CHAPTER 1
INTRODUCTION
1
1. INTRODUCTION
Analytical chemistry is the study of the separation, identification, and
quantification of the chemical components of natural and artificial materials.[1]
Qualitative analysis gives an indication of the identity of the chemical species in the
sample and quantitative analysis determines the amount of one or more of these
components. The separation of components is often performed prior to analysis.
Analytical chemists perform qualitative and quantitative analysis; use the science
of sampling, defining, isolating, concentrating, and preserving samples; set error limits;
validate and verify results through calibration and standardization; perform separations
based on differential chemical properties; create new ways to make measurements;
interpret data in proper context; and communicate results. They use their knowledge of
chemistry, instrumentation, computers, and statistics to solve problems in almost all areas
of chemistry. For example, their measurements are used to assure compliance with
environmental and other regulations; to assure the safety and quality of food,
pharmaceuticals, and water; to support the legal process; to help physicians diagnose
disease; and to provide chemical measurements essential to trade and commerce.
Analytical chemistry has been important since the early days of chemistry,
providing methods for determining which elements and chemicals are present in the
world around us. During this period significant analytical contributions to chemistry
include the development of systematic elemental analysis by Justus von Liebig and
systematized organic analysis based on the specific reactions of functional groups.
Modern analytical chemistry is dominated by instrumental analysis [2]
. Many
analytical chemists focus on a single type of instrument. Academics tend to either focus
on new applications and discoveries or on new methods of analysis. The separation
sciences follow a similar time line of development and also become increasingly
transformed into high performance instruments.[5]
2
1.1. Hyphenated Techniques:
A "hybrid" or "hyphenated" technique [3-7] is the order of the present investigation.
Several examples are in popular use today and new hybrid techniques are under
development. Forexample, gas chromatography-mass spectrometry, gas chromatography-
infrared spectroscopy, liquid chromatography-mass spectrometry, liquid
chromatography-NMR spectroscopy. Liquid chromagraphy-infrared spectroscopy and
capillary electrophoresis-mass spectrometry.
A hyphenated separation technique refers to a combination of two (or more)
techniques to detect and separate chemicals from solutions. Most often the other
technique is some form of chromatography. Hyphenated techniques are widely used in
chemistry and biochemistry. A slash is sometimes used instead of hyphen, especially if
the name of one of the methods contains a hyphen itself.
1.2. Classification of Drugs
A drug may be defined as a substance meant for diagnosis, cure, mitigation and
prevention, treatment of diseases in human beings or animals, for altering in structure or
function of the body of human beings or animals [8]
. Pharmaceutical chemistry [9-13]
is a
science that makes use of general laws of chemistry to study drugs i.e. their preparation,
chemical nature, composition, structure, influence on an organism, the methods of quality
control and the conditions of their storage etc.
The family of drugs may be broadly classified as:
1. Pharmacodynamic agents
2. Chemotherapeutic agents
1.3. Pharmacodynamic agents refer to a group of drugs, which stimulate or depress
various functions of body so as to provide some relief to the body in case of body
abnormalities, without curing the disease. They are mainly used in case of noninfectious
diseases; so as to correct the abnormal body functions. Non-selective central nervous
system modifiers (depressants or stimulants), adrenergic stimulants and blocking agents,
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cholinergic and cholinergic blocking agents, cardiovascular agents, diuretics,
antihistaminic agents and anticoagulating agents are some examples of this group. These
agents have no action on infective organisms, which cause various diseases.
1.4. Chemotherapeutic agents are agents, which are selectively more toxic to the
invading organisms without harmful effect to the host. Some of the examples of this
group are organometallic agents, antimalarials, antibacterials, antiprotozoals antifungal
agents, anthelmentics, antiseptics, antitubercular agents, antineoplastics, etc.
Every country has legislation [13]
on bulk drugs and their pharmaceutical
formulations that sets standards and obligatory quality indices for them. These
regulations are presented in separate articles relating to individual drugs and are
published in the form of book called ―Pharmacopoeia‖ such as I.P [14]
, U.S.P [15]
, B.P [16]
.
And Martindale: The Extra Pharmacopoeia [17]
.
Pharmaceutical analysis [18]
deals not only with medicaments (drugs and their
formulations) but also with their precursors i.e. with the raw material on which degree of
purity and the quality of medicament depends. The quality of a drug is determined after
establishing its authenticity by testing its purity and the quality of pure substance in the
drug and its formulations. The following sampling techniques are generally adopted for
preparing sample solutions of different pharmaceutical formulations.
1.5. Sampling Techniques:
Due to the great variability of formulation, which is to be assayed, skilful
sampling in drug analysis is very essential. The extent of variation depends upon the
product and the manner of its selection. Usually the following methods are adopted for
sampling of pharmaceutical formulations.
Liquids:
These are mixed thoroughly several times by inverting the solution. If any
sediment remains behind, it should disperse in the liquid before it consider as a sample
for analysis.
Powders:
They are thoroughly mixed before a portion of the sample is taken for analysis.
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Tablet:
Tablets are thoroughly mixed before a portion of the sample is taken for analysis.
Capsules:
About ten capsules are weighed accurately. They are opened with a razor blade
and the contents are emptied in to a small beaker and mixed thoroughly. In the case of
dry filled capsules, the adhering powder to the shells is cleaned with absorbent cotton.
One of the major decisions to be made by an analyst is the choice of the most effective
procedure for a given analysis. For this, he must be familiar with the practical details, the
theoretical principles and also that he must be conversant with the conditions under
which each method is reliable, aware of possible interferences which may arise and
capable of minimizing or circumventing such problems. He must also be concerned with
question regarding accuracy and precision. In addition he must not over look factors such
as time and costing.
Important factors, which must be taken into account when selecting an appropriate
method of analysis, are
a. Nature of the information sought.
b. Size of sample available and the proportion of the constituent sought.
c. The purpose for which the analytical data are required.
1.6. Chromatography:
Chromatography involves passing a mixture dissolved in a "mobile phase"
through a stationary phase, which separates the analyte to be measured from other
molecules in the mixture based on differential partitioning between the mobile and
stationary phases. Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus changing the separation.
Chromatography became developed substantially as a result of the work of Archer
John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s.
They established the principles and basic techniques of partition chromatography, and
their work encouraged the rapid development of several types of chromatography
method: paper chromatography, gas chromatography, and what would become known as
high performance liquid chromatography. Since then, the technology has advanced
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rapidly. Researchers found that the main principles of Tsvet's chromatography could be
applied in many different ways, resulting in the different varieties of chromatography
described below. Simultaneously, advances continually improve the technical
performance of chromatography, allowing the separation of increasingly similar
molecules. The author has selected high PLC for his investigation.
1.7. High-Performance Liquid Chromatography:
High-performance liquid chromatography (or high-pressure liquid
chromatography, HPLC) is a chromatographic technique that can separate a mixture of
compounds and is used in biochemistry and analytical chemistry to identify, quantify and
purify the individual components of the mixture.
The ―HP‖ portion of the acronym is sometimes assigned to the words high
pressure (versus high performance), but it refers to the same analytical system. HPLC is
used in drug analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics to
name a few forensic applications.
Like all chromatography, HPLC is based on selective partitioning of the
molecules of interest between two different phases. Here, the mobile phase is a solvent or
solvent mix that flows under high pressure over beads coated with the solid stationary
phase. While traveling through the column, molecules in the sample partition selectively
between the mobile phase and the stationary phase. Those that interact more with the
stationary phase will lag behind those molecules that partition preferentially with the
mobile phase. As a result, the sample introduced at the front of the column will emerge in
separate bands (called peaks), with the bands emerging first being the components that
interacted least with the stationary phase and as a result moved quicker through the
column. The components that emerge last will be the ones that interacted most with the
stationary phase and thus moved the slowest through the column. A detector is placed at
the end of the column to identify the components that elute. Occasionally, the eluting
solvent is collected at specific times correlating to specific components. This provides a
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pure or nearly pure sample of the component of interest. This technique is sometimes
referred to as preparative chromatography.
1.7.1. Types of HPLC:
There are many ways to classify liquid column chromatography. If this
classification is based on the nature of the stationary phase and the separation process,
three modes can be specified.
In adsorption chromatography the stationary phase is an adsorbent (like silica gel or
any other silica based packings) and the separation is based on repeated
adsorptiondesorption steps. In ion-exchange chromatography the stationary bed has an
ionically charged surface of opposite charge to the sample ions. This technique is used
almost exclusively with ionic or ionizable samples. The stronger the charge on the
sample, the stronger it will be attracted to the ionic surface and thus, the longer it will
take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength
are used to control elution time.
In size exclusion chromatography the column is filled with material having precisely
controlled pore sizes, and the sample is simply screened or filtered according to its
solvated molecular size. Larger molecules are rapidly washed through the column;
smaller molecules penetrate inside the porous of the packing particles and elute later.
Mainly for historical reasons, this technique is also called gel filtration or gel permeation
chromatography although, today, the stationary phase is not restricted to a "gel". There
are two variants in use in HPLC depending on the relative polarity of the solvent and the
stationary phase.
1) Normal phase HPLC:
This is essentially just the same as you will already have read about in thin layer
chromatography or column chromatography. Although it is described as "normal", it isn't
the most commonly used form of HPLC. The column is filled with tiny silica particles,
and the solvent is non-polar - hexane, for example. A typical column has an internal
diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm. Polar
compounds in the mixture being passed through the column will stick longer to the polar
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silica than non-polar compounds will. The non-polar ones will therefore pass more
quickly through the column.
2) Reversed phase HPLC:
In this case, the column size is the same, but the silica is modified to make it non-
polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18
carbon atoms in them. A polar solvent is used - for example, a mixture of water and an
alcohol such as methanol. In this case, there will be a strong attraction between the polar
solvent and polar molecules in the mixture being passed through the column. There won't
be as much attraction between the hydrocarbon chains attached to the silica (the
stationary phase) and the polar molecules in the solution. Polar molecules in the mixture
will therefore spend most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the
hydrocarbon groups because of van der Waals dispersion forces. They will also be less
soluble in the solvent because of the need to break hydrogen bonds as they squeeze in
between the water or methanol molecules, for example. They therefore spend less time in
solution in the solvent and this will slow them down on their way through the column.
That means that the polar molecules will travel through the column more quickly.
Reversed phase HPLC is the most commonly used form of HPLC.
Isocratic flow and gradient elution
A separation in which the mobile phase composition remains constant throughout
the procedure is termed isocratic (meaning constant composition). The word was coined
by Csaba Horvath from Yale University, who was one of the pioneers of HPLC.
The mobile phase composition does not have to remain constant. A separation in
which the mobile phase composition is changed during the separation process is
described as a gradient elution.[20]
One example is a gradient starting at 10% methanol
and ending at 90% methanol after 20 minutes. The two components of the mobile phase
are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to
elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from
8
the column. Solvent A is often water, while B is an organic solvent miscible with water,
such as acetonitrile, methanol, THF, or isopropanol.
In isocratic elution, peak width increases with retention time linearly according to
the equation for N, the number of theoretical plates. This leads to the disadvantage that
late-eluting peaks get very flat and broad. Their shape and width may keep them from
being recognized as peaks.
Gradient elution decreases the retention of the later-eluting components so that
they elute faster, giving narrower (and taller) peaks for most components. This also
improves the peak shape for tailed peaks, as the increasing concentration of the organic
eluent pushes the tailing part of a peak forward. This also increases the peak height (the
peak looks "sharper"), which is important in trace analysis. The gradient program may
include sudden "step" increases in the percentage of the organic component, or different
slopes at different times - all according to the desire for optimum separation in minimum
time.
In isocratic elution, the selectivity does not change if the column dimensions
(length and inner diameter) change - that is, the peaks elute in the same order. In gradient
elution, the elution order may change as the dimensions or flow rate change. The driving
force is originated in reversed phase chromatography in the high order of the water
structure. The role of the organic mobile phase is to reduce this high order by reducing
the retarding strength of the aqueous component.
9
Figure 1
Mobile phase reservoir, filtering:
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. 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.
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 packings 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.
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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 control also varies with pump expense. More expensive pumps
include such state of – the - art technology as electronic feedback and multiheaded
configurations.
Modern pumps have the following parameters:
Flow rate range: 0.01 to 5 mL/min
Flow rate stability: not more than 1%
For SEC flow rate stability should be less than 0.2%
Maximum pressure: up to 300 hPa.
It is desirable to have an integrated degassing system, either helium purging, or
membrane filtering.
Figure 2: HPLC pump
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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 autosamplers
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 5 m 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. Typical sample mass with 4.6 mm ID columns
range from the nanogram level up to about 2 mg diluted in 20 ml of solvent. In general, it
will be noted that much less sample preparation is required in LC than in GC since
unwanted or interfering compounds, or both, may often be extracted, or eliminated, by
selective detection.
Column:
Typical HPLC [21-22]
columns are 5, 10, 15 and 25 cm in length and are filled with
small diameter (3, 5 or10 m) particles. The internal diameter of the columns is usually
4.6 mm; 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 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
12
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.
Figure 3
Column-packing materials:
The heart of the system is the column. In order to achieve high efficiency of
separation, the column material (micro-particles, 5-10 μm size) packed in such a way that
highest numbers of theoretical plates are possible.
Silica (SiO2 XH2O) is the most widely used substance for the manufacture of
packing materials. It consists of a network of siloxane linkages (Si-O-Si) in a rigid three
dimensional structure containing inter connecting pores. Thus a wide range of
commercial products is available with surface areas ranging from 100 to 800 m2/g. and
particle sizes from 3 to 50m.
The silanol groups on the surface of silica give it a polar character, which is
exploited in adsorption chromatography using non-polar organic eluents. Silica can be
drastically altered by reaction with organo chloro silanes or organo alkoxy silanes giving
Si-O-Si-R linkages with the surface. The attachment of hydrocarbon change to silica
produces a non-polar surface suitable for reversed phase chromatography where mixtures
of water and organic solvents are used as eluents. The most popular material is octadecyl-
silica (ODS-Silica), which contains C18 chains, but materials with C2, C6, C8 and C22
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chains are also available. During manufacture, such materials may be reacted with a
small mono functional silane (e.g. trimethyl chloro silane) to reduce further the number
of silanol groups remaining on the surface (end-capping). There is a vast range of
materials which have intermediate surface polarities arising from the bonding to silica of
other organic compounds which contain groups such as phenyl, nitro, amino and
hydroxyl. Strong ion exchangers are also available in which sulphonic acid groups or
quaternary ammonium groups are bonded to silica. The useful pH range for columns is 2
to 8, since siloxane linkages are cleaved below pH-2 while at pH values above eight,
silica may dissolve.
In HPLC, generally two types of columns are used, normal phase columns and
reversed phase columns. Using normal phase chromatography, particularly of non-polar
and moderately polar drugs can make excellent separation. It was originally believed that
separation of compounds in mixture takes place slowly by differential adsorption on a
stationary silica phase. However, it now seems that partition plays an important role, with
the compounds interacting with the polar silanol groups on the silica or with bound water
molecules.
While normal phase seems the passage of a relatively non-polar mobile phase
over a polar stationary phase, reversed phase chromatography is carried out using a polar
mobile phase such as methanol, acetonitrile, water, buffers etc., over a non-polar
stationary phase. Ranges of stationary phases (C18, C8, -NH2, -CN, -phenyl etc.) are
available and very selective separations can be achieved. The pH of the mobile phase can
be adjusted to suppress the ionization of the drug and thereby increase the retention on
the column. For highly ionized drugs ion-pair chromatography is used.
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1.8. BONDED PHASES FOR HPLC AND THEIR ABBREVIATIONS
Phase Description
Si
Silica
Classic normal phase material. Suitable for separating polar non-
ionic organic compounds.
Si OH
C1
TMS,
Tri methyl silane
Reversed phase material. Unique selectivity for polar and
multifunctional compounds. Least retentive of all alkyl group
bonded phases for non-polar solvents.
Si CH3
C2
RP-2, Dimethyl
Reversed phase material, less retentive than C4, C8, or C18. More
retentive than C1.
Si C2H5
C3
Propyl: Reversed phase material, used in hydrophobic interaction
chromatography (HIC) of proteins and peptides.
Si C3H7
15
C4
Butyl: Reversed phase material, useful for ion-pairing
chromatography offers less retention than C8 and C18 phases for non-
polar solutes. When bonded to 300 Å silica, it is an ideal phase for
analyzing large proteins and hydrophobic peptides.
Si C4H9
C6
Hexyl: Reversed phase material, useful for ion-pairing
chromatography. Less retentive than C8 and C18 phases.
Si C6H13
C8
RP-8, LC8, Octyl
CH2CH2CH2 Si
CH2CH2CH2 Si
Reversed phase material, similar selectivity to C18 but less retentive.
Wide applicability (e.g. pharmaceuticals, nucleosides, steroids).
When bonded to300 Å silica, it is an ideal phase for peptides, peptide
mapping and small hydrophilic proteins.
C18
ODS, RP-18, LC18, Octadecyl
Classic reversed phase material is most retentive for non-polar
solutes and is excellent for ion-pairing chromatography. It is having
wide applicability for the assay of nucleosides, nucleotides, steroids,
pharmaceuticals, vitamins, fatty acids and environmental compounds
when bonded to 300 Å silica, this phase is perfect for separating
small hydrophilic peptides.
Si C18H37
16
C6H5
Phenyl
It is a reversed phase material and exhibits unique selectivity. It is
useful for analyzing aromatic compounds. When bonded to 300 Å
silica, this phase is useful for HIC.
Si CH2CH2CH2
CH3CH2
NH2
APS, Amino, Amino propyl silyl
Si CH2CH2CH2NH2
Can be employed as reversed phase, normal phase or weak anion
exchange material. Reversed phase: useful for separating
carbohydrates. Normal phase: alternative selectivity to silica, not
deactivated by small amounts of water. Ion Exchange: weak anion
exchanger when used with buffers separates anions and organic
acids.
NO2
Nitro
Si NO2
Normal phase material. Separates aromatic compounds and
compounds with double bonds.
OH
Diol, Glycerol
Si CH2CH2CH2OCH2CHOHCHOH
Can be employed as either a reversed phase or normal phase
material. Reversed phase: Used for Gel Filtration Chromatography
(GFC) of proteins and peptides. Normal phase: Similar selectivity to
silica not deactivated by small amounts of water.
SAX
SB, Quaternary amine, Strong Base
Si CH2CH2CH2N (CH3)3+
Ion exchange material. Strong anion exchangers (basic) are useful
for separating nucleosides, nucleotides and organic acids.
17
SCX
SA, Sulfonic acid,Strong Acid
Si CH2CH2CH2SO2OH
Ion exchange material. Strong cation exchangers (acidic) are useful
for separating organic bases.
WAX
PEI, DEAE, Polyethyleneimine,
Diethylaminoethyl, Weak Base Si CH2CH2N(CH2CH3)2
Ion exchange material. Weak anion exchangers (basic) are most
useful for analyzing acidic proteins and peptides.
WCX
CM, Carboxymethyl,
Weak Acid Si CH2COOH
Ion exchange material. Weak cation exchangers (acidic) are most
useful for analyzing basic proteins and peptides.
CN
CPS, PCN, Cyano,
Cyanopropyl, Nitrile
Si CH2CH2CH2CN
It can be employed as either a reversed phase or normal phase
material. It is slightly polar and exhibits unique selectivity for polar
compounds in both RP and NP modes. It equilibrates very rapidly
and suitable for gradient separations. It has many pharmaceutical
applications (e.g. tricyclic antidepressants).
1.9. 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 (~ 10) 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
18
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: Photo Diode Array UV
detector (PAD), refractive index (RI), fluorescence (FLU), electrochemical (EC). The RI
detector is universal but also the less sensitive one. FLU and EC detectors are quite
sensitive (up to 10-15 per mole) but also quite selective.
1.9. Types of HPLC Detectors [23- 24, 28-33]
1. Ultraviolet (UV):
Ultraviolet (UV) detectors are cost-effective and popular and are widely used in
industry. This type of detector responds to substances that absorb light. The UV
detector is mainly used in biomedical and pharmaceutical science to separate and
identify the principal active components of a mixture. UV detectors are the most
versatile, having the best sensitivity and linearity. UV detectors cannot be used for
testing substances that are low in chromophores (colorless or virtually colorless) as
they cannot absorb light at low range.
2. Fluorescense:
The fluorescence is the most sensitive HPLC detector, used almost exclusively in
liquid chromatography (LC). This is a specific detector that senses only those
substances that emit light. This detector is popular for trace analysis in environmental
science. As it is very sensitive, its response is only linear over a relatively limited
concentration range. As there are not many elements that fluoresce (undergo
fluorescence or become fluorescent), scientists need to synthesize samples to make
them detectable.
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3. Mass Spectrometry:
The mass spectrometry detector coupled with HPLC is called HPLC-MS and
forms one of the best analytical tandems. HPLC-MS is the most powerful detector,
particularly relevant for medical science and widely used in pharmaceutical
laboratories and research and development. The principal benefit of HPLC-MS is that
it is capable of analyzing and providing molecular identity of a wide range of
components.
4. Refractive Index (RI) Detection:
The refractive index (RI) detector uses a monochromator and is one of the least
sensitive LC detectors. This detector is extremely useful for detecting those compounds
that are non-ionic, do not absorb ultraviolet light and do not fluoresce. Samples examined
with this detector are sugar, alcohol, fatty acid and polymers.
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 optimisation and deconvolution
(i.e. resolution) of overlapping peaks. Finally, software safeguards can be designed to
reduce accidental misuse of the system.
20
1.10. Parameters:
1) Internal diameter: The internal diameter (ID) of an HPLC column is an
important parameter that influences the detection sensitivity and separation
selectivity in gradient elution. It also determines the quantity of analyte that can
be loaded onto the column. Larger columns are usually seen in industrial
applications, such as the purification of a drug product for later use. Low-ID
columns have improved sensitivity and lower solvent consumption at the expense
of loading capacity.
Larger ID columns (over 10 mm) are used to purify usable amounts of material
because of their large loading capacity.
Analytical scale columns (4.6 mm) have been the most common type of columns,
though smaller columns are rapidly gaining in popularity. They are used in
traditional quantitative analysis of samples and often use a UV-Vis absorbance
detector.
Narrow-bore columns (1-2 mm) are used for applications when more sensitivity is
desired either with special UV-vis detectors, fluorescence detection or with other
detection methods like liquid chromatography-mass spectrometry
Capillary columns (under 0.3 mm) are used almost exclusively with alternative
detection means such as mass spectrometry. They are usually made from fused
silica capillaries, rather than the stainless steel tubing that larger columns employ.
2) Particle size: Most traditional HPLC is performed with the stationary phase
attached to the outside of small spherical silica particles (very small beads). These
particles come in a variety of sizes with 5 μm beads being the most common.
Smaller particles generally provide more surface area and better separations, but
the pressure required for optimum linear velocity increases by the inverse of the
particle diameter squared.[25-27]
This means that changing to particles that are half as big, keeping the size of the
column the same, will double the performance, but increase the required pressure
21
by a factor of four. Larger particles are used in preparative HPLC (column
diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase
extraction.
3) Pore size: Many stationary phases are porous to provide greater surface area.
Small pores provide greater surface area while larger pore size has better kinetics,
especially for larger analytes. For example, a protein which is only slightly
smaller than a pore might enter the pore but does not easily leave once inside.
4) Pump pressure: Pumps vary in pressure capacity, but their performance is
measured on their ability to yield a consistent and reproducible flow rate. Pressure
may reach as high as 40 MPa (6000 lbf/in2), or about 400 atmospheres. Modern
HPLC systems have been improved to work at much higher pressures, and
therefore are able to use much smaller particle sizes in the columns (<2 μm).
These "Ultra High Performance Liquid Chromatography" systems or
RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in²), or about
1000 atmospheres. The term "UPLC", though sometimes used is a trademark of
Waters Corporation and not the name for the technique in general.
1.11. HPLC method development:
The 3 critical components for a HPLC method are: sample preparation, HPLC
analysis and standardization (calculations). During the preliminary method
development [34-35]
stage, all individual components should be investigated before the
final method optimization. This gives the scientist a chance to critically evaluate the
method performance in each component and streamline the final method optimization.
The following must be considered when developing an HPLC method:
Keep it simple
Try the most common columns and stationary phases first
Thoroughly investigate binary mobile phases before going on to ternary
22
Think of the factors that are likely to be significant in achieving the desired
resolution.
Step 1: Define method objectives and understand the chemistry (10%)
Determine the goals for method development and to understand the chemistry of the
analytes and the drug product.
Step 2: Initial HPLC conditions (20%)
Develop preliminary HPLC conditions to achieve minimally acceptable separations.
These HPLC conditions will be used for all subsequent method development
experiments.
Step 3: Sample preparation procedure (10%)
Develop a suitable sample preparation scheme for the drug product
Step 4: Standardization (10%)
Determine the appropriate standardization method and the use of relative response
factors in calculations.
Step 5: Final method optimization/robustness (20%)
Identify the ―weaknesses‖ of the method and optimize the method through
experimental design. Understand the method performance with different conditions,
different instrument set ups and different samples.
Step 6: Method validation (30%)
Complete method validation according to ICH guidelines.
23
1.12. Validation of Analytical Methods and Procedures:
Introduction:
Method validation is the process used to confirm that the analytical procedure
employed for a specific test is suitable for its intended use. Results from method
validation can be used to judge the quality, reliability and consistency of analytical
results; it is an integral part of any good analytical practice.
Analytical methods need to be validated or revalidated
Before their introduction into routine use;
Whenever the conditions change for which the method has been validated (e.g.,
an instrument with different characteristics or samples with a different matrix);
and
Whenever the method is changed and the change is outside the original scope of
the method.
Method validation has received considerable attention in the literature and from industrial
committees and regulatory agencies.
The U.S. FDA CGMP [36]
requests the accuracy, sensitivity, specificity, and
reproducibility of test methods employed by the firm shall be established and
documented. These requirements include a statement of each method used in
testing the sample to meet proper standards of accuracy and reliability, as applied
to the tested product. The U.S. FDA has also proposed industry guidance for
Analytical Procedures and Methods Validation [37]
.
ISO/IEC 17025 includes a chapter on the validation of methods [38]
with a list of
nine validation parameters. The ICH [39]
has developed a consensus text on the
validation of analytical procedures. The document includes definitions for eight
validation characteristics. ICH also developed guidance with detailed
methodology [40]
.
24
The U.S. EPA prepared guidance for method’s development and validation for the
Resource Conservation and Recovery Act (RCRA) [40]
. The AOAC, the EPA and
other scientific organizations provide methods that are validated through multi-
laboratory studies.
The USP has published specific guidelines for method validation for compound
evaluation [41]
. USP defines eight steps for validation:
Accuracy
Precision
Specificity
Limit of detection
Limit of quantitation
Linearity and range
Ruggedness
Robustness
The FDA has also published guidance for the validation of bioanalytical methods [42]
.
The most comprehensive document is the conference report of the 1990 Washington
conference: Analytical Methods Validation: Bioavailability, Bioequivalence and
Pharmacokinetic Studies, which was sponsored by, among others, the American
Association of Pharmaceutical Scientists (AAPS), the AOAC and the U.S. FDA. The
report presents guiding principles for validating studies of both human and animal
subjects. The report has also been used as a basis for the FDA industry guidance
document.
Representatives of the pharmaceutical and chemical industry have published papers
on the validation of analytical methods. Hokanson [43]
applied the life cycle approach,
developed for computerized systems, to the validation and revalidation of methods.
Green [44]
gave a practical guide for analytical method validation, with a description of a
set of minimum requirements for a method. Renger and his colleagues described the
validation of a specific analytical procedure for the analysis of theophylline in a tablet
25
using high-performance thin layer chromatography (HPTLC) [45]
. The validation
procedure in this particular article is based on requirements for EU multistate registration.
Wegscheider [46]
has published procedures for method validation with a special focus
on calibration, recovery experiments, method comparison and investigation of
ruggedness. Seno et al. [47]
have described how analytical methods are validated in a
Japanese QC laboratory. The AOAC [48]
has developed a Peer-Verified Methods
validation program with detailed guidelines on exactly which parameters should be
validated. Winslow and Meyer [49]
recommend the definition and application of a master
plan for validating analytical methods. J.Breaux and colleagues have published a study on
analytical methods development and validation [50]
. The key point is to develop methods
for easy validation and revalidation. O. Krause published a guide for analytical method
transfer, comparability, and maintenance and acceptance criteria for the testing of
biopharmaceuticals [51]
.
This primer gives a review and a strategy for the validation of analytical methods for
both methods developed in-house as well as standard methods, and a recommendation on
the documentation that should be produced during, and on completion of, method
validation[52]
. It also describes what is important when transferring a method.
1.11. Strategy for the Validation of Methods:
The validity of a specific method should be demonstrated in laboratory experiments
using samples or standards that are similar to unknown samples analyzed routinely. The
preparation and execution should follow a validation protocol, preferably written in a
step-by-step instruction format. Possible steps for a complete method validation are listed
in this proposed procedure assumes that the instrument has been selected and the method
has been developed. It meets criteria such as ease of use; ability to be automated and to
be controlled by computer systems; costs per analysis; sample throughput; turnaround
time; and environmental, health and safety requirements.
26
1. Develop a validation protocol, an operating procedure or a validation master plan
for the validation.
2. For a specific validation project define owners and responsibilities.
3. Develop a validation project plan.
4. Define the application, purpose and scope of the method.
5. Define the performance parameters and acceptance criteria.
6. Define validation experiments.
7. Verify relevant performance characteristics of equipment.
8. Qualify materials, e.g. standards and reagents for purity, accurate amounts and
sufficient stability.
9. Perform pre-validation experiments.
10. Adjust method parameters or/and acceptance criteria if necessary.
11. Perform full internal (and external) validation experiments.
12. Develop SOPs for executing the method in the routine.
13. Define criteria for revalidation.
14. Define type and frequency of system suitability tests and/or analytical quality
control (AQC) checks for the routine.
15. Document validation experiments and results in the validation report.
27
1.12. Parameters for Method Validation:
The parameters for method validation have been defined in different working groups
of national and international committees and are described in the literature.
Unfortunately, some of the definitions vary between the different organizations. An
attempt at harmonization was made for pharmaceutical applications through the
ICH [39-40]
, where representatives from the industry and regulatory agencies from the
United States, Europe and Japan defined parameters, requirements and, to some extent,
methodology for analytical methods validation. The parameters, as defined by the ICH
and by other organizations and authors, are described in brief in the following
paragraphs.
Specificity
Selectivity
Precision
repeatability
intermediate precision
reproducibility
Accuracy
Trueness
Bias
Linearity
Range
Limit of detection
Limit of quantitation
Robustness
Ruggedness
(1) Included in ICH publications, [40]
included in USP.
(3) Terminology included in ICH publication but not part of required parameters.
28
Selectivity/Specificity:
The terms selectivity and specificity are often used interchangeably. A detailed
discussion of this term, as defined by different organizations, has been presented by
Vessmann [53]
. He particularly pointed out the difference between the definitions of
specificity given by IUPAC/WELAC and the ICH.
Although it is not consistent with the ICH, the term specific generally refers to a
method that produces a response for a single analyte only, while the term selective 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. The USP
monograph [41]
defines the selectivity of an analytical method as its ability to measure
accurately an analyte in the presence of interference, such as synthetic precursors,
excipients, enantiomers and known (or likely) degradation products that may be expected
to be present in the sample matrix. Selectivity in liquid chromatography is obtained by
choosing optimal columns and setting chromatographic conditions, such as mobile phase
composition, column temperature and detector wavelength. Besides chromatographic
separation, the sample preparation step can also be optimized for best selectivity.
It is a difficult task in chromatography to ascertain whether the peaks within a
sample chromatogram are pure or consist of more than one compound. Therefore, the
analyst should know how many compounds are in the sample or whether procedures for
detecting impure peaks should be used.
While in the past chromatographic parameters such as mobile phase composition
or the column were modified, now the application of spectroscopic detectors coupled on-
line to the chromatograph is being used. UV/visible diode-array detectors and mass
spectrometers acquire spectra on-line throughout the entire chromatogram. The spectra
acquired during the elution of a peak are normalized and overlaid for graphical
29
presentation. If the normalized spectra are different, the peak consists of at least two
compounds.
The principles of diode-array detection in HPLC and their application and
limitations with regard to peak purity are described in the literature [56]
. Examples of pure
and impure HPLC peaks are shown in Figure 15. While the chromatographic signal
indicates no impurities in either peak, the spectral evaluation identifies the peak on the
left as impure. The level of impurities that can be detected with this method depends on
the spectral difference, on the detector’s performance and on the software algorithm.
Under ideal conditions, peak impurities of 0.05 to 0.1 percent can be detected.
Selectivity studies should also assess interferences that may be caused by the
matrix, e.g., urine, blood, soil, water or food. Optimized sample preparation can eliminate
most of the matrix components. The absence of matrix interferences for a quantitative
method should be demonstrated by the analysis of at least five independent sources of
control matrix.
Figure 4
30
Examples of pure and impure HPLC peaks. The chromatographic signal does not indicate
any impurity in either peak. Spectral evaluation, however, identifies the peak on the left
as impure.
Precision and Reproducibility:
The precision of a method is the extent to which the individual test results of
multiple injections of a series of standards agree. The measured standard deviation can be
subdivided into 3 categories: repeatability, intermediate precision and reproducibility.
Repeatability is obtained when the analysis is carried out in a laboratory by an operator
using a piece of equipment over a relatively short time span. At least 6 determinations of
3 different matrices at 2 or 3 different concentrations should be performed, and the RSD
calculated.
The ICH [39]
requires precision from at least 6 replications to be measured at 100
percent of the test target concentration or from at least 9 replications covering the
complete specified range. For example, the results can be obtained at 3 concentrations
with 3 injections at each concentration.
The acceptance criteria for precision depend very much on the type of analysis.
Pharmaceutical QC precision of greater than 1 percent RSD is easily achieved for
compound analysis, but the precision for biological samples is more like 15 percent at the
concentration limits and 10 percent at other concentration levels. For environmental and
food samples, precision is largely dependent on the sample matrix, the concentration of
the analyte, the performance of the equipment and the analysis technique. It can vary
between 2 percent and more than 20 percent.
The AOAC manual for the Peer-Verified Methods program [48]
includes a table
with estimated precision data as a function of analyte concentration (Table.1).
Intermediate precision is a term that has been defined by ICH as the long-term
variability of the measurement process. It is determined by comparing the results of a
31
method run within a single laboratory over a number of weeks. A method’s intermediate
precision may reflect discrepancies in results obtained.
from different operators,
from inconsistent working practice (thoroughness) of the same operator,
from different instruments,
with standards and reagents from different suppliers,
with columns from different batches or
A combination of these.
Analyte% Analyte
Ratio Unit RSD%
100 1 100% 1.3
10 10-1
10% 2.8
1 10-2 1 % 2.7
0.1 10-3 0.1% 3.7
0.01 10-4 100 ppm 5.3
0.001 10-5 10 ppm 7.3
0.0001 10-6 1 ppm 11
0.00001 10-7 100 ppb 15
0.000001 10-8 10 ppb 21
0.0000001 10-9 1 ppb 30
Table.1. Analyte concentration versus precision
The objective of intermediate precision validation is to verify that in the same
laboratory the method will provide the same results once the development phase is over.
Reproducibility (Table.2), as defined by the ICH, represents the precision obtained
32
between different laboratories. The objective is to verify that the method will provide the
same results in different laboratories. The reproducibility of an analytical method is
determined by analyzing aliquots from homogeneous lots in different laboratories with
different analysts, and by using operational and environmental conditions that may differ
from, but are still within, the specified parameters of the method (interlaboratory tests).
Validation of reproducibility is important if the method is to be used in different
laboratories.
Differences in room temperature and humidity
Operators with different experience and thoroughness
Equipment with different characteristics, e.g. delay volume of an HPLC system
Variations in material and instrument conditions, e.g. in HPLC, mobile phases
composition, pH, flow rate of mobile phase
Variation in experimental details not specified by the method
Equipment and consumables of different ages
Columns from different suppliers or different batches
Solvents, reagents and other material with varying quality
33
Precision Intermediate
Precision
Reprodu-
cibility
Instrument same different different
Batches of accessories
e.g. chrome. columns same different different
Operators same different different
Sample matrices different different different
Concentration different different different
Batches of material,
e.g., reagents same different different
Environmental
conditions, e.g.,
temperature
same different different
Laboratory same same different
Table 2. Variables for measurements of precision, intermediate precision and
reproducibility
Accuracy and Recovery
The accuracy of an analytical method is the extent to which test results generated
by the method and the true value agree. Accuracy can also be described as the closeness
of agreement between the value that is adopted, either as a conventional, true or accepted
reference value, and the value found.
The true value for accuracy assessment can be obtained in several ways. One
alternative is to compare the results of the method with results from an established
reference method. This approach assumes that the uncertainty of the reference method is
known. Secondly, accuracy can be assessed by analyzing a sample with known
concentrations (e.g., a control sample or certified reference material) and comparing the
34
measured value with the true value as supplied with the material. If certified reference
materials or control samples are not available, a blank sample matrix of interest can be
spiked with a known concentration by weight or volume. After extraction of the analyte
from the matrix and injection into the analytical instrument, its recovery can be
determined by comparing the response of the extract with the response of the reference
material dissolved in a pure solvent. Because this accuracy assessment measures the
effectiveness of sample preparation, care should be taken to mimic the actual sample
preparation as closely as possible. If validated correctly, the recovery factor determined
for different concentrations can be used to correct the final results.
The concentration should cover the range of concern and should include
concentrations close to the quantitation limit, one in the middle of the range and one at
the high end of the calibration curve. Another approach is to use the critical decision
value as the concentration point that must be the point of greatest accuracy.
Active
ingredient (%)
Analyte
Ratio Unit
Mean
Recovery
(%)
100 1 100% 98-102
10 10-1
10% 98-102
1 10-2 1 % 97-103
0.1 10-3 0.1% 95-105
0.01 10-4 100 ppm 90-107
0.001 10-5 10 ppm 80-110
0.0001 10-6 1 ppm 80-110
0.00001 10-7 100 ppb 80-110
0.000001 10-8 10 ppb 60-115
0.0000001 10-9 1 ppb 40-120
Table 3. Analyte recovery at different concentrations
35
The expected recovery (Table.3) depends on the sample matrix, the sample
processing procedure and the analyte concentration. The AOAC manual for the Peer-
Verified Methods program includes a table with estimated recovery data as a function
analyte concentration.
The ICH document on validation methodology recommends accuracy to be
assessed using a minimum of nine determinations over a minimum of three concentration
levels covering the specified range (e.g., three concentrations/three replicates each).
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.
Linearity and Calibration Curve:
The linearity of an analytical method is its ability to elicit test results that are
directly proportional to the concentration of analytes in samples within a given range or
proportional by means of well-defined mathematical transformations. Linearity may be
demonstrated directly on the test substance (by dilution of a standard stock solution)
and/or by using separate weighings of synthetic mixtures of the test product components,
using the proposed procedure.
Linearity is determined by a series of 3 to 6 injections of 5 or more standards
whose concentrations span 80–120 percent of the expected concentration range. The
response should be directly proportional to the concentrations of the analytes or
proportional by means of a well-defined mathematical calculation. A linear regression
equation applied to the results should have an intercept not significantly different from 0.
If a significant nonzero intercept is obtained, it should be demonstrated that this has no
effect on the accuracy of the method.
Frequently, the linearity is evaluated graphically, in addition to or as an
alternative to mathematical evaluation. The evaluation is made by visually inspecting a
plot of signal height or peak area as a function of analyte concentration. Because
deviations from linearity are sometimes difficult to detect, two additional graphical
36
procedures can be used. The first is to plot the deviations from the regression line versus
the concentration or versus the logarithm of the concentration, if the concentration range
covers several decades. For linear ranges, the deviations should be equally distributed
between positive and negative values.
Another approach is to divide signal data by their respective concentrations,
yielding the relative responses. A graph is plotted with the relative responses on the y-
axis and the corresponding concentrations on the x-axis, on a log scale. The obtained line
should be horizontal over the full linear range. At higher concentrations, there will
typically be a negative deviation from linearity. Parallel horizontal lines are drawn on the
graph corresponding to, for example, 95 percent and 105 percent of the horizontal line.
The method is linear up to the point where the plotted relative response line intersects the
95 percent line.
The ICH recommends, for accuracy reporting, the linearity curve’s correlation
coefficient, y-intercept, slope of the regression line and residual sum of squares. A plot of
the data should be included in the report. In addition, an analysis of the deviation of the
actual data points from the regression line may also be helpful for evaluating linearity.
Some analytical procedures, such as immunoassays, do not demonstrate linearity after
any transformation. In this case, the analytical response should be described by an
appropriate function of the concentration (amount) of an analyte in a sample. In order to
establish linearity, a minimum of five concentrations is recommended. Other approaches
should be justified.
37
Figure 4
Plotting the sensitivity (response/amount) gives clear indication of the linear
range. Plotting the amount on a logarithmic scale has a significant advantage for wide
linear ranges. Rc = Line of constant response.
Range:
The range of an analytical method is the interval between the upper and lower
levels (including these levels) that have been demonstrated to be determined with
precision, accuracy and linearity using the method as written. The range is normally
expressed in the same units as the test results (e.g., percentage, parts per million) obtained
by the analytical method.
38
For assay tests, the ICH requires the minimum specified range to be 80 to 120
percent of the test concentration, and for the determination of an impurity, the range to
extend from the limit of quantitation, or from 50 percent of the specification of each
impurity, whichever is greater, to 120 percent of the specification.
Figure 5: Definitions for linearity, range, LOQ, LOD
1.13. Limit of Detection:
The limit of detection is the point at which a measured value is larger than the
uncertainty associated with it. It is the lowest concentration of analyte in a sample that
can be detected but not necessarily quantified. The limit of detection is frequently
confused with the sensitivity of the method. The sensitivity of an analytical method is the
capability of the method to discriminate small differences in concentration or mass of the
test analyte. In practical terms, sensitivity is the slope of the calibration curve that is
obtained by plotting the response against the analyte concentration or mass.
39
In chromatography, the detection limit is the injected amount that results in a peak
with a height at least two or three times as high as the baseline noise level. Besides this
signal/noise method, the ICH describes three more methods:
1. Visual inspection: 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.
2. Standard deviation of the response based on the standard deviation of the blank:
Measurement of the magnitude of analytical background response is performed by
analyzing an appropriate number of blank samples and calculating the standard
deviation of these responses.
3. Standard deviation of the response based on the slope of the calibration curve: A
specific calibration curve is studied using samples containing an analyte in the
range of the limit of detection. 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.
Figure 6: Limit of detection and limit of quantization via signal to noise
Limit of Quantitation
The limit of quantitation is the minimum injected amount that produces
quantitative measurements in the target matrix with acceptable precision in
40
chromatography, typically requiring peak heights 10 to 20 times higher than the baseline
noise.
If the required precision of the method at the limit of quantitation has been
specified, the EURACHEM [55]
(Figure 7) approach can be used. A number of samples
with decreasing amounts of the analyte are injected six times. The calculated RSD
percent of the precision is plotted against the analyte amount. The amount that
corresponds to the previously defined required precision is equal to the limit of
quantitation. It is important to use not only pure standards for this test but also spiked
matrices that closely represent the unknown samples.
For the limit of detection, the ICH recommends, in addition to the procedures as
described above, the visual inspection and the standard deviation of the response and the
slope of the calibration curve.
Figure 7: Limit of quantitation with the EURACHEM (80) method.
Any results of limits of detection and quantitation measurements must be verified
by experimental tests with samples containing the analytes at levels across the two
regions. It is equally important to assess other method validation parameters, such as
precision, reproducibility and accuracy, close to the limits of detection and quantitation.
Figure 4 illustrates the limit of quantitation (along with the limit of detection, range and
linearity). Figure 5 illustrates both the limit of detection and the limit of quantitation.
41
Ruggedness:
Ruggedness is not addressed in the ICH documents Its definition has been
replaced by reproducibility, which has the same meaning as ruggedness, defined by the
USP as the degree of reproducibility of results obtained under a variety of conditions,
such as different laboratories, analysts, instruments, environmental conditions, operators
and materials. Ruggedness is a measure of reproducibility of test results under normal,
expected operational conditions from laboratory to laboratory and from analyst to analyst.
Ruggedness is determined by the analysis of aliquots from homogeneous lots in different
laboratories.
Robustness:
Robustness tests examine the effect that operational parameters have on the
analysis results. For the determination of a method’s robustness, a number of method
parameters, for example, pH, flow rate, column temperature, injection volume, detection
wavelength or mobile phase composition, are varied within a realistic range, and the
quantitative influence of the variables is determined. If the influence of the parameter is
within a previously specified tolerance, the parameter is said to be within the method’s
robustness range.
Obtaining data on these effects helps to assess whether a method needs to be
revalidated when one or more parameters are changed, for example, to compensate for
column performance over time. In the ICH document [52]
, it is recommended to consider
the evaluation of a method’s robustness during the development phase, and any results
that are critical for the method should be documented. This is not, however, required as
part of a registration.
Stability:
Many solutes readily decompose prior to chromatographic investigations, for
example, during the preparation of the sample solutions, extraction, cleanup, phase
transfer or storage of prepared vials (in refrigerators or in an automatic sampler). Under
42
these circumstances, method development should investigate the stability of the analytes
and standards.
The term system stability has been defined as the stability of the samples being analyzed
in a sample solution. It is a measure of the bias in assay results generated during a
preselected time interval, for example, every hour up to 46 hours, using a single solution
(Figure 8). System stability should be determined by replicate analysis of the sample
solution. System stability is considered appropriate when the RSD, calculated on the
assay results obtained at different time intervals, does not exceed more than 20 percent of
the corresponding value of the system precision. If, on plotting the assay results as a
function of time, the value is higher, the maximum duration of the usability of the sample
solution can be calculated.
Figure 8: Schematics of stability testing
The effect of long-term storage and freeze-thaw cycles can be investigated by
analyzing a spiked sample immediately after preparation and on subsequent days of the
anticipated storage period. A minimum of two cycles at two concentrations should be
studied in duplicate. If the integrity of the drug is affected by freezing and thawing,
43
spiked samples should be stored in individual containers, and appropriate caution should
be employed for the study of samples.
1.14. SYSTEM SUITABILITY TESTS (SST):
Once a method or system has been validated the task becomes one of routinely
checking the suitability of the system to perform within the validated limits. The simplest
form of an HPLC system suitability [56-65]
test involves a comparison of the
chromatogram trace with a standard trace (as shown below). This allows a comparison of
the peak shape, peak width, and baseline resolution. Alternatively these parameters can
be calculated experimentally to provide a quantitative system suitability test report:
Number of theoretical plates (efficiency)
Capacity factor,
Separation (relative retention)
Resolution,
Tailing factor
Relative Standard Deviation (Precision)
These are measured on a peak or peaks of known retention time and peak width.
Plate number or number of theoretical plates (n):
This measure of the sharpness of the peaks and therefore the efficiency of the
column. This can be calculated in various ways, for example the USP uses the peak width
at the base and the BP at half the height is shown in the figure 9.
44
Where
Wh = peak width at 1/2 peak height
Wb = peak width at base
t = retention time of peak
Therefore the higher the plate numbers the more efficient the column. The plate number
depends on column length - ie the longer the column the larger the plate number.
Therefore the column's efficiency can also be quoted as:
Either- as the plate height (h), or the height equivalent to one theoretical plate (HETP).
h= L where L = length of column n
n Or- as plates/meter.
Relative Standard Deviation or precision:
For an HPLC system this would involve the reproducibility of a number of replicate
injections (i.e.,6) of an analytical solution. The USP requires that unless otherwise
specified by a method: - if a relative standard deviation of <2% is required then five
replicate injections should be used- if a relative standard deviation of >2% is required
then six replicate injections should be used
1.15. Precision Controlling Factors:
Retention time Pump f low and composition precision Column temperature
Mobile phase composition
45
Peak area Auto sampler: inj mode, inj volume
Pump: flow, pulsation
Detector: noise and drift, response
Data system: sampling rate,
Integration parameters
In most cases the system's Relative Standard Deviation is required; deciding
which of the other tests are required is not straightforward. To assist with the decision it
has been suggested that those parameters which have an affect on the system precision
should be used. For instance the resolution of two peaks with similar retention times
should be quoted, because, if it is below a critical value, the precision will be affected. In
addition "diode array" detectors allow for the determination of the relative purity factor
typically called: Peak Purity.
The retention time precision is important, because not only is retention time the
primary method for peak identification, but also variations can indicate problems within
the LC system (i.e. with the piston seals, check valves etc). Use of a column oven can
overcome laboratory temperature variations, which is the most common cause of
retention time drift .The most dominant factor controlling the repeatability of peak area is
the autosampler's precision, though the affect of noise and integration parameters will
become more significant with small peaks.
46
1.6. Scope and objectives of present study:
The present research work focuses on the development of new analytical
methods for determination of some selective active drugs. The work also includes
the validation of the developed methods as per ICH requirements and
demonstrates the suitability of developed methods.
Table: List of drugs selected for the study
S.No. Compound name Chemical structure / name Therapeutic activity
1
ZOPICLONE
(RS)-6-(5-chloropyridin-2-yl)-7-oxo-6,7-
dihydro-5H-pyrrolo[3,4-b]pyrazin-5-yl4-
methylpiperazine-1-carboxylate
Nervous system
depressant belongs to
non benzodiazepine
sedative and hypnotic
used to treat Insomnia.
2
PIZOTIFEN
4-(1-methyl-4-piperidylidine)-9,10-dihydro-
4H-benzo-[4,5]cyclohepta[1,2]-thiophene
For the prevention
of vascular
headache including mi
graine and headache.
And an antidepressant,
or for the treatment
of anxiety or social
phobia.
47
3
SIROLIMUS
(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S
,23S,26R,27R,34aS)9,10,12,13,14,21,22,23,24
,25,26,27,32,33,34,34a-hexadecahydro-9,27-
dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-
3-methoxycyclohexyl]- 1-methylethyl]-10,21-
dimethoxy-6,8,12,14,20,26- hexamethyl-
23,27-epoxy-3H-pyrido[2,1-c][1,4]-
oxaazacyclohentriacontine-1,5,11,28,29
(4H,6H,31H)-pentone.
Macro cyclic
polyketide and used as
immunosuppressive
agent.
4
STANZOLOL
17β-Hydroxy-17-methyl-5α-ndrostano[3,2-
c]pyrazole
Synthetic steroid used
to treat anaemia and
hereditary
angioedema.
5 STAVUDINE
1-((2R,5S)-5-(hydroxymethyl)-2,5-
dihydrofuran-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione
Synthetic thymidine
nucleoside analogue in
a class of reverse
transcriptase inhibitors
used to treat the human
immunodeficiency
virus type 1 (HIV-1).
48
6 BUSPIRONE
8-[4-(4-pyrimidin-2-ylpiperazin-1-yl)butyl]-8-
azaspiro[4.5]decane-7,9-dione
Psychotropic drug with
anxiolytic properties
belongs the class of
azaspirodecanediones
used primarily as an
anxiolytic, specifically
for generalized anxiety
disorder.
7 QUETIAPINE
2-(2-(4-dibenzo[b,f][1,4]thiazepine-11-yl-1-
piperazinyl)ethoxy)ethanol
Oral antipsychotic
drug belongs to class
the dibenzothiazepine
derivatives used for
treating schizophrenia
and bipolar disorder.
8
TELMESARTAN
2-(4-{[4-methyl-6-(1-methyl-1H-1,3-
benzodiazol-2-yl)-2-propyl-1H-1,3-
benzodiazol-1 yl] methyl} phenyl)
Angiotensin receptor
blockers used to treat
high blood pressure.
49
9. DARUNAVIR
(1R,5S,6R)-2,8-dioxabicyclo[3.3.0]oct-6-yl] N
[(2S,3R)- 4- [(4-aminophenyl)sulfonyl- (2
methylpropyl)
Protease inhibitor
used to treat human
immunodeficiency
virus (HIV).
10 DIACEREIN
4,5-diacetyloxy-9,10-dioxo-anthracene-2
carboxylic acid
Anti-inflammatory,
analgesic and
antipyretic drug used
in the treatment of
Osteoarthritis.
11 CARVIDILAL
(±)-[3-(9H-carbazol-4-yloxy)-2-
hydroxypropyl][2-(2-
methoxyphenoxy)ethyl]amine
Non-selective beta
blocker/alpha-1
blocker used to control
high blood pressure.
12 Levetiracetam
(S)-2-(2-oxopyrrolidin-1-yl) butanamide
Anticonvulsant
(antiepileptic) drug
used to treat Epilepsy
50
1.7. BIBILOGRAPHY
1. Holler, F. James; Skoog, Douglas A.; West, Donald M. Fundamentals of
analytical chemistry; Philadelphia; Saunders College Pub; 1996; ISBN 0-03-
005938-0.
2. Talanta; ―Review of analytical next term measurements facilitated by drop
formation technology‖; 2000; 51(5): 921-933.
3. Wilkins CL; "Hyphenated techniques for analysis of complex organic mixtures";
Science; 1983; 222 (4621): 291–296.
4. Holt RM, Newman MJ, Pullen FS, Richards DS, Swanson AG; "High-
performance liquid chromatography/NMR spectrometry/mass spectrometry:
further advances in hyphenated technology"; Journal of mass spectrometry; JMS
1997; 32 (1): 64–70.
5. Ellis LA, Roberts DJ; "Chromatographic and hyphenated methods for elemental
speciation analysis in environmental media";Journal of chromatography A; 1997;
774 (1–2): 3–19.
6. Guetens G et.al; "Hyphenated techniques in anticancer drug monitoring. I.
Capillary gas chromatography-mass spectrometry"; Journal of chromatography A;
2002; 976(1–2): 229–238.
7. Guetens G, De Boeck G, Highley MS, Wood M, Maes RA, Eggermont AA,
Hanauske A, de Bruijn EA, Tjaden UR; "Hyphenated techniques in anticancer
drug monitoring. II. Liquid chromatography-mass spectrometry and capillary
electrophoresis-mass spectrometry"; Journal of chromatography A; 2002; 976
(1–2): 239–47.
8. R. S. Satoskar, S. D. Bhandarkar and S. S. Ainapure; ―Pharmacology and
Pharmacotherapeutics‖; 17th edition, Popular Prakashan, Mumbai, India; 2001.
9. ―Burger’s Medicinal Chemistry and drug discovery‖; 6th edition, Wiley
Interscience, New Jersey; 2007.
10. ―Wilson and Gisvold’s Textbook of Organic Medicinal and Pharmaceutical
Chemistry‖; 11th edition, Lippincott Williams & Wilkins, New York; 2004.
51
11. A. Korolkovas; ―Essentials of Medicinal Chemistry‖; 2nd edition, Wiley
Interscience, New Jersey; 1988.
12. ―Goodman and Gilman’s The Pharmacological Basis of Therapeutics‖; 9th
edition, McGraw-Hill health professions division, New York; 1996.
13. Foye’s ―Principles of Medicinal Chemistry‖; 6th edition, Lippincott Williams &
Wilkins, New York; 2008.
14. Drugs & Cosmetics Act, 1940 & Rules, 1945, 2nd edition, Susmit publishers,
Mumbai, India; 2000.
15. Indian Pharmacopoeia, Ministry of Health & Family Welfare, Government of
India, New Delhi, 1996.
16. The United States Pharmacopoeia- the National Formulary, United States
Pharmacopoeial convention, Rockville, 2007.
17. British Pharmacopoeia, The Stationary Office, London, 2005.
18. ―Martindale - The Extra Pharmacopoeia‖, 33rd edition, The PharmaceuticalPress,
London, 2002.
19. Pavia, Donald L., Gary M. Lampman, George S. Kritz, Randall G. Engel;
―Introduction to Organic Laboratory Techniques (4th Ed.)‖; Thomson
Brooks/Cole; 2006; 797–817.
20. M.W. Dong, Modern HPLC for practicing scientists. Wiley, 2006.
21. D.A.Skoog, D.M.West, F.J.Holler: Fundamentals of Analytical
Chemistry,Saunders College Publishing
22. A.Gratzfeld-Husgen, R.Schuster, HPLC for Food Analysis, Hewlett Packerd.
23. D.A.Skoog, D.M.West, F.J.Holler: Fundamentals of Analytical
Chemistry,Saunders College
24. A.Gratzfeld-Husgen, R.Schuster, HPLC for Food Analysis, Hewlett PackerdS
25. Fast and Ultrafast HPLC on sub-2 μm Porous Particles — Where Do We Go
From Here? – LC-GC Europe
26. Xiang, Y.; Liu Y. and Lee M.L. "Ultrahigh pressure liquid chromatography using
elevated temperature". Journal of Chromatography A 2006; 1104 (1–2): 198–202.
52
27. Horváth, Cs.; Preiss B.A. and Lipsky S.R. "Fast liquid chromatography.
Investigation of operating parameters and the separation of nucleotides on
pellicular ion exchangers". Analytical Chemistry 1967; 39 (12): 1422–1428.
28. Library 4 Science: Liquid Chromatography.
29. United States Department of Labor, OSHA: Carbofuran.
30. Seton Hall University: Fluorescence Detectors.
31. National Center for Biotechnology Information: A Decade of HPLC-MS.
32. University of Bristol: High Performance Liquid Chromatography Mass
Spectrometry.
33. Seton Hall University: Detectors.
34. Ll-Yord R. Snyder, Joseph J. Kirkland and Joseph L. Glajch. Practical HPLC
Method development. John Wiley & Sons, INC, U.S.A. 2nd Edition, New
York, 1997.
35. Satinder Ahuja and Michael W. Dong. Handbook of Pharmaceutical Analysis
by HPLC, Elsevier academic press,
36. U.S.FDA, Title21 of the U.S.Code of Regulations: 21 CFR 211—Current good
manufacturing practice for finished pharmaceuticals.
37. U.S. FDA - Guidance for Industry (draft) Analytical Procedures and Methods
Validation: Chemistry, Manufacturing, and Controls and Documentation, 2000
38. ISO/IEC 17025, General requirements for the competence of testing and
calibration laboratories, 2005.
39. International Conference on Harmonization (ICH) of Technical Requirements for
the Registration of Pharmaceuticals for Human Use, Validation of analytical
procedures: definitions and terminology, Geneva (1996)
40. U.S. EPA, Guidance for methods development and methods validation for the
Resource Conservation and Recovery Act (RCRA) Program, Washington,
D.C.(1995).,
53
41. General Chapter 1225, Validation of compendial methods, United States
Pharmacopeia 30, National Formulary 25, Rockville, Md., USA, The United
States Pharmacopeial Convention, Inc., (2007).
42. U.S. FDA - Guidance for Industry, Bioanalytical Method Validation
43. G. C. Hokanson, A life cycle approach to the validation of analytical methods
during pharmaceutical product development, Part I: The initial validation process,
Pharm. Tech., Sept. 1994; 118–130.
44. J. M. Green, A practical guide to analytical method validation, Anal. Chem. News
& Features, 1 May 1996; 305A–309A.
45. B. Renger, H. Jehle, M. Fischer and W. Funk, Validation of analytical procedures
in pharmaceutical analytical chemistry: HPTLC assay of theophylline in an
effervescent tablet, J. Planar Chrom. 1995; 8: 269–278.
46. Wegscheider, Validation of analytical methods, in: Accreditation and quality
assurance in analytical chemistry, edited by H. Guenzler, Springer Verlag, Berlin
(1996).
47. S. Seno, S. Ohtake and H. Kohno, Analytical validation in practice at a quality
control laboratory in the Japanese pharmaceutical industry, Accred. Qual. Assur.
1997, 2:140–145.
48. AOAC Peer-Verified Methods Program, Manual on policies and procedures,
Arlington,Va.,USA(1998).http://www.aoac.org/vmeth/PVM.pd.
49. P. A. Winslow and R. F. Meyer, Defining a master plan for the validation of
analytical methods, J. Validation Technology, 1997; 361–367.
50. J.Breaux, K. Jones, and P. Boulas, Pharmaceutical Technology, Analytical
Technology and Testing, 2003; 6-13.
51. S.O. Krause, A Guide for testing biopharmaceuticals, Part II: acceptance criteria
and analytical method maintenance, Pharm. Tech. Europe, 2006; 18(6): 29-38.
52. CITAC/EURACHEM, Working Group, International guide to quality in
analytical chemistry: An aid to accreditation, 2002.
54
53. J. Vessman, Selectivity or specificity? Validation of analytical methods from the
perspective of an analytical chemist in the pharmaceutical industry, J. Pharm &
Biomed Analysis 1996; 14: 867–869.
54. L. Huber and S. George, Diode-array detection in high-performance liquid
chromatography, New York, Marcel Dekker, ISBN 0-8247-4 (1993).
55. EURACHEM – The Fitness for Purpose of Analytical Methods A Laboratory
Guide to Method Validation and Related Topics, 1998
56. GLP - The United Kingdom Compliance Programme (Department of Health)
1989.
57. Code of Fed: Reg 21 Part 211.160 (Government Printing Office Washington DC
(1978).
58. BS 7501 EN 45001 General Criteria for the operation of Testing Laboratories
1989.
59. Practical Liquid Chromatography An lntroduction R W Yost, L S Ettre, R D
ConlanPerkin-Eimer 1980.
60. United States Pharmacopeia XXI I (United States Pharmacopeial
Convention,Rockvilie, MD, 1990).
61. Validation of compedial Assays-Guidelines'Pharmacopeial Convention,
Rockvilie,MD, 1985".
62. Perkin-Eimer Technical Bulletin LCTB 19 "Liquid Chromatographic
Considerations for High Sensitivity Impurity and Stability Testing of
Pharmaceuticals", Michael W Dong, etal.
63. British Pharmacopeia 1988. Appendix A84 fit D.
64. Poile A E & Conion R D J. Chromatogr. 204 149 -152.
65. http://www.standardbase.com for more experiments.