Department of pharmaceuticals Analysis Introduction

INTRODUCTION

Pharmaceutical Analysis:

Pharmaceutical analysis plays a vital role in the pharmaceutical product development.

Pharmaceutical analysis is a specialized branch of analytical chemistry. Analytical chemistry
involves separating, identifying, and determining the relative amounts of components in a
sample matrix. Pharmaceutical analysis derives its principles from various branches of
sciences like physics, microbiology, nuclear science, and electronics etc. Qualitative analysis
reveals the chemical identity of the sample. Quantitative analysis establishes the relative
amount of one or more of these species or analytes in numerical terms. Qualitative analysis is
required before a quantitative analysis can be undertaken. A separation step is usually a
necessary part of both a qualitative and quantitative analysis. The results of typical
quantitative analysis can computed from two measurements. One is the mass or volume of
sample to be analyzed and second is the measurement of some quantity that is proportional to
the amount of analyte in that sample and normally completes the analysis. Instruments play a
key role in the quantitative analysis of pharmaceutical chemistry[1].

Instrumental methods are exciting and fascinating part of chemical analysis that
interacts with all areas of chemistry and with many other areas of pure and applied sciences.
Analytical instrumentation plays an important role in the production and evaluation of new
products and in the protection of consumers and environment. This instrumentation provides
lower detection limits required to assure safe foods, drugs, water and air. Instrumental
methods are widely used by Analytical chemists to save time, to avoid chemical separation
and to obtain increased accuracy. Most instrumental techniques fit into one of the four-
principle areas mentioned below[2].

A) Spectrophotometric techniques:

UV and Visible Spectrophotometry

Fluorescence and Phosphorescence Spectrophotometry

Atomic Spectrophotometry (emission &absorption)

Infrared Spectrophotometry

Raman Spectrophotometry

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X-Ray Spectrophotometry

Nuclear Magnetic Resonance Spectroscopy

Mass Spectroscopy

Electron Spin Resonance Spectroscopy

B) Electrochemical Techniques:

Potentiometry

Voltametry

Electrogravimetry

Conductometry

Amperomertry

C) Chromatographic Techniques:

High Performance Liquid Chromatography

Gas chromatography

High Performance Thin Layer Chromatography

Thin Layer Chromatography

GC- MS (Gas chromatography - Mass Spectroscopy

LC-MS (Liquid Chromatography - Mass Spectroscopy)

D) Miscellaneous techniques:

Thermal analysis

Kinetic techniques

Chromatographic techniques are predominantly used in the pharmaceutical industry

for a large variety of samples. HPLC is one of the chromatographic techniques is widely used
for checking the purity of new drug candidates, monitoring changes or scale ups of synthetic

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procedures, evaluating new formulations, and scrutinizing quality control/assurance of final

drug products.

High Performance Liquid Chromatography:

High performance liquid chromatography is basically a highly improved form of

column chromatography. Instead of a solvent being allowed to drip through a column under
gravity, it is forced through under high pressures of up to 400 atm. That makes it much faster.
It also allows using a very much smaller particle size for the column packing material which
gives a much greater surface area for interactions between the stationary phase and the
molecules flowing past it. This allows a much better separation of the components of the
mixture. The other major improvement over column chromatography concerns the detection
methods which can be used. These methods are highly automated and extremely sensitive.

HPLC employs a liquid mobile phase and a very finely divided stationary phase. In
order to obtain satisfactory flow rate liquid must be pressurized to a few thousands of pounds
per square inch. The rate of distribution of drugs between stationary and mobile phase is
controlled by diffusion process, if diffusion is minimized, a faster and effective separation
can be achieved. The technique of high performance liquid chromatography is so called
because of its improved performance when compared to classical column chromatography.
Advances in column technology, high-pressure pumping system and sensitive detectors have
transformed liquid column chromatography into high speed, efficient, accurate and highly
resolved method of separation [3, 4].

The HPLC is the method of choice in the field of analytical chemistry, since this
method is specific, robust, linear, precise and accurate and the limit of detection is low and
also it offers the following advantages [5].

 Speed (analysis can be accomplished in 20 min 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 analysis).
 Ideal for the substances of low viscosity.
 Easy sample recovery, handling and maintenance.

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 Instrumentation leads itself to automation and quantification (less time and less
labour).
 Precise and reproducible.
 Integrator itself does calculations.

Separation Principles of HPLC:

Adsorption chromatography employs high-surface area particles that absorb the solute
molecules. Usually a polar solid such as a silica gel, alumina or porous glass beads and a non-
polar mobile phase such as heptane, octane or chloroform are used in adsorption
chromatography. In adsorption chromatography, adsorption process is described by
competition model and solvent interaction model. Competition model assumes that entire
surface of the stationary phase is covered by mobile phase molecules as result of competition
for absorption site. In solvent interaction model the retention results from the interaction of
solute molecule with the second layer of adsorbed mobile phase molecules. The differences
in affinity of solutes for the surface of the stationary phase account for the separation
achieved.

In partition chromatography, the solid support is coated with a liquid stationary phase.
The relative distribution of solutes between the two liquid phases determines the separation.
The stationary phase can either be polar or non polar. If the stationary phase is polar and the
mobile phase is non polar, it is called normal phase partition chromatography. If the opposite
case holds, it is called reversed-phase partition chromatography. In normal phase mode, the
polar molecule partition preferentially in to the stationary phase and are retained longer than
non-polar compounds. In reverse phase partition chromatography, the opposite behavior is
observed.

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Types of HPLC techniques:

Based on modes of chromatography:

 Normal phase chromatography

 Reverse phase chromatography

Based on principle of separation:

 Adsorption chromatography
 Ion exchange chromatography
 Size exclusion chromatography
 Affinity chromatography
 Chiral phase chromatography

Base on elution technique:

 Isocratic separation
 Gradient separation

Based on the scale of operation:

 Analytical HPLC
 Preparative HPLC

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Reversed phase chromatography:

In 1960s, chromatographers started modifying the polar nature of the silanol group by
chemically reacting silicon with organic silanes. The object was to make silica less polar or
non-polar so that polar solvents can be used to separate water-soluble polar compounds.
Since the ionic nature of the chemically modified silica in now reversed i.e., it is non-polar or
the nature of the phase is reverted, the chromatographic separation carried out with such
silica is referred to as reversed-phase chromatography [6].

Reversed phase liquid chromatography (RPLC) is considered as the method of choice

for the analysis of pharmaceutical compounds for several reasons, such as its compatibility
with aqueous and organic solutions as well as with different detection systems and its high
consistency and repeatability. Sensitive and accurate RPLC analysis, whether in the
pharmaceutical or bioanalytical field, necessitates the use of stationary phases which give
symmetrical and efficient peaks. Therefore, manufacturers of stationary phases are
continuously improving and introducing new RPLC products, and the selection of various
types of reversed phase stationary phases is high. The needs for consistency as well as the
globalization of the pharmaceutical companies require that the methods will be transferred
from site to site, using either the same column brands or their equivalents. Therefore, an
extensive categorization or characterization of the rich selection of stationary phases has been
done in recent years.

The stationary phase in the Reversed Phase chromatographic columns is a

hydrophobic support that is consisted mainly of porous particles of silica gel in various
shapes (spherical or irregular) at various diameters (1.8, 3, 5, 7, 10 µm etc.) at various pore
sizes (such as 60, 100, 120, 300). The surface of these particles is covered with various
chemical entities, such as various hydrocarbons (C 1, C6, C4, C8, C18, etc). In most methods
used currently to separate medicinal materials, C 18 columns are used, which sometimes are
called ODS (octedecylsilane) or RP-18. A polar solvent is used as mobile phase [7].

The parameters that govern the retention in Reversed Phase systems are the following:

A. The chemical nature of the stationary phase surface.

B. The type of solvents that compose the mobile phase.

C. pH and ionic strength of the mobile phase.

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A. The chemical nature of the stationary phase:

The chemical nature is determined by the size and chemistry of hydrocarbon bonded
on the silica gel surface, its bonding density (units of µmole/m 2), and the purity and quality of
the silica gel support. As a rule, the more carbons in a bonded hydrocarbon the more it
retains organic solutes (as long as similar % coverage is compared). The higher the bonding
density the longer the organic solutes are retained. A column is considered relatively
hydrophobic if its bonding density exceeds 3µmole/m2.

Very important modifiers of the stationary phase's surface are surface-active

substances used as mobile phase's additives, acting as ion-pair reagents. These are substances
such as tri-ethylamine or tetrabutylamine or hexyl, heptyl, octyl sulfonate. They are
distributed between the mobile phase and the hydrophobic surface and cover it with either
positive (alkylamines) or negative (alkylsulfonates) charges. This change of the surface into
charged surface affects the retention significantly, especially on charged species in the
sample [8].

B. Composition of the mobile phase:

As a rule, the weakest solvent in Reversed Phase is the most polar one, water. The
other polar organic solvents are considered stronger solvents, where the order of solvent
strength follows more or less their dielectric properties, or polarity. The less polar the solvent
added to the mobile phase, the stronger it gets, shortening the retention times.

C. PH and ionic strength of the mobile phase:

When the samples contain solutes of ionisable functional groups, such as amines,
carboxyl’s, phosphates, phosphonates, sulphates and sulfonates, it is possible to control their
ionization degree with the help of buffers in the mobile phase. As a rule, the change of an
ionizable molecule to an ion makes it more polar and less available to the stationary phase [9,
10]
.

Normal phase chromatography:

In normal phase chromatography, the stationary phase is polar adsorbent. The mobile
phase is generally a mixture of non-aqueous solvents. The silica structure is saturated with
silanol group at the end in normal phase separations. These OH groups are statistically

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distributed over the whole of the surface. The silanol groups represent the active sites (very
polar) in the stationary phase.

This forms a weak bond with many molecules in the vicinity when any of the
following interactions are present. Dipole-induced dipole, dipole-dipole, hydrogen bonding,
-complex bonding. These situations arise when the molecule has one or several atoms with
lone pair electrons or a double bond. The adsorption strengths and hence ‘K’ value (elution
series) increase in the following order. Saturated hydrocarbon < olefins < aromatic < 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 stearic factors. If a molecule has several
functional groups, then the most polar one determines the reaction properties.

Chemically modified silica, such as aminopropyl, cyanopropyl and diol phases are the
stationary phases alternative to silica gel in normal phase chromatography.

The aminopropyl and cyanopropyl phases provide opportunities for specific

interactions between the analyte and the stationary phase and thus offer additional options for
the optimizations of separations. Other advantages of bonded phases lie in the increased
homogeneity of the stationary phase surface.

Polar modifiers such as acetic acid or triethylamine (TEA) are added to the mobile
phase, to deactivate the more polar adsorption sites on the surface of stationary phase, which
in turn will improve peak shape as well as the reproducibility of the retention times.

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Table no.1.1 Differences b/w normal and reverse phase

Properties Normal phase Reverse phase

Polarity of Stationary phase High Low

Polarity of Mobile phase Low to medium Low to high

Sample Elution Order Lower polar first Most polar first

Retention will increase by Increasing surface of Increasing surface of

stationary phase stationary phase
Increasing of n-alkyl chain
length of stationary phase
Decreasing polarity of Increasing polarity of mobile
mobile phase phase
Increasing polarity of Decreasing polarity of
sample molecules sample molecules

Adsorption chromatography:

The stationary phase is an adsorbent (like silica gel or any other silica based packing)
and the separation is based on repeated adsorption-desorption steps.

Ion-exchange chromatography:

Separation is based on the charge-bearing functional groups, anion exchange for

sample negative ion, or cation exchange - for sample positive ion. Gradient elution by pH is
common.

Affinity chromatography:

Separation is based on a chemical interaction specific to the target species. The more
popular reversed phase mode uses a buffer and an added counter-ion of opposite charge to the
sample with separation being influenced by pH, ionic strength, temperature, concentration of
and type of organic co-solvent(s). Affinity chromatography, common for macromolecules,
employs a ligand (biologically active molecule bonded covalently to the solid matrix) which

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interacts with its homologous antigen (analyte) as a reversible complex that can be eluted by
changing buffer conditions.

Chiral chromatography:

Separation of the enantiomers can be achieved on chiral stationary phases by

formation of diastereomers via derivatizing agents or mobile phase additives on a chiral
stationary phase. When used as an impurity test method, the sensitivity is enhanced if the
enantiomeric impurity elutes before the enantiomeric drug.

Isocratic separation:

In this technique, the same mobile phase combination is used throughout the process
of separation. The same polarity or elution strength is maintained the process.

Gradient separation:

In this technique, a mobile phase combination of lower polarity or elution strength is

used followed by gradually increasing the polarity or elution strength.

Analytical HPLC:

In this only analysis of the samples are done. Recovery of the samples for reusing is
normally not done, since the samples used are very low.

Preparative HPLC:

Where analysis of the individual fractions of pure compounds can be collected using
fraction collector. The collected samples are reused.

Ion-pair chromatography:

Ion Pair Chromatography (IPC) is used to separate ionic analytes on a Column. An

Ion Pair reagent is added to modulate retention of the ionic analytes. Ion-pair chromatography
is commonly used in combination with UV detection, in which case it is referred as reverse
phase ion-pair chromatography (RPIPC) [11, 12].

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Principle of ion-pairing:

With the aid of ion pair chromatography it is possible to separate the same analytes as
in ion exclusion chromatography, but the separation mechanism is completely different. The
stationary phases used are completely polar reversed phase materials such as are used in
distribution chromatography. A so-called ion pair regent is added to the eluents; this consists
of anionic or cationic surfactants such as tetraalkylammonium salts or n-alkylsulfonic acids.
Together with the oppositely charged analyte ions the ion pair reagents form an uncharged
ion pair, which can be retarded at the stationary phase by hydrophobic interactions.
Separation is possible because of the formation constants of the ion pairs and their different
degrees of adsorption. Figure 2 shows a simplified static ion exchange model in which it is
assumed that interactions with the analytes only occur after adsorption of the ion pair reagent
at the stationary phase.

Fig: no. 1.1 Mechanism of ion pair formation

Ion Pair Chromatography (IPC) is used to separate ionic analytes on a reversed phase
column. An Ion Pair reagent is added to modulate retention of the ionic analytes.

Separation mechanism:

Ion-exchange selectivity is mediated by both the mobile phase and stationary phase.
In contrast the selectivity of an ion-pair separation is determined primarily by the mobile
phase. The two major components of aqueous mobile phase are the ion-pair reagent and the
organic solvent; the type and concentration of each component can be varied to achieve
desired separation. The ion-pair reagent is a large ionic molecule that carries a charge
opposite to analyte of interest. It usually has both hydrophobic region to interact with the
analyte. Stationary phases used for ion-pair are neutral, hydrophobic resins such as

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polystyrene, divinyl benzene or bonded silica. A single stationary phase can be used for either
anion or cation analysis.

Although the retention mechanism of ion-pair chromatography is not fully understood, three
major theories have been proposed:

 Ion pair formation

 Dynamic ion exchange

In first model, the analyte and ion-pair reagent form a neutral pair, which is then
partitions between the mobile phase and stationary phase. Retention can be controlled by
varying the concentration of organic solvent in the mobile phase as in reverse phase
chromatography.

The dynamic ion-exchange model maintains that the hydrophobic portion of ion-pair
reagent adsorbs to the hydrophobic stationary phase to form a dynamic ion-exchange surface.
The analyte is retained on this surface, as it would in classic IC. Using this scenario, solvents
used In the mobile phase can be used to impede interaction of ion-pair reagent with the
stationary phase, there by altering the capacity of the column [13].

A third model describes an electrical double layer that is formed when ion-pair
reagent permeates the stationary phase, carrying with it an associated counter-ion. Retention
of analyte ion in this model is dependent upon a combination of factors including those
described in first two models.

Typical IP reagents are divided into two categories:

 Cationic: These are used for anion analysis. Cationic ion-pair reagents include
ammonium and tetra methyl-, tetraethyl-, tetra propyl-, and tetra butyl ammonium.

 Anionic: These reagents used for cation analysis. Anionic ion-pair reagents include
hydrochloric, perchloric, and perfluorocarboxylic acids and pentane-, hexane-,
heptane-, and octane sulfonic acids.

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Instrumentation Of HPLC:

The individual components HPLC and their working functions are described below [14].

 Mobile phase and reservoir

 Solvent degassing system

 Pump

 Injector

 Column

 Detector

 Data system

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Fig: no. 1.2 Block diagram of HPLC [15].

Mobile phase and reservoir:

The most common type of solvent reservoir is a glass bottle. Most of the
manufacturers supply these bottles with special caps, Teflon tubing and filters to connect to
the pump inlet and to the purge gas (helium) used to remove dissolved air.

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
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.

Mobile phases used for HPLC typically are mixtures of organic solvents and water or
aqueous buffers. Isocratic methods are preferable to gradient methods. Gradient methods will

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sometimes be required when the molecules being separated have vastly different partitioning
properties. When a gradient elution method is used, care must be taken to ensure that all
solvents are miscible [16, 17].

The following points should also be considered when choosing a mobile phase:

It is essential to establish that the drug is stable in the mobile phase for at least the
duration of the analysis.

Excessive salt concentrations should be avoided. High salt concentrations can result in
precipitation, which can damage HPLC equipment.

The mobile phase should have a pH 2.5 and pH 7.0 to maximize the lifetime of the
column.

Reduce cost and toxicity of the mobile phase by using methanol instead of acetonitrile
when possible minimize the absorbance of buffer. Since trifluoroacetic acid, acetic acid or
formic acid absorb at shorter wavelengths, they may prevent detection of products without
chromophores above 220nm. Carboxylic acid modifiers can be frequently replaced by
phosphoric acid, which does not absorb above 200nm.

Use volatile mobile phases when possible, to facilitate collection of products and
LC-MS analysis. Volatile mobile phases include ammonium acetate, ammonium phosphate,
formic acid, acetic acid, and trifluoroacetic acid. Some caution is needed as these buffers
absorb below 220 nm.

Ionizable compounds in some cases can present some problems when analyzed by
reverse phase chromatography. Two modifications of the mobile phase can be useful in
reverse phase HPLC for ionizable compounds. One is called ion suppression and other ion
pairing chromatography. In both techniques, a buffer is used to ensure that the pH of the
solution is constant and usually at least 1.5 pH units from a pKa of the drug to ensure that one
form predominates. If pH is approximately equal to pKa peak broadening can occur. In ion
suppression chromatography, the pH of the aqueous portion of the mobile phase is adjusted to
allow the neutral form of the drug to predominate. This ensures that the drug is persistent in
only one form and results in improvement of the peak shape and consistency of retention
times. In ion pairing chromatography, the pH of the mobile phase is adjusted so that the drug
is completely ionized. If necessary to improve peak shape or lengthen retention time, an alkyl

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sulfonic acid salt or bulky anion such as trifluoroacetic acid is added to the ion pair to
cationic drugs or a quaternary alkyl ammonium salt is added to ion-pair to anionic drugs. Ion
pairing chromatography also allows the simultaneous analysis of both neutral and charged
compounds.

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 signification or
purging or combination of these methods. Helium purging and storage of the solvent under
helium is not sufficient for degassing aqueous solvents. It is useful to apply a vacuum for 5-
10 min and then keep the solvent under a helium atmosphere. HPLC systems are also
provided an online degassing system, which continuously removes the dissolved gases from
the mobile phase.

Pump:

High pressure pumps are needed to force solvents through packed stationary phase
beds. Smaller bed particles require higher pressures. There are many advantages to using
smaller particles, but they may not be essential for all separations.

The most important advantages are higher resolution, faster analyses and increased
sample load capacity. However, only the most demanding separations require these advances
in significant amounts. Many separation problems can be resolved with larger particle
pickings that require less pressure. Flow rate stability is another important pump feature that
distinguishes pumps. Very stable flow rates are usually not essential for analytical
chromatography.

However, if the user plans to use a system in size exclusion mode, then there must be
a pump which provides an extremely stable flow rate. An additional feature found on the
more elaborate pumps is external electronic control. Although it adds to the expense of the
pump, external electronic control is a very desirable feature when automation or
electronically controlled gradients are to be run. Alternatively, this becomes an undesirable
feature (since it is an unnecessary expense) when using isocratic methods. The degree of flow

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control also varies with pump expense. More expensive pumps include such state of-the-art
technology as electronic feedback and multiheaded configurations. It is desirable to have an
integrated degassing system, either helium purging or membrane filtering.

Injector:

Sample introduction can be accomplished in various ways. The simplest method is to

use an injection valve. In more sophisticated LC systems, automatic sampling devices are
incorporated where the sample is introduced with the help of auto samplers and
microprocessors. In liquid chromatography, liquid samples may be injected directly and solid
samples need only be dissolved in an appropriate solvent. The solvent need not be the mobile
phase, but frequently it is judiciously chosen to avoid detector interference,
column/component interference, and loss in efficiency or all of these. It is always best to
remove particles from the sample by filtering over a 5μm filter, or centrifuging, since
continuous injections of particulate material will eventually cause blockages in injection
devices or columns. Sample sizes may vary widely. The availability of highly sensitive
detectors frequently allows use of the small samples which yield the highest column
performance. Sample introduction techniques can be used with a syringe or an injection
valve.

Column:

The heart of the system is the column. The choice of common packing material and
mobile phases depends on the physical properties of the drug. Many different reverse phase
columns will provide excellent specificity for any particular separation. It is therefore best to
routinely attempt separations with a standard C 8 or C18 column and determine if it provides
good separations. If this column does not provide good separation or the mobile phase is
unsatisfactory, alternate methods or columns should be explored. Reverse phase columns
differ by the carbon chain length, degree of end capping and percent carbon loading. Diol,
cyano and amino groups can also be used for reverse phase chromatography. Typical HPLC
columns are 5, 10, 15 and 25cm in length and are filled with small diameter (3, 5 or 10μm)
particles. The internal diameter of the columns is usually 4.6mm; this is considered the best
compromise for sample capacity, mobile phase consumption, speed and resolution. However,
if pure substances are to be collected (preparative scale), then larger diameter columns may
be needed. Packing the column tubing with small diameter particles requires high skill and

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specialized equipment. For this reason, it is generally recommended that all but the most
experienced chromatographers purchase prepacked columns, since it is difficult to match the
high performance of professionally packed LC columns without a large investment in time
and equipment. In general, LC columns are fairly durable and one can expect a long service
life unless they are used in some manner which is intrinsically destructive, as for example,
with highly acidic or basic eluents or with continual injections of 'dirty' biological or crude
samples. It is wise to inject some test mixture (under fixed conditions) into a column when
new, and to retain the chromatogram. If questionable results are obtained later, the test
mixture can be injected again under specified conditions. The two chromatograms may be
compared to establish whether or not the column is still useful.

Type of columns:

 Normal phase
 Reverse phase
 Size exclusion
 Ion exchange
 Bio-affinity
 Chiral
 Monolithic slica
 Chip HPLC
 Two Dimensional HPLC

Detector:

Today, optical detectors are used most frequently in liquid chromatographic systems.
These detectors pass a beam of light through the flowing column effluent as it passes through
a low Volume (~ 10μl) flow cell. The variations in light intensity caused by UV absorption,
fluorescence emission or change in refractive index, from the sample components passing
through the cell, are monitored as changes in the output voltage. These voltage changes are
recorded on a strip chart recorder and frequently are fed into a computer to provide retention
time and peak area data. The most commonly used detector in LC is the ultraviolet absorption
detector. A variable wavelength detector of this type, capable of monitoring from 190 to 400
nm, will be found suitable for the detection of the majority samples. Other detectors in
common use include
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Type of Detector

 Photo Diode Array (PDA),

 UV detector
 Refractive index (RI),
 Fluorescence (FLU),
 Electrochemical (EC).
 Mass spectromass
 Fluorescence
 Light scattering
 Electro chemical
 Radio activity
 Conductivity

The RI detector is universal but also the less sensitive one. FLU and EC detectors are
quite sensitive (up to 10-15pmole) but also quite selective.

Data system:

Since the detector signal is electronic, using modern data collection techniques can
aid the signal analysis. In addition, some systems can store data in a retrievable form for
highly sophisticated computer analysis at a later time. The main goal in using electronic data
systems is to increase analysis accuracy and precision, while reducing operator attention.
There are several types of data systems, each differing in terms of available features. In
routine analysis, where no automation (in terms of data management or process control) is
needed, a pre-programmed computing integrator may be sufficient. If higher control levels
are desired, a more intelligent device is necessary, such as a data station or minicomputer.
The advantages of intelligent processors in chromatographs are found in several areas. First,
additional automation options become easier to implement. Secondly, complex data analysis
becomes more feasible. These analysis options include such features as run parameter
optimization and deconvolution (i.e. resolution) of overlapping peaks. Finally, software
safeguards can be designed to reduce accidental misuse of the system.

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Ultra Performance Liquid Chromatography (UPLC) :

UPLC refers to Ultra Performance Liquid Chromatography. It improves in three

areas: chromatographic resolution, speed and sensitivity analysis. It uses fine particles and
saves time and reduces solvent consumption. UPLC comes from HPLC. UPLC is used in
many laboratories all over the world. One of the main advantage of this technique is growth
and development is due to the advancement of materials used for packaging is used in
stimulating the separation. The Causal ideology of this advancement is governed by what is
called the Van Deemter equation. This Technology takes full benefit of Chromatographic
principles to encourage separations utilizing columns chock full of tinier particles and/or
superior flow rates for higher speed with exceptional resolution and excellent sensitivity.
Today, because of in vivo doses or low sample size doses, new technology using narrow,
high density columns boasting higher resolution and more precise sensitivities. Speed and
precision became much higher and could be expected when using UPLC.

Advantages of UPLC
The advantages of UPLC are:
 Decreases run time and increases sensitivity
 Provides the selectivity, sensitivity, and dynamic range of LC analysis
 Maintaining resolution performance.
 Expands scope of Multiresidue Methods
 UPLC’s fast resolving power quickly quantifies related and unrelated compounds
 Faster analysis through the use of a novel separation material of very fine particle size
 Operation cost is reduced
 Less solvent consumption
 Reduces process cycle times, so that more product can be produced with existing
resources
 Increases sample throughput and enables manufacturers to produce more material that
consistently meet or exceeds the product specifications, potentially eliminating
variability, failed batches, or the need to re-work material
 Delivers real-time analysis in step with manufacturing processes

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Use of UPLC system

Elevated-temperature chromatography also allows for high flow rates by lowering the
viscosity of the mobile phase, which significantly reduces the column backpressure 9, 10.
Monolithic columns contain a polymerized porous support structure that provides lower flow
resistances than conventional particle-packed columns.

Disadvantages
The dis advantages of UPLC are
 Due to increased pressure requires more maintenance and reduces the life of the
columns of this type.
 So far performance similar or even higher has been demonstrated by using stationary
phases of size around 2 μm without the adverse effects of high pressure.
 In addition, the phases of less than 2 μm are generally non-regenerable.

Draw Backs
 Cost mixing
 Solvent pumping
 Lack of variety in commericial columns at 1.7 μm

Difference between HPLC - UPLC

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Gas chromatography-mass spectrometry (GC-MS):

Gas chromatography-mass spectrometry (GC-MS) is used to analyze complex organic

and biochemical mixtures. The GC-MS instrument consists of two main components. The
gas chromatography portion separates different compounds in the sample into “pulses” of
pure chemicals based on their volatility by flowing an inert gas (mobile phase), which carries
the sample, through a stationary phase fixed in the column. Spectra of compounds are
collected as they exit a chromatographic column by the mass spectrometer, which identifies
and quantifies the chemicals according their mass-to-charge ratio (m/z). These spectra can
then be stored on the computer and analyzed.
As stated above, the instrument is used to analyze complex organic and biochemical
mixtures. The Agilent 5975C GC-MS can inject various kinds of samples, including
microliter volumes of liquids, vapor from the headspace of a closed sample bottle, or
chemicals adsorbed on a solid phase microextraction (SPME) fiber. The samples must be of a
suitable size and are introduced as a “plug” of vapor. Sample sizes range from a few tenths of
a microliter to 20 µL for ordinary packed analytical columns. Capillary columns, which
require samples that are smaller by a factor of 100 or more, often require a sample splitter,
which delivers a small known fraction of the injected sample, while the rest goes to waste.

Figure 1 shows a block diagram of the GC-MS. The mobile-phase gas is called the
carrier gas, which must be chemically inert. This gas is most often helium, however, argon,
nitrogen, and hydrogen are also used. These gases are held in pressurized tanks and use
pressure regulators, gauges, and flow meters to control the flow rate of the gas. Flow rates

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usually range from 25-150 mL/min with packed columns and 1-25 mL/min for open tubular
capillary columns, and are assumed to be constant if inlet pressure is constant. This is often
accompanied by a molecular sieve to purify the gas before it is used.
Samples are introduced as a “plug” of vapor. Liquid samples are introduced using
calibrated micro syringes to inject sample through a septum and into a heated sample port
which should be about 50°C above the boiling point of the least volatile constituent of the
sample. After the sample is introduced, it is carried to the column by the mobile phase.
Packed and open tubular or capillary columns are two types of columns used in gas
chromatography. Packed columns are 1-5 m long, while capillary columns range from a few
meters to 100 meters long. The Agilent 5975C GC-MS uses a 30 meter capillary column.
Columns are usually constructed of fused silica or stainless steel, and coiled to fit into an
oven, with the coils ranging from 10-30 cm in diameter. The temperature of the column is an
important variable, so the oven is equipped with a thermostat that controls the temperature to
a few tenths of a degree. Boiling point of the sample and the amount of separation required
determines the temperature the sample should be run with. As a rough estimate, a temperature
equal to or slightly above the average boiling point of the sample should give reasonable
results.
Temperatures can also be increased in steps as separation proceeds, for samples with a
broad boiling range. As the mobile phase carrying the sample is passed through the stationary
phase in the column, the different components of the sample are separated.
After being separated, the sample is run through a detector. The detector in a GC-MS
is, not surprisingly, a mass spectrometer. The type of mass spectrometer used in the Agilent
5975C GC-MS is a quadrupole mass analyzer (4), which ionizes the sample and then
separates the ions based on their mass-to-charge ratio.
This data is then sent to a computer to be displayed and analysed. The computer
linked to the Agilent 5975C GC-MS has a library of samples to help analyze this data.
Data for the GC-MS is displayed in several ways. One is a total-ion chromatogram, which
sums the total ion abundances in each spectrum and plots them as a function of time. Another
is the mass spectrum at a particular time in the chromatogram to identify the particular
component that was eluted at that time. A mass spectra of selected ions with a specific mass
to charge ratio, called a mass chromatogram, can also be used.
Instrument parameters include flow rate, sample size, type of column, as well as
column length and temperature, and the library used to analyze the data. As stated above,
flow rates range from 25-150 mL/min with packed columns and 1-25 mL/min for open

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tubular capillary columns. Sample sizes range from a few tenths of a microliter to 20 µL for
ordinary packed analytical columns. Capillary columns require samples that are smaller by a
factor of 100 or more. The Agilent 5975C GC-MS uses a capillary column that is 30 meters
long (4). The temperature of the oven can be varied depending on the boiling point of the
sample. The column can withstand temperatures of -60°C to 360°C. There are also several
different libraries available to analyze data.
Samples can be both identified and quantified. The mass spectrum is used to identify
various components of the sample based on mass to charge ratio, however, the components of
the sample can also be quantified using the peak height or peak area of an eluate from the GC
column. Under carefully controlled conditions and using a standard, an accuracy of 1%
relative error can be obtained.

Liquid Chromatography – mass Spectrometry

LC/MS is ahyphenated technique, which combines the separating power of High
Performance Liquid Chromatography (HPLC), with the detection power of mass
spectrometry.

Mass Spectrometry is a wide-ranging analytical technique, which involves the

production and subsequent separation and identification of charged species.

The associated acronym, LCMS (Liquid Chromatography – mass Spectrometry)

covers a broad range of application areas. This module will explore the instrument acquisition
methods used.

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HPLC THEORY

System Suitability Parameters:

High performance liquid chromatography is defined as a separation of mixtures of

compounds due to differences in their distribution equilibrium between two phases, the
stationary phase packed inside columns and the mobile phase, delivered through the columns
by high pressure pumps. Components whose distribution into the stationary phase is higher,
are retained longer, and get separated from those with lower distribution into the stationary
phase. The theoretical and practical foundations of this method were laid down at the end of
1960s and at the beginning of 1970s. The theory of chromatography has been used as the
basis for System- Suitability tests, which are set of quantitative criteria that test the suitability
of the chromatographic system to identify and quantify drug related samples by HPLC at any
step of the pharmaceutical analysis [18].

Retention Time (Rt), Capacity Factor k' and Relative Retention Time (RRT):

The time elapsed between the injection of the sample components into the column and
their detection is known as the Retention Time (Rt). The retention time is longer when the
solute has higher affinity to the stationary phase due to its chemical nature. For example, in
reverse phase chromatography, the more lypophilic compounds are retained longer.
Therefore, the retention time is a property of the analyte that can be used for its identification.
A non retained substance passes through the column at a time t0, called the Void Time.

Retention factor is calculated as follows

The Capacity Factor describes the thermodynamic basis of the separation and its
definition is the ratio of the amounts of the solute at the stationary and mobile phases within
the analyte band inside the chromatographic column

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Where Cs is the concentration of the solute at the stationary phase and C m is its
concentration at the mobile phase and phi is the ratio of the stationary and mobile phase
volumes all within the chromatographic band.

The Retention Factor is used to compare the retention of a solute between two
chromatographic systems, normalizing it to the column's geometry and system flow rate. The
retention factor value should be in between 1-20. The need to determine the void time can be
tricky sometimes, due to the instability of the elution time of the void time marker, t 0,
therefore, when the chromatogram is complex in nature, and one known component is always
present at a certain retention time, it can be used as a retention marker for other peaks. In
such cases the ratio between the retention time of any peak in the chromatogram and the
retention time of the marker is used (RT (Peak) / RT (Marker) ) and referred to as the Relative
Retention Time (RRT). RRT is also used instead of the capacity ratio for the identification of
the analyte as well as to compare its extent of retention in two different chromatographic
systems.

The sharpness of a peak relative to its retention time is a measure of the system's
efficiency, calculated as N, plate count. Band-broadening phenomena in the column such as
eddy diffusion, molecular diffusion, and mass-transfer kinetics and extra-column effects
reduce the efficiency of the separation. The sharpness of a peak is relevant to the limit of
detection and limit of quantification of the chromatographic system. The sharper the peak for
a specific area, the better is its signal-to-noise; hence the system is capable of detecting lower
concentrations. Therefore, the efficiency of the chromatographic system must be established
by the system suitability test before the analysis of low concentrations that requires high
sensitivity of the system, such as the analysis of drug impurities and degradation products.

Efficiency: Plate Count N and Peak Capacity Pc:

The efficiency of the separation is determined by the Plate Count N when working at
isocratic conditions, whereas it is usually measured by Peak Capacity P c when working at
gradient conditions. The following equation for the plate count is used by the United States
Pharmacopoeia (USP) to calculate N

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Where w is measured from the baseline peak width calculated using lines tangent to
the peak width at 50 % height. European and Japanese Pharmacopoeias use the peak width at
50% of the peak height, hence the equation becomes

Peak Capacity Pc is defined as number of peaks that can be separated within a

retention window for a specific pre-determined resolution. In other words, it is the runtime
measured in peak width units (34). It is assumed that peaks occur over the gradient
chromatogram. Therefore, Peak Capacity can be calculated from the peak widths w in the
chromatogram as follows

Where n is the number of peaks at the segment of the gradient selected for the
calculation, tg. Thus peak capacity can be simply the gradient run time divided by the average
peak width. The sharper the peaks the higher is the peak capacity, hence the system should
be able to resolve more peaks at the selected run time as well as detect lower concentrations.

Another measure of the column's chromatographic efficiency is the Height Equivalent

to Theoretical Plate (HETP) which is calculated from the following equation

HETP = (L/N)

Where L is column length and N is the plate count. HETP is measured in micrometer.

The behaviour of HETP as function of linear velocity has been described by various
equations. It is frequently called "The Van-Deemter curve", and it is frequently used to
describe and characterize various chromatographic stationary phases' performance and
compare them to each other. The lower are the values of HETP, the more efficient is the
chromatographic system, enabling the detection of lower concentrations due to the enhanced
signal-to-noise ratio of all the peaks in the chromatogram.

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Peak Asymmetry Factor Af and Tailing Factor T:

The chromatographic peak is assumed to have a Gaussian shape under ideal

conditions, describing normal distribution of the velocity of the molecules populating the
peak zone migrating through the stationary phase inside the column. Any deviation from the
normal distribution indicates non-ideality of the distribution and the migration process
therefore might jeopardize the integrity of the peak's integration, reducing the accuracy of the
quantitation. This is the reason why USP Tailing is a peak's parameter almost always
measured in the system suitability step of the analysis.

The deviation from symmetry is measured by the Asymmetry Factor, Af or Tailing

Factor T. The calculation of Asymmetry Factor, Af is described by the following equation

Where A and B are sections in the horizontal line parallel to the baseline, drawn at
10% of the peak height

The calculation of Tailing Factor, T, which is more widely used in the pharmaceutical
industry, as suggested by the pharmacopeia’s, is described by the following equation

Where A and B are sections in the horizontal line parallel to the baseline, drawn at 5%
of the peak height. The USP suggests that Tailing Factor should be in the range of 0.5 up to 2
to assure a precise and accurate quantitative measurement.

Selectivity Factor: Alfa and Resolution Factor Rs:

The separation is a function of the thermodynamics of the system. Substances are

separated in a chromatographic column when their rate of migration differs, due to their
different distribution between the stationary and mobile phases. The Selectivity Factor, α,
and Resolution Factor, Rs, measure the extent of separation between two adjacent peaks. The
Selectivity Factor accounts only for the ratio of the Retention Factors, k', of the two peaks

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(k'2/k'1), whereas the Resolution Factor, Rs, accounts for the difference between the retention
times of the two peaks relative to their width

The equation that describes the experimental measurement of the Resolution Factor,
Rs, is as follows

Where Rt is the retention time of peaks 1 and 2 respectively and w is their respective
peak width at the tangents' baseline. According to the pharmacopeia’s Rs should be above
1.5 for an accurate quantitative measurement. The resolution is a critical value when
working with complex samples such as drug impurities and degradation products, or when
the formulation is complex and excipients might interfere with the quantitative
measurements. Therefore, it is an essential part of the system suitability measurement stage
before the quantitative work of these types of samples.

The sample used for the measurements of Rs during the system suitability runs is
sometimes called Resolution Solution, It usually contains the components that are the most
difficult to resolve. The theoretical description of the Resolution Factor Rs equation is shown
in Equation. It includes some of the above parameters, the plate count N, the selectivity α and
the average of the two peaks' capacity factors k'

It can be clearly seen from this equation that the plate count is the most effecting
parameter in the increase of the chromatographic resolution. Since the plate count increases
with the reduction in particle diameter, it explains the reduction in particle diameter of the
stationary phase material during the last 3 decades of HPLC. This is also the rational behind
the recent trend in HPLC, the use of sub 2 micron particle columns and the development of a
specially design of ultra performance HPLC systems to accommodate such columns.

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ANALYTICAL METHOD DEVELOPMENT

Method development has following steps

Collect information on sample, define separation goals

Need for special HPLC procedure, sample pre-treatment, etc.

Choose detector and detector settings

Choose LC method, preliminary run, select best separation conditions

Optimize separation conditions

Check for problems, requirement for special procedure

Recover purified material Quantitative calibration Qualitative method

Validate method for release to routine laboratory

A good method development strategy should require only as many experimental runs
as are necessary to achieve the desired final result. Finally method development should be as
simple as possible, and it should allow the use of sophisticated tools such as computer
modeling [19].

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The important factors, which are to be taken into account to obtain reliable quantitative
analysis, are:
1. Careful sampling and sample preparation.
2. Appropriate choice of the column.
3. Choice of the operating conditions to obtain the adequate resolution of the mixture.
4. Reliable performance of the recording and data handling systems.
5. Suitable integration/peak height measurement technique.
6. The mode of calculation best suited for the purpose.
7. Validation of the developed method.

Careful Sampling and Sample Preparation:

Before beginning method development, it is need to review what is known about the
sample in order to define the goals of separation. The sample related information that is
important is summarized in following Table 1.2

The sample related summarized information:

Table no 1.2 Sample information

1. Number of compounds present

2. Chemical structures

3. Molecular weights of compounds

4. pKa values of compounds

5. UV spectra of compounds

6. Concentration range of compounds in samples of

interest

7. Sample solubility

The chemical composition of the sample can provide valuable clues for the best
choice of initial conditions for an HPLC separation.

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Separation goals:

The goals of HPLC separation need to be specified clearly, which include:

 The use of HPLC to isolate purified sample components for spectral identification or
quantitative analysis.
 It may be necessary to separate all degradants or impurities from a product for reliable
content assay or not.
 In quantitative analysis, the required levels of accuracy and precision should be
known (a precision of ± 1 to 2% is usually achievable).
 Whether a single HPLC procedure is sufficient for raw material or one or more
different procedures are desired for formulations.
 When the number of samples for analysis at one time is greater than 10, a run time of
less than 20 minutes often will be important.

Sample preparation:

Samples come in various forms:

 Solutions ready for injection.

 Solutions that require dilution, buffering, addition of an internal standard or other
volumetric manipulation.
 Solids must be dissolved or extracted.
 Samples that require pre-treatment to remove interferences and/or protect the column
or equipment from damage.

Most samples for HPLC analysis require weighing and /or volumetric dilution before
injection. Best results are often obtained when the composition of the sample solvent is close
to that of the mobile phase since this minimizes baseline upset and other problems. Some
samples require a partial separation (pretreatment) prior to HPLC, because of need to remove
interferences, concentrate sample analytes or eliminate “column killers”.

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Selecting HPLC Method and initial conditions:

The mode of HPLC can be selected based on the following chart [20].

Reverse
phase
Hydrocarbon
soluble
Adsorption

Organic soluble
Normal phase
Alcohol soluble
Adsorption
MW<2000
Cation
Electrolyte exchange

Anion exchange
Water soluble
Reverse phase
Sample
Non electrolyte

Normal phase

Organic soluble SFC

MW>2000

Water soluble SEC

In many cases the development of an adequate sample pretreatment can be

challenging for achieving a good HPLC separation. The samples may be of two types, regular
or special. The regular samples are typical mixtures of small molecules (<2000Da) that can
be separated by normal starting conditions. Whereas special samples are better separated
under customized conditions given.

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SPECIAL SAMPLES AND CUSTOMIZED CONDITIONS:

Table no.1.3 Special samples & customized conditions

Sample Requirements

Inorganic ions Detection is primary problem; use ion chromatography.

Some isomers can be separated by reversed-phase HPLC and are

then classified as regular samples; better separations of isomers are
Isomers
obtained using either (1) normal-phase HPLC or (2) reversed-phase
separations with cyclodextrin-silica columns.

Enantiomers These compounds require “chiral” conditions for their separation.

Several factors make samples of this kind “special”: molecular

Biological conformation, polar functionality, and a wide range of
hydrophobicity.
“Big” molecules require column packings with large pores (>>10-
Macromolecules nm diameters); in addition, biological molecules require special
conditions as noted above.

Choice of the Column:

The selection of the column in HPLC is somewhat similar to the selection of columns
in G.C, in the sense that, in the adsorption and partition modes, the separation mechanism is
based on inductive forces, dipole-dipole interactions and hydrogen bond formation. In case of
ion-exchange chromatography, the separation is based on the differences in the charge, size
of the ions generated by the sample molecules and the nature of ionisable group on the
stationary phase. In the case of size-exclusion chromatography the selection of the column is
based on the molecular weight and size of the sample components [21].

Choice of the Operating Conditions to Obtain the Adequate Resolution Of the mixture:

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Most of the drugs come under the category of regular samples. Regular samples mean
typical mixtures of small molecules (<2000Da) that can be separated using more or less
standardized starting conditions. Regular samples can be further classified as neutral or ionic.
Samples classified as ionic include acids, bases, amphoteric compounds and organic salts. If
the sample is neutral, buffers or additives are generally not required in the mobile phase.
Acids or bases usually require the addition of a buffer to the mobile phase. For basic or
cationic samples, less acidic reverse phase columns are recommended. Based on
recommendations of the conditions, the first exploratory run is carried and then improved
systematically. On the basis of the initial exploratory run isocratic or gradient elution can be
selected as most suitable. If typical reverse-phase conditions provided inadequate sample
retention, it suggests the use of either ion-pair or normal phase HPLC. Alternatively, the
sample may be strongly retained with 100% Acetonitrile as mobile phase suggesting the use
of non-aqueous reverse phase chromatography or normal phase HPLC.

Getting Started On Method Development:

One approach is to use an isocratic mobile phase of some average organic solvent
strength (50%). A better alternative is to use a very strong mobile phase first (80-100%)
then reduce %B as necessary. The initial separation with 100% B results in rapid elution of
the entire sample, but few groups will separate. Decreasing the solvent strength shows the
rapid separation of all components with a much longer run time, with a broadening of latter
bands and reduced retention sensitivity. Goals that are to be achieved in method development
are briefly summarized in Table 1.4[22].

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GOALS THAT ARE TO BE ACHIEVED IN METHOD DEVELOPMENT:

Table no.1.4 Goals of method development

Goal Comment

Separation time <5-10 min is desirable for routine procedures.

£2% for assays; £5% for less-demanding analyses £15% for trace
Quantitation
analyses.
<150 bars is desirable, <200 bars is usually essential (new column
Pressure
assumed).

Peak height Narrow peaks are desirable for large signal/noise ratios.

Solvent consumption Minimum mobile-phase use per run is desirable.

Roughly in order of decreasing importance but may vary with analysis requirements.

Separation or resolution is a primary requirement in quantitative HPLC. The

resolution (Rs) value should be maximum (Rs > 2) favors maximum precision. Resolution
usually degrades during the life of the column and can vary from day to day with minor
fluctuations in separation conditions. Therefore, values of Rs=2 or greater should be the goal
during method development for sample mixtures. Such resolution will favor both improved
assay precision and greater method ruggedness. Some HPLC assays do not require base line
separation of the compounds of interest (qualitative analysis). In such cases only enough
separation of individual components is required to provide characteristic retention times for
peak identification. The time required for a separation (runtime = retention time for base
band) should be as short as possible and the total time spent on method development is
reasonable (runtimes 5 to 10 minutes are desirable).

Conditions for the final HPLC method should be selected so that the operating
pressure with a new column does not exceed 170 bars (2500 psi) and an upper pressure limit
below 2000 psi is desirable. There are two reasons for this pressure limit, despite the fact that
most HPLC equipment can be operated at much higher pressures. First, during the life of a
column, the backpressure may rise by a factor of as much as 2 due to the gradual plugging of
the column by particulate matter. Second, at lower pressures (<170 bars) pumps, sample
valves and especially auto samplers operate much better, seals last longer, columns tend to

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plug less and system reliability is significantly improved. For these reasons, a target pressure
of less than 50 % of the maximum capability of the pump is desirable. When dealing with
more challenging samples or if the goals of separation are particularly stringent, a large
number of method development runs may be required to achieve acceptable separation.

Repeatable separation:

As the experimental runs described above are being carried out, it is important to
confirm that each chromatogram can be repeated. When we change conditions (mobile phase,
column, and temperature) between method development experiments, enough time must
elapse for the column to come into equilibrium with the new mobile phase and temperature.

Usually column equilibration is achieved after passage of 10 to 20 volumes of the new

mobile phase through the column. However, this should be confirmed by repeating the
experiment under the same conditions. When constant retention times are observed in two
such back-to-back repeat experiments (± 0.5% or better), it can be assumed that the column is
equilibrated and the experiments are repeatable.

Optimization of HPLC method:

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 factor, elution time, detection limits, limit of quantitation
and overall ability to quantify the specific analyte of interest.
Optimization of a method can follow either of two general approaches

 Manual
 Computer driven
The manual approach involves varying one experimental variable at a time, while
holding all other constant and recording changes in response. The variables might include
flow rate, mobile or stationary phase composition, temperature, detection wavelength and P H.
This univariate approach to system 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.

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In the second approach, computer driven automated method 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 all instrumental method development.

The various parameters that include to be optimized during method development are
 Selection of mode of separation.
 Selection of stationary phase.
 Selection of mobile phase.
 Selection of detector.

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

Buffers 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.
 Acetic acid buffers prepared using CH3COOH.
The retention also depends 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

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to pull analyte from the column. It is generally controlled by the concentration of the solvent
with the highest strength.

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 silioxane linkages are cleaved below pH 2.0, while pH values above 8.0 silica
may dissolve.

Mobile Phase Composition:

Most chromatographic separations can be achieved by choosing the optimum mobile

phase composition. This is due to the 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 should be conducted with mobile phases having buffers with
different pH and different organic phases to check for the best separations of analyte peak. A
mobile phase which gives separation of analyte peak and which is rugged for variation of
both aqueous and organic phase by at least ± 0.2% of the selected mobile phase composition
should be used.

Selection of Detector:

The detector was chosen depending upon some characteristic property of the analyte
like UV absorbance, florescence, conductance, oxidation, reduction etc. The 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.
 Inexpensive to purchase and operate.
Pharmaceutical ingredients do not absorb all 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.

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For the greatest sensitivity λmax should be used. Ultra violet wavelengths below 200nm
should be avoided because detector noise increases in this region. Higher wavelengths give
greater selectivity.

Performance Calculations:

Carrying out system suitability experiment does the performance calculations. System
suitability experiments can be defined as tests to ensure that the method can generate results
of acceptable accuracy and precision. The requirements for system suitability are usually
developed after method development and validations have been completed. The criteria
selected will be based on the actual performance of the method as determined during its
validation. For example, if sample retention times form part of the system suitability criteria,
their variation SD can be determined during validation. System suitability might then require
that retention times fall within a ±3 SD range during routine performance of the method. The
USP (2000) defines parameters that can be used to determine system suitability prior to
analysis. These parameters include plate number (n), tailing factor (T), resolution (R S) and
relative standard deviation (RSD) of peak height or peak area for respective injections. The
RSD of peak height or area of five injections of a standard solution is normally accepted as
one of the standard criteria. For assay method of a major component, the RSD should
typically be less than 1% for these five respective injections.
The plate number and/ or tailing factor are used if the run contains only one peak. For
chromatographic separations with more than one peak, such as an internal standard assay or
an impurity method expected to contain many peaks, some measure of separations such as R S
is recommended. Reproducibility of Rt or k value for a specific compound also defines
system performance.

The column performance can be defined in terms of column plate number. As the
plate count is more the column is more efficient.

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ANALYTICAL METHOD VALIDATION

Method validation can be defined as (ICH) “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 by which a
method is tested by the developer or user for reliability, accuracy and preciseness of its
intended purpose and demonstrating that analytical procedures are suitable for their intended
use that they support the identity, quality, purity, and potency of the drug substances and drug
products Data thus generated become part of the methods validation package submitted to
Center for Drug Evaluation and Research (CDER). Simply, method validation is the process
of proving that an analytical method is acceptable for its intended purpose.

Methods should be reproducible when used by other analysts, on other equivalent

equipment, on other days or locations, and throughout the life of the drug product. Data that
are generated for acceptance, release, stability, or pharmacokinetic will only be trustworthy if
the methods used to generate the data are reliable [23, 24].

Though many types of HPLC techniques are available, the most commonly used
method, the reversed-phase HPLC with UV detection, is selected to illustrate the parameters
for validation. The criteria for the validation of this technique can be extrapolated to other
detection methods and chromatographic techniques.

 System suitability
 Specificity
 Accuracy
 Precision
 Linearity
 Limit of Detection
 Limit of Quantitation
 Ruggedness
 Robustness

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System Suitability:

According to the USP, system suitability tests are an integral part of chromatographic
methods. These tests are used to verify that the resolution and reproducibility of the system
are adequate for the analysis to be performed. System suitability tests are based on the
concept that the equipment, electronics, analytical operations, and samples constitute an
integral system that can be evaluated as a whole. The purpose of the system suitability test is
to ensure that the complete testing system (including instrument, reagents, columns, analysts)
is suitable for the intended application.

Similar to the analytical method development, the system suitability test Strategy
should be revised as the analysts develop more experience with the assay. In general,
consistency of system performance. (E.g.: Replicate injections of the standard) and
chromatographic suitability. (Eg: Tailing factor, column efficiency and resolution of the
critical pair) are the main components of system suitability.

During the early stage of the method development process some of the more
sophisticated system suitability tests may not be practical due to the lack of experience with
the method. In this stage, usually a more "generic" approach is used. For example, evaluation
of the tailing factor to check chromatographic suitability, and replicate injections of the
system suitability solution to check injection precision may be sufficient for an HPLC
impurities assay. As the method matures more experience is acquired for this method, a more
sophisticated system suitability test may be necessary.

System suitability is the checking of a system to ensure system performance before or

during the analysis of unknowns. Parameters such as plate count, tailing factors; resolution
and reproducibility (%RSD retention time and area for six repetitions) are determined and
compared against the specifications set for the method. These parameters are measured
during the analysis of system suitability "sample" that is a mixture of main components and
expected by-products.

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System suitability parameters and recommendations:

Table no.1.5 System suitability parameters

Parameter Recommendation

The peak should be well-resolved from other peaks and the void
Capacity Factor (k’)
volume, generally k’ 1 to 20

Repeatability RSD < 1% for N > 5 is desirable.

Relative retention Not essential as long as the resolution is stated.

Rs of > 2 between the peak of interest and the closest eluting

Resolution (Rs) potential interfering (impurity, excipient, degradation product,
internal standard, etc.

Tailing Factor (T) T of >0.5 and < 2

Theoretical Plates (N) N > 2000

Specificity/Selectivity:

The terms selectivity and specificity are often used interchangeably. According to
ICH, the term specific generally refers to a method that produces a response for a single
analyte only while the term selectivity refers to a method that provides responses for a
number of chemical entities that may or may not be distinguished from each other. If the
response is distinguished from all other responses, the method is said to be selective. Since
there are very few methods that respond to only one analyte, the term selectivity is usually
more appropriate [25].

Specificity is the ability of a method to discriminate between the analyte(s) of interest

and other components that are present in the sample. Studies are designed to evaluate the
degree of interference, if any, which can be attributed to other analytes, impurities,

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degradation products, reagent "blanks" and excipients. This provides the analyst with a
degree of certainty that the response observed is due to the single analyte of interest. The
degree of specificity testing varies depending on the method type and the stage of validation.
Specificity should be evaluated continually through the drug development process.
Specificity is sometimes used interchangeably with the term "selectivity". The argument over
which term is more correct is one of semantics. Although there is some dissention, the term
"specificity" has been adopted by the regulatory guidance documents and should be used to
prevent further confusion.

Non-Interference of Placebo:

This portion of specificity evaluation applies to the finished drug product only.
Excipients present in the formulation should be evaluated and must not interfere with the
detection of the analyte. Individual solutions of each excipient prepared at several times the
normal concentration of the component in the drug product ensure that any detector response
from the excipient will be readily visible. Injecting individual solutions of each excipient into
the HPLC system in comparison with a standard solution of the analyte is one means of
performing this experiment. The absence of a peak eluting at the retention time of the active
ingredient is sufficient to demonstrate specificity for excipients.

Challenge Study:

Injecting solutions of known process impurities, degradation products, intermediates,

homologues, dimers, etc. further challenges the specificity of a method. Identification of
these compounds may require an extensive search in order to identify all possible species that
may be present in the sample. For new chemical entities (NCE), this information may not be
readily available. Probable suspects should be identified by careful review of the synthetic
route and manufacturing process to identify any likely species that may be present in the
sample.

Forced Degradation Studies:

Degradation studies involve exposing the sample to a variety of stressed conditions to

further evaluate the specificity of degradation products. In this study, the drug substance,
drug product, and the combined excipients (or placebos) are each exposed to the stressed
conditions. These may include, but are not limited to, heat, light, acidic media, alkaline
media, and oxidative environments. Other conditions may be used depending on the nature

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and chemistry of the test subject. Forced degradation is usually evaluated with not more than
20% degradation of the drug substance, although more may be acceptable depending on the
particular properties of the drug. A reasonable effort should be made to degrade samples in
order to identify possible degradation products. If the planned experiments do not show any
appreciable degradation, the strength and/or exposure time of the stress condition may be
increased, but degradation is not required for every condition studied. There is a point beyond
which the stress condition becomes extreme and unrealistic. Sound scientific judgment
should be used to determine the extent and degree of degradation studies.

Acid stress study:

Blank, placebo and sample solutions were prepared as per test method. Blank, placebo
and sample were refluxed in 1N Hcl. They were neutralized by adding same amount of 1N
NaOH. The final volume was made up with diluent. These preparations were heated for 2hrs
at 600C and injected into the chromatographic system to observe the net degradation.

Base stress study:

Blank, placebo and sample solutions were prepared as per test method. Blank, placebo
and sample were refluxed in 1N NaOH. They were neutralized by adding same amount of 1N
HCl. The final volume was made up with diluent. These preparations were kept on bench top
for 2min and injected into the chromatographic system to observe the net degradation.

Peroxide stress study:

Blank, placebo and sample solutions were prepared as per test method and refluxed
with 30%H2O2. They were heated for 1hr at 60 oC at injected into the chromatographic system
to observe the net degradation.

Water stress study:

Blank, placebo and sample were refluxed with water and kept for 1hr on bench top
and then injected into the chromatographic system to observe the net degradation.

Sun light exposure study (visible light):

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Placebo and sample were exposed to sunlight for 55hrs and injected into the
chromatographic system to observe the net degradation.

UV light exposure study:

Placebo and sample were exposed to 200Wts/hr for 55hrs and injected into the
chromatographic system to observe the net degradation.

Heat stress study:

Placebo and sample were exposed to 105 oC for 48hrs and injected into the
chromatographic system to observe the net degradation.

Humidity stress study (about 90% RH at 250C):

Placebo and sample were exposed to 90% humidity for 7 days and injected into the
chromatographic system to observe the net degradation.

Accuracy:

Accuracy is the measure of how close the experimental value is to the true value.
Accuracy should be established across the specified range of the analytical procedure.

Drug Substance:

Several methods of determining accuracy are available:

a) Application of an analytical procedure to an analyte of known purity (e.g. reference

material);
b) Comparison of the results of the proposed analytical procedure with those of a second
well-characterized procedure, the accuracy of which is stated and/or defined.
c) Accuracy may be inferred once precision, linearity and specificity have been
established.

Drug Product:

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Several methods for determining accuracy are available:

a. Application of the analytical procedure to synthetic mixtures of the drug product

components to which known quantities of the drug substance to be analyzed have
been added.
b. 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.
c. Accuracy may be inferred once precision, linearity and specificity have been
established.

Impurities (Quantitation):

Accuracy should be assessed on samples (drug substance/drug product) spiked with

known amounts of impurities. In cases where it is impossible to obtain samples of certain
impurities and/or degradation products, it is considered acceptable to compare results
obtained by an independent procedure. The response factor of the drug substance can be used.
It should be clear how the individual or total impurities are to be determined e.g.,
weight/weight or area percent, in all cases with respect to the major analyte.

Recommendations:

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

Precision:

Precision is the measure of how close the data values are to each other for a number
of measurements under the same analytical conditions. ICH has defined precision to contain
three components: repeatability, intermediate precision and reproducibility. Ruggedness as
defined in USP XXII <1225>, 1990 incorporates the concepts described under the terms
"intermediate precision", "reproducibility" and "robustness" of this guide.

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Repeatability:
Injection Repeatability:

Sensitivity is the ability to detect small changes in the concentration of the analyte in
the sample. Sensitivity can be partially controlled by monitoring the specification for
injection reproducibility (system suitability testing).

The sensitivity or precision as measured by multiple injections of a homogeneous

sample (prepared solution) indicates the performance of the HPLC instrument under the
chromatographic conditions and day tested.

The information is provided as part of the validation data and as a system suitability
test. The specification, as the coefficient of variation in % or relative standard deviation
(RSD), set here will determine the variation limit of the analysis. The tighter the value, the
more precise or sensitive to variation one can expect the results. This assumes that the
chromatograph does not malfunction after the system suitability testing has been performed.
Keep in mind, however, that it does not consider variations due to the drug product
manufacturing and laboratory sample preparation procedures. The set of four duplicate
samples were injected sequentially. Variations in peak area and drift of retention times are
noted.

Precision refers to the reproducibility of measurement within a set, that is, to the
scatter of dispersion of a set about its central value. The term ‘set’ is defined as referring to a
number (N) of independent replicate measurements of some property. One of the most
common statistical terms employed is the standard deviation of a population of observation.
Standard deviation is the square root of the sum of squares of deviations of individual results
for the mean, divided by one less than the number of results in the set. The standard deviation
S, is given by

Standard deviation has the same units as the property being measured.

The square of standard deviation is called variance (S 2). Relative standard deviation is the
standard deviation expressed as a fraction of the mean, i.e., S/x. It is sometimes multiplied by

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100 and expressed as a percent relative standard deviation. It becomes a more reliable
expression of precision.

% Relative standard deviation = S x 100 / x

Recommendations:

As part of methods validation, a minimum of 10 injections with an RSD of 2% is

recommended. With the methods for release and stability studies, an RSD of 2% for precision
of the system suitability tests for at least five injections (n=5) for the active drug either in
drug substance or drug product is desirable. For low-level impurities, higher variations may
be acceptable.

Analysis Repeatability:

Determination, expressed as the RSD, consists of multiple measurements of a sample

by the same analyst under the same analytical conditions.

For practical purpose, it is often combined with accuracy and carried out as a single study.

Intermediate Precision:

Intermediate precision was previously known as part of ruggedness. The attribute

evaluates the reliability of the method in a different environment other than that used during
development of the method. The objective is to ensure that the method will provide the same
results when similar samples are analyzed once the method development phase is over.
Depending on time and resources, the method can be tested on multiple days, analysts,
instruments, etc.

Intermediate precision in the test method can be partly assured by good system suitability
specifications. Thus, it is important to set tight, but realistic, system suitability specification.

Linearity:

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The linearity of a method is its ability to obtain test results that are directly
proportional to the analyte concentration over a given range. For HPLC methods, the
relationship between analyte concentration and detector response (peak area or height) is used
to make this determination.

Concentration Ranges:
The concentration range used for linearity should be large enough to encompass the
desired range of the method. A minimum of five concentration ranges should be investigated
and a plot of the detector response vs. the sample concentration should be generated. It is
important that the concentration ranges selected for the linearity study are relatively equally
spaced throughout the range of the method (e.g., 50%, 75%, 100%, 125% and 150%), and not
clustered, as this will provide a skewed estimation of linearity.

Acceptance Criteria:
Acceptance criteria should be evaluated to ensure that they are meaningful when
compared with the performance of the method. Table 6 gives a list of suggested acceptance
criteria for use in evaluating method linearity. The ranges in Table 6 are suggestions only and
should be adjusted to ensure that all specification limits are within the validated linear range
for any given method. Under most circumstances, regression coefficient (r) is 0.999. Intercept
and slope should be indicated.

Statistical Analysis:
Linearity data should be evaluated using appropriate statistical methods. A simple
regression line of the detector response vs the analyte concentration is the most common
means of evaluation. Regulatory agencies require the submission of the correlation
coefficient, y-intercept, slope of the regression line, and the residual sum of squares for
linearity evaluation. A graphical representation of the linearity data should also be generated.
Additional analysis of the deviation of the actual values from the regression line is suggested,
especially when the method uses a single-point calibration standard. The percent y-intercept
is calculated by dividing the y-intercept by the detector response at the nominal concentration
expressed as a percentage. For single-point calibration, this value should be less than 1-2% to
ensure accurate results.
Table no.1.6 Linearity

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Leve
Test Range Acceptance criteria
l

Assay 5 50% to 150% R> 0.999,

Dissolution 5-8 10% to 150% R > 0.99,

Impurity 5 LOQ to 2% R > 0.98

Limit of Detection:
These limits are normally applied to related substances in the drug substance or drug
product. Specifications on these limits are submitted with the regulatory impurities method
relating to release and stability of both drug substance and drug product.

Limit of detection is the lowest concentration of analyte in a sample that can be

detected, but not necessarily quantitated, under the stated experimental conditions. With UV
detectors, it is difficult to assure the detection precision of low-level compounds due to
potential gradual loss of sensitivity of detector lamps with age, or noise level variation by
detector manufacturer. At low levels, assurance is needed that the detection and quantitation
limits are achievable with the test method each time. With no reference standard for a given
impurity or means to assure detectability, extraneous peak(s) could "disappear/appear." A
crude method to evaluate the feasibility of the extraneous peak detection is to use the
percentage claimed for detection limit from the area counts of the analyte. Several approaches
for determining the detection limit are possible, depending on whether the procedure is a non-
instrumental or instrumental.

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.

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

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signal-to-noise ratio between 3:1 or 2:1 is generally considered acceptable for estimating the
detection limit.

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

The limit of detection (LOD) may be expressed as

LOD = 3.3 σ / S

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 estimate
of s may be carried out in a variety of ways, for example

Based on the Standard Deviation of the Blank:

Analyzing an appropriate number of blank samples and calculating the standard

deviation of these responses perform measurement of the magnitude of analytical background
response.

Based on the Calibration Curve:

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

the range of LOD. 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.

Recommendations:

The detection limit and the method used for determining the detection limit should be
presented. If is determined based on visual evaluation or based on signal to noise ratio, the
presentation of the relevant chromatograms is considered acceptable for justification.

In cases where an estimated value for the detection limit is obtained by calculation or
extrapolation, this estimate may subsequently be validated by the independent analysis of a
suitable number of samples known to be near or prepared at the detection limit.

Limit of Quantification:

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Limit of quantitation is the lowest concentration of analyte in a sample that can be

determined with acceptable precision and accuracy under the stated experimental conditions.

Several approaches for determining the quantitation limit are possible, depending on
whether the procedure is a non-instrumental or instrumental.

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.

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 samples and by
establishing the minimum concentration at which the analyte can be reliably quantified. A
typical signal-to-noise ratio is 10:1.

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

The quantitation limit (LOQ) may be expressed as:

LOQ = 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 estimate of s
may be carried out in a variety of ways.

Based on Standard Deviation of the Blank:

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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.

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.

Recommendations:

The quantitation limit and the method used for determining the quantitation limit
should be presented. The limit should be subsequently validated by the analysis of a suitable
number of samples known to be near or prepared at the quantitation limit. Otherwise the
information that is expressed as % area or height of the drug substance peak from the same
HPLC chromatogram will be biased. It should also be noted that the extraneous peak using
area count does not consider the detection response that depends on the UV extinction
coefficient or absorptivity of the compound.

Ruggedness:

The ruggedness of an analytical method is the degree of reproducibility of test results

obtained by the analysis of the same samples under a variety of conditions, such as different
laboratories, analysts, instruments, reagents, elapsed assay times, assay temperatures, or days.
It is normally expressed as the lack of influence on test results of operational and
environmental variables of the analytical method. Method Ruggedness is defined as the
reproducibility of results when the method is performed under actual use conditions. Method
ruggedness may not be known when a method is first developed, but insight is obtained
during subsequent use of that method.

Recommendations:

The ruggedness of an analytical method is determined by analysis of aliquots from

homogeneous lots in different laboratories, by different analysts, using operational and
environmental conditions that may differ but are still within the specified parameters of the
assay. The degree of reproducibility of test results is then determined as a function of the

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assay variables. This reproducibility may be compared to the precision of the assay under
normal conditions to obtain a measure of the ruggedness of the method.

Robustness:

ICH defines robustness as a measure of the method's capability to remain unaffected

by small, but deliberate variations in method parameters. Robustness can be partly assured by
good system suitability specifications. The evaluation of robustness should be considered
during the development phase and depends on the type of procedure under study. It should
show the reliability of an analysis with respect to deliberate variations in method parameters.
If measurements are susceptible to variations in analytical conditions, the analytical
conditions should be suitably controlled or a precautionary statement should be included in
the procedure. One consequence of the evaluation of robustness should be that a series of
system suitability parameters (e.g., resolution test) is established to ensure that the validity of
the analytical procedure is maintained whenever used.

Examples of typical variations are

 Stability of analytical solutions

 Extraction time
In the case of liquid chromatography, examples of typical variations are

 Influence of variations of pH in a mobile phase

 Influence of variations in mobile phase composition
 Different columns (different lots and/or suppliers)
 Temperature
 Flow rate
In the case of gas chromatography, examples of typical variations are
 Different columns (different lots and/or suppliers)
 Temperature
 Flow rate

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Recommendations:

Data obtained from studies for robustness, though not usually submitted, are
recommended to be included as part of method validation.

General Recommendation:
System suitability testing is essential for the assurance of the quality performance of
the chromatographic system. The amount of testing required will depend on the purpose of
the test method. For dissolution or release profile test methods using an external standard
method, k', T and RSD are minimum recommended system suitability tests. For acceptance,
release, stability, or impurities/degradation methods using external or internal standards, k',
T, RS and RSD are recommended as minimum system suitability testing parameters. In
practice, each method submitted for validation should include an appropriate number of
system suitability tests defining the necessary characteristics of that system. Additional tests
may be selected at the discretion of the applicant or the reviewer.

HPLC methods for drug substance and drug product, methods should not be validated
as a one-time situation, but methods should be validated and designed by the developer or
user to ensure ruggedness or robustness throughout the life of the method. The variations due
to the drug product manufacturing process, the laboratory sample preparation procedure and
the instrument performance contribute to the accuracy of the data obtained from the analysis.
With proper validation and tight chromatographic performance (system suitability) criteria,
an improvement in the reliability of the data can be obtained. Variations except from the drug
product-manufacturing process will be minimized only with good reliable validated methods
can data that are generated for release, stability, and pharmacokinetic is trust-worthy.

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General points to consider:

Some basic points to note in the test method are

 The sample and standard should be dissolved in the mobile phase. If that is not
possible, then avoid using too high a level of the organic solvent as compared to the
level in the mobile phase.
 The sample and standard concentrations should be close if not the same.
 The samples should be bracketed by standards during the analytical procedure.
 Filtration of the samples before injection is occasionally observed Filtration Will
remove particulates (centrifugation performs the same function) that may Clog
columns.
 Adhesion of the analyte to the filter can also happen. This will be of importance
especially for low-level impurities. Data to validate this aspect should be submitted by
the applicant.

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