as shown in the Table 1 below

Table1. Classification of Chromatographic methods

General Type of
Specific Method Stationary Phase
classification Equilibrium

6
Chapter 1 Introduction

1.Gas a.Gas-liquid Liquid adsorbed or Partition

chromatography bonded to a solid between gas and
Chromatography
(GLC). surface. liquid.

(GC)
b. Gas-solid Solid Adsorption

2.Liquid a. Liquid-liquid, or Liquid adsorbed or Partition

partition bonded to a solid between
Chromatography
surface immiscible
(LC)
liquids

b. Liquid-Solid, or Solid Adsorption

adsorption

c. Ion Exchange Ion Exchange resin Ion Exchange

d. Size Exclusion Liquid in interstices Partition/sieving

of a polymeric solid

e. Affinity Group specific liquid Partition

bonded to a solid between surface
surface liquid and
mobile liquid.

3. Super critical a. Separation and Organic species Partition

Purification bonded to a solid between
Fluid
surface. supercritical
Chromatography
Fluid
andbondedsurfac

7
Chapter 1 Introduction

1.4 HPLC SYSTEM (9-15)

The components of a basic High Performance Liquid chromatography (HPLC) system

are shown in the simple diagram below:

Fig. 2. HPLC System

The importance of Chromatography is increasing rapidly in pharmaceutical analysis. The

exact differentiation selective identification and quantitative determination of structurally
closely related compounds. Another important field of application of chromatographic
methods is the purity testing of final products and intermediates (detection of decomposition
products and by-products). As a consequence of the above points, chromatographic methods
are occupying an ever-expanding position in the latest editions of the pharmacopoeias and
other testing standards.

The modern form of column chromatography has been called high performance, high
pressure, and high-resolution and high-speed liquid chromatography.

High-Performance Liquid Chromatography (HPLC) is a special branch of column

chromatography in which the mobile phase is forced through the column at high speed. As a
result the analysis time is reduced by 1-2 orders of magnitude relative to classical column
chromatography and the use of much smaller particles of the adsorbent or support becomes
possible increasing the column efficiency substantially.

The essential equipment consists of an eluent, reservoir, a high-pressure pump, and an

injector for introducing the sample, a column containing the stationary phase, a detector and
recorder. The development of highly efficient micro particulate bonded phases has increased

8
Chapter 1 Introduction

the versatility of the technique and has greatly improved the analysis of multi component
mixtures.

The systems used are often described as belonging to one of four mechanistic types,
adsorption, partition, ion exchange and size-exclusion. Adsorption chromatography arises
from interaction between solutes on the surface of the solid stationary phase. Partition
chromatography involves a liquid stationary phase, which is immiscible with the eluent and
coated on an inert support. Adsorption and partition systems can be normal phase (stationary
phase more polar than eluent) or reversed phase (stationary phase less polar than eluent). Ion-
exchange chromatography involves a solid stationary phase with anionic or cationic groups
on the surface to which solute molecules of opposite charge are attracted. Size-exclusion
chromatography involves a solid stationary phase with controlled pore size. Solutes are
separated according to their molecular size, the large molecules enable to enter the pores
eluting first.

The various components of a HPLC system are herewith described.

Fig.3. Instrumentation (Components) of HPLC System

1.4.1 System Components

1.4.1.1 Solvent delivery system

The mobile phase is pumped under pressure from one or several reservoirs and flows
through the column at a constant rate. With micro particulate packing, there is a high-pressure

9
Chapter 1 Introduction

drop across a chromatography column. Eluting power of the mobile phase is determined by
its overall polarity, the polarity of the stationary phase and the nature of the sample
components. For normal phase separations eluting power increases with increasing polarity of
the solvent but for reversed phase separations, eluting power decreases with increasing
solvent polarity. Optimum separating conditions can be achieved by making use of mixture of
two solvents. Some other properties of the solvents, which need to be considered for a
successful separation, are boiling point, viscosity, detector compatibility, flammability and
toxicity.

The most important component of HPLC in solvent delivery system is the pump,
because its performance directly effects the retention time, reproducibility and detector
sensitivity. Among the several solvent delivery systems (direct gas pressure, pneumatic
intensifier, reciprocating etc.) reciprocating pump with twin or triple pistons is widely used,
as this system gives less baseline noise, good flow rate reproducibility etc.

1.4.1.2 Solvent degassing system

The constituents of the mobile phase should be degassed and filtered before use.
Several methods are employed to remove the dissolved gases in the mobile phase. They
include heating and stirring, vacuum degassing with an aspirator, filtration through 0.45 filter,
vacuum degassing with an air-soluble membrane, helium purging ultra sonication or purging
or combination of these methods. HPLC systems are also provided an online degassing
system, which continuously removes the dissolved gases from the mobile phase.

1.4.1.3 Gradient elution devices

HPLC columns may be run isocratically, i.e., with constant eluent or they may be run
in the gradient elution mode in which the mobile phase composition varies during run.
Gradient elution is a means of overcoming the problem of dealing with a complex mixture of
solutes.

1.4.1.4 Sample introduction systems

Two means for analyte introduction on the column are injection in to a flowing stream
and a stop flow injection. These techniques can be used with a syringe or an injection valve.
Automatic injector is a microprocessor-controlled version of the manual universal injector.
Usually, up to 100 samples can be loaded in to the auto injector tray. The system parameters
such as flow rates, gradient, run time, volume to be injected, etc. are chosen, stored in
memory and sequentially executed on consecutive injections.

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Chapter 1 Introduction

1.4.1.5 Liquid chromatographic detectors

The function of the detector in HPLC is to monitor the mobile phase as it emerges
from the column. Generally, there are two types of HPLC detectors, bulk property detectors
and solute property detectors.

1.4.1.5.1 Bulk property detectors

These detectors are based on differential measurement of a property, which is

common to both the sample and the mobile phase. Examples of such detectors are refractive
index, conductivity and dielectric constant detectors.

1.4.1.5.2 Solute property detectors

Solute property detectors respond to a physical property of the solute, which is not
exhibited by the pure mobile phase. These detectors measure a property, which is specific to
the sample, either with or without the removal of the mobile phase prior to the detection.
Solute property detectors which do not require the removal of the mobile phase before
detection include spectrophotometric (UV or UV-Vis) detector, fluorescence detectors,
polarographic, electro-chemical and radio activity detectors, whilst the moving wire flame
ionisation detector and electron capture detector both require removal of the mobile phase
before detection.

UV-Vis and fluorescent detectors are suitable for gradient elution, because many
solvents used in HPLC do not absorb to any significant extent.

1.4.1.6 Column and Column-packing materials

The heart of the system is the column. In order to achieve high efficiency of
separation, the column material (micro-particles, 5-10 μm size) packed in such a way that
highest numbers of theoretical plates are possible.

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Chapter 1 Introduction

Fig.4. Column Used In HPLC System

Silica (SiO2 x H2O) is the most widely used substance for the manufacture of packing
materials. It consists of a network of siloxane linkages (Si-O-Si) in a rigid three dimensional
structure containing inter connecting pores. Thus a wide range of commercial products is
available with surface areas ranging from 100 to 800 m2/g. and particle sizes from 3 to 50
μm.

The silanol groups on the surface of silica give it a polar character, which is exploited
in adsorption chromatography using non-polar organic eluants. Silica can be drastically
altered by reaction with Organochloro-silanes or Organoalkoxy silanes giving Si-O-Si-R
linkages with the surface. The attachment of hydrocarbon change to silica produces a non-
polar surface suitable for reversed phase chromatography where mixtures of water and
organic solvents are used as eluants.

The most popular material is octadecyl-silica (ODS-Silica), which contains C 18

chains, but materials with C2, C6, C8 and C22 chains are also available. During manufacture,
such materials may be reacted with a small mono functional silane (e.g. trimethylchloro
silane) to reduce further the number of silanol groups remaining on the surface (end-capping).
There is a vast range of materials which have intermediate surface polarities arising from the
bonding to silica of other organic compounds which contain groups such as phenyl, nitro,
amino and hydroxyl. Strong ion exchangers are also available in which sulphonic acid groups
or quaternary ammonium groups are bonded to silica. The useful pH range for columns is 2
to 8, since siloxane linkages are cleaved below pH-2 while at pH-8 or values above silica
may dissolve.

In HPLC, generally two types of columns are used, normal phase columns and reverse
phase columns. Using normal phase chromatography, particularly of non-polar and
moderately polar drugs can make excellent separation. It was originally believed that
separation of compounds in mixture takes place slowly by differential adsorption on a

12
Chapter 1 Introduction

stationary silica phase. However, it now seems that partition plays an important role, with the
compounds interacting with the polar silanol groups on the silica or with bound water
molecules.

While normal phase seems the passage of a relatively non-polar mobile phase over a
polar stationary phase, reversed phase chromatography is carried out using a polar mobile
phase such as methanol, acetonitrile, water, buffers etc., over a non-polar stationary phase.
Ranges of stationary phases (C18, C8, -NH2, -CN, -phenyl etc.) are available and very selective
separations can be achieved. The pH of the mobile phase can be adjusted to suppress the
ionisation of the drug and thereby increase the retention on the column. For highly ionised
drugs ion-pair chromatography is used.

1.4.1.7 Derivatization

In HPLC derivatization is used to enhance the sensitivity and selectivity of detection

when available detectors are not satisfactory for the underivatized compounds. Both ultra
violet absorbing and fluorescence derivatives have been widely used. Ultra violet
derivatization reagents include N-succinimidyl p-nitro phenyl acetate, phenyl hydrazine and
3, 5-dinitro benzyl chlorides, while fluorescent derivatives can be formed with reagents such
as dansyl chloride, 4-bromo methyl-7-methoxy-coumarin and fluorescamine. Derivative
formation can be carried out before the sample is injected on to the column or by online
chemical reactions between the column outlet and the detector.

1.5 STRATEGY FOR METHOD DEVELOPMENT OF HPLC

Selection of suitable chromatography for organic compounds,
▪ First reverse phase should be tried.
▪ If not successful, then, normal phase should be taken into consideration.
Before making experimentation with ion-exchange or ion-pair chromatography, ion
suppression by pH controls and reverse phase chromatography should be tried for ion
forming organic compounds. Ion-pair chromatography should be preferred to Ion Exchange
chromatography.

1.6 METHOD DEVELOPMENT AND OPTIMIZATION ( 15-20)

During the optimization stage, the initial sets of conditions that have evolved from the
first stages of development are improved or maximized in terms of resolution and peak shape,
plate counts asymmetry, capacity, elution time, detection limits, limit of quantitation, and
overall ability to quantify the specific analyte of interest.

13
Chapter 1 Introduction

Optimization of a method can follow either of two general approaches -

1. Manual

2. Computer driven

The manual approach involves varying one experimental variable at a time, while
holding all others constant, and recording changes in response. The variables might include
flow rates, mobile or stationary phase composition, temperature, detection wavelength, and
pH this univariate approach to system optimization is slow, time consuming and potentially
expensive. However, it may provide a much better understanding of the principles and theory
involved and of interactions of the variables.

In the second approach, computer driven automated methods development,

efficiency is optimized while experimental input is minimized. Computer driven automated
approaches can be applied to many applications. In addition, they are capable of
significantly reducing the time, energy and cost of virtually all-instrumental methods
development.

The various parameters that include to be optimized during method development,

1. Mode of separation

2. Selection of stationary phase

3. Selection of mobile phase

4. Selection of detector

1.6.1 Selection of mode of separation

In reverse phase mode, the mobile phase is comparatively more polar than the
stationary phase. For the separation of polar or moderately polar compounds, the most
preferred mode is reverse phase. The nature of the analyte is the primary factor in the
selection of the mode of separation. A second factor is the nature of the matrix.

1.6.2 Selection of stationary phase / column

Selection of the column is the first and the most important step in method
development .The appropriate choice of separation column includes three different
approaches

Selection of separation system

1. The particle size and the nature of the column packing

14
Chapter 1 Introduction

2. The physical parameters of the column i.e. the length and the diameter

3. Some of the important parameters considered while selecting chromatographic columns

are

▪ Length and diameter of the column.

▪ Packing material.

▪ Shape of the particles.

▪ Size of the particles.

▪ % of Carbon loading

▪ Pore volume.

▪ Surface area.

▪ End capping.

The column is selected depending on the nature of the solute and the information
about the analyte. Reversed phase mode of chromatography facilitates a wide range of
columns like dimethyl silane (C2), butylsilane (C4), octylsilane (C8), octadecylsilane (C18),
base deactivated silane (C18) BDS phenyl, cyanopropyl (CN), nitro, amino etc. C 18 was
chosen for this study since it is most retentive one. The sample manipulation becomes easier
with this type of column.

Generally longer columns provide better separation due to higher theoretical plate
numbers. As the particle size decreases the surface area available for coating increases.
Columns with 5-µm particle size give the best compromise of efficiency, reproducibility and
reliability. In this case, the column selected had a particle size of 5 µm and an internal
diameter of 4.0 mm.

Peak shape is equally important in method development. Columns that provide

symmetrical peaks are always preferred while peaks with poor asymmetry can result in,

▪ In accurate plate number and resolution measurement

▪ Imprecise quantisation

▪ Degraded and undetected minor bands in the peak tail

▪ Poor retention reproducibility

A useful and practical measurement of peak shape is peak asymmetry factor and peak
tailing factor. Peak asymmetry is measured at 10% of full peak height and peak tailing factor

15
Chapter 1 Introduction

at 5%. Reproducibility of retention times and capacity factor is important for developing a
rugged and repeatable method.

A column which gives separation of all the impurities and degradants from each other
and from analyte peak and which is rugged for variation in mobile phase shall be selected.

1.6.3 Selection of mobile phase

The primary objective in selection and optimization of mobile phase is to achieve

optimum separation of all the individual impurities and degradants from each other and from
analyte peak

In liquid chromatography, the solute retention is governed by the solute distribution

factor, which reflects the different interactions of the solute – stationary phase, solute –
mobile phase and the mobile phase – stationary phase .For a given stationary phase, the
retention of the given solute depends directly upon the mobile phase, the nature and the
composition of which has to be judiciously selected in order to get appropriate and required
solute retention. The mobile has to be adapted in terms of elution strength (solute retention)
and solvent selectivity (solute separation) Solvent polarity is the key word in
chromatographic separations since a polar mobile phase will give rise to low solute retention
in normal phase and high solute retention in reverse phase LC. The selectivity will be
particularly altered if the buffer pH is close to the pKa of the analytes; the solvent strength is
a measure of its ability to pull analyte from the column. It is generally controlled by the
concentration of the solvent with the highest strength.

The following are the parameters, which shall be taken into consideration while
selecting and optimizing the mobile phase.

▪ Buffer

▪ pH of the buffer

▪ Mobile phase composition.

1.6.3.1 Buffer, if any and its strength

Buffer and its strength play an important role in deciding the peak symmetries and
separations. Some of the most, commonly employed buffers are

▪ Phosphate buffers prepared using salts like KH2PO4, K2HPO4, NaH2PO4, Na2HPO4, etc

▪ Phosphoric acid buffers prepared using H3PO4.

▪ Acetate buffers – Ammonium acetate, Sodium acetate, etc.

16
Chapter 1 Introduction

▪ Acetic acid buffers prepared using CH3COOH.

The retention times also depend on the molar strengths of the buffer – Molar strength
is increasingly proportional to retention times. The strength of the buffer can be increased, if
necessary, to achieve the required separations.

The solvent strength is a measure of its ability to pull analytes from the column. It is
generally controlled by the concentration of the solvent with the highest strength.

1.6.3.2 pH of the buffer

pH plays an important role in achieving the chromatographic separations as it controls

the elution properties by controlling the ionization characteristics. Experiments were
conducted using buffers having different pH to obtain the required separations.

It is important to maintain the pH of the mobile phase in the range of 2.0 to 8.0 as most
columns does not withstand to the pH which are outside this range. This is due to the fact that
the siloxane linkages area cleaved below pH 2.0, while pH valued above 8.0 silica may
dissolve.

Fig.5. Relationship between Polarity and Elution Times for NP and RP

Chromatography

1.6.3.3 Mobile phase composition

Most chromatographic separations can be achieved by choosing the optimum mobile

phase composition. This is due to that fact that fairly large amount of selectivity can be
achieved by choosing the qualitative and quantitative composition of aqueous and organic
portions. Most widely used solvents in reverse phase chromatography are Methanol and
Acetonitrile. Experiments were conducted with mobile phases having buffers with different

17
Chapter 1 Introduction

pH and different organic phases to check for the best separations between the impurities. A
mobile phase which gives separation of all the impurities and degradants from each other
from analytic peak and which is rugged for variation of both aqueous and organic phase by at
least ±0.2 % of the selected mobile phase composition.

1.6.4 Selection of detector

The detector was chosen depending upon some characteristic property of the analyte
like UV absorbance, fluorescence, conductance, oxidation, reduction etc. characteristics that
are to be fulfilled by a detector to be used in HPLC determination are,

▪ High sensitivity, facilitating trace analysis

▪ Negligible baseline noise. To facilitate lower detection

▪ Large linear dynamic range

▪ Low dead volume

▪ Non destructive to sample

▪ Inexpensive to purchase and operate

Pharmaceutical ingredients do not all absorb UV light equally, so that selection of

detection wavelength is important. An understanding of the UV light absorptive properties of
the organic impurities and the active pharmaceutical ingredient is very helpful.

For the greatest sensitivity λmax should be used. UV wavelengths below 200 nm should be
avoided because detector noise increases in this region. Higher wavelengths give greater
selectivity. Here, PDA detector Used.

1.7 METHOD VALIDATION (16-18)

Method validation can be defined as establishing documented evidence, which

provides a high degree of assurance that a specific activity will consistently produce a desired
result or product meeting its predetermined specifications and quality characteristics.

Method validation is an integral part of the method development; it is the process of

demonstrating that analytical procedures are suitable for their intended use and that they
support the identity, quality, purity, and potency of the drug substances and drug products.
Simply, method validation is the process of proving that an analytical method is acceptable
for its intended purpose.

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Chapter 1 Introduction

Method Validation, however, is generally a one-time process performed after the

method has been developed to demonstrate that the method is scientifically sound and that it
serves the intended analytical purpose.

All the variables of the method should be considered, including sampling procedure,
sample preparation, chromatographic separation, and detection and data evaluation. For
chromatographic methods used in analytical applications there is more consistency in
validation practice with key analytical parameters including:

▪ Specificity /Selectivity

▪ System suitability

▪ Precision

o Repeatability

o Intermediate precision

o Reproducibility

▪ Accuracy

▪ Linearity

▪ Range

▪ Limit Of Detection

▪ Limit Of Quantitation

▪ Robustness

1.7.1 SPECIFICITY/SELECTIVITY

For chromatographic methods, developing a separation involves demonstrating

specificity, which is the ability of the method to accurately measure the analyte response in
the presence of all potential sample components. The response of the analyte in test mixtures
containing the analyte and all potential sample components (placebo formulation, synthesis
intermediates, excipients, degradation products, process impurities, etc.) is compared with the
response of a solution containing only the analyte. Other potential sample components are
generated by exposing the analyte to stress conditions sufficient to degrade it to 80-90%
purity. For bulk pharmaceuticals, stress conditions such as heat (50|AoC), light (600 FC),
acid (0.1 N HCl), base (0.1 N NaOH), and oxidant (3% H 2O2) are typical. For formulated
products, heat, light, and humidity (85%) are often used.

19
Chapter 1 Introduction

The resulting mixtures are then analysed, and the analyte peak is evaluated for peak
purity and resolution from the nearest eluting peak. If an alternate chromatographic column is
to be allowed in the final method procedure, it should be identified during these studies. Once
acceptable resolution is obtained for the analyte and potential sample components, the
chromatographic parameters, such as column type, mobile-phase composition, flow rate, and
detection mode, are considered set. An example of specificity criteria for an assay method is
that the analyte peak will have baseline chromatographic resolution of at least 1.5 from all
other sample components. If this cannot be achieved, the unresolved components at their
maximum expected levels will not affect the final assay result by more than 0.5%. An
example of specificity criteria for an impurity method is that all impurity peaks that are 0.1%
by area will have baseline chromatographic resolution from the main component peak(s) and,
where practical, will have resolution from all other impurities.

1.7.2 SYSTEM SUITABILITY

System suitability testing is an integral part of many analytical procedures to

determine to overall system performance. In System suitability testing various parameters to
be established for a particular procedure depends on the type of procedure to be validated.
Calculating the following values used to access overall system performance.

1. Relative retention

2. Theoretical plates

3. Capacity factor

4. Resolution

5. Peak asymmetry

6. Plates per meter

The following formulae shows the parameters used to calculate these system
performance values for the separation of two chromatographic components. (Note: Where the
terms W and t both appear in the same equation they must be expressed in the same units).

Relative retention
α = (t2 - ta) / (t1 - ta)
Theoretical plates
n = 16 (t / W) 2
Capacity factor

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K' = (t2 / ta) - 1

Resolution
R = 2 (t2 – t1) / (W2 + W1)
Peak asymmetry
T = W0.05 / 2f
Plates per meter
N=n/L
Height Equivalent to Theoretical Plate
HETP = L/n

Where,
α = Relative retention.
t2 = Retention time of the second peak measured from point of injection.
t1 = Retention time of the first peak measured from point of injection.
ta = Retention time of an inert peak not retained by the column, measured from point
of injection.
n = Theoretical plates.
t = Retention time of the component.
W = Width of the base of the component peak using tangent method.
K' = Capacity factor.
R = Resolution between a peak of interest (peak 2) and the peak preceding it (Peak 1).
W2 = Width of the base of component peak 2.
W1 = Width of the base of component peak 1.
T = Peak asymmetry, or tailing factor.
W0.05 = Distance from the leading edge to the tailing edge of the peak, measured at a point
5 % of the peak height from the baseline.
f = Distance from the peak maximum to the leading edge of the peak.
N = Plates per meter.
L = Column length, in meters.

1.7.3 PRECISION

The precision of a method is the extent to which the individual test results of multiple
injections of a series of standards agree It is expressed as the percentage coefficient of
variation (%CV) or relative standard deviation (RSD) of the replicate measurements.

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The measured standard deviation can be subdivided into three categories:

1.7.3.1 Repeatability

Repeatability is obtained when the analysis is carried out in one laboratory by one
operator using one piece of equipment over a relatively short time span. At least 5 or 6
determinations of three different matrices at two or three different concentrations should be
done and the relative standard deviation calculated. The acceptance criteria for precision
depend very much on the type of analysis.

1.7.3.2 Intermediate precision

Intermediate precision is a term that has been defined by ICH as the long-term
variability of the measurement process and is determined by comparing the results of a
method run within a single laboratory. A method’s intermediate precision may reflect
discrepancies in results obtained by different operators, from different instruments, with
standards and reagents from different suppliers, with columns from different batches or a
combination of these. The objective of intermediate precision validation is to verify that in
the same laboratory the method will provide the same results once the development phase is
over.

1.7.3.3 Reproducibility

Reproducibility as defined by ICH represents the precision obtained between

laboratories. The objective is to verify that the method will provide the same results in
different laboratories. The reproducibility of an analytical method is determined by analysing
aliquots from homogeneous lots in different laboratories with different analysts and by using
operational and environmental conditions that may differ from but are still within the
specified parameters of the method (interlaboratory tests). Validation of reproducibility is
important if the method will used in different laboratories.

1.7.4 ACCURACY

The accuracy of an analytical method is the extent to which test results generated by
the method and the true value agree. The true value for accuracy assessment can be obtained
in several ways. One alternative is to compare results of the method with results from an
established reference method. This approach assumes that the uncertainty of the reference

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method is known. Secondly, accuracy can be assessed by analysing a sample with known
concentrations, for example, a certified reference material, and comparing the measured value
with the true value as supplied with the material. If such certified reference material is not
available, blank a blank sample matrix of interest can be spiked with a known concentration
by weight or volume. After extraction of the analyte from the matrix and injection into the
analytical instrument, its recovery can be determined by comparing the response of the
extract with the response of the reference material dissolved in a pure solvent. Because this
accuracy assessment measures the effectiveness of sample preparation, care should be taken
to mimic the actual sample preparation as closely as possible. The concentration should cover
the range of concern and should particularly include one concentration close to the
quantitation limit. The expected recovery depends on the sample matrix, the sample
processing procedure and on the analyte concentration.

1.7.5 LINEARITY

A linearity study verifies that the sample solutions are in a concentration range where
analyte response is linearly proportional to concentration. For assay methods, this study is
generally performed by preparing standard solutions at five concentration levels, from 50 to
150% of the target analyte concentration. Five levels are required to allow detection of
curvature in the plotted data. The standards are evaluated using the chromatographic
conditions determined during the specificity studies.

Standards should be prepared and analysed a minimum of three times. The 50 to

150% range for this study is wider than what is required by the FDA guidelines. In the final
method procedure, a tighter range of three standards is generally used, such as 80, 100, and
120% of target; and in some instances, a single standard concentration is used.

Validating over a wider range provides confidence that the routine standard levels are
well removed from non-linear response concentrations, that the method covers a wide enough
range to incorporate the limits of content uniformity testing, and that it allows quantitation of
crude samples in support of process development. For impurity methods, linearity is
determined by preparing standard solutions at five concentration levels over a range such as
0.05-2.5 wt%.

Acceptability of linearity data is often judged by examining the correlation coefficient

and y-intercept of the linear regression line for the response versus concentration plot. A
correlation coefficient of > 0.999 is generally considered as evidence of acceptable fit of the
data to the regression line.

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1.7.6 RANGE

The range of an analytical method is the concentration interval over which acceptable
accuracy, linearity, and precision are obtained. In practice, the range is determined using data
from the linearity and accuracy studies. Assuming that acceptable linearity and accuracy
(recovery) results were obtained as described earlier, the only remaining factor to be
evaluated is precision. This precision data should be available from the triplicate analyses of
spiked samples in the accuracy study.

7. LIMIT OF DETECTION (LOD)

ICH defines the detection limit of an individual analytical procedure asthe lowest
amount of analyte in a sample which can be detected but not necessarily quantitated as an
exact value.
The limit of detection (LOD) is the point at which a measured value is larger than the
uncertainty associated with it. It is the lowest concentration of analyte in a sample that can be
detected but not necessarily quantified. The limit of detection is frequently confused with the
sensitivity of the method.

LOD = 3.3 (SD)/S

where, SD = Standard deviation of Y intercept

S = Slope
The limit of detection (LOD) was found to be 0.99 µg/ml.

8. LIMIT OF QUANTITATION (LOQ)

ICH defines the limit of quantitation (LOQ) of an individual analytical procedure as

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 issued particularly for the determination of impurities or
degradation products.
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.

LOQ = 10 (SD)/ S

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Where, SD = Standard deviation Y intercept

S = Slope
The limit of quantitation (LOQ) was found to be 2.98 µg/ml

9 ROBUSTNESS

The concept of robustness of an analytical procedure has been defined by the ICH as
“a measure of its capacity to remain unaffected by small but deliberate variations in method
parameters”.

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