07 Chapter 1shodhganga.inflibnet.ac.in/bitstream/10603/79276/7/07_chapter 1.pdfDep artment of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University Aurangabad 3 Chapter

____________________________________________________________________________ Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University Aurangabad

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Chapter : 1

Introduction


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I n t r o d u c t i o n :

Efficiency and speed of research and development in order to meet growing

competition in the pharmaceutical industry is a challenge. Any registered

medicine represents approx. 10 years of research and development and an

investment of approx. 500 million US dollars.

Moreover, a new product is no guarantee of commercial success typically in

the pharmaceutical industry, only one in four new products brought onto the

market recoup its research and development investment [1].

Sales of pharmaceutical products in the last decade have increased i.e. in 1997

world wide sales of pharmaceutical products was total of $329 billion, an

increase of approximately 6% compared to the sales from 1996. From 1992 to

1997, global sales of pharmaceutical products increased by approximately

40%, which is an annual growth rate of 7%. Moreover, it is estimated that

world wide sales will rise to $420 billion in the year 2002.

Research and development of pharmaceuticals

Research and development of new drugs is a difficult and time consuming

process. Because of the arguments described in the introduction (Investment

of 500 $ million, high risk) the time pressure on research, development and

investment is large. The research and development pathway takes a

pharmaceutical compound through three stages; the drug discovery research

phase, the pre clinical development phase and the clinical development phase.

The drug discovery research phase primarily involves the design of a

biological model. In the pre and early clinical development phase safety is the

crucial issue. Also pharmacokinetics, dosage forms and stability are studied in

this stage.

In clinical phase- I the compound is tested in healthy volunteers. Given

dosages which for safety reasons, start much lower on a mg/kg basis than

those used to study the safety, pharmacokinetics and pharmacodynamics in

animals. After behaviour of the compound has been assessed in a limited

number of volunteers, resulting in a detailed profile, the compound is studied

on a larger scale in patients.


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In phase- II a group of usually 100-200 carefully selected patients receive the

lead compound in its expected therapeutic dose to establish the efficacy and

safety.

In phase- III the medication efficacy and safety is compared against placebo

and active compounds in typically a group of 3000- 5000 patients who

represent the population that will ultimately receive the treatment. This leads

on to submitting detailed files to registration authorities with a request for

marketing authorization.

In phase- IV the registration of the medication is followed with a view to

identify rare, unforeseen side effects in a real clinical situation by means of

post marketing surveillance, cost effectiveness and real life efficacy studies[2].

Past decade has witnessed some path breaking discoveries in the field of

chemistry and biology. These discoveries have almost significantly changed

the way pharmaceutical research was conducted. In the field of organic

chemical synthesis, combinatorial chemistry has tremendously increased the

throughput of chemical synthesis. Advances in the field of computational

chemistry and other branches of chemo metrics had made it possible to design

the drugs that give maximum therapeutic effect and minimum adverse effect.

In the field of biology discoveries in genomic, decoding of human gene, has

led to better understanding about the source of various ailments and will

ultimately help in designing better drugs in future. Thus the current trends in

drug development emphasize high volumes approaches to accelerate lead

candidate generation and evaluation. The impact of these developments on the

overall drug development cycle has been significant, creating unprecedented

opportunities for growth and focus particularly in analytical science.

Novel drug delivery system is another important area of pharmaceutical

research where most thrust is now placed. It aims at developing new and

improved drug delivery methods by which the maximum therapeutic effect

minimum toxic effect of a drug is experienced. The rate and extent of drug

released is precisely controlled. Some of these delivery systems or dosage

forms (pharmaceutical preparation) are usually prepared using different types

of polymers which form complex matrix. These matrices make quantification


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of drugs difficult, and analytical methods used for their analysis need to be

selective, accurate and robust.

Analytical chemistry in pharmaceutical research and development

Development of a pharmaceutical product involves both the optimization of

the chemistry and manufacturing, as well as determination of the bio medical

profile. The quality of drug substances (active compounds) and drug products

(e.g. tablet, liquid for injection) is determined by the design, development and

in process- control. Additionally, tests, procedures and acceptance criteria,

play a major role in assuring the quality. For monitoring the quality of the

drug substances and drug products the global tests and specifications has been

established in the form of guidelines and pharmacopeias [3-6]. The biomedical

profile is determined through e.g. pharmacokinetic and metabolism studies.

These development activities are supported by analytical techniques which can

be divided into specific (on a case by case basis) and universal (generally

applicable) techniques. Different analytical techniques are applicable to

characterize drug substances and drug products. Separation methods play an

important role in determining impurities and active compounds and coupled

on – line with spectroscopic techniques structural information can be obtained.

Limits for the presence of impurities in drugs are established by the

international conference on harmonization (ICH).

For metabolism studies separation methods are coupled on line with

techniques such as mass spectrometry for selective and sensitive detection.

From the separation methods, in the pharmaceutical industry reversed phase

liquid chromatography (RPLC) is most widely applied because of the broad

range of compounds that can be analysed i.e. non-ionic, ionisable and ionic

compounds [7]. Moreover, when a pharmaceutical product is on the market,

LC is the most widely used analytical technique in quality control to determine

the identity and content of drugs and impurities in production batches.

Pharmaceutical industry is highly regulated industry. It is under increased

scrutiny from the government regulatory authorities and public interest groups

to curtail costs and consistent delivery of safe and efficacious products to

market. Quality of drug products has become the focus of both industry and

regulatory authorities. Physico-chemical properties of drug substance and drug


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product are more closely monitored, both at the time of finished product

release and throughout the shelf life of product. Determining stability of drug

substance and drug product under accelerated and real time stability study

conditions is a mandatory requirement for registration of product. Very

sensitive and specific stability indicating analytical methods are required for

analysis of stability study samples. Thus faster drug discovery and drug

product development programs coupled with greater requirement from

industry and regulatory authorities, have resulted in increased pressures on

pharmaceutical analyst to deliver accurate and precise analytical data in a

shortest possible time.

Wet chemical and the classical methods of chemical are inadequate to meet

this challenge faced by pharmaceutical analyst. Today apart from high

throughput, better sensitivity and selectivity are desired from analytical

methods. Various instrumental methods used for chemical analysis have often

provided better sensitivity, selectivity, and high throughput as compared to

classical methods. Excellent progress in the field of analytical instrumentation

coupled with advances in the field of information technology has led to

automation of many instrumental methods of analysis. This has further

resulted into more precise and accurate analysis.

Research in analytical chemistry has also become very rapid with major

discoveries being reported almost on regular basis. Some of the more

important discoveries in recent times are in the area of Electrospray and

Nanoelectrospray (Ion source interface for LC-MS), Capillary electrophoresis,

Solid phase microextraction, matrix assisted laser desorption/ionization and

DNA analysis by CE. All these developments have unprecedented application

to pharmaceutical research. Research in analytical chemistry has also resulted

in significant improvement in other modern instrumental analytical techniques

like HPLC, HPTLC, GC, spectrophotometry etc. These developments have

revolutionized chemical analysis in many areas and more particularly in the

field of pharmaceutical analysis.

Some of the modern instrumental techniques, that find application for analysis

of different physico- chemical properties of drugs are,

� Ultraviolet, Visible Spectrophotometry


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� Infrared Spectrophotometry

� Nuclear magnetic resonance Spectroscopy

� Electron paramagnetic resonance

� X- ray diffraction and X-ray fluorescence methods

� Thermo analytical methods ( DSC, DTA and TGA)

� Electo-analytical methods (Voltametry, Amperometry,

Potentiometry, conductometry and ion- selective electrodes)

� Capillary elcetrophoresis and related techniques

� Thin layer chromatography

� Gas chromatography

� High performance liquid chromatography

� Supercritical fluid chromatography

� Liquid chromatography- mass spectrometry.

Of the above listed instrumental techniques, chromatographic methods are the

most important analytical techniques available to the today’s analytical

chemist for accurate quantitative work. Presently most of the pharmaceutical

analysis (nearly 80%) and most of the pharmacopoeial assay methods are

based on chromatographic techniques. The widespread use of

chromatographic techniques is because they posses a dual capability, the

mixture is separated into its components and simultaneously each component

are quantified accurately and precisely. In fact, all forms of chromatography

are primarily separation techniques, but by employing detectors with linear

response to monitor the eluents leaving from the chromatographic system, the

amount of each component present can also be determined. As different types

of sensitive detectors are now available, even trace components of mixture can

be accurately quantified. The simultaneous determination of multicomponent

drugs in the pharmaceutical products with impurities and degradants are

achieved by the new chromatographic techniques.

Considering the need of the pharmaceutical industry to analyse and released

the products in a shortest period attempt have been made to develop some new

chromatographic methods for the analysis of active ingredients in combined


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dosage forms and are presented in this thesis. To develop new analytical

methods for the estimation of the drugs are from antidiabetic category.

The brief discussion of the instruments and chromatographic methods used in

the present work viz. reverse phase high performance and micellar liquid

chromatography is presented below.

Discussion on chromatographic techniques used in the present work

Chromatography has been classically defined as separation process that is

achieved by the distribution of substances between two phases, a stationary

phase and a mobile phase. Those analytes distributed preferentially in the

mobile phase will move more rapidly through the system as compared to those

distributed preferentially in the stationary phase, and are thus separated from

each other. The analytes will elute or move with mobile phase in order of their

increasing distribution coefficients with respect to the stationary phase. This

distribution is affected by various phenomenon like, adsorption, partition, ion-

exchange and size exclusion.

To understand the nature of chromatographic separation, the process of

analyte migration through a chromatographic column needs to be considered.

Consider the progress of a analyte through a chromatographic column as

depicted in diagram (Fig 1).

It is seen that as a result of slightly greater displacement of analyte in mobile

phase, the concentration of analyte in the mobile phase at the front of the peak

exceeds the equilibrium concentration with respect to that in the stationary

phase. It follows that there is a net transfer of analyte from the mobile phase in

the front part of the peak to the stationary phase to re-establish equilibrium as

the peak to the peak progresses along the column. At the rear of the peak, the

converse occurs. As the concentration profile moves forward, the

concentration of analyte in the stationary phase at the rear of the peak is now

in excess of the equilibrium concentration. Thus, to re-established the

equilibrium, again there is net transfer of analyte from stationary phase to

mobile phase occurs.

This process is continuously repeated and ultimately the analyte is completely

transferred from stationary phase in mobile phase and elutes


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Fig 1 : Showing passage of analyte band along a chromatographic column

Following important chromatographic techniques uses in the pharmaceutical

analysis are.

� Thin layer chromatography (TLC) and High Performance thin layer

chromatography (HPTLC).

� Gas Chromatography (GC).

� High Performance Liquid Chromatography (HPLC)

-Reverse phase high performance liquid chromatography (RP-

HPLC).

- Normal phase high performance liquid chromatography.

- Ion – exchange chromatography (IEC)

- Micellar liquid chromatography (MLC)

- Size- exclusion chromatography (SEC)

� Super critical fluid chromatography (SFC)

Of the above mentioned techniques used, RP-HPLC is mostly used in

pharmaceutical analysis. Micellar liquid chromatography is another

important variant of liquid chromatography, which has been shown to be useful

for analysis of drugs of different physico-chemical characteristics. The other

important advantages of micellar mobile phases, they are significantly less

polluting biodegradable, less inflammable, and inexpensive when compared to

hydro-organic mobile phases. These techniques have been used in the present

work to develop new analytical methods and are discussed in more detail

below.


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Reversed Phase High Performance Liquid Chromatogrphy

Instrumentation :

HPLC system consists of a solvent reservoir, a pump, an injector system or

autosampler, a column , a detector and a data collection and processing unit

( mostly microcomputer using a appropriate software).

Solvent Reservoir

A modern HPLC unit is equipped with one or more glass or stainless steel

reservoirs, each of which contains 500 ml or more of solvent. Provisions are

often included to remove dissolved gases and dust from the liquids.

An elution with a single solvent mixture of constant composition is termed as

isocratic elution. In gradient elution two or more solvent systems that differ

significantly in polarity are employed. The ratios of the two solvents are varied

in a preprogrammed way to improve the separation efficiency.

Pump

A HPLC pump should satisfy the following basic requirements

1. Pulse free output

2. Flow rates ranging from 0.1 to 10 ml/min

3. Flow rated reproducibilities of 0.5% relative or better

4. Resist to corrosion by a variety of solvents.

These requirements can be satisfied by a number of different designs but most

systems use either reciprocating or diaphragm pumps, out of which

reciprocating pumps are most widely used. The pumping action is achieved by

a cam – driven reciprocating piston driven by a constant speed motor. It is dual

headed pump with two pistons.

Sample injection system and Autosamplers

The most widely used method for sample introduction is based on sampling

loop. Interchangeable loops that provide a choice of sample size ranging from 5

to 500 µl are commercially available. Now–a-days autosamplers with

calibrated syringes are used to inject variable volumes of sample. This mode of

injection is also called as variable loop injection system. Auto samplers can be

programmed to inject sample at regular time intervals or at predetermined time.

In built column ovens in auto samplers provide means of controlling column

temperature.


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Columns

a ) Analytical columns : HPLC columns are constructed from smooth bore

stainless steel tubing. The majority of the columns range in length from 10 to

30 cm. The inside diameter of the columns often range from 4 to 10 mm. the

common particle sizes of the packing are 3,5 and 10 µm. The most commonly

used packing material is silica, alkyl bonded silica and polymer based particles.

b) Guard columns :

Often a short guard column is introduced before the analytical column to

increase the life of the analytical column by removing particulate matter and

contaminants from the solvent. The composition of the guard column packing

should be similar to the analytical column.

Detectors

The important characteristics sought in a HPLC detector are sensitivity,

reproducibility, selectivity, stability, low operating cost and a wide linear range.

Commonly used detectors are,

a) UV- Visible spectrophotometry : Majority of organic compounds can be

detected by their absorption of UV or visible light. Most of the drugs have

chromophores and auxochromes, like conjugated double bonds, aromatic rings,

unsaturated bonds, aromatic rings, carbonyl, ester, nitro, nitrile, amine

functional groups and heterocyclic rings, etc. In spectrophotometric detection

the sensitivity and response, depends primarily on the extinction coefficient of

the analyte at the wavelength used for the detection. It differs markedly for

different compounds depending on their chromophores. The advantage of the

spectrophotometric detector is that it responds to most compounds and hence is

known as pseudo- universal detector. The various types of UV- visible

detectors used in HPLC are,

i ) Single wavelength detector

ii) Variable wavelength detector

iii) Photo-diode array detector

These utilize a deuterium or xenon lamp that emits light over the UV spectral

range. The light from lamp is focused by means of an achromatic lens through

the sample cell onto a holographic grating. The dispersed light from the grating

is arranged to fall on a linear diode array. The diode array gives signal in


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proportion of the intensity of radiation incident on it and this entire spectrum of

the analyte eluting from the column can be continuously recorded. By

examining the spectrum of analyte peak from beginning to end, peak purity

(peak homogeneity) can be evaluated. If a single component is present in the

peak (i.e. no co-eluting component is present), the UV spectra obtained across

the peak should be superimposable. Chromatography softwares used for

intergration and data processing usually provide spectral analysis option, which

compares the spectrum obtained over the entire peak and provides peak purity

data.

b) Refractive index (RI) detector :

These detectors sense the difference in refractive index between the column

eluent and a reference stream of pure mobile phase. As any analyte can be

detected as long as there is a difference between RI of analyte and mobile

phase, hence it is a universal detector. But these detectors are less sensitive as

compared to UV detectors and are only used for analysis of compounds that do

not absorb UV radiation.

c) Fluorescence detector :

Analytes that show fluorescence properties can be analyze using these

detectors. As relatively few compounds possessed this property the use of this

detector is limited. But some analytes can be converted to fluorescent

derivatives by pre-column or post column derivatization. Due to the

spectroscopic and chemical specificity, fluorescence detection is very selective

and very useful for analyzing compounds from complex matrix like biological

fluids. As fluorescence is a emission technique in which the background signal

in the absence of fluorophore is virtually zero, therefore the sensitivity is more

in this technique.

d) Electrochemical detector :

Electrochemical detectors measure either the conductance of the eluent or

current associated with the oxidation or reduction of analytes. To be capable of

detection using the first method the analytes must be ionic and using the second

method the analytes must be relatively easy to oxidize or reduce.

Data collection and processing unit


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Signals from detectors are integrated using integrator or microcomputers.

Now – a – days microcomputers (PC’s) are widely used for data collection and

processing. The collected data is processed using various chromatography

softwares. These softwares provide variety of options for peak integration. The

spectral analysis option provides for determining the chromatographic peak

purity, comparing spectrums of analyte peaks acquired using diode array

detector, and matching them with library spectra for better identification of

unknown peaks.

Technique

RP-HPLC is generally considered as a type of partition chromatography in

which the analytes distribute between the non polar bonded stationary phase

and polar mobile phase. It is similar to extraction of different compounds from

water into an organic solvent such as octanol, where more hydrophobic (non-

polar) compounds preferentially extract into the non polar octanol phase. In RP

HPLC the stationary phase is packed into a column and is generally made up of

a silica support modified with a C8 or C18 bonded phase. More recently, the use

of macro-porous polymer resins as a support material is also gaining more

acceptance. These resins are generally formed by copolymerization of

polystyrene and divinylbenzene. Resin based packing material has advantage

over silica based ones, in that they can be used with mobile phases having very

high and low pH (pH 1 to 13) which is required in the analysis of peptides and

proteins. But columns packed with macro- porous polymer resins have less

efficiency as compared to those packed with silica particles. Semi polar

stationary phases consisting of silica support modified with either cyanoalkyl

groups (CN) are used for analysis of hydrophilic compounds (polar) in RP-

HPLC as stationary phase. The stationary phases used in RP HPLC are less

polar than water-organic mobile phase because of which the analyte molecules

partition between the polar mobile phase and non polar C8 or C18 stationary

phase. The hydrophobic (non polar) compounds are retained more strongly on

the stationary phase while the relatively hydrophilic (polar) compounds weakly

retained on stationary phase and elute first with the mobile phase while the

most hydrophobic compounds elute last thus for a given mobile phase


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composition , the result is a differential retention of analyte according to their

hydrophobocity .

The retention in RP HPLC is also determined by the other experimental

conditions like mobile phase composition and pH , temperature , particle size of

column packing material (silica or polymer) used , and dimensions of column (

length and diameter) . The effects of these parameters on retention of analyte

on different types have been well studied and they can lead to a systematic

approach of RP HPLC method development.

Some of these important parameters are discussed below.

i) Mobile phase composition and pH :

Mobile phases used in RP HPLC consist of water (or aqueous buffer) and

organic solvents. The retention of analytes can be changed by changing the

phase composition i.e. proportion of water and organic solvent. The retention is

less stronger for mobile phase which is less polar i.e. containing a higher

proportion of organic solvent. Initial goal of method development is to obtain

adequate retention of all the analytes. An adequate retention range is mostly k’

values in the range of 1-10 , i.e 1 < k’< 10.

The retention of analytes is studied with change in proportion of organic

solvent and values of k’ are plotted against the percentage of organic solvent in

mobile phase to determine the mobile phase composition in which most of the

analytes are separated and have retention in the above mentioned range. The

strength of mobile phase to elute the analytes also depends on the type of

organic solvent used. The most commonly used solvents are acetonitrile,

methanol and tetrahydrofuran. Among these solvents, acetonitrile has the

highest solvent strength (elution strength) primarily because its low viscosity,

higher polarity index, and its ability to solubalise most of the organic

compounds. Generally, better peak shape is also achieved with the use of

acetonitrile as compared to methanol or tetrahydrofuran.

Organic compounds used as drugs often have one or more functional groups

like COOH, -NHR, -CONH2, SH, -COOR etc and /or also contain heterocyclic

rings. These compounds can have their pka values in the working pH range or

silica based column (pH 2 .0 – 8.0). Thus with the change in pH a

transformation of analytes between ionized and non ionized can occur. For


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acidic compounds, a pH higher than their pka value will result in ionized

species, whereas for basic compounds a pH lower than their pka value will

result in ionization. As ionized species are weakly retained as compared to

their unionized once on stationary phases used in RP HPLC, changing the pH

of mobile phase used for elution can modify retention of analytes. The situation

is somewhat complex for compounds containing more than one functional

group. Thus to optimize the elution retention behaviour of analytes are studied

with change of pH of mobile phase and the k’ values of different analytes

present in mixture are plotted against the pH of mobile phase. From this plot

the optimum pH of mobile phase at which most of the analytes are separated

and have reasonable retention times is selected.

ii) Column type and temperature:

A change in the type of column used, often produces significant changes in

retention of analytes. The columns most frequently used in RP HPLC are C8,

C18 , Phenyl and CN . Hydrophobic (non polar) analytes are better retained on

non polar stationary phases like C18, C8 and phenyl whereas more hydrophilic

( polar) analytes are retained on semi polar stationary phases like CN. Change

in selectivity by changing the column type may also be advantageous if only

one organic solvent used in mobile phase. Thus a change in column type can be

useful parameter for attaining difficult separation. Retention of analytes

generally decreases with increase in column temperature. However, this change

usually does not modify the selectivity significantly for non ionic compounds.

For ionic compounds, the change in temperature can result in large variation in

selectivity as in this case retention is dependent on more than one processes and

this respond differently to change in temperature. But the use of higher

temperatures in combination with low or high mobile phase pH can lead to a

rapid loss of bonded phase with most columns. Thus the use of temperature for

modifying selectivity should be made with caution.

iii) Column packing materials of different particle sizes have different

applications. There are three types of silica particles used in column packing,

a) Micropellicular particles


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The size of these particles ranges from 1.5 to 2.5 µm. Micropellicular particles

have very thin outer skin of interacting stationary phase that allows fast mass

transfer and thus displace outstanding efficiency for micromolecules.

b) Perfusion particles

The size of these porous particles are in 0.4 to 0.8 µm. Perfusion particles

allow high flow rates with less band broadening and pressure drop. However,

experiences with this type of particles are limited and hence their practical

implication are incomplete.

c) Totally porous microspheres

The particle diameters are in the range of 3 or 5 µm. Totally porous particles

are the most commonly used particle type because they provide of favourable

compromise of desired properties: efficiency, sample loading, durability,

convenience, and availability. The particles are available in a variety of

diameters, pore sizes, and surface areas, so that all types of HPLC methods can

be developed with this packing material. Totally porous particles of diameter

about 5 µm represent a good compromise for analytical columns in terms of

column efficiency, back pressure, and lifetime. The analytical columns

containing silica packing material having 5 µm diameters are used to develop

the methods in the present thesis.

Micellar Liquid chromatography

The instrumentation used in MLC is same as that used for RP-HPLC

Technique

Micellar liquid chromatography used surfactant solution, above its critical

micellization concentration (c.m.c) as the mobile phase instead of hydro-

organic mobile phase used in RP-HPLC. These aqueous solutions of surfactants

at concentrations above their cmc contain micelles, along with monomers,

dimmers, etc. and constitute a complex mobile phase modifier. The analytes are

eluted from column with micellar aggregates and bulk solvent (water).


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Micellar solutions are microscopically heterogeneous, being composed of the

amphillic micellar aggregates and the bulk surrounding solvent that contains

surfactants whose concentration is approximately equal to the cmc. Micelles

are not static but exist in equilibrium with surfactant monomer. The monomer

adsorbs on alkyl bonded stationary phases (e.g. C1, C18 and C8) in at least two

ways, i) the hydrophobic adsorption where the alkyl tail of the surfactant would

be adsorbed and the ionic group would then be in contact with the polar

solution, giving stationary phase some ion exchange capacity for charged

analytes., ii) sylanophillic adsorption, where the ionic head group of the

surfactant would be adsorbed. The amount of surfactant adsorbed on the

stationary phase remains constant after equilibration once the concentration is

above cmc and such a stationary phase is also called a surfactant modified

stationary phase.

Different types of interactions are possible in MLC (electrostatic, hydrophobic

and steric) between analyte and micellar mobile phase and surfactant modified

stationary phase. None of these interactions can occur with hydro-organic

system. The analytes are separated based on their differential partitioning

between the bulk aqueous phase and the micelllar aggregates in the mobile

phase and between the bulk aqueous phase and the surfactant –coated

stationary phase. For water insoluble species, partitioning can also occur via

direct transfer of the analyte in the micellar aggregates to the surfactant

modified stationary phase. Hence, the elution of analytes in MLC depends on

three partition coefficients, that between stationary phase and water (PSM),

between stationary phase and micelle (Psm) and between the micelle and water

(PMW).

First Armstrong and Nome and late Arunyanar and Clive Love proposed

different models to describe the relation between retention of analytes and

micelle concentration in purely micellar mobile phase. The equation as

mentioned by Garcia Alvarez- Coque etal .

1 = KAM [M] + 1 k’ ΦPSW ΦPSW

Where k’ is the capacity factor, (M) is the total concentration of surfactant in

the mobile phase minus the cmc. In this equation Φ is the ratio of the volume of


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stationary phase Vs, to the volume of mobile phase Vm, in the column, KAM is

the analyte micelle binding constant and PSW is analyte stationary phase-water

partition constant.

This equation can be used to describe the retention of apolar, polar and even

ionic analytes, chromatographed with anionic, cationic and non-ionic

surfactants. The non- homogenous nature of the micelle creates a unique

situation in which different analytes can experience various micro-environment

polarities in a given mobile phase. Retention of analyte will depend on the type

of interaction it has with the micelle and the surfactant modified stationary

phase. Non-polar analytes should be affected by hydrophobic interactions but

for analytes that are charged two distinct situations can be considered.

i) charge on the analyte and surfactant have same sign,

ii) charge on analyte and surfactant have opposite sign.

Fig 2 (a), (b) and (c) show diagrammatically the analyte micelle and analyte

and analyte stationary phase interactions.

Figure 2


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Analyte- micelle and analyte –stationary phase hydrophobic ( ) and

electrostatic interactions ( ) with an anionic surfactant (a) apolar analyte,

(b) anionic analyte, and (c) cationic analyte.

The first situation is encountered when a anionic analyte is chromatographed

with anionic surfactant or a cationic analyte with cationic surfactant.

Electrostatic repulsion from the micelle should not affect retention as the

analyte would still reside in the bulk of mobile phase and therefore will still

move down the column. In contrast, repulsion from surfactant modified

stationary phase should cause a decrease in retention. Analyte may be eluted

in void volume. However, they may also be retained due to hydrophobic

interaction with the stationary phase. The second situation appears when a

analyte is chromatographed with an oppositely charged surfactant, where

electrostatic attraction occurs between both species. If the electrostatic

attraction with the micelle is complemented by a hydrophobic interaction the

analytes remain in the mobile phase for a longer time and retention will

decrease. However electrostatic and hydrophobic interactions with the

stationary phase may be sufficiently large to offset the increase the micellar

attraction and would increase retention.

Factors affecting retention of analyte :


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i) Concentration of surfactant: For many analytes, an increase in concentration

of surfactant in mobile phase results in decrease in retention. Thus, elution

strength of mobile phase increases with increase in concentration of surfactant.

However, this is possible only when the analyte interacts with the mobile

phase. Different analytes show any of the following three types of interaction

a) binding interaction- the compound binds to micelle in mobile phase and its

retention decreases with increase in concentration of micelle, (Kam> 0)

b) non binding interaction compound does not bind to micelle and thus

retention remains unaltered with increase in concentration of micelle,

( Kam=0 )

c) Anti-binding interaction-compounds is strongly excluded from micelle and

thus results in increase in retention with increase in micelle concentration

(Kam <0) .

Non binding and anti- binding interaction are rare as compared to binding

interactions. Non binding interaction occurs only when analyte and micelle

have similar type of charge (i.e. both are either positively or negatively

charged). However, in this case sometimes binding is observed. Antibinding

can only be observed when analyte and micelle have similar type of charge

and stationary phase does not adsorb significant amount of surfactant. Thus,

anti-binding can only be observed with stationary phase like C1 or CN.

ii) pH of mobile phase : Retention of weak acid or bases are affected by

change in pH of micellar mobile phase. As the dissociated and undissociated

forms of anaytes have different analyte-micelle partition constant, retention is

altered significantly only when pH of mobile phase is near to pka value of

analyte. C18 and CN bonded phase interact very differently with surfactant

monomers, resulting in a different elution behavior of organic acids and bases

as a function of pH and concentration of micelle.

iii) Organic additives : Organic additives, mostly short chained alcohols to

micellar mobile phase alter the chromatographic retention and efficiency

significantly. Small amounts of alcohols like propan-2-ol , butan-1-ol penta-1-

ol etc., bring about significant decrease in the amount of surfactant coated on

the stationary phase and thickness of surfactant monomamer layer, which

results in better mass transfer and hence better efficiency and reduced


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retention . The effect of modifier increases with increase in its concentration

and hydrophobocity. Thus reduction in retention of analytes is greater with

pentan-1-ol, followed by butan-1-ol and propan-1-ol. The reduction in

retention of analyte and increased efficiency is also attributed better solubility

of analyte in mobile phase in presence of alcohols.

iv) Ionic strength: An increase in ionic strength of mobile phase (by addition

of salt like NaCl) decreases the thickness of charged double layer surrounding

the micelle. Thus analytes which are excluded out of micelle due to the

charged double layer (non-binding and anti-binding interaction), can interact

hydrophobically with micelle with the increase in ionic strength and thus can

have lesser retention.

Validation of Method

Once a particular method is optimised, it is necessary to validate the method.

Valdiation is the process of establishing the suitability of a method for

authorization for its use in the laboratory. Validation of a chromatographic

method is not limited to pharmaceuticals, it is applicable to all sample types

and all chromatographic techniques. A huge validation work is done in the

pharmaceutical industry because it is initiated by various agencies that to

monitor the development, manufacture, and sale of pharmaceutical products.

These requirements are essential so that only safe and effective products are

made available for the sale to the public. Ensuring that the analytical methods

for assay and control of these products are operating correctly is one way to

protect the safety of the patients using the product. The developed analytical

method needs to be validated for following common parameters.

a) Specificity- selectivity

Specificity is the ability of the method to measure the analyte without

interference from other sample matrix components. The term specificity is

used to express the quality of the separation when not all peaks are of equal

importance. The term selectivity is generally used when all the peaks are of

equal importance. This is determined by spiking a solution with all the known

possible interfering components of the sample. In the case of drug substance

and drug products these would include synthetic precursors and degradation

products.


23

b) Linearity

The linearity of an analytical procedure is its ability to produce responses that

are directly proportional to the concentration of the analytes in the sample

within a given working range. The range of the procedure is an expression of

the lowest and highest levels of the analytes that the method can determine

with reasonable accuracy and precision. Results of linearity test are analysed

by a method of linear regression and represented in terms of the coefficient of

regression (r).

c ) Precision

The precision of the analytical method relates to the degree of agreement

among individual test results and how individual results are scattered from the

mean value. This is generally determined by assaying multiple replicates from

a homogenous sample and calculating the standard deviation of the results.

The precision of the method is a combination of several factors including the

homogeneity of the samples, the sample preparation techniques and the actual

separation and detection of the components. Also replicate analysis to be done

in different labs, with different analysts, different day and different columns to

determine the actual variability of the method over a period of time. This

determination is often called as method ruggedness.

d) Recovery

Recovery is simply a determination that all the species of interest are carried

through the sample preparation scheme without loss. This determination is

also referred as the accuracy of the method. Recovery is determined by

assaying samples of matrix to which known amounts of analyte have been

added and comparing results of their assay to those of standards prepared

without matrix. The recovery is the amount of analyte recovered from the

spiked samples compared to that from the standards. The acceptance criteria

for recovery must depend on the nature of the samples.

e) Limit of detection

It is defined as the lowest concentration of substance in a sample can be

detected, but not necessarily determined quantitatively, under the stated

experimental conditions. ICH guidelines recommended three types of method


24

for determination of LOD. In the present work slope method is used in which

LOD is determined by the following equation

LOD= 3.3 x Standard deviation of residuals (�)

Slope (S)

f) Limit of quantitation

This is defined as the lowest concentration of a substance that can be

determined quantitatively with acceptable accuracy and precision using the

recommended procedure of analysis. The LOQ is determined by the following

equation

LOQ= 10 x Standard deviation of residuals (�)

Slope (S)

g) Robustness

Robustness of the method is established by carrying out analysis of the same

sample by deliberately varying certain experimental conditions such as pH,

composition of the mobile phase and temperature in chromatographic analysis,

sample preparation using different volume of solvent and different time of

sonication etc.

h) Solution stability

The extended use of auto-injectors for HPLC assays has certainly increased

the number of samples that one analyst may run in a given length of time.

However these automated systems are not infallible, and occasionally a

problem will occur while the instrument is running unattended that will

prevent the completion of the run. In this event, it is useful to know if the

samples and standards may simply be reused and run again. Testing the

stability of these samples solutions provide an assurance that the integrity of

the analytes has not changed. Solution stability is established by analyzing a

standard solution or sample solution at different time intervals and comparing

it with a freshly prepared standard solution of comparable concentration.

Typically solution is considered to be stable for the time till its assay is within

+ 2.0% of its original assay level.

i) System suitability


25

Both the FDA and the pharmacopoeia have recommended the use of system

suitability tests for chromatographic assays. The purpose of this test is to

demonstrate that the chromatographic system is properly operating at that

particular time. This generally entails a test of both the precision and

chromatographic performance of the system. The precision is demonstrated by

the injections of one standard or sample solution five times. The response of

the peak of interest is measured and its reproducibilities is calculated.

Bioanalytical Method development and Validation

Bioanalytical chemistry is the qualitative and quantitative analysis of drug

substances in biological fluids (mainly plasma and urine) or tissue. It plays a

significant role in the evaluation and interpretation of bioavailability,

bioequivalence and pharmacokinetic data [16]. The main analytical phases

that comprise bioanalytical services are, method development, method

validation and sample analysis (method application).

Owing to increased interdependence among countries in recent times it has

become necessary for results of many analytical methods to be accepted

internationally. Consequently, to assure a common level of quality, the need

for and use of validated methods has increased [17].

Analytical methods are used for product research, product development,

process control and chemical quality control proposes. Each of the techniques

used, chromatographic or spectroscopic, have their own special features and

deficiencies, which must be considered. Whatever way the analysis is done it

must be checked to see whether it does what it was intended to do i.e. it must

be validated. Each step in the method must be investigated to determine the

extent to which environment, matrix, or procedural variables can affect the

estimation of analyte in the matrix from the time of collection up to the time of

analysis.

A full validation requires a high workload and should therefore only start

when promising results are obtained from explorative validation performed

during the method development phase. The process of validating a method

cannot be separated from the actual development of method conditions,

because the developer will not know whether the method conditions are


26

acceptable until validation studies are performed [18]. Method development

clears the way for the further processes on the validation stage. It must be

recognized that proper validation requires a lot of work. However, this effort is

repaid by the time saved when running the method routinely during sample

analysis.

Method development

A bioanalytical method is a set of all the procedures involved in the collection,

processing, storing and analysis of a biological matrix for an analyte [19].

Analytical methods employed for quantitative determination of drugs and their

metabolites in biological fluids are the key determinations in generating

reproducible and reliable data that in turn are used in the evaluation and

interpretation of bioavailability , bioequivalency and pharmacokinetics [20].

Method development involves evaluation and optimization of the various

stages of sample preparation, chromatographic separation, detection and

quantification. To start these work an extensive literature survey, reading work

done on the same or similar analyte and summarizing main starting points for

future work is of primary importance. Based on the information from this

survey, the following can be done.

The choice of instrument that is suitable for the analysis of analyte of interest.

This include the choice of the column associated with instrument of choice ,

the detector, the mobile phase in the HPLC, and the choice of carrier in gas

chromatography (GC).

Choice of internal standard, which is suitable for study, must have similar

chromatographic properties to the analyte.

Choice of extraction procedure, which is time economical, gives the highest

possible recovery without interference at the elution time of the analyte of

interest and has acceptable accuracy and precision.

Different procedures for the extraction of sample are as follows

Sample Preparation

In sample preparation there are two major objectives: to remove as much and

as many of the endogenous components of the bio-fluid as possible, while at

the same time concentrating the analyte in a small volume as possible. In


27

general, precipitation techniques are used for analytes at higher concentration

where it is possible to inject the sample into a liquid chromatography (LC)

system and obtain adequate sensitivity.

Precipitation Techniques

The simplest sample preparation procedure for plasma is precipitation of the

proteins. Where whole plasma is processed, acetonitrile (x 1 vol.) or methanol

(x 2 vol.) can be added and the precipitated plasma proteins removed by

centrifugation. The supernatant containing the analyte of interest can be

processed further depending upon the end point detection system to be used.

In general, precipitation techniques are used for analytes at higher

concentration where it is possible to inject the sample into a liquid

chromatography system and obtain adequate sensitivity.

Liquid-Liquid extraction

Liquid-liquid extraction is still the most widely used method for extracting

analytes from aqueous media and separating them from endogenous

interference. In addition it provides a simple means of concentrating the drug

by evaporation of the solvent.

An essential characteristic of a solvent used for this purpose is its

immiscibility with water. The most widely used solvents in increasing solvent

strength are: ethyl acetate, methylene chloride, chloroform, methyl tertiary

butylether (MTBE), chloroform, butyl chloride, hexane, petroleum ether and

pentane. The actual order may vary depending upon the criteria used to

determine solvent strength. Ideally the polarity of the solvent used should be

sufficient to remove the drug from the aqueous phase without removing

closely related endogenous compounds. In addition it is important to consider

the volatility, density and toxicity of solvents.

The procedure that is usually followed is to adjust the pH of the sample such

that the ionization of the analyte is suppressed and it therefore exists in its

more lipophilic form. The amount of organic solvent should be as low as

possible compatible with the partition coefficient. Although the partition

process is essentially a rapid and immediate process the presence of proteins,

which physically hinder the mixing process, coupled with protein binding of


28

the drug, may necessitate shaking for 1 hr or more in order to maximize

recovery.

Endogenous interferences may still be present after one extraction. Further

clean-ups can be effected by back-extracting the analyte into an aqueous layer

(pH depends on analyte) followed by re-extraction into an organic solvent

which then is evaporated to dryness and re-dissolved in an appropriate solvent

prior to analysis. For LC methods, the aqueous back-extraction usually

provides an adequate clean up. Provided that the back-extraction phase is of

the appropriate pH and molarity, it is possible to inject this extract onto the LC

systems.

Solid-Phase Extraction (SPE)

Solid-phase extraction (SPE) is a technique, which can avoid many of the

problems associated with liquid-liquid extraction.

The general approach to solid-phase (liquid-solid) extraction is adsorption of

the drug from a liquid onto a solid adsorbent or stationary phase immobilized

on a solid support. SPE using silica, alumina, celite, talc, charcoal, ion-

exchanger or hydrophobic resins has long been a common practice in the

clinical laboratory. The manufacture of modern HPLC stationary phases has

led to new methods in the solid-phase extraction technique. Nowadays, the

materials available for SPE are myriad and silica gels bonded with a variety of

functional groups, e.g. alkyl, phenyl, cyano and diol-moieties, are commonly

employed to provide specific interactions with analytes.

SPE is usually carried out in small columns packed with a material similar to

those used for analytical separations.

Method performance is determined primarily by the quality of the procedure

itself. The two factors that are most important in determining the quality of the

method are selective recovery and standardization. Analytical recovery of a

method refers to whether the analytical method in question provides response

for the entire amount of analyte that is contained in a sample. Recovery is

usually defined as the percentage of the reference material that is measured, to

that which is added to a blank. This should not be confused with the test of

matrix effect in which recovery is defined as the response measured from the

matrix (e.g.plasma) as a percentage of that measured from the pure solvent


29

(e.g. water) . Results of the experiment that compare matrix to pure solvent is

referred to as relative recovery and true test of recovery is referred to as

absolute recovery [21].

Another important issue in method development stage is the choice of

internal versus external standardization. Internal standardization is common

in bioanalytical methods especially with chromatographic procedures. The

assumption for the use of internal standard is that the partition coefficient of

the analyte and the internal standard are very similar [21]. For internal

standardization, a structural or isotopic analogue of the analyte is added to the

sample prior to sample pre-treatment and the ratios of the response of the

analyte to that of the internal standard is plotted against the concentration [22].

Another important point is that the tests performed at the stage of method

development should be done with the same equipment that will actually be used

for subsequent routine analysis. The differences found between individual

instruments representing similar models from the same manufacturer is not

surprising and should be accounted for [23].

The following two parameters must be determined at the method development

stage as they are the benchmark for further work.

i. LOD and LOQ

The US pharmacopoeia define LOD as the lowest concentration of an analyte

in a sample that can be detected but not necessarily quantitated. LOQ is the

lowest amount of a sample that can be determined with acceptable precision

and accuracy under the stated operational condition of the method [24].

In the case of LOD, analyst often use S/N (signal to noise ratio) of 2:1 or 3:1,

while a S/N of 10:1 is often considered to be necessary for the LOQ.

The ICH Q2B guideline on validation methodology lists two options in

addition to the S/N method of determining limits of detection and

quantification: visual non- instrumental methods and limit calculations. The

calculation is based on the standard deviation of the response (�) and the slope

of the calibration curve (S) at levels approaching the limits according to

equations below [24].

LOD = 3.3 x (�/S)


30

LOQ =10 x (�/S)

The standard deviation of the response can be determined based on the standard

deviation of the blank, based on the residual standard deviation of the

regression line, or the standard deviation of the y- intercept of the regression

line. This method can reduce the bias that sometimes occurs when determining

the S/N. The bias can result because of difference in opinion about how to

determine and measure noise.

ii. Calibration curve

A calibration line is a curve showing the relation between the concentration of

the analyte in the sample and the detected response. It is necessary to use a

sufficient number of standards to define adequately the relationship between

response and concentration. The relationship between response and

concentration must be demonstrated to be continuous and reproducible. The

number of standards to be used will be a function of the dynamic range and

nature of the concentration –response relationship. In many cases, five to eight

concentrations (excluding blank values) may define the standard curve. More

standard concentrations may be necessary for non-linear relationships than

would be for a linear relationship [19].

The difference between the observed y- value and fitted y- value is called a

residual. One of the assumptions involved in linear regression analysis is that

the calculated residuals are independent, are normally distributed and have

equal variance, which is termed as homoscedasticity. If the variance is not

equal, the case is termed as heteroscedasticity, in which case a weighted

regression may be performed. The most appropriate weighting factor is the

inverse of the variance of the standard, although 1/x, 1/x2, 1/y and 1/y2 (x=

concentration and y= response) are suitable approximations [25].

It is important to use a standard curve that will cover the entire range of the

concentration of the unknown sample. Estimation of the unknown by

extrapolation of standard curve below the lower standard and above the higher

standard is not recommended. Instead, it is suggested that the standard curve be

re-determined or sample re-assayed after dilution [19].

Method validation


31

The search for reliable range of method and continuous application of this

knowledge is called validation [23]. It can be defined as the process of

documenting that the method under consideration is suitable for its intended

purpose [17]. Method validation involves all the procedures required to

demonstrate that a particular method for quantitative determination of the

concentration of an analyte ( or a series of analytes) in a particular biological

matrix is reliable for the intended application [19]. Validation is also a proof of

the repeatability, specificity and suitability of the method.

Bioanalytical methods must be validated if the results are used to support the

registration of a new drug or a new formulation of an existing one. Validation

is required to demonstrate the performance of the method and reliability of

analytical results [26]. If a bioanalytical method is claimed to be for

quantitative biomedical application, then it is important to ensure that a

minimum package of validation experiments has been conducted and yields

satisfactory results [22].

Fundamental parameters for bioanalytical method are accuracy, precision,

selectivity, sensitivity, reproducibility, recovery and stability. For a

bioanalytical method to be considered valid, specific acceptance criteria should

be set in advance and achieved for accuracy and precision for the validation of

the QC samples.

1 Selectivity

A method is said to be specific when there is no interference from the matrix at

the retention times of analytes. Interferences in biological samples arise from a

number of endogenous (analyte metabolite, degradation products, co-

administered drugs and chemicals normally occurring in biological fluids) and

exogenous sources (impurities in reagents and dirty lab-ware). Zero level

interference of the analyte is desired but it is hardly ever the case. The

interference is acceptable at the 20% of LLOQ.

2. Precision

It is expressed as the percentage of coefficient of variance (%CV) or relative

standard deviation (%RSD) of the replicate measurements.

a. Intra assay precision


32

This is also known as repeatability i.e. the ability to repeat the same procedure

with the same analyst, using the same reagent and equipment in a short interval

of time.

b. Inter assay precision

The ability to repeat the same method under different conditions e.g. change of

analyst, reagent, or equipment or on subsequent occasions.

A minimum of three concentrations in the range of expected concentrations is

recommended. The % CV determined at each concentration level, should not

exceed 15% except for the LOQ, where it should not exceed 20%. [27].

3. Accuracy

It is defined as agreement between the measured value and the true value.

Accuracy is best reported as percentage bias that is calculated from the

expression

% Bias= (measured value-true value) x 100 true value

Accuracy should be measured using a minimum of five determinations per

concentration. A minimum of three concentrations in the expected range is

recommended. The mean value should be within 15% of the actual value

except at LLOQ, where it should not deviate by more than 20% [27].

4 Recovery

Absolute recovery of a bioanalytical method is the measured response of a

processed spiked matrix standard expressed as a percentage of the response of a

pure standard, which has not been subjected to sample pre-treatment and

indicates whether the method provides a response for the entire amount of the

analyte that is present in the sample [16].

Absolute recovery = ( Response of processed sample) x 100 Response of unprocessed standard

5 Stability

An essential aspect of method validation is to demonstrate that analyte(s) is

(are) stable in the biological matrix and in all solvents encountered during the

sample work up process, under the conditions to which study samples will be

subjected [28].

According to the recommendations on the Washington conference report by

Shah et al., (1992), the stability of the analyte in matrix at ambient temperature


33

should be evaluated over a time that encompass the duration of typical sample

preparation, sample handling and analytical run time.

Acceptable stability is 2% change in standard solution or sample solution

response relative to freshly prepared standard. Acceptable stability at the

LLOQ for standard solution and sample solution is 20% change in response

relative to a freshly prepared sample (Green, 1996).

Chromatographic Parametres

Chromatographic parameters used in the present work are defined below

a ) Capacity factor

The capacity factor is an important experimental parameter that is widely used

to describe the migration rates of analytes on columns. The capacity factor k’ is

expressed as

k’ = t1- t0 t0 Where, t1 = Retention time of the first component

t0 = Retention time of the nonretained component

b). Resolution

The resolution (RS) of two components in a chromatogram is determined from

the difference in their retention times and the widths of the peaks.

RS = 2 (tR2- tR1) tw1 + tw2

C) Plate height and number

Ideally, an analyte placed on a column , as a sharp band should spread out as

little as possible during the separations. Any broadening of the peaks can cause

overlap and loss of resolution. The efficiency of the column is

the measure of the broadening of sample peak as it passed through the column.

Plate height H and plate number of theoretical plates N are the two widely used

terms for the quantitative measures of the efficiency of chromatographic

columns. The two terms are related by the equation :

N= L/H

Where L= length of the column

The mathematical expression of the number of theoretical plates N in a

chromatographic system is obtained from the width of the peak in relation to

the retention time


34

N= 16 [tR/tW]2

Where, tW= peak width at base

tR= retention time of the peak

The more difficult separation problems can be solved with greater plate number

of a chromatographic system. In principle number of plates can be increased by

using a longer column.

d) Tailing factor or asymmetry factor

Determination of the shape of the chromatographic peak can be performed

using various methods. Generally the asymmetry factor, which is determined at

10% of the peak height is used however. However in the pharmaceutical

industry often the tailing factor calculated by USP, which is determined at 5%

peak height.

It is determined by following formula

T= W 0.05

2f

Where,

W 0.05 = width of peak at 5% height.

f = distance from the peak maximum to the leading edge of the peak, the

distance being measured at a point 5% of the peak height from the baseline.


35

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19. Shah, P.V., Midha, K.K., Dighe, S., McGilveray, J.I, Skelly, P.J,

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128,93-106.

24. Krull J., & Swartz, M. Validation view point 1998, 16(10).

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07 Chapter 1shodhganga.inflibnet.ac.in/bitstream/10603/79276/7/07_chapter 1.pdfDep artment of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University Aurangabad 3 Chapter