1

1.1 PREFACE

The number of new drugs being designed and introduced for therapy is

constantly increasing. Consequently, the dosage forms that include these drugs are

introduced into the market in huge numbers. So, there is always a necessity for

developing newer and efficient methods for determining these drugs in bulk samples

and formulations. The introduction of large number of newer drugs and their

formulations may also lead to widespread distribution of substandard or even

counterfeit drugs and their formulations in the market. Quality control and quality

assurance of pharmaceutical chemicals and their formulations are essential for

ensuring the availability of safe and effective drug formulations to the consumers and

safeguarding the general public against the hazards of substandard drugs.

Pharmaceutical analysis is indispensable in the process of quality control for statutory

certification of drugs and their formulations either by the industry or by the regulatory

authorities. Thus, constant development of new and improved analytical methods for

accurate determination of drugs in raw materials and in pharmaceutical dosage forms

is essential for quality control, pharmacokinetic, bioequivalence and toxicological

studies.

Pharmaceutical analysis deals with the analysis of not only the drugs but also

their formulations. It is also necessary to check the quality of the raw materials

including the bulk drugs that go into the formulation of the dosage forms. There are

several valid reasons for developing new analytical methods. The existing methods

may be erratic or unreliable i.e. having poor accuracy and precision. The existing

method may be time consuming or may be too expensive. The advent of new

techniques and improved instrumentation in the field of analysis may give way to

more sensitive, precise and accurate methods.

In order to develop a newer or improved analytical method, the analyst has to

set some goals. It is necessary to determine the analyte at trace levels accurately. The

method should be precise to the drug under study. The method should be simple

consuming minimum analysis time and using cheaper chemicals and materials. The

method should yield reproducible results, when carried out by different analysts and

in different laboratories. It should also be robust giving accurate results even there are

2

slight variations in the conditions of the method. It is not sufficient to develop and

optimize analytical methods using the pure standard drugs but, it is necessary that

these methods be validated appropriately and the methods should be applicable for

estimation of these drugs in their dosage forms.

High Performance Liquid Chromatography is the fastest growing analytical

technique for the analysis of drugs. Its simplicity and wide range of sensitivity and

short analysis time makes it ideal for analysis of many drugs in both dosage forms and

biological fluids. With the development of more sophisticated instrumentation,

efficient column materials1, 2 and moderate pricing, the HPLC technique has now

become more reliable and indispensable. In view of this the author has chosen to

develop HPLC methods for determination of some of the recent drugs.

The present study incorporated in the thesis was taken up by the author with

an aim to develop more efficient and validated new high performance liquid

chromatographic methods for estimation of fourteen important drugs namely

Eszopiclone, Lamotrigine, Dextromethorphan hydrobromide,Quinidine sulfate,

Fenofibrate, Pregabalin, Pramipexole dihydrochloride monohydrate,

Memantine, Donepazil hydrochloride, Levodopa, carbidopa, Entacapone in their

bulk samples as well as in dosage forms. The study design involves the development

of new reverse phase HPLC methods for estimation of the selected drugs either

individually or in combination with other drugs and validation of the methods thus

developed and testing their suitability for estimation of the drugs in their

pharmaceutical dosage forms. All the methods were carried out by adopting reverse

phase HPLC technique. The methods were validated as per ICH3 guidelines.

A literature survey on the analytical methods of Eszopiclone, Lamotrigine,

Dextromethorphan hydrobromide,Quinidine sulfate, Fenofibrate, Pregabalin,

Pramipexole dihydrochloride monohydrate, Memantine, Donepazil

hydrochloride, Levodopa, carbidopa, Entacapone revealed that a few HPLC

methods are available for their estimation in dosage forms in addition to other

techniques. Some of these methods have certain drawbacks like gradient elution

technique, long run time, less resolution and lack of sufficient sensitivity, precision

and accuracy. Furthermore, some methods lacked proper validation and

3

documentation. Hence, the author had attempted to develop simple, fast, accurate and

precise HPLC methods for determination of these drugs. The methods proposed by

the author are economical, quick and the solvents used in them are of moderate cost

and are thus easily affordable by the laboratories equipped with standard HPLC

systems. The proposed methods can be used as alternative methods to those reported

by the earlier workers and provide good choice for the routine determination of the

chosen drugs in their formulations and also in their clinical, pharmacokinetic and

biological studies.

The thesis incorporates the results of experimentation carried out by the author

for determination of the drugs listed above in pure form, validation of the method so

developed and applicability of the method for the estimation of the drugs in their

dosage forms by HPLC.

The thesis has been presented in two sections. Section 1 incorporates

introductory information about HPLC and its technique. This is followed by the

general guidelines and methodology to be followed for developing a new method for

estimation of drugs by HPLC. Later, the procedures adopted to determine various

parameters for validation of the developed method have been given.

Section 2 of the thesis deals with the details of the author’s experimentation

and results obtained in the HPLC method development for the assay of the following

fourteen selected drugs namely Eszopiclone, Lamotrigine, Dextromethorphan

hydrobromide,Quinidine sulfate, Fenofibrate, Pregabalin, Pramipexole

dihydrochloride monohydrate, Memantine, Donepazil hydrochloride, Levodopa,

carbidopa, Entacapone. The data in section 2 have been divided into nine chapters,

each chapter being devoted to one drug. The contents in each part have been

presented under the following heads.

1) Drug profile (s)

2) Review of the past work on the analytical methods

3) Experimental and results

4

a) Material and methods

b) Optimization of chromatographic conditions and method development

c) Validation of the proposed method

4) Summary of the results and Conclusion

5) References

The results obtained in these experiments have been thoroughly discussed.

The references cited in the text of the thesis have been given at the end of each part.

The following table shows the list of drugs taken up for the study and the source of

their monographs. The figures in the cells indicate the page number (*) / monograph

number (#).

S.No. Name of the

Drug *IP1 *USP2 *Martindale3

#Merck

Index4

#Drug

Bank5

1 Eszopiclone - - 1755 DB00402

2 Lamotrigine 1566 375 5368 DB00555

3 Dextrometharpha

n hydrobromide - - 1066 1392 DB00514

4 Quinidine sulfate 2016 - - DB00908

5 Fenofibrate 1337 3160 1307 679 DB1039

6 Pregabalin 1960 - - 1327 DB00230

7 - 4384 - 1755 DB00413

5

Name of the Drug *IP1 *USP2 *Martindale3 #Merck

Index4

#Drug

Bank5

Memantine - - 1165 1007 DB01043

Donepezil

hydrochloride 1249 2968 - 578 DB00843

Levodopa 1576 1636 1888 946 DB01235

Carbidopa - 1636 1888 291 DB00190

Entacapone - 3048 1159 613 DB00494

REFERENCES

1. Indian Pharmacopoiea 2010, Govt. of India, Ministry of Health and Family

Welfare. Delhi: Indian Pharmacopoeial Commission, Ghaziabad, 2010.

2. USP 35 NF 30, United States Phamacopoeia, The United States Pharmacopoeial

Convention, Rockville, MD, 2010.

3. Sweetman SC. Martindale, the Complete Drug Reference. 31st ed. London:

Pharmaceutical Press, 1996.

4. M.J. O’ Neil, The Merck Index: An encyclopedia of Chemicals, Drugs and

Biologicals, 13th Edn., The Merck and Co, NJ, 2001.

5. http://www.drugbank.ca/drugs

6

1.2. INTRODUCTION TO HPLC AND ITS TECHNIQUE

High-performance liquid chromatography1-2 (HPLC) is the fastest growing

analytical technique for analysis of drugs. Its simplicity, high sensitivity and

specificity make it ideal for the analysis of many drugs in pharmaceutical dosage

forms and biological fluids.

High Performance Liquid Chromatography is used to describe liquid

chromatography in which the liquid mobile phase is forced through the column at

high pressure and as a result the analysis time is reduced by 1-2 orders of the

magnitude relative to classical column chromatography. As it uses much smaller

particles of the adsorbent or stationary support it becomes possible for increasing the

column efficiency substantially. The importance of chromatography is increasing

rapidly in pharmaceutical analysis for exact differentiation, selective identification

and quantitative determination of structurally related compounds. Another important

field of application of chromatographic methods is the purity testing of final products

in a bulk drug industry. The reasons for the popularity of the method are its

sensitivity, its ready adaptability to accurate quantitative determinations, its suitability

for separating non-volatile and thermolabile species. Sensitive detectors have

transformed liquid column chromatography into high speed, efficient, accurate and

highly resolved method of separation.

The HPLC is the method of choice in the field of analytical chemistry, since

the methods developed using HPLC are specific, robust, linear, precise and accurate

and the limit of detection is low. HPLC technique offers the following advantages.

� Speed (many analyses can be accomplished within 20 min.)

� Greater sensitivity (various detectors can be employed)

� Improved resolution (wide variety of stationary phases can be used)

� Reusable columns (columns are expensive but can be used repeatedly)

� Easy sample handling and recovery.

� Automated instrumentation (less time and less labour)

7

� Precise and reproducible

� Computer monitored analysis and data interpretation.

PRINCIPLES OF SEPARATION

Adsorption chromatography employs high-surface area particles that

adsorb the solute molecules. Usually a polar solid such as silica gel, alumina or

porous glass beads and non-polar solvents such as heptane, octane or chloroform are

used as the stationary phase and mobile phase respectively in adsorption

chromatography. In adsorption chromatography, adsorption process is described by

competition model and solvent interaction model. Competition model assumes that

entire surface of the stationary phase is covered by mobile phase molecules as result

of competition for adsorption site. In solvent interaction model the retention results

from the interaction of solute molecule with the second layer of adsorbed mobile

phase molecules. The differences in affinity of solutes for the surface of the stationary

phase account for the separation achieved.

In partition chromatography, the solid support is coated with a liquid stationary

phase. The relative distribution of solutes between the stationary and the mobile phases

determines the separation. The stationary phase can either be polar or non polar. If the

stationary phase is polar and the mobile phase is non polar, it is called normal phase

partition chromatography. If the opposite case holds, it is called reversed-phase

partition chromatography. In normal phase mode, the polar molecule partition

preferentially in to the stationary phase and are retained longer than non-polar

compounds. In reverse phase partition chromatography, the opposite behavior is

observed.

8

INSTRUMENTATION

Schematic diagram of a typical HPLC unit

The individual components of HPLC and their functions are described below.

SOLVENT DELIVERY SYSTEM

The mobile phase is pumped under pressure from one or several reservoirs and

flows through the column at a constant rate. With micro particulate packing, there is a

high-pressure drop across a chromatography column. Eluting power of the mobile

phase is determined by its overall polarity, the polarity of the stationary phase and the

nature of the sample components. For normal phase separations, eluting power

increases with increasing polarity of the solvent but for reversed phase separations,

eluting power decreases with increasing solvent polarity. Optimum separating

conditions can be achieved by making use of mixture of two solvents. Some other

properties of the solvents, which need to be considered for a successful separation, are

the boiling point, viscosity, detector compatibility, flammability and toxicity.

The most important component of HPLC in solvent delivery system is the

pump, because its performance directly affects the retention time, reproducibility and

detector sensitivity. Among the several solvent delivery systems, (direct gas pressure,

pneumatic intensifier, reciprocating etc.) reciprocating pump with twin or triple

pistons is widely used, as this system gives less baseline noise, good flow rate and

reproducibility etc.

9

MOBILE PHASE

Typically, the mobile phases used for HPLC are mixtures of organic solvents

and water or aqueous buffers. Table given below lists the physical properties of

organic solvents commonly used for HPLC. Isocratic methods are preferable to

gradient methods. Gradient methods will sometimes be required when the molecules

being separated have vastly different partitioning properties. When a gradient elution

method is used, care must be taken to ensure that all solvents are miscible.

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

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

in the duration of the analysis.

2. Excessive salt concentrations in the buffers should be avoided. High salt

concentrations can result in precipitation of the analytes, which can damage

the column.

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

of the column.

4. Cost and toxicity of the mobile phase can be reduced by using methanol

instead of acetonitrile where possible.

5. Absorbance of the buffer is to be minimized. Since trifluoroacetic acid, acetic

acid or formic acid absorb at shorter wavelengths, they may prevent detection

of products without chromophores above 220 nm. Carboxylic acid modifiers

can be frequently replaced by phosphoric acid, which does not absorb above

200 nm.

6. Volatile mobile phases should be used when possible, to facilitate collection of

products and LC-MS analysis. Volatile mobile phases include ammonium

acetate, ammonium phosphate, formic acid, acetic acid, and trifluoroacetic

acid. Some caution is needed as these buffers absorb below 220 nm.

10

PHYSICAL PROPERTIES OF COMMON HPLC SOLVENTS

Solvent MW BP RI

(25oC)

UV*

Cut-off

(nm)

Density

g/ml

(25oC)

Viscosity

cP

(25oC)

Dielectric

Constant

Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8

Dioxan 88.1 101 1.420 215 1.034 1.26 2.21

Ethanol 46.1 78 1.359 205 0.789 1.19 24.5

Ethyl acetate 88.1 77 1.372 256 0.901 0.450 6.02

Methanol 32.0 65 1.326 205 0.792 0.584 32.7

CH2Cl2 84.9 40 1.424 233 1.326 0.44 8.93

Isopropanol 60.1 82 1.375 205 0.785 2.39 19.9

n-propanol 60.1 97 1.383 205 0.804 2.20 20.3

THF 72.1 66 1.404 210 0.889 0.51 7.58

Water 18.0 100 1.333 170 0.998 1.00 78.5

* wavelength at which the absorbance of 1cm cell is 1.0

SOLVENT DEGASSING SYSTEM

The mobile phase should be degassed and filtered before use. Several methods

are employed to remove the dissolved gases in the mobile phase. They include heating

and stirring, vacuum degassing with an aspirator, filtration through 0.45 filter,

vacuum degassing with an air-soluble membrane, helium purging and ultra sonication

or combinations of these methods. HPLC systems are also provided an online

degassing system, which continuously removes the dissolved gases from the mobile

phase.

11

GRADIENT ELUTION DEVICES

HPLC column elution may be in the isocratic mode i.e., with a constant

composition of the mobile phase or in the gradient elution mode in which the mobile

phase composition varies during run. Gradient elution overcomes the problem of

analysis when dealing with a complex mixture of solutes.

COLUMNS

The heart of the system is the column. The choice of common packing

material and mobile phases depends on the physical properties of the drug. Many

different reverse phase columns will provide excellent specificity for any particular

separation. It is therefore preferable to routinely attempt separations with a standard

C8 or C18 column and determine if it provides good separations. If this column does

not provide good separation or the mobile phase is unsatisfactory, alternate methods

or columns should be explored. Reverse phase columns differ by the carbon chain

length, degree of end capping and percent carbon loading. Diol, cyano and amino

groups can also be used in the matrix for reverse phase chromatography.

SAMPLE INTRODUCTION SYSTEM

The two methods of analyte introduction on the column are by injection into a

flowing stream and by stop flow injection. These techniques can be used with a

syringe or an injection valve. Automatic injector is a microprocessor-controlled

version of the manual universal injector. Usually, up to 100 samples can be loaded in

to the auto injector tray. The system parameters such as flow rates, gradient, run time,

volume to be injected, etc. are chosen, stored in memory and sequentially executed on

consecutive injections.

LIQUID CHROMATOGRAPHIC DETECTORS

The function of the detector in HPLC is to monitor the mobile phase as it

emerges from the column. Generally, there are two types of HPLC detectors, bulk

property detectors and solute property detectors.

12

a. Bulk property detectors: These detectors are based on differential measurement of

a property, which is common to both the sample and the mobile phase. Examples

of such detectors are refractive index, conductivity and dielectric constant

detectors.

b. Solute property detectors: Solute property detectors respond to a physical property

of the solute, which is not exhibited by the pure mobile phase. These detectors

measure a property, which is specific to the sample, either with or without the

removal of the mobile phase before the detection. Solute property detectors which

do not require the removal of the mobile phase before detection include

spectrophotometric (UV or UV-Visible) detector, fluorescence detectors,

polarographic, electro-chemical and radio activity detectors, where flame

ionization detector and electron capture detector both require removal of the

mobile phase before detection.

UV-Visible and fluorescent detectors are suitable for gradient elution, because

many solvents used in HPLC do not absorb to any significant extent.

Detectors

Optical detectors are most frequently used.

These detectors pass a beam of light through the flowing column effluent as it passes

through a low volume (~ 10 ml) flow cell.

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 460-

600 nm, will be found suitable for the detection of the majority samples.

Other types of detectors:

Refractive Index detector: Universal analyte detector. Solvent must remain the same

throughout separation. It is temperature sensitive.

Fluorescence detector: Excitation wavelength generates fluorescence emission at a

higher wavelength. Analytes must have fluorophore group. Very sensitive and

selective. Results vary depending upon separation conditions.

Mass Spectroscopic detectors: Mass to charge ratio (m/z). Allow identification of

specific compounds. Several types of ionization techniques include: electro-spray,

atmospheric pressure chemical ionization, electron impact. The detector usually

13

contains low volume cell through which the mobile phase passes carrying the sample

components.

INJECTORS

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 sample introduction is done with the help of

auto samplers and microprocessors.

In liquid chromatography, liquid samples may be injected directly and solid

samples need only be dissolved in an appropriate solvent. The solvent need not be the

mobile phase, but frequently it is judiciously chosen to avoid detector interference,

column/component interference, and loss in efficiency or all of these. It is always best

to remove particles from the sample by filtering, or centrifuging since continuous

injections of particulate material will eventually cause blockage of injection devices

or columns.

DATA SYSTEMS

The main goal in using electronic data systems is to increase the accuracy and

precision of the analysis, while reducing operator attention. In routine analysis, where

no automation (in terms of data management or process control) is needed, a pre-

programmed computing integrator may be sufficient. For higher control levels, a more

intelligent device is necessary, such as a data station or minicomputer.

The advantages of intelligent processors in chromatographs:

� additional automation options become easier to implement;

� complex data analysis becomes more feasible;

� Software safeguards can be designed to reduce accidental misuse of the

system.

REFERENCES

1. Josefsson M, Zackrisson; A.L. Norlander B, J of Chromatography B, 1995, 672,

310-313.

2. Stopher D.A. Beresford A.P. Macrae, P.V. Humphrey M.J., of Cardiovasc.

Pharmacol, 1988, 12, Supp 7, S55-S59.

14

1.3. HPLC METHOD DEVELOPMENT AND OPTIMIZATION

The development of any new or improved method for the analysis of an analyte

usually depends on tailoring the existing analytical approaches and instrumentation.

Method development usually involves selecting the method requirements and on the

type of instrumentation. In the development stage of a HPLC method, decision

regarding the choice of column, mobile phase, detector and method of quantitation

must be addressed.

Once the instrumentation has been selected, it is important to determine the

chromatographic parameters for the analyte of interest. It is necessary to consider the

properties of the analyte(s) that may be useful to select the nature of the column to be

used, establish the approximate composition and pH of the mobile phase for

separation of the components wave length to be employed or mass/charge ratio to be

scanned at for detection of the compound, the concentration range to be followed and

choice of a suitable internal standard for quantification purpose etc. Such information

may be already available in the literature for the analyte or related compounds.

This is followed by optimization and preliminary evaluation of the method.

Optimization criteria must be determined with cognizance of the goals common to any

new method. Initial analytical parameters of merit like sensitivity (measured as

response per amount injected), limit of detection, limit of quantitation and linearity of

calibration plots are to be determined. As a precautionary measure, it is important that

method development be performed using only the analytical standards that are highly

pure and have been well identified and characterized and whose purity is known.

During the optimization stage, the initial sets of conditions that have evolved

from the first stages of development are improved or optimised in terms of resolution,

peak shape, plate counts, peak asymmetry, capacity, elution time, detection limits,

limit of quantitation, and overall ability to quantify the specific analyte of interest.

Results obtained during optimization must be evaluated against the goals of the

analysis set forth by the analytical figures of merit. This evaluation reveals if

additional improvement and optimization are needed to meet the initial method

requirements.

15

Optimization of the method should yield maximum sensitivity, good peak

symmetry, minimum detection and quantitation levels, a wide linearity range, and a

high degree of accuracy and precision. Other potential optimization goals include

baseline resolution of the analyte of interest from other sample components, unique

peak identification, on-line demonstration of purity and interfacing of computerized

data for routine sample analysis. Absolute quantitation should use simplified methods

that require minimal sample handling and analysis time.

Optimization of the method may follow either manual or computer driven

approaches. The manual approach involves varying one experimental condition at a

time, while holding all others constant and evaluating the changes in response. The

variables might include flow rate, mobile or stationary phase composition,

temperature, detection wavelength and pH. This univariate approach of system

optimization is usually time consuming and expensive. However, it may provide a

much better understanding of the principle involved and of the interactions of the

variables. In computer-driven automated method development efficiency is optimized

while experimental input is minimized. This approach can be applied to many types of

methods. It significantly reduces the time of analysis, energy, and cost of analysis.

SYSTEMATIC APPROACH TO THE REVERSE PHASE CHROMATOGRAPHIC

SEPARATION OF PHARMACEUTICAL COMPOUNDS

Classifying the sample

The first step in the method development is to characterize the drug whether it

is regular or special. The regular compounds are those that are neutral or ionic. The

inorganic ions, bio-molecules, carbohydrates, isomers, enantiomers and synthetic

polymers etc are called special compounds. The selection of initial conditions for

regular compounds depends on the sample type. The general approach for the reverse

phase chromatographic method development is based on the following considerations.

The regular samples like pharmaceuticals (either ionic or neutral) respond in

predicable fashion to changes in solvent strength (%B) and type (e.g. acetonitrile or

methanol) or temperature. A 10% decrease in %B increases retention by about three

fold and selectivity usually changes as either %B or solvent type is varied. An

increase in temperature causes a decrease in retention as well as changes in

16

selectivity. It is possible to separate many regular samples just by varying solvent

strength and type. Alternatively, varying solvent strength and temperature can

separate many ionic samples and some non-ionic samples.

The choice of the initial column, mobile phase and temperature is quite

important. The initial conditions for RP HPLC method developed are given in Table.

Separation variable Preferred initial choice

Column Packing C8 or C18 column, less acidic silica columns; if

temperatures > 50 C are planned, more stable,

sterically protected packings are preferred

Column configuration 15 X 0.46 cm column; 5µm particles

Flow rate 2.0 mL/min

Mobile phase Acetonitrile-water (neutral samples) or

acetonitrile-buffer (ionic samples); buffer is 25-

50 mM potassium phosphate at pH 2-3 (lower

pH preferrable if column is stable).

Temperatue 35 or 400 C

Sample size < 50 µL; 50-100 g

The Column and Flow rate

To avoid problems from irreproducible sample retention during method

development, it is important that columns be stable and reproducible. A C8 or C18

column made from specially purified less acidic silica and designed specifically for

the separation of basic compounds is generally suitable for all samples and is strongly

recommended. If temperatures >50 0 C are used at low pH, stearically protected

bonded-phase column packing is preferred. The column should provide reasonable

resolution in initial experiments, short run times and an acceptable pressure drop for

different mobile phases. A 5µ, 150 X 4.6 mm column with a flow rate of 2 mL/min is

good for different mobile phases as initial choice. These conditions provide

reasonable plate number (N=8000), a run time of < 15 min for a capacity factor k < 20

and a maximum pressure drop < 2500 psi for any mobile phase made from mixtures

of water, acetonitrile and/or methanol.

17

The mobile phase

The preferred organic solvent (B) for the mobile phase mixture is acetonitrile

(ACN) because of its favorable UV transmittance and low viscosity. However,

Methanol (MeOH) is a reasonable alternative. Amine modifiers like tetra hydro furan

(THF) are less desirable because they may require longer column equilibration times,

which can be a problem in method development and routine use of the method. They

may occasionally introduce additional problems like erratic base line and poor peak

shape. However, some samples may require the use of amine modifiers when poor

peak shapes or low plate number are encountered.

The pH of the mobile phase should be selected with two important

considerations. A low pH that protonates column silanols and reduces their

chromatographic activity is generally preferred. A low pH (<3) is usually quite

different from the pKa values of common acidic and basic functional groups.

Therefore, at low pH the retention of these compounds will not be affected by small

changes in pH and the reverse phase liquid chromatographic method will be more

rugged. For columns that are stable at low pH, a pH of 2 to 2.5 is recommended. For

less stable columns, a pH of 3.0 is a better choice.

Separation temperature

Mostly the temperature controllers operate best above ambient (>300 C).

Higher temperature operation also gives lower operating pressures and higher plate

numbers, because of decrease in mobile phase viscosity. A temperature of 35-400 is

usually a good starting point.

Sample size

Initially, a 25-50 µL injection (25-50 µg) can be used for maximum detection

sensitivity. Smaller injection volumes are required for column diameters of below 4.5

mm and /or particles smaller than 5 µm. The sample should be dissolved initially in

water (1mg/mL) or dilute solution of acetonitrile in water. For the final method

development stage, the best sample solvent is the mobile phase. The samples which

cannot be dissolved in water or the mobile phase should be dissolved initially in either

acetonitrile or methanol and then diluted with water or mobile phase before injection.

18

Equilibration of the column with the mobile phase

The analytical column is completely equilibrated with the mobile phase before

injecting the sample for analysis and retention data are collected for interpretation.

This is done for ensuring accurate retention data. Equilibration is required whenever

the column, mobiles phase or temperature is changed during method development;

usually by flow rate at least 10 column volumes of the new mobile phase before the

first injection. Some mobile phases may require a much longer column equilibration

time (e.g. mobile phases that contain THF amine modifiers such tri ethylamine and

tetra butyl amine and any ion pair reagent).

Column equilibration and reproducible data can be confirmed by first washing

the column with at least 10 columns volumes of the new mobile phase and injecting

the sample and then a second washing with at least 5 column volumes of the new

mobile phase and reinjection of the sample. If the column is equilibrated, the retention

times should not change by more than 0.02 min between the two runs.

Column Performance

The following values are used to assess overall system performance.

1. Relative retention

2. Theoretical plates

3. Capacity factor

4. Resolution

5. Peak tailing factor

6. Plates per meter

The chromatographic peak shape and plate number are calculated to assess

column performance. The asymmetry factor AS should fall between 0.9-10.5 and

number of theoretical plates should be >4000 for a 15 cm; 5 µm column at a flow rate

of 2 mL/min. The number of theoretical plates for well packed HPLC columns under

optimized test conditions is given in the Table.

19

Particle

Diameter (µm)

Column

length (cm) Plate Number N

10 15 6000-7000

10 25 8000-10000

5 10 7000-9000

5 15 10000-12000

5 25 17000-20000

3 5 6000-7000

3 7.5 9000-11000

3 10 12000-14000

3 15 17000-20000

Evaluating peak shape and plate number

The requirements for a given separation usually determine the type and

configuration of the column to be used. There are different suppliers for a given type

of column. These columns vary generally in performance. Therefore, certain

information concerning column specifications and performance is needed for use in

method development and their routine performance.

The column plate number (N) is an important characteristic of a column. N

signifies the ability of the column to produce sharp, narrow peaks for achieving good

resolution of band pairs with small α values. The table 2.2 shows the typical plate

numbers (small, neutral sample molecules) for well packed HPLC columns of various

lengths and particle sizes. A 15 or 25 cm column of 5 µ particles are preferred as a

starting point for method development. This configuration provides a large enough N

value for most separations and such columns are quite reliable. A column which gives

large N value can easily recognize closely over lapping peaks. Short columns of 3 µ

particles are useful for carrying out very fast separation (< 5 min). But these columns

are less used because they are more susceptible to sampling problems, more operator

dependent and more affected by band-broadening.

20

Peak asymmetry and Peak tailing

Columns and experimental conditions that provide symmetrical peaks always

preferred. Peaks with poor symmetry can result in inaccurate plate number and

resolution measurement, imprecise quantitaion, degraded resolution and poor

retention reproducibility.

Peak shape is measured in terms of peak asymmetry factor (AS) and peak

tailing factor (PTF). Peak asymmetry factor (AS) is measured at 10% of full peak

height. Good columns produce with AS values of 0.95 to 1.1. For accurate

measurement of symmetry bands should be measured with a magnified time scale.

Asymmetrical bands often appear symmetrical when observed in a compressed

chromatogram. Calculation of peak asymmetry factor and peak tailing factor are

represented is Fig.2.1. As per U.S.P., the peak asymmetry factor is calculated at 5% of

full height. Peak asymmetry and peak tailing factor are easily inter convertible as

shown below.

Retention

The time between the sample injection point and the analyte reaching the

detector is called the retention time tR.

21

Capacity factor k’

It measures how many times the analyte is retained to an unretained component.

Capacity factor, k’ = tR-t0/t0

Where tR = retention time of the peak

t0 = void time,

A k’ value zero means that the compound is not retained and elutes with the

solvent front. A k’ value of 1 means that the component is slightly retained by the

column while k’ value of 20 means that component is highly retained and spends

much time interacting with the stationary phase.

Selectivity α

Separation between tow components is only possible if they have different

migration rates through column. Selectivity or separation factor is a measure of

differential retention of two analytes It is defined as the ratio of the capacity factors

(k’) of two peaks.

α = k1’/k2’

Column efficiency (N) or number of theoretical plates

The term plate number N is a quantitative measure of the efficiency of the

column and is related to the ratio of the retention time and the standard deviation of

the peak width σ. Since ti is difficult to measure σ or Wb (width at base of the peak), a

relationship using width at half height or w1/2 is often used to calculate N as described

in the USP.

N = 5.54 (tR/ w1/2)2

Height equivalent of a Theoretical Plate (HETP) or Plate Height (H)

HETP = L/N

L= length of the column

N = number of theoretical plates

22

Resolution RS

It is the degree of separation of two adjacent peaks and is defined as the

difference in retention times of the two peaks divided by the average peak width. As

the peak width of adjacent peaks tends to be similar, the average peak width can be

equal to the width of one of the two peaks.

Resolution RS = tR1- TR2/ Wb1+wb2

Where,

Wb2 = Width of the base of component peak 2.

Wb1 = Width of the base of component peak 1.

Tailing factor T f

It is a measure of peak asymmetry. In this calculation peak width at 5% peak

height W0.05 is used.

T f = W0.05/2f

Tailing factors for most peaks should fall between 0.9 and 1.4 with a value of

1.0 indicating a perfectly symmetrical peak.

The following table furnishes the formulae for calculating the different system

performance parameters (Note: Where the terms W and t both appear in the same

equation they must be expressed in the same units).

Relative retention (Selectivity): α = ( t2 - ta ) / ( t1 - ta )

Capacity factor k' = ( t2 / ta ) - 1

Tailing factor T = W0.05 / 2f

Resolution R = 2 ( t2 - t1 ) / ( W2 + W1 )

Theoretical plates: n = 16 ( t / W )2

Plates per meter: N = n / L

HETP L/n

23

where

α = Relative retention.

t2 = Retention time of the second peak measured from point of

injection.

t1 = Retention time of the first peak measured from point of injection.

ta = Retention time of an inert peak not retained by the column

measured from point of injection.

n = Theoretical plates.

t = Retention time of the component.

W = Width of the base of the component peak using tangent method.

k' = Capacity factor.

R = Resolution between a peak of interest (peak 2) and the peak

preceding it (peak 1).

W2 = Width of the base of component peak 2.

W1 = Width of the base of component peak 1.

T = Tailing factor.

W0.05 = Distance from the leading edge to the tailing edge of the peak,

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

f = Distance from the peak maximum to the leading edge of the peak.

N = Plates per meter.

L = Column length, in meters.

24

1.4. METHOD VALIDATION

Introduction

Validation is the process of ensuring that a test procedure performs within

acceptable standards of reliability, accuracy and precision for its intended purpose. It

is the act of confirming that a method does what it is intended to do.

It is difficult to completely separate method development and optimization

from validation; these areas often overlap. In the validation stage, an attempt should

be made to demonstrate that the method works with samples of the given analyte, at

the expected concentration in the matrix, with a high degree of accuracy and precision.

Complete method validation can occur only after the method is developed and

optimized. In validation studies, suitability of the final method for the given analyte

and a select sample matrix is demonstrated, using specified instrumentation, samples,

and data handling; ultimately, the method can be transferred from one laboratory to

another that is suitably equipped and staffed. A method that provides all or most of the

original method requirements is deemed optimized and becomes ready for validation.

There is no single validation approach that must always be employed for a

new method; the analyst’s primary concern should be to select an approach that will

prove to be a true validation. Acceptance of any new method by others in the field will

depend on the specific validation approaches used. It is the responsibility of the

individual analyst to select the correct validation method(s). Validation approaches

include the zero-, single-, and double-blind spiking methods; inter-laboratory

collaborative studies; and comparison with a currently accepted (compendium)

method.

The Zero-Blind Method

The zero-blind approach involves a single analyst using the method with

samples at known levels of analyte to demonstrate recovery, accuracy, and precision.

The method is subject to analyst bias, and though the method is, in general, fast,

simple, and useful, it leads to subjective results and doubt on the part of the unbiased

reviewer or end user. However, as a first approximation and a demonstration of

validation potential requiring minimal time, manpower, samples, and cost, a zero-

25

blind study is a good place to start the overall validation process. Clearly, if this

approach fails to validate a method, then there is no reason to proceed with further

validation of the method.

The Single-Blind Method

The single-blind approach involves one analyst preparing samples at varying

levels unknown to a second analyst, who also analyzes the samples. The results are

then complied and compared by the first analyst. Although this approach is unbiased

at the start, it loses its blindness at the most crucial stage − when both sets of data are

compared. While perhaps more valuable and believable than the zero-blind approach,

the single-blind approach still invites bias on the part of the first analyst to bring two

sets of data into better agreement. This approach is appropriate at the very start of the

method validation, after the single-blind approach has proven successful, but before

one decides to involve additional analysts or management.

The Double-Blind Method

The double-blind approach involves three analysts. The first analyst prepares

samples at known levels, the second does the actual analysis, and the third analyst (or

administrator) compares both sets of data received separately from the first two

analysts. Neither the first or second analyst has access to the set of data generated by

the other. This double-blind approach is the most objective approach, assuming no

bias on the part of the third analyst.

The Analysis of Standard Reference Materials

The analysis of a standard reference material (SRM) or an authenticated

sample is a generally accepted method of validation. The USP, NIST, and other,

private organizations specialize in preparing, guaranteeing, and marketing standard

reference materials of various analyte species in different sample matrices. It may be

necessary, however, to contract the preparation of a unique sample in a particular

matrix in order to utilize this procedure for method validation. When using SRMs, the

analyst must demonstrate that the method provides accurate and precise measurements

of the analyte in a particular sample matrix. Analyst bias can also be an issue,

especially when the analyst knows the amounts and levels of the SRM.

26

The Inter-laboratory Collaborative Study

The inter-laboratory collaborative study is perhaps the most widely accepted

procedure to validate any new analytical method, but it suffers from serious practical

drawbacks. The collaborative approach is costly and time consuming; it can take years

from start to finish. During that time, the analysts may have to expend considerable

effort coordinating the process, shipping samples and receiving results, statistically

analyzing and interpreting the results, and then finally interpreting and verifying the

data. Although the approach is operator dependent (generating laboratory-to-

laboratory variability), when all laboratories involved come up with over-lapping

quantitative values in comparison with known levels present, the method is generally

accepted as full validation. This approach is rarely employed when a method is being

described for the first time in the literature.

Comparison with a Currently Accepted Method

Comparison with a currently accepted analytical method is yet another

validation approach. This is usually done by a singly analyst, but it can be done by

two analysts using a split sample. This approach uses results from the currently

accepted method as verification of the new method’s results. Agreement between

results initially suggests validation. Disagreement is a serious cause for concern of

future acceptability of the new method. However, disagreement could also suggest

that the currently accepted method is invalid, creating additional problems. If the

analyst can prove that the currently accepted method is indeed invalid, the analyst

must then initiate an alternative approach to validate the new method.

The question will eventually arise as to how many samples should be analyzed

in any validation approach. In general, the more the better, and the greater the variety

of samples and variation in the concentration range the better. Ideally, the method

should be validated for the analyte using several different sample types, with several

of each type determined separately for statistical and validation purposes. A single,

zero-blind or a single, single-blind study is obviously less meaningful and less

acceptable than an inter-laboratory collaborative, true double-blind study of several

sample matrices at widely different concentration levels. Initial validation approaches

are generally less rigorous and demanding than ones performed for standard reference

material (SRM) development.

27

Validation is an act of providing evidence that any procedures, process, equipments,

materials, activity or system perform as expected under given set of conditions and

also give the required accuracy, precision.

Assay procedures are intended to measure the analyte present in a given

sample. In the context of this document, the assay represents a quantitative

measurement of the major component(s) in the drug substance. For the drug product,

similar validation characteristics also apply when assaying for the active or other

selected component(s). The same validation characteristics may also apply to assays

associated with other analytical procedures (e.g., dissolution).

The objective of the analytical procedure should be clearly understood since

this will govern the validation characteristics which need to be evaluated. Typical

validation characteristics which should be considered are accuracy, precision,

(repeatability, intermediate precision), specificity, detection limit, quantitation limit,

linearity range, robustness and stability of analytical solution3.

28

METHOD VALIDATION PROCEDURES

1. SPECIFICITY

An investigation of specificity should be conducted during the validation of

identification tests, the determination of impurities and the assay. The procedures

used to demonstrate specificity will depend on the intended objective of the analytical

procedure.

It is not always possible to demonstrate that an analytical procedure is specific for a

particular analyte (complete discrimination). In this case a combination of two or

more analytical procedures is recommended to achieve the necessary level of

discrimination.

Identification

Suitable identification tests should be able to discriminate between compounds

of closely related structures which are likely to be present. The discrimination of a

procedure may be confirmed by obtaining positive results (perhaps by comparison

with a known reference material) from samples containing the analyte, coupled with

negative results from samples which do not contain the analyte. In addition, the

identification test may be applied to materials structurally similar to or closely related

to the analyte to confirm that a positive response is not obtained. The choice of such

potentially interfering materials should be based on sound scientific judgement with a

consideration of the interferences that could occur.

Assay and Impurity Test(s)

For chromatographic procedures, representative chromatograms should be

used to demonstrate specificity and individual components should be appropriately

labelled. Similar considerations should be given to other separation techniques.

Critical separations in chromatography should be investigated at an appropriate level.

For critical separations, specificity can be demonstrated by the resolution of the two

components which elute closest to each other.

In cases where a non-specific assay is used, other supporting analytical

procedures should be used to demonstrate overall specificity. For example, where a

29

titration is adopted to assay the drug substance for release, the combination of the

assay and a suitable test for impurities can be used.

The approach is similar for both assay and impurity tests:

Impurities are available

For the assay, this should involve demonstration of the discrimination of the

analyte in the presence of impurities and/or excipients; practically, this can be done by

spiking pure substances (drug substance or drug product) with appropriate levels of

impurities and/or excipients and demonstrating that the assay result is unaffected by

the presence of these materials (by comparison with the assay result obtained on

unspiked samples).

For the impurity test, the discrimination may be established by spiking drug

substance or drug product with appropriate levels of impurities and demonstrating the

separation of these impurities individually and/or from other components in the

sample matrix.

Impurities are not available

If impurity or degradation product standards are unavailable, specificity may

be demonstrated by comparing the test results of samples containing impurities or

degradation products to a second well-characterized procedure e.g.: pharmacopoeial

method or other validated analytical procedure (independent procedure). As

appropriate, this should include samples stored under relevant stress conditions: light,

heat, humidity, acid/base hydrolysis and oxidation.

For the assay, the two results should be compared;

For the impurity tests, the impurity profiles should be compared.

Peak purity tests may be useful to show that the analyte chromatographic peak is not

attributable to more than one component (e.g., diode array, mass spectrometry).

2. LINEARITY

The linear relationship should be evaluated across the range of the analytical

procedure. It may be demonstrated directly on the drug substance (by dilution of a

standard stock solution) and/or on synthetic mixtures of the drug product components,

30

using the proposed procedure. The latter aspect can be studied during investigation of

the range of the method.

Linearity should be evaluated by visual inspection of a plot of signals as a

function of analyte concentration or content. If there is a linear relationship, test

results should be evaluated by appropriate statistical methods, for example, by

calculation of a regression line by the method of least squares. In some cases, to

obtain linearity between assays and sample concentrations, the test data may need to

be subjected to a mathematical transformation prior to the regression analysis. Data

from the regression line itself may be helpful to provide mathematical estimates of the

degree of linearity.

The correlation coefficient, y-intercept, slope of the regression line and

residual sum of squares should be submitted. A plot of the data should be included. In

addition, an analysis of the deviation of the actual data points from the regression line

may also be helpful for evaluating linearity. For the establishment of linearity, a

minimum of 5 concentrations is recommended. Other approaches should be justified.

3. RANGE

The specified range is normally derived from linearity studies and depends on

the intended application of the procedure. It is established by confirming that the

analytical procedure provides an acceptable degree of linearity, accuracy and

precision when applied to samples containing amounts of analyte within or at the

extremes of the specified range of the analytical procedure.

The following minimum specified ranges should be considered:

For the assay of a drug substance or a finished (drug) product: normally from 80 to

120 percent of the test concentration

For content uniformity, covering a minimum of 70 to 130 percent of the test

concentration, unless a wider more appropriate range, based on the nature of the

dosage form (e.g., metered dose inhalers), is justified;

For dissolution testing: ± 20 % over the specified range;

31

e.g., if the specifications for a controlled released product cover a region from 20%,

after 1 hour, up to 90%, after 24 hours, the validated range would be 0-110% of the

label claim.

For the determination of an impurity: from the reporting level of an impurity1 to

120% of the specification;

For impurities known to be unusually potent or to produce toxic or unexpected

pharmacological effects, the detection/quantitation limit should be commensurate

with the level at which the impurities must be controlled;

Note: for validation of impurity test procedures carried out during development, it

may be necessary to consider the range around a suggested (probable) limit.

If assay and purity are performed together as one test and only a 100% standard is

used, linearity should cover the range from the reporting level of the impurities1 to

120% of the assay specification.

4. ACCURACY

Accuracy should be established across the specified range of the analytical

procedure.

Assay of Drug Substance: Several methods of determining accuracy are available:

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

material);

b) Comparison of the results of the proposed analytical procedure with those of a

second well-characterized procedure, the accuracy of which is stated and/or

defined

c) Accuracy may be inferred once precision, linearity and specificity have been

established.

Assay of Drug Product: Several methods for determining accuracy are available

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

components to which known quantities of the drug substance to be analysed have

been added;

32

b) In cases where it is impossible to obtain samples of all drug product components, it

may be acceptable either to add known quantities of the analyte to the drug

product or to compare the results obtained from a second, well characterized

procedure, the accuracy of which is stated and/or defined

c) Accuracy may be inferred once precision, linearity and specificity have been

established.

Assay of Impurities (Quantitation)

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

with known amounts of impurities.

In cases where it is impossible to obtain samples of certain impurities and/or

degradation products, it is considered acceptable to compare results obtained by an

independent procedure. The response factor of the drug substance can be used.

It should be clear how the individual or total impurities are to be determined e.g.,

weight/weight or area percent, in all cases with respect to the major analyte.

Recommended Data

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

minimum of 3 concentration levels covering the specified range (e.g., 3

concentrations /3 replicates each of the total analytical procedure).

Accuracy should be reported as percent recovery by the assay of known added

amount of analyte in the sample or as the difference between the mean and the

accepted true value together with the confidence intervals.

5. PRECISION

Validation of tests for assay and for quantitative determination of impurities

includes an investigation of precision.

Repeatability

Repeatability should be assessed using:

a) a minimum of 9 determinations covering the specified range for the procedure (e.g.,

3 concentrations/3 replicates each) or

b) a minimum of 6 determinations at 100% of the test concentration.

33

Intermediate Precision

The extent to which intermediate precision should be established depends on the

circumstances under which the procedure is intended to be used. The applicant should

establish the effects of random events on the precision of the analytical procedure.

Typical variations to be studied include days, analysts, equipment, etc. It is not

considered necessary to study these effects individually. The use of an experimental

design (matrix) is encouraged.

Reproducibility

Reproducibility is assessed by means of an inter-laboratory trial. Reproducibility

should be considered in case of the standardization of an analytical procedure, for

instance, for inclusion of procedures in pharmacopoeias. These data are not part of the

marketing authorization dossier.

Recommended Data

The standard deviation, relative standard deviation (coefficient of variation) and

confidence interval should be reported for each type of precision investigated.

6. LIMIT OF DETECTION

Several approaches for determining the detection limit are possible, depending

on whether the procedure is a non-instrumental or instrumental. Approaches other

than those listed below may be acceptable.

Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be used

with instrumental methods.

The detection limit is determined by the analysis of samples with known

concentrations of analyte and by establishing the minimum level at which the analyte

can be reliably detected.

Based on Signal-to-Noise

This approach can only be applied to analytical procedures which exhibit baseline

noise.

Determination of the signal-to-noise ratio is performed by comparing measured

signals from samples with known low concentrations of analyte with those of blank

34

samples and establishing the minimum concentration at which the analyte can be

reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered

acceptable for estimating the detection limit.

Based on the Standard Deviation of the Response and the Slope

The detection limit (DL) may be expressed as:

3.3 σ

DL = ---------

S

where σ = the standard deviation of the response

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte. The estimate

of σ may be carried out in a variety of ways, for example:

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.

Based on the Calibration Curve

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

the range of DL. 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.

Recommended Data

The detection limit and the method used for determining the detection limit should be

presented. If DL is determined based on visual evaluation or based on signal to noise

ratio, the presentation of the relevant chromatograms is considered acceptable for

justification.

In cases where an estimated value for the detection limit is obtained by calculation or

extrapolation, this estimate may subsequently be validated by the independent

analysis of a suitable number of samples known to be near or prepared at the detection

limit.

35

7. LIMIT OF QUANTITATION

Several approaches for determining the quantitation limit are possible,

depending on whether the procedure is a non-instrumental or instrumental.

Approaches other than those listed below may be acceptable.

Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be used

with instrumental methods.

The quantitation limit is generally determined by the analysis of samples with known

concentrations of analyte and by establishing the minimum level at which the analyte

can be quantified with acceptable accuracy and precision.

Based on Signal-to-Noise Approach

This approach can only be applied to analytical procedures that exhibit baseline noise.

Determination of the signal-to-noise ratio is performed by comparing measured

signals from samples with known low concentrations of analyte with those of blank

samples and by establishing the minimum concentration at which the analyte can be

reliably quantified. A typical signal-to-noise ratio is 10:1.

Based on the Standard Deviation of the Response and the Slope

The quantitation limit (QL) may be expressed as:

10 σ

QL = ----------

S

where σ = the standard deviation of the response

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte. The estimate

of σ may be carried out in a variety of ways for example:

Based on 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.

36

Based on the Calibration Curve

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

the range of QL. The residual standard deviation of a regression line or the standard

deviation of y-intercepts of regression lines may be used as the standard deviation.

Recommended Data

The quantitation limit and the method used for determining the quantitation

limit should be presented. The limit should be subsequently validated by the analysis

of a suitable number of samples known to be near or prepared at the quantitation limit.

8. ROBUSTNESS

The evaluation of robustness should be considered during the development

phase and depends on the type of procedure under study. It should show the reliability

of an analysis with respect to deliberate variations in method parameters.

If measurements are susceptible to variations in analytical conditions, the analytical

conditions should be suitably controlled or a precautionary statement should be

included in the procedure. One consequence of the evaluation of robustness should be

that a series of system suitability parameters (e.g., resolution test) is established to

ensure that the validity of the analytical procedure is maintained whenever used.

Examples of typical variations are:

Stability of analytical solutions and extraction time.

In the case of liquid chromatography, examples of typical variations are:

Influence of variations of pH in a mobile phase, influence of variations in mobile

phase composition, different columns (different lots and/or suppliers), temperature

and flow rate.

9. SYSTEM SUITABILITY TESTING

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

The tests are based on the concept that the equipment, electronics, analytical

operations and samples to be analyzed constitute an integral system that can be

evaluated as such. System suitability test parameters to be established for a particular

procedure depend on the type of procedure being validated.

37

References

1. Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajah

Practical HPLC method development, 2nd Edition, New York, 1997.

2. Satinder Ahuja and Michael W. Dong

Hand book of Pharmaceutical Analysis by HPLC, Vol. 6, 1st Edition, Elsevier

academic Press, 2005.

3. Validation of analytical procedures: Text and Methodology, ICH Hormonised

Tripartite Guideline Q2 (R1), Commission of the European Communities (2005).

38

1.5 STABIITY INDICATING ASSAY

THE ICH REQUIREMENTS OF STRESS TESTING FOR OF DRUGS

AND ESTABLISHMENTS OF STABILITY –INDICATING ASSAYS

Determining Inherent Stability

The ICH guidelines entitled “stability testing of new drug substances and

products” (QIAR) requires that stress testing be carried out to elucidate the inherent

stability characteristics of the active substances. It suggests that the degradation

products that are formed under a variety of conditions should be identified and

degradation pathways established. It is stated that testing should include the effect of

temperature, humidity, (where appropriate); oxidation, photolysis, and susceptibility

to hydrolysis across a wide range of pH values. The study of effect of temperature is

suggested to be done in 10ºC increments above the accelerated temperature test

condition (e.g.50ºC, 60ºC etc) and that of humidity at a level of 75%RH or greater.

The ICH guidelines QIAR also emphasizes that the testing of these features,

which are susceptible to change during storage and are likely to influence quality,

safety, and or efficacy, must be done by stability –indicating testing methods. The

ICH guidelines Q3B entitled impurities in new drug products emphasizes on

providing documented evidence that analytical procedure are validated and suitable

for the detection and quantification of degradation products. It is also required that

analytical method should validated to demonstrate that impurity unique to the new

drug substances do not interfere with or are separated from specified or unspecified

degradation products in the drug product .The ICH guidelines Q6A, which provides

notes for guidance on specification, also mention the requirement of stability –

indicating assays under universal test criteria for both the drug substances and

products.

The same is also a requirement in the guidelines Q5C on stability testing of

biotechnological /biological products. Since there are no single assays or parameter

that profiles the stability characteristics of such products, stability- indicating profile

39

that provides assurance on detection of change in identity, purity, and potency of the

product.

Regulatory and Compendial Status of Stress Testing And Stability Indicating

Assays

The requirements of stress testing and establishment of stability indicating assays are

also covered in other international guidelines and compendia. The notes for guidelines

stability testing: stability testing of existing active substances and related finished

products (CPMP.QWP/122/02) issued by European committee for proprietary

medicinal products (CPMP) states that when a drug substance is described in an

official monograph (European pharmacopoeia or a European union member state), no

data required on the degradation products if they are named under the headings

“purity test “and or “impurities”; In this case no stress testing is required . That means

no forced decomposition required under the pharmacopoeial monographs. On other

hand, stress testing is to be done when no data available in the scientific literature or

the official pharmacopoeia. The route of stress testing for determining intrinsic

stability of drug is also mentioned as requirement in Canadian therapeutic products

directorate’s (TDP) draft guidelines entitled‘ stability testing of existing drug

substances and products ‘The requirements of establishment of stability indicating

assay is listed in guidelines on stability.

Testing of well established or existing drug substances and drug products issued by

CPMP, TDP, and WHO. Even the United States pharmacopoeia has a products should

be assayed for the potency of the use of a stability indicating assay. The requirement

is such explicit manner is, however, absent in other pharmacopoeias. Current ICH

guidelines on good manufacturing for active pharmaceutical ingredients (Q7A),

Which under adoption by WHO, also clearly mentions that the test procedure used in

stability testing should be validated and be stability indicating .The ICH guidelines do

not provide an exact definition of a stability indicating method.

Elaborate definitions of stability indicating methodology are, however provided in the

US-FDA stability guidelines of1998. Stability indicating methods, according to 1987

guideline, were defined as the quantitative analytical methods that are based on

characteristics structural, chemical, or biological properties of each ingredient of a

drug product that will distinguish each active ingredient from its degradation products

so that the active ingredient content can be accurately measured; This definition in

draft guidelines of 1998 read as:-

40

1. Validated quantitative analytical methods that can be detect change with time in

the chemical, physical, or microbiological properties of a drug substances and

drug products and that are specific so that the contents of active ingredients,

degradation products and other components of interest can be accurately measured

without interference The major change brought in the new guidelines are with

respect to a. introduction of the requirements of validation.

2. The requirements of analysis of degradation products and other components, apart

from the active ingredients.

Survey of The Literature For Reaction Conditions and Approaches

For Stress Testing

The practical aspect concerning the conduct of stress testing is addressed

neither by ICH nor by any other regulatory guidelines. Leaving the performance of

these studies to the discretion of the applicant. Therefore to determine the nature of

condition of stress testing, a survey was carried out of the monographs given in

various volumes of analytical profiles of drug substances .The monographs carry a

suitable reaction, which usually records the inherent stability behaviour of the drug

and the reaction condition used. Good amount of information on stress condition was

also found in the literature reports on establishment of stability indicating assays. The

information reveals that HCl at strength 0.1N is mostly used for stress decomposition

of drugs in acid condition. There are many studies where 1N has been exploited.

In some cases, HCl of higher normalities has been used. In few cases, mention is only

found of ‘acid’ without defining the type used. Also, large variation exists in the

reaction (temperature) condition, periods of study and the extent of decomposition.

While the temperature of study various between 25ºC and 116ºC the periods of

studies range from few minutes to as long as 2 months. The extent of drug

decompositions falls between two extremes, i.e. from nil to total degradation. This

clearly reflects that conditions for stress testing vary strongly, depending upon the

inherent stability of the drug. In alkaline degradation, sodium hydroxide either at

strength 0.1N or 1 N has been mostly employed for stress testing. Potassium

hydroxide or ammonium hydroxide is rarely used. Like the acidic degradation, a lot of

variation. Depending upon the inherent stability characteristics, some drugs did not

41

degrade even after refluxing in 0.1 N NaOH for one week while others underwent

almost complete degradation when kept at 25ºC in 0.1N alkali for just 210 min.

In another example, boiling with 1N NaOH for 5-20 min resulted in extensive

degradation or almost total loss of potency of Acyclovir, Cephalexin etc; while

boiling of Norfloxacin in alkali of same strength for 15 hr did not cause any change.

Not many reports could be found on stress studies in neutral pH. As may be seen

from the examples given in Table, the testing is generally done in water and no

significant degradation was observed; while temp. Ranged from 37-40ºC or refluxing

condition was employed. The slow rate of decomposition in neutral conditions in

most of the cases is understandable because reactions at neutral pH are non-catalytic

and hence very long periods under exaggerated temperature condition required to get

sufficient degradation products.

Approaches suggested in literature for performing stress testing

A few approaches have been purposed recently for performing forced

decomposition studies, as a sequel to introduction of requirements in ICH guidelines.

The first one by Hong and Shah suggests selection of stress testing condition, as

described in below Table. The concentration of the reagents and time of study are

given. It is recommended that stress tests should be carried out under a specific

condition for a time period enough to yield about 20-30% degradation would be too

strenuous and could possibly cause secondary degradation, yielding products of the

degradation product 42, which are not likely to be found under normal storage

condition.

Another approaches for forced decomposition studies have been put forth lately by

Reynolds etal. Following table provides the suggested protocol.

According to this approach, sufficient exposure of a drug substance degrade by ∼105

from its initial amount or after an exposure in excess of energy provided by

accelerated storage, whichever comes first .The duration of storage required at a given

temperature can be estimated by making conservative kinetic assumption For drug

products, non-drug substances related peaks should be distinguished from drug

substance related compounds which can be accomplished through comparative

analysis of stress samples drug substance plus excipients, and of excipients alone.

42

General Protocol For Stress Testing of Drug Substances and Products

Condition Drug Substances Drug Product

Solid Solution/

Suspension

Solid (Tablets,

capsules, blends)

Solution

i.v.,oral solution,

suspension)

Acid/Base √

Oxidative √ √ √

Photostability √ √ √

Thermal √ √ √

Thermal/

Humidity √ √

A further approach for automating multiple degradation experiments and performing

online HPLC analysis of the resultant impurities using combined diode array UV

detection and mass spectrometry has been exploited in another recent publication.

It involves the use of an automated workstation, wherein ten samples are refluxed

with stirring in a single heating block .The robot arm is equipped with a sampling

device capable of removing aliquots, during reflux experiment and them to HPLC

injector after suitable neutralization and dilution .The automation process does not

only benefit in terms of analyst time but also allow extra high quality data to be

collected with a minimal time delay between sample preparation and analysis .The

combination of HPLC and mass detection in the system also allows structural

information to be obtained on the degradants along with kinetic data on the

appearance and disappearance of degradants during the course of experiments.

43

REFERENCES

1) D.W. Reynolds, K.L. Fachieve, J.F. Mullaney, K.M. Alsante, T.D. Hatajik and

M.G. Motto, Available guidance and test practice for conducting forced

degradation studies, Pharm. Technol 20 (2002) pp48-56.

2) J.L.Sims, J.K. Roberts, A.G. Bateman, J.A.Carreiran and M.J. Hardy, an

automated workstation for forced degradation of active pharmaceutical

ingredients, J of Pharm. Science, 2002 (91) 884-892.

3) L. Chafetz, stability indicating assay method for drug and their dosage forms,

J of Pharm.Science, 1971 (60) 335-341.

4) ICH, Stability testing Photostability testing of new drug substances for

products, International conference on harmonization, IFPMA, Geneva 1996.

5) ICH, Stability testing of new drug substances & product, International

conference on harmonization, IFPMA, Geneva 1993.