METHOD DEVELOPMENT AND VALIDATION FOR

THE SIMULTANEOUS ESTIMATION OF DMM &

DTT BY USING RP-HPLC METHOD
 Introduction

1. INTRODUCTION

Pharmaceutical Analysis is the branch of Chemistry involved in separating,

identifying and determining the relative amounts of the components making up a
sample of matter. It is mainly involved in the qualitative identification or detection
of compounds and quantitative measurements of the substances present in Bulk drug
and Pharmaceutical preparations (Sharma B.K, 2000)

Pharmaceutical Analysis simply means analysis of a Pharmaceutical(s). It is

generally known that a Pharmaceutical is a chemical entity of Therapeutic interest.
A more appropriate term for a Pharmaceutical is Active Pharmaceutical Ingredient
(API) or Active Ingredient. Even though the term Active Ingredient is more
frequently used, the preferred term is Active Pharmaceutical Ingredient. To
distinguish it from the formulated product or drug product, API is also called Drug
substance. The drug product is prepared by formulating a drug substance with inert
ingredients (excipients) to prepare a drug product that is suitable for administration
to patients.

However, it should be recognized that there are situations where a drug

substance can be administered after simple dissolution in a solvent such as water.
Even in these situations, a suitable Pharmaceutical treatment has to be conducted to
assure availability and other safety considerations.

It is well known in the Pharmaceutical industry that Pharmaceutical Analysts

in Research and Development (R&D) play a very comprehensive role in new drug
development and follow up activities to assure that a new drug product meets the
established standards, is stable and continues to meet the purported quality
throughout its shelf life. After the drug product is approved by regulatory
authorities, assuring that all batches of drug product are made to the specified
standards, utilization of approved ingredients and production methods becomes the
responsibility of Pharmaceutical Analysts in the Quality Control (QC) or Quality
Assurance (QA) department. The methods are generally developed in an analytical
R&D department and transferred to QC or other departments, as needed. At times,
they are transferred to other divisions located nationally or abroad or to outsourced

1 C.L.Baid Metha College of Pharmacy

companies. By now it should be quite apparent that Pharmaceutical Analysts play a
major role in assuring the identity, safety, efficacy, and quality of a Drug product.
Safety and efficacy studies require that drug substance and drug product meet two
critical requirements:

Established Identity and Purity.

Established Bioavailability / Dissolution (Satinder A & Stephen S,

2001)

Quality Assurance plays a central role in determining the safety and efficacy
of medicines. Highly specific and sensitive analytical techniques hold the key to the
design, development, standardization and quality control of medicinal products.
They are equally important in pharmacokinetics and in drug metabolism studies,
both of which are fundamental to the assessment of bioavailability and the duration
of clinical response. However, modern concepts of quality differs and concerned not
only with chemical purity, but also with those other characteristics of
Pharmaceutical materials which may influence safety, efficacy, formulation and
processing of medicines (Beckett AH & Stenlake JB 1997).

1.1 INSTRUMENTAL ANALYSIS

The instrument is only one component of the total analysis. Often, it is necessary
to use several instrumental techniques to obtain the information required to solve an
analytical problem. Instrumental method may be used by analytical chemists to save
time, to avoid chemical separation or to obtain increased accuracy.

Based on Principle Types of Chemical Instrumentation:

A) Spectrometric Techniques:

Atomic Spectrometry (Emission and Absorption)

Electron Spin Resonance Spectroscopy

Fluorescence and phosphorescence Spectrophotometry

 Infrared Spectrophotometry

Nuclear Magnetic Resonance Spectroscopy

Radiochemical Techniques including activation analysis

Raman Spectroscopy

Ultraviolet and visible Spectrophotometry

X-Ray Spectroscopy

B) Electrochemical techniques

 Potentiometry

 Voltametry

 Stripping techniques

 Amperometric techniques

 Coulometry

 Electrogravimetry

 Conductance techniques.

C) Chromatographic Techniques:

Gas Chromatography

High performance Liquid Chromatography

Thin Layer Chromatography

D) Miscellaneous Techniques:
 Kinetic Techniques

Mass Spectrometry

Thermal Analysis

D) Hyphenated Techniques:

GC-MS (Gas Chromatography - Mass Spectrometry)

ICP-MS (Inductivity Coupled Plasma - Mass Spectrometry)

GC-IR (Gas Chromatography - Infrared Spectroscopy)

MS-MS (Mass Spectrometry - Mass Spectrometry (Willard H.H. et al

1986)

1.1. CHROMATOGRAPHY

Chromatography (from Greek: chroma, color and:"graphein" to write) is

essentially a group of techniques for the separation of the compounds of mixtures by
their continuous distribution between two phases, one of which is moving fast the
other that depends on differential affinities of the solute between two immiscible
phases, one of which will be fix with large surface area, while the other is fluid
which moves through or over the surface of the fixed phase. (Beckett AH &
Stenlake JB 1997)

Definitions for Chromatography:

1. Tswett gave the first definition of chromatography. Chromatography is a

method in which the compounds of a mixture are separated on an adsorbent
column in a flowing system.
2. Chromatography defined as a method of separating a mixture of components
into individual components through equilibrium distribution between two
phases. (Gurdeep R Chatwal & Sham K.Anand 2002)

3. IUPAC: chromatography is a physical method of separation in which the

compound to be separated are distributed between two phases, one of which is
stationary (stationary phase) while the other (the mobile phase) moves in a
definite direction (IUPAC, 1993)
CLASSIFICATION OF CHROMATOGRAPHIC METHODS

(Gurdeep R. Chatwal & Sham K.Ananad2002)

STATIONARY MOBILE PHASE NAME

PHASE

Plane Chromatography

Paper Chromatography

Thin layer Chromatography

SOLID LIQUID
Adsorption Column Chromatography

High Performance Liquid

Chromatography

SOLID Ion exchange Chromatography

LIQUID
(Ion exchange resin)

SOLID Gas-Solid Chromatography

GAS

Gel permeation Chromatography

SOLID MATRIX LIQUID
(Exclusion Chromatography)

LIQUID GAS Gas-Liquid Chromatography

LIQUID LIQUID Liquid-Liquid Chromatoraphy

1.3. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

 High Performance Liquid Chromatography (HPLC) was developed in the
late 1960s and early 1970s. Today it is widely applied for separations and
purifications in a variety of areas including pharmaceuticals, biotechnology,
environmental, polymer and food industries.

HPLC has over the past decade become the method of choice for the analysis
of a wide variety of compounds. Its main advantage over GC is that the analytes do
not have to be volatile, so macromolecules are suitable for HPLC analysis.

PRINCIPLE:

HPLC is accomplished by injection of a small amount of liquid sample into a

moving stream of liquid (called the mobile phase) that passes through a column
packed with particles of stationary phase. Separation of a mixture into its
components depends on different degrees of retention of each component in the
column. Since the compounds have different mobilities, they exit the column at
different times; i.e., they have different retention times, Rt. The retention time is the
time between injection and detection. There are numerous detectors which can be
used in liquid chromatography. It is a device that senses the presence of components
different from the liquid mobile phase and converts that information to an electrical
signal.

Reversed phase HPLC

In this case, the column size is the same, but the silica is modified to make it
non-polar by attaching long hydrocarbon chains to its surface - typically with either
8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of
water and an alcohol such as methanol. There will be a strong attraction between
the polar solvent and polar molecules in the mixture being passed through the
column. There won't be as much attraction between the hydrocarbon chains attached
to the silica (the stationary phase) and the polar molecules in the solution. Polar
molecules in the mixture will therefore spend most of their time moving with the
solvent. Non-polar compounds in the mixture will tend to form attractions with the
hydrocarbon groups because of vander Waals dispersion forces. They will also be
less soluble in the solvent because of the need to break hydrogen bonds as they
squeeze in between the water or methanol molecules, for example. They therefore
spend less time in solution in the solvent and this will slow them down on their way
through the column. That means that now it is the polar molecules that will travel
through the column more quickly. (David C. Lee &Michael Webb, 2003)(Synder
L.R &Kirkland J.J.,1997)

The majority of the HPLC separations are done with Reversed phase
separation, probably over 90%. In reversed phase separations organic molecules are
separated based on their degree of hydrophobicity. There is a correlation between
the degree of lipophylicity and retention in the column.

Types of HPLC techniques

 Based on Principles of Separations

 Partition Chromatography

 Adsorption (liquid-solid) Chromatography

 Ion exchange Chromatography

 Size exclusion Chromatography

 Based on Modes of Chromatography

 Normal Phase Chromatography

 Reverse Phase Chromatography

 Based on Elution Techniques

 Isocratic Separation

 Gradient Separation

 Based on the Scale of Operation

  Analytical HPLC

 Preparative HPLC

Flow chart: 1. Selection of HPLC methods depending upon Nature of samples

INSTRUMENTATION:

1. COLUMN:

HPLC columns are made of high quality Stainless steel, polished internally to a
mirror finish. Standard analytical columns are 4-5 m internal diameter and 10-30 cm
in length, shorter column (3-6 cm in length) containing a small particle size packing
material (3 or 5 µm). ( Beckett AH& Stenlake J B 1997) Figure 1 : Shcematic
representation of HPLC

Column packing:

Three forms of column packing material are available based on a rigid

structure. These are

i. Microporous supports

ii. Pellicular supports

iii. Bonded phase supports (Gurdeep R.Chatwal &Sham K.Anand,2002)

2. MOBILE PHASE RESERVIOR :

The mobile phase reservior can be any clean, inert containers made up of
stainless steel and glass. Precaution should be taken to present solvents spills in case
of breakage of the reservoir and it should be placed in plastic container. Solvent
bottles are available that are coated with a resin material that resist breaking. It
usually contain 1 or 2 liter of solvent and it should have a cap that allows the tubing
inlet line to pass through it. (James W. Munson,2001)

The choice of mobile to be used in any separation depend on the type of

separation to be achieved. Isocratic separation may be made with a single
solvent, or two or more solvents mixed in fixed proportion. Alternatively a
gradient elution system may be used where the composition of the developing
solvent is continuously changed by use of a suitable gradient programmer. All
solvents for using HPLC systems must be specially purified since traces of
impurities can affect the column and interfere with the detection system. It is
also essential that all solvents are degassed before use other wise gassing tends
to occur in most pumps. Gassing can alter column resolution and interfere with
the continues monitoring of the column effluent. Degassing may be carried out
in several way; by warming the solvents, by stirring it vigorously with a
magnetic stir, subjecting it to a vacuum, ultrasonic vibrations or by bubbling
helium gas through the solvent reservoir.(Gurdeep R.Chatwal & Sham
K.Ananad,2002)

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 the duration of the analysis.

2. Excessive salt concentrations should be avoided. High salt concentrations

can result in precipitation, which can damage HPLC equipment.

3. The Mobile phase should have a pH between 2.5 to pH 7.0 to maximize the
lifetime of the column.

4. Reduce cost and toxicity of the Mobile phase by using methanol instead of
acetonitrile when possible.

5. Minimize the absorbance of buffer. Since trifluroacetic acid or formic acid

absorb at shorter wavelengths, they may prevent detection of products with
out chromophores above 220 nm. Carboxylic acid modifiers can be
 frequently replaced by phosphoric acid, which does not absorb above 200
nm.

6. Use volatile Mobile phases when possible to facilitate collection of products

and LC - MS analysis. Volatile Mobile phases include ammonium acetate,
ammonium phosphate, and formic acid, acetic acid and trifluroacetic acid.
Some caution is needed as these buffers absorb below 220 nm.

3. INJECTORS:

Injection ports are of two basic types

a. Those in which the sample is injected directly into the column

b. Those in which the sample is deposited before the column inlet and
then swept by a vying action into the column by the mobile phase.

On –column injection involves the injection of the sample by means of a

syringe through a septum into the center of the packing material. The column and
the capacity of the packing material is typically 5-25µl for analytical column.

High- pressure syringes that can be used at pressure up to 650 atmospheres

allow the injection of the sample while the mobile phase is flowing. While using
Low- pressure syringes the flow must be stopped.

Modern injectors are based on injection valves which allow the sample at
atmospheric pressure to be transferred to the high-pressure mobile phase
immediately before the column inlet. With the injection in LOAD position, the
sample is injected from a syringe through a needle port into the loop.

The valve lever is then turned through 60o to the inject position and the sample is
swept into the flowing mobile phase. An excess of sample is flushed through the
loop in the LOAD position, the volume injected is the volume of the loop, which is
typically 10-20µl for analytical separation. (Beckett AH& Stenlake J B 1997)
4. PUMPS:

The pumping system is one of the most important features of a HPLC system.
There is a high resistance to solvent due to the narrow columns packed in small
particles, high pressure are required to achieve satisfactory flow rate.

The requirements for an HPLC pumping system are several;

They include

a. The generation of pressures of up to 6000 psi (lbs/in2)

b. Pulse- free output

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

d. Good flow control capacity

e. All materials in the pump should be chemically resistance to all solvents

These pumping system available which operate on the principle of constant

pressure or constant displacement.

Constant pressure pumps produce a pulse less flow through the column, but
any decrease in the permeability of the column will result in lower flow rates for
which the pumps will not compensate. These pumps operate by the introduction of
high pressure gas into the pump, and the gas in turn forces the solvent from the
pump chamber in to the column. The intermediate solvent between the gas and the
eluting solvent reduce the chances of dissolved gas directly enter in the eluting
solvent and causing problems during the analysis.

Constant displacement pump maintain a constant flow rate through the

column irrespective of changing conditions with in the column. One form of
constant displacement pump is a motor-driven syringe type pump where a fixed
volume of solvent is forced from the pump to the column by a piston driven by a
motor. Such pumps, as well as providing uniform solvents flow rates, also yields a
pulse less solvent flow which is important as certain in detectors are sensitive to
change in solvent flow rate.

The reciprocating pump is most commonly used form of constant

displacement pump. The piston is moved by a motorized crank and entry of solvent
from the reservoir to the pump chamber and exit of solvent to the column is
regulated by check valves. On the compression stroke solvent is forced from the
pump chamber in to the column. During the return stroke the exit check valve closes
and solvent is drawn in via entry valve to t e pump chamber, ready to be pumped on
to the column on the next compression stroke. (Gurdeep R.chatwal &Sham K.
Anand,2002)

5. DETECTORS:

The detector for an HPLC is the compound that emits a response due to the
eluting sample compound and subsequently signals a peak on the chromatogram. It
is positioned immediately posterior to the stationary phase on order to detect the
compounds as they elute from the column. The bandwidth and height of the peaks
may usually be adjusted using the coarse and fine tuning controls, and the detection
and sensitivity parameters may also be controlled (in most cases). There are many
types of detectors that can be used with HPLC

Types of Detectors

1. Solute specific detectors (UV, visible, fluorescence, electrochemical, infra-

red, radioactivity).

2. Bulk property detectors (refractive index, viscometer, conductivity).

3. Desolvation detectors (flame ionization etc.).

4. LC-MS detectors.

5. Reaction detectors.
Absorbance Detectors

Absorbance detectors is a typical, Z- shaped, flow through cell for

absorbance measurements on eluent from chromatographic column. Volumes are
limited to 1 to 10 µl and cell lengths to 2 to 10 mm, and the pressure not greater than
600 psi. Many Absorbance detectors are double-beam devices in which one beam
passes through the eluent cell and the other through a filter to reduce the intensity.

Ultraviolet Absorbance Detectors

UV Absorbance Detectors are available in two types, UV Absorbance

Detectors with Filter and with monochromators. Most HPLC manufactures offer
detectors that consist of a scanning spectrophotometer with grating optics. Some are
limited to UV radiation; others encompass both UV and Visible radiation. The most
powerful UV Spectrophotometric detectors are diode - array instruments.

Refractive Index Detectors

RI Detectors are also called as Universal analyte detector. RI detectors have

the significant advantage of responding to nearly all solutes. That is they are general
detectors analogous to flame detectors in gas chromatography. In addition they are
reliable and unaffected by flow rate. They are highly temperature sensitive and must
be maintained at a constant temperature to a few thousands of a degree centigrade.
They are not as sensitive as most other type of detectors and generally cannot be
used with gradient elution.

Fluorescence Detectors

Excitation wavelength generates fluorescence emission. Analytes must

contain a Flurophore group it reacts with the same group of the reagent. The
inherent advantage of fluorescence methods is their high sensitivity. Results are
dependent up on the separation condition. (Gennaro A.R.Remigton, 2000)
1.4. STEPS FOR ANALYTICAL DEVELOPMENT

Methods are developed for new products when no official methods are
available. Alternate methods for existing (non-pharmacopoeial) products are
developed to reduce the cost and time for better precision and ruggedness. Trial runs
are conducted, method is optimized and validated.

1. Analyte standard characterization:

a) All information about the analyte i.e., physical and chemical properties, toxicity,

b) The standard analyte (100% purity) is obtained. Made an arrangement for the
proper storage (refrigerator, desiccators and freezer).

c) When multiple components are to be analyzed in the sample matrix, the number
of components is noted, data is assembled and the availability of standards for
each one is determined.

d) Only those methods (MS, GC, HPLC etc.,) that are compatible with sample
stability are considered.

2. Method requirements:

The goals of the analytical method that need to be developed are considered.
The detection limits, selectivity, linearity, range, accuracy and precision are defined.

3. Literature search and prior methodology:

The information related to the analyte is surveyed for synthesis, physical and
chemical Properties, solubility and relevant analytical methods. Books, periodicals
and USP / NF, and publications are reviewed. Chemical Abstracts Service (CAS)
automated computerized literature searches are convenient.

4. Choosing a method:

a) Using the information in the literatures, methodology is adapted. The

methods are modified wherever necessary. Sometimes it is necessary to acquire
additional instrumentation to reproduce, modify, improve or validate existing
methods for in-house analytes and samples.

b) If there are no prior methods for the analyte in the literature, from
analogy, the compounds that are similar in structure and chemical properties are
investigated and are worked out. There is usually one compound for which
analytical method already exist that is similar to the analyte of interest.

5. Instrumental setup and initial studies:

a) The required instrumentation is setup Installation, operational and

performance qualifications of instrumentation verified by using laboratory Standard
Operating Procedures (SOP’s).

b) Always new solvents, filters are used, for example, method development
is never started, on a HPLC column that has been used earlier.

c) The analyte standard in a suitable injection / introduction solution and in

known concentrations and solvents are prepared. It is important to start with an
authentic, known standard rather than with a complex sample matrix. If the sample
is extremely close to the standard (e.g., Bulk drug), then it is possible to start work
with the actual sample.

d) Analysis is done using analytical conditions described in the existing

literature.

6. Optimization:

During optimization one parameter is changed at a time, and set of

conditions are isolated, rather than using a trial and error approach. Work has been
done from an organized methodical plan, and every step is documented (in a lab
notebook) in case of dead ends.

7. Documentation of analytical figures of merit:

 The originally determined analytical figures of merit Limit of Quantitation
(LOQ), Limit of Detection (LOD), linearity, time per analysis, cost, sample
preparation etc., are documented.

8. Evaluation of method development with actual samples:

The sample solution should lead to unequivocal, absolute identification of

the analyte peak of interest apart from all other matrix components.

9. Determination of percent recovery of actual sample and demonstration

of quantitative sample analysis:

a) Percent recovery of spiked, authentic standard analyte into a sample

matrix that is shown to contain no analyte is determined. Reproducibility of
recovery (average +/- standard deviation) from sample to sample and whether
recovery has been optimized is determined. It is not necessary to obtain 100%
recovery as long as the results are reproducible and known with a high degree of
certainty.

b) The validity of analytical method can be verified only by laboratory

studies. Therefore documentation of the successful completion of such studies is a
basic requirement for determining whether a method is suitable for its intended
applications.( Michael E & Schartz IS)

1.5. OPTIMIZATION OF CHROMATOGRAPHIC CONDITION

Optimization can be started only after a reasonable chromatogram has been

obtained. A reasonable chromatogram means that all the compounds are detected by
more or less symmetrical peaks on the chromatogram. By a slight change of the
mobile phase composition, the shifting of the peaks can be expected. From a few
experimental measurements, the position of the peaks can be predicted within the
range of investigated changes. An optimized chromatogram is the one in which all
the peaks are symmetrical and are well separated in less run time. (Munson
J.W,1994 )

The peak resolution can be increased by using a more efficient column with
higher theoretical plate number, N.

The parameters that are affected by the changes in chromatographic

conditions are,

 Resolution (Rs),

 Capacity factor (k’),

 Selectivity (),

 Column efficiency (N) and

 Peak asymmetry factor (As).

i) Resolution (Rs)

The resolution, Rs, of two neighboring peaks is defined by the ratio of the
distance between the two peak maxima. It is the difference between the retention
times of two solutes divided by their average peak width. For baseline separation,
the ideal value of Rs is 1.5. It is calculated by using the formula,

Rt2  Rt1
Rs =
0.5W1  W2


Where, Rt1 and Rt2 are the retention times of components 1 and 2 and

W1 and W2 are peak widths of components 1 and 2.

ii) Capacity factor (k’)

Capacity factor, k’, is defined as the ratio of the number of molecules of

solute in the stationary phase to the number of molecules of the same in the mobile
phase. Capacity factor is a measure of how well the sample molecule is retained by a
column or TLC plate during an isocratic separation. The ideal value of k’ ranges
from 2-10. Capacity factor can be determined by using the formula,

V1  V0
k’ = S
V0

Where, V1 = retention volume at the apex of the peak (solute) and

V0 = void volume of the system.

The values of k’of individual band increase or decrease with changes in

solvent strength. In reverse phase HPLC, solvent strength increases with the increase
in the volume of organic phase in the water / organic mobile phase. Typically an
increase in percentage of the organic phase by 10 % by volume will decrease k’ of
the bands by a factor of 2-3.

iii) Selectivity ()

The selectivity (or separation factor), , is a measure of relative retention of

two components in a mixture. The ideal value of selectivity is 2. It can be calculated
by using the formula,

V2  V0
α = V1  V0

Where, V0 is the void volume of the column and V2 and V1 are the retention
volumes of the second and the first peak respectively.
iv) Column efficiency (N)

Efficiency, N, of a column is measured by the number of theoretical plates

per meter. It is a measure of band spreading of a peak. Smaller the band spread,
higher is the number of theoretical plates, indicating good column and system
performance. Columns with N ranging from 2000 - 100,000 plates/meter are ideal
for a good system. Efficiency is calculated by using the formula,

Rt 2
N = 16 ,
2
W

Where, Rt is the retention time and W is the peak width.

v) Peak asymmetry factor (As)

Peak asymmetry factor, can be used as a criterion of column performance.

The peak half width, b, of a peak at 10 % of the peak height, divided by the
corresponding front half width, a, gives the asymmetry factor.

b
As =
a

For a well packed column, an asymmetry factor of 0.9 to 1.1 should be

achievable. (Jeffery G.H et al , 2003)

1.6. VALIDATION

The word “validation” means “Assessment” of validity or action of validity

or action of providing effectiveness’. Validation is, of course, a basic requirement to
ensure quality and reliability of the results for all analytical applications. However,
in comparison with Analytical Chemistry, in Pharmaceutical Analysis, some special
aspects and conditions exist that need to be taken into consideration. Validation of
an analytical method is the process by which it is established by laboratory studies,
that the performance characteristics of the method meet the requirements for the
intended analytical applications.

Definitions:

Validation is a systematic approach to gathering and analyzing sufficient

data which will give reasonable assurance (documented evidence), based upon
scientific judgment, that a process, when operating within specified parameters, will
consistently produce results within predetermined specifications.

Validation is defined as follows by different agencies:

European Committee (EC):

Action of providing in accordance with the principles of Good

Manufacturing Practice (GMP) that any procedure, process, equipment, material,
activity or system actually leads to the expected results. In brief validation is a key
process for effective Quality Assurance

Food and Drug Administration (FDA):

Provides a high degree of assurance that specific process will consistently

produce a product meeting its predetermined specification and quality attributes.

World Health Organization (WHO):

Action of providing that any procedure, process, equipment, material,

activity, or system actually leads to the expected results.

History:

Since the mid-1970s validation has become an increasingly dominant

influence in the manufacturer and quality assurance of pharmaceutical products. In
1976 the FDA proposed a whole set of current GMP regulations which were revised
several times.

Objective of the Validation

 There are two important reasons for validating assays in the Pharmaceutical
Industry.

 The first, and by for the most important, is that assay validation is an
integral part of the quality control system.

 The second is that current Good Manufacturing Practice (GMP)

regulation requires assay validation. In industry it would be difficult
to confirm that the product being manufactured is uniform and that
meet the standards set to assure fitness for use. The varying nature of
the differences between the analytical development laboratory and
quality control laboratory is a good reason for validation program.
(USP 1985) ( Joachim Ermer& Miller2005)

ANALYTICAL METHOD VALIDATION

Method Validation is the process of proving that an analytical method is

acceptable for its intended purpose. Methods need to be validated or revalidated-
Before their introduction into routine use, whenever the conditions change for which
the method has been validated, whenever the method is changed and the change is
outside the original scope of the method.

 United States Pharmacopoeia (USP).

 Food and Drug Administration (FDA).

 World Health Organization (WHO).

 International Conference on Harmonization (ICH).

These guidelines provide a framework for performing Validation. In general,

methods for routine analysis, standardization or regulatory submission must include
studies on specificity, linearity, accuracy, precision, range, limit of detection, limit
of Quantitation and robustness.
 In the early stages of drug development, it is usually not necessary to
perform all of the various validation studies. Many researchers focus on specificity,
linearity, accuracy, and precision studies for drugs in the preclinical through Phase II
(preliminary efficacy) stages. The remaining studies are performed when the drug
reaches the Phase III (efficacy) stage of development and has a higher probability of
becoming a marketed product. The process of validating a method cannot be
separated from the actual development of the method conditions, because the
developer will not know whether the method conditions are acceptable until
validation studies are performed. The development and validation of a new
analytical method may therefore be an iterative process. Results of validation studies
may indicate that a change in the procedure is necessary, which may then require
revalidation.

During each validation study, key method parameters are determined and
then used for all subsequent validation steps. To minimize repetitious studies and
ensure that the validation data they are generated under conditions equivalent to the
final procedure. (Mark JG.)

Benefits of Method Validation:

A fully validated process may require less in-process control and end product
testing. It deepens the understanding of processes, decrease the risks of processing
problems, and thus assure the smooth running of the process.(WHO 1999)

Validation Parameters of Analytical Method:

According to ICH guidelines, typical analytical performance characteristics

that should be considered in the validation of the types of methods are

Typical Validation Characteristics which should be considered are:

 Figure 5: The USP and ICH Method Validation Parameter

1. Accuracy:

The accuracy of an analytical procedure expresses the closeness of

agreement between the value, which is accepted either as a conventional true value
or an accepted reference value and the value found.

The ICH documents recommended that accuracy should be assessed using a

minimum of nine determinations over a minimum of three concentrations levels the
specified range (i.e., three concentrations and three replicates of each concentration).
Accuracy was tested (% Recovery and % RSD of individual measurements) by
analyzing samples at least in triplicate, at each level (80,100 and 120 % of label
claim) is recommended. For each determination fresh samples were prepared and
assay value is calculated. Recovery was calculated from following regression
equation obtained in linearity study.

The % recovery was calculated using the formula,

(a  b)  a
% Re cov ery 
bX100
 Where,

a – Amount of drug present in sample

b – Amount of standard added to the sample

2. Precision:

The precision of an analytical procedure expresses the closeness of

agreement (degree of scatter) between series of measurements obtained from
multiple sampling of the same homogenous sample under the prescribed conditions.

Precision may be considered at three levels: repeatability, intermediate

precision and reproducibility. Precision should be investigated using homogeneous,
authentic samples. However, if it is not possible to obtain a homogeneous sample it
may be investigated using artificially prepared samples or a sample solution. The
precision of an analytical procedure is usually expressed as the variance, standard
deviation or coefficient of variation of a series of measurements. The ICH
documents recommend the repeatability should be assessed using a minimum of
nine determinations covering specified range of procedure.

2.1) Repeatability:

Repeatability expresses the precision under the same operating conditions

over a short interval of time. Repeatability is also termed intra-assay precision.

2.2) Intermediate Precision:

Intermediate precision expresses with in laboratories variations: different

days, different analyst and different equipment.

2.3) Reproducibility:

When the procedure is carried out by different analyst in different

laboratories using different equipment, regents and laboratories setting
reproducibility was determined by measuring repeatability and intermediate
precision. Reproducibility is assessed by means of an inter-laboratory trial.

3. Specificity:

An ICH document defines Specificity is the ability to assess unequivocally

the analyte in the presence of components which may be expected to be present.
Typically these might include impurities, degradants, matrix, etc. Lack of specificity
of an individual analytical procedure may be compensated by other supporting
analytical procedure(s).

The definition has the following implications:

Identification test:

To ensure identity of an analyte.

Purity test:

To ensure that all the analytical procedures performed allow an accurate

statement of the content of impurity of the content of impurity of an analyte i.e.
related substances test, heavy metals, residual solvents etc.

Assay:

To provide an exact result, this allows an accurate statement on the content

or potency of the analyte in a sample.

5. Linearity:

The Linearity of an analytical procedure is its ability (within a given range) to

obtain test results, which are directly proportional to the concentration (amount) of
analyte in the sample.

The linearity is determined from 60% of the ICH reporting level to 140 % of
the proposed shelf life specifications of the related substance as a minimum.
6. Range:

The range of an analytical procedure is the interval between the upper and
lower of analyte, which is studied.

The range of an analytical procedure was the concentration interval over

which acceptable accuracy, precision and linearity were obtained. In practice, the
range was determined using data from the linearity and accuracy studies. Assuming
that acceptable linearity and accuracy (recovery) results were obtained as described
earlier. The only remaining factor to be evaluated was precision. To confirm the
‘range’ of any analytical procedure, linearity studies alone are not sufficient, and
accuracy at each concentration (minimum three concentration levels covering lower
and upper levels) should be proved.

7. Ruggedness:

Degree of reproducibility of test results obtained by the analysis of the same

samples under a variety of condition such as different laboratories, different
analysts, different instruments etc, normally expressed as the lack of influence on
test results of operational and environmental variable of the analytical method.
Ruggedness is a measurement of reproducibility of test results under the variation in
condition normally expected from laboratory to laboratory and from analyst to
analyst. Degree of representative of test results is then determined as a function of
the assay variable.

8. Robustness:

Robustness of an analytical method is measure of its capacity to remain

unaffected small but deliberate variations in method parameters and provides an
indication of its reliability during normal usage. (www.waters.com, USP
specification) (ICH Guidelines 1996)
 Table C: Acceptance criteria of validation for HPLC

S.No. Characteristics Acceptance criteria

1. Accuracy Recovery 98-102% with 80,90,100,120
spiked sample
2. Precision RSD < 2
a) Repeatability RSD < 2
b) Intermediate precision RSD < 2
3. Specificity / Selectivity No interference
4. Detection limit S/N > 2 or 3
5. Quantitation limit S/N > 10
6. Linearity r2 > 0.999
7. Range 80 - 120%
8. Stability >24hr or < 8hr
 Literature Review

2. LITERATURE REVIEW

30 C.L.Baid Metha College of Pharmacy

 Aim and Objective of Work

3. AIM AND OBJECTIVE OF WORK

31 C.L.Baid Metha College of Pharmacy

 Plan Of Work

4. PLAN OF WORK

To develop and validate an effective RP – HPLC method for the estimation

of DMM & DTT in bulk and its pharmaceutical dosage forms.

So ,the plan of work for the designed study was as follows:

 Gathering physical chemical properties of drug

 From the UV- analysis ,selection of λ max

 Selection of chromatographic condition

 Selection of stationary phase

 Selection of mobile phase

 Selection of flow rate

 Selection of Initial separation condition

 Optimization of chromatographic condition

 Validation of proposed method

 Applying developed method to the marketed formulation.

 Summarize methodology, finalize documentation.

32 C.L.Baid Metha College of Pharmacy

 5. DRUG PROFILE

DMM

Structure :

Chemical name :

Description :
Solubility :

Molecular formula :
Molecular mass :
pKa :
BCS Classification :
Melting point :
Solubility :
Pregnancy Category :

Elimination Half life :

M.O.A:

Adverse reaction

Dose :

Pharmacokinetics

Pharmacodynamics

33 C.L.Baid Metha College of Pharmacy

6. MATERIALS AND

INSTRUMENTS Instruments used:

 System : HPLC(Shimadzu)

 Pump : I80 ( LC – 10 AT Vp series)

 Detector : UV/Visible E2469

 Column : Intertsil ODS-3 C18 (300mm x 3.9 mm, 5 µ

i.d.)

 Semi-Micro balance : LC/GC

 Analytical balance : Sartorious

 Hot air oven : Technico

 Mechanical shaker : Remi RS-24 Plus

 Sonicator : PCI Analytics

Reagents and Chemicals

 Acetonitrile : HPLC grade

 Water : HPLCgrade

 Hydrogen peroxide : AR

 Sodium hydroxide : AR grade

 Hydrochloric acid : EMPARTA

34 C.L.Baid Metha College of Pharmacy

Reference Standards

1. DMM: : Purity 99.25%

2. DTT : Purity 99.48%

3. Propyl paraben : Purity 100%

Brand Used : Megace

Label claim :

35 C.L.Baid Metha College of Pharmacy

Preparation of buffer :

Preparation of mobile phase :

Standard Preparation:

DMM (10µg/ml) & DTT (5 µg/ml)

50 mg DMM ------->. 100 ml -------> 2ml -------> 100ml

50 mg DTT -------->. 100 ml -------> 1ml -------> 100ml

Sample Preparation: label claim of DMM 1mg and DTT 0.5 mg

1g sample made up to 100 ml sonicated for 15 min and then volume make up with
mobile phase -----> The final concentration of DMM(10 µg/ml) & DTT (5 µg/ml)

METHOD DEVELOPMENT AND OPTIMIZATION OF

CHROMATOGRAPHIC CONDITIONS

SELECTION OF CHROMATOGRAPHIC CONDITION

Proper selection of the method depends upon the nature of the sample (ionic /
ionisable / neutral molecule), its molecular weight and solubility. The drugs selected
in the present study are polar in nature and hence reversed phase or ion-pair or ion
exchange chromatography method may be used. The reversed phase HPLC was
selected for the separation because of its simplicity and suitability.

36 C.L.Baid Metha College of Pharmacy

SELECTION OF WAVELENGTH ( max)

10 mg of DMM and DTT was dissolved in mobile phase. The solution was
scanned from 200-400 nm the spectrum was obtained. The overlay spectrum was
used for selection of wavelength for DMM and DTT. The isobestic point was taken
as detection wavelength. In the entire UV visible region both the drugs were
strongly absorbed at 254 nm.So this wavelength was selected for further studies.

7.1. METHOD DEVELOPMENT TRIALS

Trial –1, 2,3,4,5,6.

37 C.L.Baid Metha College of Pharmacy

6.4 Results Optimized Condition

Chromatographic conditions
 System : HPLC(Shimadzu)

 Column : Intertsil ODS-3 C18 (300mm x 3.9 mm, 5 µ i.d.)

 Flow rate : 1.2 ml/ min

 UV Detection : 254 nm
 Injection Volume : 20 µl
 Run Time : 15 min

 Mobile phase :
 PH :
 Diluents : Mobile phase

38 C.L.Baid Metha College of Pharmacy

VALIDATION

Method validation can be defined as per ICH

Establishing documented evidence, which provides a high degree of

assurance that a specific activity will consistently produce a desired result or product
meeting its predetermined specifications and quality characteristics.

The developed method was validated according to ICH guidelines.

• System suitability

• Specificity

• Linearity

• Range

• Accuracy

• Precision

• LOD, LOQ

• Robustness and ruggedness.

39 C.L.Baid Metha College of Pharmacy

SYSTEM SUITABILITY

System suitability was performed by five replicate injection of standard solution

with the concentration of 10µg/mL of DMM and 5µg/mL of DTT was injected. The
parameters like retention time, theoretical plate and peak area were shown in the
table

Figure 1. System suitability chromatogram of DMM and DTT

Injection DMM DTT

ID USP Plate RT(min) Area USP Plate RT(min) Area

Count Count

1 30563 2.61 2322845 45714 9.64 973418

2 30128 2.65 2303613 45072 9.62 961548

3 31265 2.63 2329012 45639 9.63 972640

4 30987 2.60 2312915 44986 9.62 968365

5 30302 2.66 2298810 45428 9.67 980139

Average 2.63 2313439 9.64 971222

SD 0.03 12667.12 0.02 6587.38

40 C.L.Baid Metha College of Pharmacy

%RSD 0.97 0.55 0.22 0.71

SPECIFICITY

Specificity is the ability to check unequivocally the analyte in the presence of

components which may expect to be present. Typically these might include
impurities, degradant and matrix. There was no interference from excipient and
other component with the drug peak. So, the developed method has been found
to be specific.

Figure 2. chromatogram of Blank

Figure 3. chromatogram of Placebo

41 C.L.Baid Metha College of Pharmacy

 Figure 4. chromatogram of Std

Quantitative estimation

S.no Sample Retention Peak area Amount present

name time

DMM DTT DMM DTT DMM DTT

1 Standard 2.61 9.64 2313439 971222 101.21% 100.58%

(1.01 mg) (0.50 mg)
2 Sample 1 2.65 9.65 2342581 992643 100.73 % 100.18%
(1.01mg) (0.50 mg)
3 Sample 2 2.63 9.63 2331537 988672 100.97% 100.38%
(1.01 mg) (0.50 mg)

ACCEPTANCE CRITERIA:
Assay value should be in the range of 90% to 110%
Result
Test result is showing that the test method is precise. The percentage assay of
DMM and DTT found to be 100.97% and 100.38%.Results are within the limits

42 C.L.Baid Metha College of Pharmacy

Accuracy

The closeness of agreement between the true values which is accepted either
conventional new value or an accepted reference value and the value found.
Based on the comparison to reference standard method.
Procedure:

The accuracy was calculated the present method to the analysis of tablet and
standard at low, medium, and high concentration level. The accuracy was estimate
from three replicate injection and calculated as the µg/mL drug recovered from the
drug matrix.

Acceptance criteria:

The percentage Recovery for each level should be between 98.00 to 102.00%

Figure 5. chromatogram of Low level

43 C.L.Baid Metha College of Pharmacy

 Figure 6. chromatogram of Middle level

Figure 7. chromatogram of High level

Accuracy

Accuracy table of DMM

% Amount Spiked Amount % Mean %

Level (mg) recovered (mg) Recovery Recovery

50 % 1 1.0145 100.72 100.35 %

1 1.0207 101.34

1 0.9971 99.00

100% 2 2.0315 100.85 100.36%

2 2.0088 99.72

44 C.L.Baid Metha College of Pharmacy

 2 2.0245 100.50

150% 3 3.0301 100.28 100.07%

3 3.0156 99.80

3 3.0255 100.13

Accuracy table of DTT

% Amount Spiked Amount recovered % Mean %

Level (µg/ml) (µg/ml) Recovery Recovery

50 % 0.5 0.5024 100.48 100.56%

0.5 0.5070 101.40

0.5 0.4990 99.80

100% 1.0 1.0041 100.41 100.45%

1.0 1.0164 101.64

1.0 0.9931 99.31

150% 1.5 1.5133 100.89 100.51%

1.5 1.5233 101.55

1.5 1.4862 99.08

45 C.L.Baid Metha College of Pharmacy

PRECISION
Precision of an analytical method is the degree of agreement among individual test
results when the procedure is applied repeatedly to multiple sampling of a
homogenous sample. Precision of analytical method is usually expressed as the
standard deviation and relative standard deviation.

Determination:
The precision of the analytical method was determined by assaying sufficient
number of samples and relative standard deviation was calculated.
The precision of the instrument was determined by assaying the samples
consecutively, number of time and relative standard deviation was calculated.
Inject 20μl of the blank solution and the standard solution of for five times and
calculate the %RSD for the area of six replicate injections.

Acceptance Criteria:
The % RSD for the area of six injections results should not be more than 2%.

DMM

S.NO Parameter Mean peak area %RSD Mean RT %RSD

1 System precision 2373733 0.380 2.61 0.69

2 Method precision 2380506 0.728 2.63 0.71

3 Intermediate precision 2318839 0.469 2.64 0.26

46 C.L.Baid Metha College of Pharmacy

DTT

S.NO Parameter Mean peak area %RSD Mean RT %RSD

1 System precision 970997 0.230 9.64 0.58

2 Method precision 978478 1.431 9.61 0.64

3 Intermediate precision 968137 0.812 9.65 0.41

7.2.6 LINEARITY AND RANGE

LINEARITY: Linearity is the ability of the method to obtain test results that are
directly
Proportional to the analyte concentration within a given range.

RANGE: Range of analytical procedure is the interval between the upper and lower
concentration of analyte in the sample (including concentrations) for which it has
been
Demonstrated that the analytical procedure has a suitable level of precision,
accuracy, and linearity.
Preparation of Standard Solution:
The linearity of the method was performed by preparing the concentration range of
40-120µg/mL and 20-60 µg/mL for DMM and DTT, from standard stock solution.
Calibration curves were constructed by plotting concentration versus area of DMM
and DTT.

47 C.L.Baid Metha College of Pharmacy

DMM

DTT

48 C.L.Baid Metha College of Pharmacy

 Linearity Results:

PARAMETER DMM DTT

Concentration 40-120 20-60

Correlation 0.999 0.999

Slope 29061 805.4

Intercept 2855 107

No. of data points 5 5

49 C.L.Baid Metha College of Pharmacy

ROBUSTNESS

The robustness of a method was analysed by changing experimental,

chromatographic condition. Altering in flow rate (1.2±1 mL/min), changes in
column oven temperature (40±5 °C), Changes mobile phase buffer pH (3.5±0.2),
changes in mobile phase composition and changes in wavelength allowable limits
from actual chromatographic condition. It was noted that there is no recognizable
change in mean RT and RSD and parameters fall within the limit of ≤ 2. The
theoretical plate, tailing factor, resolution is found to be good of DMM and DTT.
This method is robust with variability condition. The analytical condition result is
shown in table

DMM

Proposed Mean %RSD Mean %RSD

peak
variations RT
area

Normal 2373733 0.380 2.61 0.69

Variation in flow rate 1.1 ml 2350211 0.776 2.64 0.42

±0.1(optimized
1.2ml/min) 1.3 ml 2371026 0.534 2.65 0.71

Variation in 252 2375364 0.550 2.63 0.63

50 C.L.Baid Metha College of Pharmacy

wavelength 256 2369712 0.670 2.61 0.57
±2(optimized 254)

DTT

Proposed Mean %RSD Mean %RSD

peak
variations RT
area

For Optimized Condition 970997 0.230 9.64 0.66

Variation in flow rate 1.1 980956 1.102 9.65 0.64

±0.1(optimized 1.2ml/min) ml

1.3 984194 0.789 9.64 0.38

ml

Variation in wavelength 252 976556 0.814 9.64 0.41

±2(optimized 254)

256 974081 0.774 9.63 0.53

51 C.L.Baid Metha College of Pharmacy

SOLUTION STABILITY

Evaluate the stability of analytical solution by injecting the standard

and sample solution at 48 hours. The results are summarized in the table (13:14) for
standard and sample solutions.

Table No. 13 Solution stability for standard

Time in hours % Assay % Difference

Initial 100.2 -

48 Hours 100.0 0.2

Table No. 13 Solution stabiity for sample

Time in hours % of Assay % Diff for % of Assay % Diff for

For 1mg sample For 0.5mg sample

[DMM] [DTT]

Initial 99.5 - 100.2 -

52 C.L.Baid Metha College of Pharmacy

48 Hours 99.2 0.3 99.8 0.4

Acceptance Criteria

The % difference for % assay of standard and sample preparation between initial
and different time intervals should be within ±2.0

SUMMARY

An innovative RP-HPLC method was developed for the simultaneous estimation of

DMM and DTT in semisolid dosage forms and validated according to the ICH
guidelines.

OPTIMIZED CHROMATOGRAPHIC CONDITION

 System : HPLC(Shimadzu)

 Column : Intertsil ODS-3 C18 (300mm x 3.9 mm, 5 µ i.d.)

 Flow rate : 1.2 ml/ min

 UV Detection : 254 nm
 Injection Volume : 20 µl
 Run Time : 15 min

 Mobile phase :
 pH :
 Diluents : Mobile phase

QUANTITATIVE ESTIMATION

SAMPLE AMOUNT PRESENT (%) ACCEPTANCE LIMIT

53 C.L.Baid Metha College of Pharmacy

DMM 100.97% 90%-110%

DTT 100.38%

VALIDATION REPORTS OF OPTIMIZED METHOD

S.NO PARAMETERS ACCEPTANCE RESULTS OBTAINED

CRITERIA

1 System Theoretical plate NLT DMM - 30649

suitability 2000
DTT - 45368

2 specificity No interference was No interference was

observed between the observed between the
placebo and blank placebo and blank with the
principal peak

3 Accuracy 90-110 % DMM - 100.26%

DTT - 100.51%

4 Precision % RSD NMT 2 % %RSD NMT 2 %

5 Linearity Correlation co efficient DMM - 0.999

NLT 0.996 DTT - 0.999

54 C.L.Baid Metha College of Pharmacy

6 Solution % RSD NMT 2 % Stable for 48 hours
stability

7 Assay 90-110 % DMM - 100.97%

DTT - 100.38%

8 Robustness %RSD NMT 2 % %RSD NMT 2 %

55 C.L.Baid Metha College of Pharmacy