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,

3

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

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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

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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.

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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.

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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

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