Stability-indicating High performance liquid

chromatography method development and validation of

Trabectedin in bulk drug and pharmaceutical dosage
forms
Dissertation

Submitted in partial fulfilment of the requirement for the

Award of the degree of

MASTER OF PHARMACY
IN
PHARMACEUTICAL CHEMISTRY
SUBMITTED TO
SAVITRIBAI PHULEPUNE UNIVERSITY

Research Guide
Dr. P.D.Pawar
M. Pharm; Ph.D.
Research Student
Ms. Ravankole Pratiksha Rajkumar
B. Pharmacy

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

Shri Wagheshwar Gramvikas Pratishthan's
LOKNETE SHRI DADAPATIL PHARATE COLLEGE OF PHARMACY
Mandavgan Pharata, Tal-Shirur, Dist-Pune, 412211
2023-2024
 Stability-indicating High performance liquid
chromatography method development and validation of
Trabectedin in bulk drug and pharmaceutical dosage
forms
Dissertation

Submitted in partial fulfilment of the requirement for the

Award of the degree of

MASTER OF PHARMACY
IN
PHARMACEUTICAL CHEMISTRY
SUBMITTED TO
SAVITRIBAI PHULEPUNE UNIVERSITY

Research Co-Guide
Dr.P.D.Pawar
M. Pharm, PhD
Research Student
Ms. Ravankole Pratiksha Rajkumar
B. Pharmacy

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

Shri Wagheshwar Gramvikas Pratishthan's
LOKNETE SHRI DADAPATIL PHARATE COLLEGE OF PHARMA
Mandavgan Pharata, Tal-Shirur, Dist-Pune, 412211
2023-2024
Dr. HemantKamble

Principal,

Loknete Shri Dadapatil Pharate College of Pharmacy,

Mandavgan Pharata, Pune-412211.

CERTIFICATE
This is to certify that the research work embodied in this thesis entitled
“Stability-indicating High performance liquid chromatography

method development and validation of Trabectedin in bulk drug and

pharmaceutical dosage forms” was carried out by Ms. Ravankole

Pratiksha Rajkumar at Loknete Shri Dadapatil Pharate College of

Pharmacy, for partial fulfillment of M. Pharm degree to be awarded by

Savitribai Phule Pune University. The research work has been carried out

under the guidance Dr. P. D. Pawar.

The thesis is now ready for examination.

Date: Dr. HemantKamble

Place: Principal

Seal of College
Dr. P. D. Pawar

Assistant Professor,

Loknete Shri Dadapatil Pharate College of Pharmacy,

Mandavgan Pharata, Pune-412211.

CERTIFICATE
This is to certify that the research work embodied in this thesis entitled
“Stability-indicating High performance liquid chromatography

method development and validation of Trabectedin in bulk drug and

pharmaceutical dosage forms” was carried out by Ms. Ravankole

Pratiksha Rajkumar at Loknete Shri Dadapatil Pharate College of Pharmacy

for partial fulfillment of M. Pharm degree to be awarded by Savitribai Phule

Pune University. The research work has been carried out under my
supervision and to my satisfaction.

The thesis is now ready for examination.

Date: Dr. P. D. Pawar

Place: Research Guide

Seal of College
 ACKNOWLEDGEMENT

Atfirst,I wouldliketothankstoAlmightyGODforbeingkind giving strength to me to carry

on and complete my research
work.ThenIwouldliketothankstoMyParentsforsupporting me a lot by eachmeans.

I would like to express my sincere appreciation, deep sense of gratitude and cordial
thanks to my thesis Supervisor, Prof.Santosh A. Waghmare&
Prof.S.R.GhodakeAssistant Professor, Department of Pharmacy, for his valuable
instructions, continuous guidance, Encouraging, constructive criticisms and thoughtful
advice related to the experiments and theoretical
studiesduringpursuingthisresearchandpreparationofthis report.

Thestudiesandfindingspresentedinthisreportaretheresult of a great effort by many

persons at Savitribai Phule Pune University, Pune and a lot of research article during the
period of studies were conducted as well as when the paper and the thesis framework
wereproduced.

Iamalsothankfultowriterofdifferentresearcharticlesand journals for getting valuable

information to prepare my thesis.
Special thanks are extended to Teaching, non-Teaching, my classmatesofPharmaceutical
Chemistry,SavitribaiPhule Pune University for their helping hand, and continuous
support cooperation during this researchwork.

Ms. Ravankole Pratiksha Rajkumar

5
 ABBREVATIONS

UV: Ultra Voilet

nm: Nano meter

cm: Centi meter

i.e: That is
V: Volt

HPLC: High Pressure Liquide Chromatography

Maximum Wavelength
𝝀max:
e.g.: For example

etc: Extra
%: Percentage

Std: Standard

LC: Liquid Chrmatography

US: United State

Fig: Figure

No.: Number
°C: Degree Celsius

MS: Mass Spectroscopy

GC: Gas Chromatography

ICH: International Conference on Harmonization

SD: Standard Deviation

RSD: Relative Standard Deviation

ppm: Parts Per Million

USP: United State Pharmacopoeia

HETP: Height Equivalent to Theoretical Plate

FDA: Food and Drug Affairs

DoE: Design of Experiment

CQA: Critical Quality Attribute

μg/ml: Micro gram per milli litre

mm: Milli meter

v/v: Volume/volume

min: Minute

BP: Boiling Point

ACN: Acetonitrile

HCL: Hydrochloric acid

NaOH: Sodium Hydoxide

Conc: Concentration

API: Active Pharmaceutical Ingredient

HPTLC: High Pressure Thin Layer Chromatography

P: Probability

A.R.: Analytical Reagent

International Union of Pure and Applied

IUPAC:
Chemistry
pKa: Acid Dissociation Constant
QA: Quality Assurance

Rt: Retention Time

QC: Quality Control

WHO: World Health Organization

 INDEX

Sr.
Name of Chapter Page No.
No.
1. Introduction 1-55

2 Drug Profile 56-59

3 Literature Survey 60-65

4 Aim and Objective 66

5 Plan of Work 67-68

CHROMATOGRAPHIC METHOD
7.1 Material and Method
7.2 Result and Discussion
7. 7.2.1 Result of Trials
7.2.2 Optimization
7.2.3 Validation
7.2.3 Conclusion
8. Summary

9. References
APPENDIX
I. Presentation and Publications
II. Errata
 LIST OF TABLES

Table No. Name of Tables Page No.

1.1 Table showing list of instrumental methods 2

Combined parameters for operational and

1.2 48
performance qualification

6.1 Active Pharmaceutical Drug 69

6.2 List of Chemicals use in Research work 69

6.3 List of instruments 70

Spectroscopic Conditions Suggested by Software for

6.4 72
each Solvent
 LIST OF FIGURES

Fig. No. Name of Figures Page No.

1.1 UV Spectrophotometer 3

1.2 Components of a UV Spectrophotometer 8

Overlain spectra of two drugs X and Y and its

1.3 11
mixture
Zero (a), First (b), Second(c), Third (d) and
1.4 Fourth (e) order derivative spectra of 1.4
gaussian peak.

1.5 Representation of Tswett's LC analysis 16

1.6 HPLC Components 17

1.7 Resolution Chromatogram 33

INTRODUCTION

1. INTRODUCTION
 CHAPTER I INTRODUCATION

1.1 ANALYTICAL CHEMISTRY:

The science and art of determining the composition, purity, safety and quality of materials by
application of analytical procedures is called as Analytical chemistry. It is the art of
recognizing different substances & determining their constituents and it involves separating,
identifying and determining the relative amounts of components in sample of matter.
Analytical chemistry mainly consists of two branches as Qualitative and Quantitative
analysis.
 Qualitative analysis reveals the chemical identity of the components in the sample.
 Quantitative analysis provides numerical information as to the relative amounts of
these components or analytes.
Most manufacturing industries rely upon Qualitative and Quantitative analysis to ensure that
the raw material used meets certain specification and also to check the quality of the final
product. The results of some analysis are qualitative and yield useful clues from which the
molecular or atomic species, the structural features or the functional group in the sample can
be detected. Other analysis is quantitative in which the results are expressed in form of
numerical data. In both types of analysis the required information is obtain by measuring
physical and chemical properties that are characteristically related to the components of
interest.

1.1.1 Need of Development of Analytical Methods:

Newer analytical methods are developed for the drugs or the drug combination because of the
following reasons:
 The drug or the drug combination may not be official in any pharmacopoeia.
 A literature search may not reveal an analytical procedure for the drug or its
combination.
 Analytical methods may not be available for the drug combination form of the
formulation due to interference caused by the excipients.
 Analytical methods for the quantification of the drug or the drug combination from
biological fluids may not be available.
 Method may be available for simultaneous estimation but it may be tedious, time
consuming & expensive.
 There may be a need for an alternative method to confirm for legal or scientific
reasons, analytical data originally obtained by existing methods.
 CHAPTER I INTRODUCATION

The newly developed methods find their importance in various fields such as:
 Research institutions
 Quality control departments
 Approved testing laboratories
 Biopharmaceutics & bioequivalence studies
The combination dosage forms are complex in nature because of different additives and
individual drug components present in it, in the process of estimation it is important to
confirm that one component does not interfere with the estimation of the other. Hence the
analytical approach should be different.

1.2 Techniques used in Instrumental Methods:

There are many techniques available for the analysis of materials; however, they all are based
on the materials interaction with energy. This interaction permits the creation of a signal that
is subsequently detected and processed for its information content. Chemical instrumentation
includes the following principle types:

1.2.1 Spectroscopic techniques:

Spectroscopy measures the interaction of the material with electromagnetic radiation.
Different types are ultraviolet and visible spectrophotometry, fluorescence and
phosphorescence spectrophotometry, atomic spectrometry (emission and absorption), infrared
spectrophotometry, raman spectroscopy, X-ray spectroscopy, nuclear magnetic resonance
spectroscopy and electron spin resonance spectroscopy.

1.2.2 Electrochemical techniques:

Electrochemistry measures the interaction of the material with an electric field. Different
types are potentiometry, voltametry, amperometric technique, coulometry electrogravimetry
and conductometric techniques.

1.2.3 Chromatographic techniques:

Separation processes are used to decrease the complexity of material mixtures. The most
utilized separation method is chromatography. Mainly following type of techniques are used
High –performance liquid chromatographic techniques (HPLC), High –performance thin
layer chromatography (HPTLC) and Gas chromatography (GC).

1.2.4Mass Analysis:
 CHAPTER I INTRODUCATION

Mass spectrometry measures the interaction of charged materials and electric and magnetic
fields.

1.2.5Thermal Analysis:
Calorimetric and thermo gravimetric analysis measure the interaction of material and heat. In
order to utilize the techniques available currently, complex material mixtures must be
separated into simpler samples for individual analysis.

1.2.6 Hyphenated Techniques:

Combinations of the above techniques are called as "hyphenated" techniques. Several
examples are in popular use today and new hybrid techniques are under development. Few
examples of these techniques are: GC-MS (Gas chromatography – mass spectrometry), GC-
IR (Gas chromatography-infrared spectroscopy), MS-MS (Mass spectrometry-mass
spectrometry) and LC – MS (Liquid chromatography-mass spectroscopy). When a sample is
available for analysis, then attention must be given on most suitable and effective technique
to be employed from many of the available methods for the required determination and this
main decision is taken by the analyst.

1.3 INTRODUCTION OF CHROMATOGRAPHY:12-14

Chromatography is the technique by which solutes of two or more component are separated
by dynamic differential migration process, in a system consist of two phases, one of which
moves continuously in a given direction and in which the individual component exhibits
different mobilites due to difference in adsorption or partition, molecular size etc. The word
chromatography is derived from Greek letters chromos meaning color and graphy means
color writing. The purpose of preparative chromatography is to separate the components of a
mixture. Analytical chromatography is done normally with smaller amounts of material and
is for measuring the relative proportions of analytes in a mixture.

1.3.1 Introduction of High Performance Liquid Chromatography (HPLC):

HPLC is a popular method of analysis because it is easy to learn and use and is not limited by
the volatility or stability of the sample compound. It is used for speedy resolution of complex
mixtures, separation and determination of species in a variety of organic, inorganic and
biological materials. The time of retention is important and is depend upon the nature of the
components. The interaction of the solute with mobile and stationary phase can be
manipulated through different choices of both solvents and stationary phases.
 CHAPTER I INTRODUCATION

Instrumentation of HPLC:

Figure 1.1: Basic Instrumentation of HPLC

The basic parts of HPLC: -

1) Mobile Phase Supply System:

The solvent reservoirs can be filled with a range of solvents of different polarities, provided
they are miscible or they can be filled with solutions of different pH and are mixed in buffer
volume. The solvents must be pure and be degassed to avoid formation of gas bubbles.
Typical flow rates are 1 to 2 ml/min for conventional 4.6mm i.d. columns. Solvents used for
HPLC must be of “HPLC” grade, that is, the solvents that have been filtered through 0.2 µm
filters. This extends the pump life by preventing scoring and reduces contamination and
plugging of the column. Because the particles that are used to pack HPLC columns are small
enough (<50µm) to prevent solvent flow by gravity, pumps that develop pressure up to 5000
psi are needed to force the mobile phase through the column. Two types are available12:
Mechanical pumps and Pneumatic pumpsof the mechanical pumps, the most frequently used
is the reciprocating pump. Pneumatic pumps are either gas displacement type, which uses
direct pressure from highly compressed gas to force the solvent out of the tube, or the
pneumatic amplifier type in which the compressed gas at a lower pressure impinges on the
large end of the piston to force the smaller end to deliver the liquid. The pneumatic pumps
have the advantage of pulse less operation. The most commonly used pump for the HPLC
system reciprocating pump (Fig No. 6)
 CHAPTER I INTRODUCATION

Figure 1.2: Reciprocating Pump

2) Sample Injection System:

Samples can be introduced manually in to the valve with the syringe to fill the sample loop.

Figure 1.3: Manual Sample Injection System

3) Column:
Straight lengths of stainless steel tubing make excellent columns. These come invarious
diameter and lengths depending upon the particular application. Usually the internal diameter
is 3.9 or 4.6mm. A well packed 4.6 mm column of 5 µm diameter (d p) should give a plate
count on the order of 60,000 to 90,000 plates/meter at flow rates of 1 ml/min.

A small 3 to 10cm guard column or precolumn is placed between the injector and the
analytical column.

4) Detectors:
The function of the detector in HPLC is to monitor the mobile phase as it merges from the
column. Detectors are usually of two types:

1. Bulk property detectors:

 CHAPTER I INTRODUCATION

It compares overall changes in a physical property of the mobile phase with and without an
eluting solute. e.g. refractive index, dielectric constant or density.

2. Solute property detectors:

It responds to a physical property of the solute which is not exhibited by the pure mobile
phase. e.g. UV absorbance, fluorescence or diffusion current. Such detectors are about 1000
times more sensitive giving a detectable signal for a few nanograms of sample.

Modes of HPLC:
1) Normal phase chromatography: In normal phase mode, the nature of stationary phase
is polar and the mobile phase is non-polar. In this technique, non-polar compounds travel
faster and are eluted first because of the lower affinity between the non-polar compounds
and stationary phase. Polar compounds are retained for longer time and take more time to
elute because if their higher affinity with the stationary phase. Normal phase mode of
separation is, therefore, not generally used pharmaceutical applications because most of the
drug molecules are polar in nature and hence take longer time toelute.

2) Reversed phase chromatography: Reversed phase mode is the most popular mode for
analytical and preparative separations of compounds of interest inchemical,biological,
pharmaceutical, food and biomedical sciences. In this mode, the stationary phase is non-
polar hydrophobic packing with octyl and octadecyl functional group bonded to silica gel
and the mobile phase is a polar solvent, often a partially or fully aqueous mobile phase.
Polar substances prefer the mobile phase and elute first. As the hydrophobic character of
the solutes increases, retention increases. Generally, the lower the polarity of the mobile
phase, the higher is its eluent strength. The elution order of the classes of compounds is
reversed (thus the name reverse-phase chromatography).

1.4 Method Development on HPLC

Method development and optimization in liquid chromatography is still an attractive field
of research for theoreticians (researchers) and attracts also a lot of interest from practical
analysts. Among all, the liquid chromatographic methods, the reversed phase systems based
on modified silica offers the highest probability of successful results. However, a large
number of (system) variables (parameters) affect the selectivity and the resolution.
Alternate analytical methods are developed for the drug product to reduce the cost and time.
When alternative analytical methods are indented to replace the existing procedure, analyst
should collect the literature for all types of information related to Analyte and define the
 CHAPTER I INTRODUCATION

separation goal. Then estimate the best separation condition from trial runs. After
optimizing the separation condition, validate the method for release to routine laboratory.

Getting Started on Method Development

“Best column, best mobile phase, best detection wavelength, efforts in separation can make a
world ofdifference while developing HPLC method for routine analysis
Determining the ideal combination of these factors assures faster delivery of desired results
was validated method of separation.”

1.4.1 The Mobile Phase: In reverse-phase chromatography, the mobile phase is more polar
than the stationary phase. Mobile phase in these systems is usually mixtures of two or more
individual solvents with or without additives or organic solvent modifiers. The usual
approach is to choose what appears to be the most appropriate column, and then to design a
mobile phase that will optimize the retention and selectivity of the system. Separations in
these systems are considered to be due to different degrees of hydrophobicity of the solutes.
The simple alteration of composition of the mobile phase or of the flow rate allows the rate
of the elution of the solutes to be adjusted to an optimum value and permits the separation
of a wide range of the chemical types. First isocratic run followed by gradient run
ispreferred.

1.4.2 The Detector: The next consideration should be the choice of detector. UV-visible
detectors are the most popular as they can detect a broad range of compounds and have a
fair degree of selectivity for someanalyses.

1.4.3 The Column Length: Many chromatographers make the mistake of simply using
what is available. Often this is a 250 cm × 4.6 mm C18 column. These columns are ableto
resolve a wide variety of compounds. While many reverse phase separations can be carried
out on such column. Method development can be streamlined by starting with shorter
columns; 150, 100 or even 50 cmlong.

1.4.4 The Stationary Phase: Selecting an appropriate stationary phase can also help to
improve the efficiency of method development. For example, a C8 phase (reversed phase)
can provide a further time saving over a C18, as it does not retain analytes as strongly as the
C18 phase. For normal phase applications, cyano (nitrile) phases are mostversatile.

1.4.5 The Internal Diameter: By selecting a shorter column with an appropriate phase, run
 CHAPTER I INTRODUCATION

times can be minimized so that an elution order and an optimum mobile phase can be
quicklydetermined.

1.4.6 Gradient Programming: The fastest and easiest way to develop a method is to use a
mobile phase gradient. Always start with a weak solvent strength and move to a higher
solvent strength. To begin, use a very fast gradient (e.g.10 minutes) and then
modifythestartingand finishing mobilephasestoachieveasuitableseparation.

1.4.7 Retention: Analytes may be too strongly retained (producing long run times). If this
occurs, the solvent strength should be increased. In reverse phase analysis this means a
higher % of organic solvent in the mobile phase.

1.4.8 Poor Separation: Analytes often co-elute with each other or impurities. To overcome
this, the analysis should be run at both higher and lower solvent strengths so the best
separation conditions may bedetermined.

1.4.9 Peak Shape: This is often a problem, especially for basic compounds analyzed by
reversed phase HPLC. To minimize any potential problems always use a high purity silica
phase such as Wakosil II. These modern phases are very highly deactivated so secondary
interactions with the support are minimal. To maximize the reproducibility of a method, it is
best to use a column heater to control the temperature of the separation. A temperature of 35
– 40°C isrecommended.

1.4.10 Buffer selection: In reverse phase HPLC, the retention of analytes is related to their
hydrophobicity. The more hydrophobic the analyte, the longer it is retained. When an
analyte is ionized, it becomes less hydrophobic and, therefore, its retention decreases. When
separating mixtures containing acid and/or bases by reversed phase HPLC, it is necessary to
control the pH of mobile phase using appropriate buffer in order to achieve reproducible
results. Buffers play an additional role in the reproducibility of a separation. The buffer
salts reduce peak tailing for basic compounds by effectively masking silanols.
They also reduce potential ion-exchange interactions with unprotonated silanols (Figure 8).
 CHAPTER I INTRODUCATION

Figure 1.4: Peak Tailing Interaction.

1.4.11 Selection of pH: The pH range most often used for reversed-phase HPLC is 1 - 8
and can be divided into low pH (1 - 4) and intermediate pH (4 - 8) ranges. Each range has a
number of advantages. Low pH has the advantage of creating an environment in which peak
tailing is minimized and method ruggedness is, maximized. For this reason, operating at
low pH isrecommended.
At a mobile phase pH greater than 7, dissolution of silica can severely shorten the lifetime
of columns packed with silica-based stationary phases.
The pKa value (acid dissociation [ionization] constant) for a compound is the pH at which
equal concentrations of the acidic and basic forms of the molecule are present in aqueous
solutions. Analytes may sometimes appear as broad or tailing peaks when the mobile phase
pH is at, or near, their pKa values. A more rugged mobile phase pH will be at least 1 pH
unit different from the analyte pKa. This shifts the equilibrium so that 99% of the sample
will be in one form. The result is consistent chromatography.

1.4.12 Optimization of chromatography conditions:

An optimized chromatogram is the one in which all the peaks are symmetrical and are well
separated in less run time. The peak resolution can be increased by using a more efficient
column with higher theoretical plate number.

1.4.13 The following are the system suitability parameters:

I) Retention:
The retention of a drug with a given packing material and eluent can be expressed as
retention time or retention volume, but both of this are dependent on flow rate, column,
length and column diameter. The retention is best described as column capacity ratio (k’),
which is independent of these factors. (KA) is defined as,

KA = VA-V0 /V0 = t A – t 0 /t0

Where, VA = Elution time of A and V0 = Elution volume of non retained compound. (Void
volume)

At a constant flow rate, retention times t A – t0 can be used instead of retention volumes.
Retention data is sometimes expressed, relative to a known internal standard (B). The ratio of
 CHAPTER I INTRODUCATION

retention times tA/ tB can be used, but the ratios of adjusted retention times (t A- t0 / tB-t0) is
better when data need to be transferred between different chromatographs.

II) Resolution:

Resolution is a measure of the extent of separation of two components and the baseline
separation achieved. Resolution is equal to the distance between the peaks centers divided by
the average band width. The ideal value of R s for baseline separation is 1.5. It is calculated by
using the formula,

Rs = 2 (t2 –t1) / W1 +W2

Where, t1 and t2 are the retention times of the first and second adjacent bandwidth.

I) Capacity factor (K’):

Capacity factor (K’) is a measure of the efficiency of the separation process occurring at the
phase boundary. It is defined as the ratio of the number of molecules of solute

K’ = V1 –V0/ V0

in the stationary phase to the number of molecules of the same in the mobile phase. The ideal
value of K’ ranges from 2-10. Capacity factor can be determined by testing the formula,

Where, V1 = retention volume at the apex of the system and V0 = Void volume of the system.

When conditions are changed so that K’ is made larger, resolution usually improves.
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.

IV) Selectivity (α):

The selectivity (α) is a measure of relative retention of two components in a mixture. The
ideal value of selectivity is 2. It calculated by using the formula,

α = V2 –V0 / V1 -V0

Where, V2 &V1 = retention volume at the apex of the system and V 0 = Void volume of the
column.
 CHAPTER I INTRODUCATION

V) Column Efficiency: 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.
Efficiency is calculated by using the formula,

N = 16 (TR) 2 / W

VI) Peak asymmetry:

The asymmetry is a tool for quickly determining how much if any, of an eluting peak profile
deviates in shape from a normal distribution.

Ax =b/a The
equation for determining peak asymmetry is, Where, b = the distance between the
perpendicular connecting the baseline to peak maximum and the latest eluting portion of the
curve and a = the distance between the perpendicular connecting the baseline to peak
maximum and the earliest eluting portion of the curve.

1.5 INTRODUCTION TO METHOD VALIDATION13-14

“Doing thorough method validation can be tedious, but the consequences of not doing
it right are wasted time, money, and resources.”

Validation is a process of 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. Method validation is
the process of demonstrating that analytical procedures are suitable for their intended use
and that they support the identity, quality, purity, and potency of the drug substances and
drug products. The real goal of validation process is to challenge the method and

determine limits of allowed variability for the conditions needed to run the method. [10]
1.5.1 Type of analytical procedures to be validated: Validation of analytical procedures
is directed to the four most common types of analyticalprocedures.

 Identificationtest.
 Quantitative test for impuritiescontent.
 Limit test for the control ofimpurities.
 Quantitative test of the active moiety in samples of drug substance on drug product
 CHAPTER I INTRODUCATION

on other selected components in the drugproduct.

In our method of validation, we are following last type.
Assay procedures are intended to measure the analyst present in given sample, assay
represent a quantitative measurement of the major component(s) in the drug sample.
Two steps are required to evaluate an analytical method.
1) First determine the classification of themethod.
2) The second step is to consider the characteristics of theanalytical method.
For analytical method validation of pharmaceuticals, guidelines from the International
Conference on Harmonization (ICH), United States Food and Drug Administration (US
FDA), American Association of Official Analytical Chemists (AOAC), United States
Pharmacopoeia (USP) and International Union of Pure and Applied Chemists (IUPAC)
provide a framework for performing such validations in efficient and productive manner.

1.5.2 Validation of analytical methods as per ICH and USP guidelines:

Method validation is the process used to confirm that the analytical procedure employed for a
specific test is suitable for its intended use. Results from method validation can be used to
judge the quality, reliability and consistency of analytical results; it is an integral part of any
good analytical practice. Analytical methods need to be validated or revalidated:-
 before their introduction into routine use;
 whenever the conditions change for which the method has been validated (e.g., an
instrument with different characteristics or samples with a different matrix);
 Whenever the method is changed and the change is outside the original scope of the
method.
Once the method has been developed and validated, a validation report should be prepared
that includes the following:

 Objective and scope of the method (applicability, type).

 Summary of methodology.
 Type of compounds and matrix.
 All chemicals, reagents, reference standards, QC samples with purity, grade, their
source or detailed instructions on their preparation.
 Procedures for quality checks of standards and chemicals used.
 A plan and procedure for method implementation from the method development lab
to routine analysis.
 Method parameters.
 CHAPTER I INTRODUCATION

 Safety precautions.
 Critical parameters taken from robustness testing.
 Listing of equipment and its functional and performance requirements, e.g., cell
dimensions baseline noise and column temperature range. For complex equipment, a
picture or schematic diagram may be useful.
 Detailed conditions on how the experiments were conducted, including sample
preparation. The report must be detailed enough to ensure that it can be reproduced by
a competent technician with comparable equipment.
 Statistical procedures and representative calculations.
 Procedures for QC in routine analyses, e.g. system suitability tests.
 Representative plots, e.g., chromatograms, spectra and calibration curves.
 Method acceptance limits performance data. The expected uncertainty of
measurement results.
 Criteria for revalidation.
 The person(s) who developed and validated the method.
 References (if any).
 Summary and conclusions.

1.5.3 Significance of the method validation:

The quality of analytical data is a key factor in the success of a drug development
programme. The process of method development and validation has a direct impact on the
quality of these data.
 To trust the method
 Regulatory requirement

A direct consequence and most significant outcome from any method validation exercise is
“the development of meaningful specifications can be predicted upon the use of validated
analytical procedures that can assess changes in a drug substance or drug product during its
lifetime.”

Reasons for method 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 regulation requires
assay validation.
 CHAPTER I
INTRODUCATION

1.5.4 Important terms and properties to be considered for measurements:

a) Accuracy:
It is the measure of how close the experimental value is to the true value. It is measured as the
% of analyte recovered by assay or by spiking samples in a blind study. Accuracy should be
established across the specified range (that is, line of working range) of the analytical
procedures.

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.
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 of 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 difference between the mean and
the accepted true value together with the confidence intervals.

b) Precision:
The precision of the analytical method is related to the degree of the agreement among
individual test results when the procedure or method is applied repeatedly to multiple
sampling of homogenous sample. It is expressed as standard deviation or as coefficient of
variation or relative standard deviation and it is possible to calculate statistically valid
estimates of standard deviations or relative standard deviation(S).

Where, X = individual value, X = arithmetic mean, n = number of samples

Relative standard deviation (R.S.D.) = S/X x 100

Precision may be considered at three levels according to ICH:

i) Repeatability
 CHAPTER I INTRODUCATION

The result of the methods operating over a short time interval under the same conditions
(within a laboratory over a short period of time using the same analyst with the same
equipment). Repeatability is also termed as intra-assay precision.

Repeatability should be assessed using:

a) A minimum of 9 determinations covering the specified range for the procedure (e.g. 3
concentrations/3 replicate each).
b) A minimum of 6 determinations at 100 % of the test concentration.
ii) Intermediate precision

Intermediate precision expresses within laboratory variation (as on different days, or with
different analyst, or equipment within the same laboratory).

iii) Reproducibility
It expresses the precision between laboratories and is often determined in collaborative
studies or method transfer experiments.

c) Linearity:
The linearity of an analytical procedure is its ability to obtain test results that are directly
proportional to the concentration of the analyte in the sample within a given range. Linearity
should be evaluated by visual inspections of a plot of signals as a function of analyte
concentration or content. Data from regression line itself may helpful to provide
mathematical estimates of the degree of linearity. For establishing the linearity, minimum of
5 concentrations are recommended.

d) Range:
The range of an analytical method is the interval between the upper and lower concentration
levels of analyte for which it has been demonstrated that the analytical range has a suitable
level of precision, accuracy and linearity.

e) Specificity:
It is the ability to asses unequivocally the analyte in the presence of the components which
may be expected to be present in the sample matrix. It is obtained by analysis of sample
containing added impurities, degradation products, related chemical compounds or placebo
ingredients when compared to test results from samples without added substance. The
 CHAPTER I
INTRODUCATION

impurity should not interfere in detection. Specificity has been divided into two separate
categories by ICH:

 Identification test
 Assay and impurity tests

f) Limit of Detection (LOD):

The Limit of detection is the lowest concentration of the analyte that can be detected but not
necessarily quantified, under the stated experimental conditions.

Approaches for determining the LOD are:

 Based on visual evaluation
LOD 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:
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
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 Limit of detection may be calculated based on the standard deviation (S. D.) of the
response and slope (S) of the calibration curve. The S. D. of the response can be determined
from the S. D. of the blank, the residual S. D. of regression line or the S. D. of the y- intercept
of the regression line.

The Limit of detection = 3.3 X S.D. / S

g) Limit of Quantitation (LOQ):

It is the lowest concentration of the substance (analyte) in a sample that can be determined
with acceptable precision and accuracy under the stated experimental conditions of the
method. This is a parameter of the quantitative assays for low concentrations of compounds
in sample such as impurities in bulk drugs substances and degradation products in finished
products.

Approaches for determining the LOQ are:

 CHAPTER I INTRODUCATION

 Based on visual evaluation:

LOQ can be 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 ratio:

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.
Signal to noise ratio of 10:1 is generally considered.

 Based on standard deviation of the response and slope:

LOQ may be calculated based on the standards deviation (S. D.) of the response and Slope
(S) of the calibration curve,

The Limit of Quantitation= 10 X S.D. / S

h) Robustness:
It is the measure of its capacity to remain unaffected by small, but deliberate variation in
method parameters and provides indication of its reliability during its normal usage.

i) Ruggedness:
This is reproducibility of the results when the method is performed under actual use
conditions. This includes different analyst, laboratories, column, instruments, sources of
reagents, chemicals, solvents and so on.

j) Stability:
In order to make reliable measurements, the intensity of solution must remain constant for
long-times.

Table 1.1: System Suitability Parameters and their recommended limits.

Sr. No Parameter Recommendation

Capacity Factor The peak should be well-resolved from other

1
(K’) peaks and the void volume, generally K’ > 2
RSD ≤ 1%
2 Repeatability
N ≥ 5 is desirable

3 Relative Retention Not essential as the resolution is stated

CHAPTER I INTRODUCATION

Rs of > 2 between the peak of interest and the

4 Resolution(Rs)
closest eluting potential interferent

5 Tailing Factor(T) T≤2

Theoretical
6 In general should be > 2000
Plates(N)

Table 1.2: Characteristics to be validated in HPLC.

Sr. No Characteristics Acceptance Criteria

1 Accuracy/trueness Recovery 98-102% (individual)

2 Precision RSD < 2%

3 Repeatability RSD < 2%

4 Intermediate Precision RSD < 2%

5 Specificity / Selectivity No interference

6 Detection Limit S/N > 2 or 3

7 Quantitation Limit S/N > 10

8 Linearity Correlation coefficient R2 >

0.999
9 Range 80 –120 %
 CHAPTER I INTRODUCATION

1.6 INTRODUCTION TO QUALITY BY DESIGN (QBD) 15-24

1.6.1 History
QualitybyDesign (QbD) is increasinglybecomingan
importantandwidelyusedterminthepharmaceuticalindustryqualitysystem.QbDcanbeconsidered
tobeaholistic,system-basedapproachto the
designinganddevelopingformulationandmanufacturingprocesseswhichensurespredefinedprod
uctspecifications.

In2002,inordertoestablishamoresystematicandriskbasedapproachtothedevelopmentofpharmac
euticalproducts,usingtheprogressesinscienceandtechnology,FoodandDrugAdministration(FDA

)announcedthe“cGMPforthe21stCentury:ARiskbasedApproach”Initiative.Thisinitiative,focus
edonQbD,andthepublicationoftheProcessAnalyticalTechnology(PAT)Guidancein2004bytheFD
Acontributeddecisivelyforthemodernizationofthepharmaceuticalindustryandchallengedthemto
lookbeyondthetraditionalapproachofQualitybyTesting(QbT).Inadditiontothesenewideas,threei
mportantguidancedocumentswerepublished
aspartofInternationalConferenceonHarmonization(ICH)guidelines:Q8PharmaceuticalDevelop
mentandQ9 Quality
RiskManagement,in2005,andICHQ10PharmaceuticalQualitySystem,in2008.Theseguidancedo
cumentsimplementedtogether,inaholisticmanner,providesaneffectivesystemthatemphasizesahar
monizedscienceandrisk-
basedapproachtoproductdevelopment,assuringanimprovinginQualityinpharmaceuticalindustry.

InICHQ8guidance,theconceptofQbDwasmentioned,statingthat“qualitycannotbetestedintoprod
ucts,i.e.,qualityshouldbebuiltinbydesign”.In2009,theICHQ8guidancewasreviewed,clarifyingk
eyconceptsoftheoriginalguidance.Additionally,theprinciplesofQbDweredescribesandQbDdefi
ningas“asystematicapproachtodevelopmentthatbeginswithpredefinedobjectivesandemphasizes
productandprocessunderstanding and process control,based on sound science and quality risk
management”.

Thisframework represents a moveawayfromthetraditionalapproachinthe

industryofQbDandwasrelatively new tothepharmaceuticalindustryatthebeginningofthetwenty-
firstcentury.However,itcanbefoundtheapplicationofsomeprinciplesofQbDacrosstheindustrylon
gbeforethen,butinanisolatedway.Table6comparesthecurrentstatetothe desiredQbDstate.
 CHAPTER I INTRODUCATION

Infact,QbDisacomprehensiveapproachtargetingallphasesofdrugdiscovery,manufacture,anddeli
very.The aimistoimprovethequality and reduce thecostsofmedicinesfortheconsumer.
Thismaybeaninteractivesystematicapproachandthusthe circular designasshown
inFigure9.Thiscircle ofQbDcan bedividedintotwogeneral
areas,productknowledgeandprocessunderstanding.
Thesetwoareasmeetinthedesignspaceandtheinteractionofproductknowledgeandprocessundersta
ndingallowsforcontinuousimprovement.

Figure 1.5:QualitybyDesignconcept

QbDbeginsbydefiningthedesiredproductperformanceandalsobydefiningaproductthatmeetsthos
eperformancerequirements.Thecharacteristicsofthedesiredproductarethebasisfordesigningthem
anufacturingprocess,whichneedstobemonitoredintermsofperformance.Eachofthesestepsinflue
nceseachother,continuingthecycle.Theinnercircleinteractswithmanyotherspecificmeasuresofph
armaceuticalmanufacturing,suchasspecifications,criticalprocessparameters,ensuringtheproduc
tknowledgeandprocessunderstanding.

TheunderlyingprinciplesofQbDareexplainedinthequalityguidelinesofinternational
conferenceonharmonizationi.e.ICH
Q8PharmaceuticalDevelopment,ICHQ9QualityRiskManagement,andICHQ10Pharmaceutical
QualitySystem.Fig. No.10presentstheguidelines that explainQbD.
 CHAPTER I INTRODUCATION

Quality

byDesig
n
ICHQ9

QualityRiskM
anagement

ICHQ8 ICHQ10
Pharmaceutica Pharmaceutical
lDevelopment QualitySystem

Figure 1.6: ICHQ8/Q9/Q10triangleinQbDparadigm

TheapplicationofQbDpresentsseveraladvantagesandcanbesummarizedas:
 Patientsafetyandproductefficacyarefocused
 Scientificunderstandingofpharmaceuticalprocessandmethodsisdone
 Itinvolvesproductdesignandprocessdevelopment
 Sciencebasedriskassessmentiscarried
 Criticalqualityattributesareidentifiedandtheireffectonfinalqualityofproductisanalyzed
 Itoffersrobustmethodorprocess
 BusinessbenefitsarealsodrivingforcetoadoptQbD.

1.6.2 Quality by Design

Means that product and process performance characteristics are scientifically designed to
meet specific objectives, not merely empirically derived from performance of test batches.

Target measurement

Select Technique

Risk Assessment
 CHAPTER I INTRODUCATION

Method Development /
Validation

Figure 1.7: Flow diagram of QbD approach to method development

The approach provides an in-depth knowledge and enables the creation of a chromatographic
database than can be utilized to provide alternative method if required, in future

 Development of a robustmethod.
 Understand, reduce and control sources of variability.
 Applicable throughout the life cycle of the method.
 Regulatory flexibility Movements within “Analytical Design Space”.

1.6.3 There are two types of QbD?

1) Manually
2) Software

1.6.4 QbD to HPLC Method development

a. Dependent factors
i) Instrument operating parameters
 Column
 Flow rate

ii) Method development Parameters

 Mobile Phase
 Mobile phase Composition
 pH

b. Independent factors
i) Sample preparation variations
 Solvents of stock solution
 Solvents of sub-dilutions

1.6.5 Elements of QualityDesign

ICH guideline Q8 refers all elements of pharmaceutical development included in QbD. In a
marketing authorization application, the Pharmaceutical Development section is projected
to provide a complete understanding of the product and manufacturing process. The aim of
this section is to design a quality product and its manufacturing process to consistently
 CHAPTER I INTRODUCATION

deliver the intended performance of product. The information and knowledge gained from
pharmaceutical development studies and manufacturing experience provide scientific
understanding to support the establishment of the specifications, and
manufacturingcontrols.Duringpharmaceuticaldevelopment,QbD
suggeststhatitshouldincludethefollowingelements:
 Definingthequalitytargetproductprofile(QTPP)
 Identifyingpotentialcriticalqualityattributes(CQAs)
 LinkrawmaterialattributesandprocessparameterstoCQAsandperformriskassessment
 Developingadesignspace
 Designingandimplementingcontrolstrategy
 Continuousimprovement

1) DefiningProductDesignRequirementsandCriticalQualityAttributes
Theproductdesignrequirementsmustbewellunderstoodintheearlydesignphase,andtheycanbefo
undinaQualityTargetProductProfile(QTPP).TheQTPPisderivedfromthedesiredproductinform
ationandithasbeendefinedas“aprospectivesummaryofthequalitycharacteristicsofadrugproductt
hatideallywillbeachievedtoensurethedesiredquality,takingintoaccountsafetyandefficacyofthed
rugproduct”.
Therefore,pharmaceuticalcompaniesconstructatargetproductprofilethatdescribes:
Intendeduseinclinicalsetting,routeofadministration,dosageform,deliverySystems
Dosagestrength(s),Containerclosuresystem
Therapeutic moiety release or delivery and attributes
affecting,Pharmacokineticcharacteristics(e.g.,dissolution,aerodynamicperformance) Drug
product quality criteria likesterility,purity,stability and drug release
asappropriatefordosageformtheintendedformarketing.
TheQTPPguidesscientiststoestablishstrategiesandkeeptheproductdeveloping
effortfocusedandefficient.

Inadditiontodefiningtherequirementstodesigntheproduct,theQTPPwillhelpidentifycriticalqual
ityattributes(CQAs).ICHQ8definesCQAas“aphysical,chemical,biological,ormicrobiologicalp
ropertyorcharacteristicthatshouldbewithinanappropriatelimit,range,ordistributiontoensurethe
desiredproductquality”.CQAsaregenerallylinkedwiththedrugsubstance,excipients,intermediat
es(in-
processmaterials)anddrugproduct.Qualityriskmanagementtools,foundintheICHQ9guideline,
 CHAPTER I INTRODUCATION

areoftenusedtoidentifyandprioritizethepotentialCQAs.RelevantCQAscanbeidentifiedbyadyna
micprocessqualityriskmanagementandexperimentationthatevaluatestheextenttowhichtheirvar
iationcanhaveanimpactontheultimatequalityproduct.Theaccumulatedexperience,theknowledg
e obtained from similarproductsandfrom
literaturereferencesareessentialtomaketheseriskassessments.Takentogether,thisdataprovidesara
tionalethatlinkstheCQAwiththesafetyandefficacyoftheproduct.Theoutcomeoftheriskassessme
ntwouldbealistofCQAsrankedinorderofimportance.ThepotentialCQAscanbemodifiedwhenth
eformulationandmanufacturingprocessesareselectedandasproductknowledgeandprocessunder
standingincrease.

2) Quality Risk Management inQbD

Riskmanagementhasbecomeapriorityprocessinthepharmaceuticalindustrywiththeadvancesinth
eQbD.Asseenbefore,
QbDisbasedonsoundscienceandqualityriskmanagement.Itisasystematicapproachtodevelopmen
tthatbeginswithpredefinedobjectivesandanemphasisonproductprocessunderstandingandproces
scontrol.Inordertoachievethis,ariskmanagementprocesshastobeapriority.
Qualityriskmanagementisasystematicprocessfortheassessment,
control,communicationandreviewofriskstothequalityofthedrugproductacrosstheproductlifecycl
e.ICHQ9discussestheroleofriskmanagementinpharmaceuticalindustry.Forpharmaceuticaldevel
opment,ICHQ9suggeststheapplicationoftheprinciplesandtoolsofqualityriskmanagementto:
Selecttheoptimalproductdesignandprocessdesign.
Enhanceknowledgeofproductperformanceoverawide
rangeofmaterialattributes,processingoptions,andprocessparameters Assess the critical
attributes of rawmaterials, solvents,Active
PharmaceuticalIngredient(API),startingmaterials,APIs,excipients,orpackagingmaterials
toestablish appropriate specifications, identify critical processparameters and
establishmanufacturingcontrols decreasevariabilityofqualityattributes
Assesstheneedforadditionalstudiesrelatingtoscaleupandtechnologytransfer make use of
the“design space” concept(see ICHQ8).

Qualityriskmanagementsupportsascientificandpracticalapproachtodecision-
making,assessingtheprobability,severityandsometimesdetectabilityoftherisk.Inpharmaceutical
development,riskassessmentisimportantinidentifyingwhichmaterialattributesandprocessparam
eterspotentiallyhaveaneffectonproductCQAs–
 CHAPTER I INTRODUCATION

CriticalMaterialAttributes(CMAs)andCriticalProcessParameters(CPPs).Riskassessmentistypi
callyperformedearlyinthepharmaceuticaldevelopmentprocessandisrepeatedasmoreinformation
becomesavailableandgreaterknowledgeisobtained.

Riskstoqualitycanbeassessedinavarietyofinformalways(empiricaland/orinternalprocedures)bas
edon,forexample,compilationofobservations,trendsandotherinformation.Suchapproachesconti
nuetoprovideusefulinformationthatmightsupporttopicssuchashandling of complaints,quality
defects,deviationsandallocation of
resources.Additionally,thepharmaceuticalindustrycanevaluatetheriskusingrecognizedriskmana
gementtools.Someofthesetoolsare:

Basic risk management facilitation methods (flowcharts,checksheets,cause and

effectdiagram,etc.)

 FailureModeEffectsAnalysis(FMEA)
 FailureMode,EffectsandCriticalityAnalysis(FMECA)
 FaultTreeAnalysis(FTA)
 HazardAnalysisandCriticalControlPoints(HACCP)
 HazardOperabilityAnalysis(HAZOP)
 PreliminaryHazardAnalysis(PHA)
 Riskrankingandfiltering.
Thesetoolsmightbeadaptedforuseinspecificareastodrugsubstanceanddrugproductquality.Also,q
ualityriskmanagementmethodsandsomesupportingstatisticaltoolscanbeusedincombination.Co
mbineduseprovidesflexibilitythatcanfacilitatetheapplicationofqualityriskmanagementprinciple
s.

Thestatisticaltoolscansupportandfacilitatequalityriskmanagement.Theycanenableeffectivedata
assessment,aidindeterminingthesignificanceofthedataset(s),andfacilitatemore reliable decision
making.Exampleof statisticaltool are Designof Experiments,ControlCharts,Histograms,etc.

1.6.6 Designof Experiments(DoE)

Traditionalpharmaceuticaldevelopmentapproachesareoftenlimitedbyexperiments thattestone-
at-a-
timevariability.ComprehensiveDesignofExperimentsusesmultidisciplinaryteamstodesignande
xecutesoundlybasedstatisticaldesignstogainafullunderstandingoftheproductanditsmanufacturin
CHAPTER I INTRODUCATION

gprocess.TheoutputofDoEconfirmsCQAsandCPPsthatneedtobecontrolledinthemanufacturing
process.

Inanexperiment,oneormorefactorsaredeliberatelychangedinordertoobservetheeffectononeormo
reresponsevariables.Thismayleadtoanextendnumberofexperiments.InDoE,itisensuredthatthese
lectedexperimentsproducethemaximumamountofrelevantinformation,keepingcostslowbycond
uctingfewexperiments.

CreatedbySirRonalA.Fisherinthe1920sand1930s,DoEisdefinedasastructuredandefficientstatist
icalmethodforplanningexperiments,sothatthedataobtainedcanbeanalyzedto yield valid and
objective conclusions andfordetermining therelationships
amongthefactorsaffectingaprocessanditsoutput.

DoEinitiateswithdefiningtheobjectivesofanexperimentandselectingtheprocessfactorsforthestud
y.Anexperimentaldesignisthelayingoutofadetailedexperimentalplaninadvanceofdoingtheexper
iment.

ThestatisticaltheoryunderlyingDoEgenerallybeginswiththeconceptofprocessmodels,andthemo
stcommonitistheprocessmodelofthe“blackbox”type,withseveraldiscreteorcontinuousinputfacto
rsthatcanbecontrolledandoneormoremeasuredoutputresponses,asshowninFigure12.Themeasur
edresponsesdescribethepropertiesoftheinvestigatedsystem.Bychangingthemostinfluentialfacto
rs(e.g.amountofdisintegrant,timeofmixture,force
ofcompression)thefeaturesofthesystemmightbealtered
accordingtoaresponse(e.g.disintegrationtime,contentuniformity,hardness).Frequently,theexperi
mentsareaffectedbyanumberofuncontrolledfactorsthatmaybediscrete,suchasdifferentmachines
oroperators,and/orcontinuoussuchasambienttemperatureor humidity.

Oncefactorshavebeenchosenandresponsesmeasured,itisdesirabletogetanunderstandingoftherel
ationshipbetweenthem,thatis,linkingthechangesinthefactorstothechangesintheresponseswitha
mathematicalmodel.Infact,thebaseforDoEisanapproximationofrealitywiththehelpofamathemat
icalmodel.Thismodelisnever100%right,butsimplyhelpstotransportthecomplexityoftherealityint
oanequationwhichiseasytohandle.Themostcommonempiricalmathematicalmodelsfittotheexper
imentaldatatakearepolynomialfunctions,usuallyinalinearformorquadraticform.

ThechoiceofanexperimentaldesignisanimportantpartofaDoEprocess,
beingcriticalforthesuccessofthestudy.Thischoicedependsonanumberofaspects,includingthenatu
reoftheproblemandstudy(e.g.,ascreening,optimization,or robustness
 CHAPTER I INTRODUCATION

study),thefactorsandinteractionstobestudied(e.g.,four,six,orninefactors,andmaineffectsortwo-
wayinteractions),andavailableresources(e.g.,time,labour,cost,andmaterials).Numerousstatistica
lexperimentaldesigns areknown.Thefollowinglist gives thecommonlyuseddesigntypes:

1. Fullfactorialdesign
2. Box-Behnkendesign
3. Centralcompositedesign
4. Doehlert design

1. Full two-level factorial designs

Full two-level factorial design is an experimental matrix that has limited application in RSM
when the factor number is higher than 1 because the number of experiments required for this
design (calculated by expression N = 2k, where N is experiment number and k is factor
number) is very large, thereby losing its efficiency in the modeling of quadratic functions.
Because a complete three-level factorial design for more than two variables requires more
experimental runs than can usually be accommodated in practice, designs that present a
smaller number of experimental points, such as the Box–Behnken, central composite, and
Doehlert designs, are more often used. However, for two variables, the efficiency is
comparable with designs such as central composite. The majority of applications of three-
level factorial designs are in the area of chromatography.

2. Box–Behnken designs
Box and Behnken suggested how to select points from the three-level factorial arrangement,
which allows the efficient estimation of the first and second-order coefficients of the
mathematical model. These designs are, in this way, more efficient and economical then their
corresponding 3k designs, mainly for a large number of variables. Its principal characteristics
are: (1) Requires an experiment number according to N = 2k (k − 1) + cp, where k is the
number of factors and (cp) is the number of the central points; (2) All factor levels have to be
adjusted only at three levels (−1, 0, +1) with equally spaced intervals between these levels.
This experimental design has been applied for the optimization of several chemical and
physical processes; however, its application in analytical chemistry is still much smaller in
comparison with central composite design.
 CHAPTER I INTRODUCATION

3. Central composite design

The central composite design was presented by Box and Wilson. This design consists of the
following parts:

 A full factorial or fractional factorial design;

 An additional design, often a star design in which experimental points are at a
distance α from its center;
 A central point.
Fig. 13 (a) and (b) illustrates the full central composite design for optimization of two and
three variables. Full uniformly routable central composite designs present the following
characteristics:
1. Require an experiment number according to N = k 2 + 2k + cp, where k is the factor
number and (cp) is the replicate number of the central point;
2. α- values depend on the number of variables and can be calculated by α = 2(k − p)/4. For
two, three, and four variables, they are, respectively, 1.41, 1.68, and 2.00;
3. All factors are studied in five levels (−α, −1, 0, +1, + α).

4)Doehlert design
Developed by Doehlert, the design is a practical and economical alternative in relation to
other second-order experimental matrices. This design describes a circular domain for two
variables, spherical for three variables, and hyper spherical for more than three variables,
which accents the uniformity of the studied variables in the experimental domain. Although
its matrices are not routable as previous designs, it presents some advantages, such as
requiring few experimental points for its application and high efficiency. Other characteristics
are presented below:

1. Requires an experiment number according to N = k2 + k + cp, where k is the factor number

and (cp) is the replicate number of the central point;
2. Each variable is studied at a different number of levels, a particularly important
characteristic when some variables are subject to restrictions such as cost and/or instrumental
constraints or when it is interesting to study a variable at a major or minor number of levels;
3. The intervals between its levels present a uniform distribution;
For two variables, the Doehlert design is represented by a central point surrounded by six
points from a regular hexagon as shown in Fig.14.
 CHAPTER I
INTRODUCATION

Applications of the Doehlert design in analytical chemistry are increasing in recent years,
mainly because of its advantageous characteristics in relation to other designs. Application of
response surface methodology in the optimization of analytical procedures is today largely
diffused and consolidated principally because of its advantages to classical one-variable-a-
time optimization, such as the generation of large amounts of information from a small
number of experiments and the possibility of evaluating the interaction effect between the
variables on the response. The central composite design is still the symmetrical second- order
experimental design most utilized for the development of analytical procedures. The
application of three-level factorial designs is not frequent, and the use of this design has been
limited to the optimization of two variables because its efficiency is very low for higher
numbers of variables. However, the Box–Behnken and Doehlert designs present more
efficient matrices and have increased the number of published works in recent years. Multiple
response optimization using desirability functions have until now had its utilization limited to
the chromatographic field, its related techniques, and to electrochemical methods. However,
its principles can be applied to the development of procedures using various analytical
techniques, which demand a search for optimal conditions for a set of responses
simultaneously.

1.6.7 DesignSpace andControl Strategy

AkeyconceptintheQbDparadigmisDesignSpace–
amultidimensionalspacethatencompassescombinationsofprocessinputs(materialattributesandp
rocessparameters)andtheCQAsthatprovideassuranceofsuitableproductperformance.ICHQ8(R2
)guidelineintroducestheconceptofDesignSpacetothepharmaceuticalindustryanddefinesitas“the
multidimensional combination and interaction of input variables (e.g., material attributes) and
process parameters that have been demonstrated to provide assurance of quality.”

ADesignSpaceisawaytorepresenttheproductandprocessunderstandingwhichwillbeestablish.Th
eproductandprocessunderstandingandDesignSpacehelpsto
identifyandexplaintheallsourcesofvariabilityandthuswayoutfromthisvariabilitybymeasuringan
dcontrollingtheCPPsandCMAsresponsibleforvariability.Finally,thisassignmentpredictstheaccu
rateandreliableproductqualityattributeswithinspecificationsintermsofquality.

Once a sufficient level of product and process understanding is achieved, through Design
Space, a Control Strategy should be developed that assures that the process will remain
incontrolwithin the normal variation in material attributes and process operating ranges.
 CHAPTER I INTRODUCATION

Figure 8 shows how Control Strategy are connected and interact with Design Space and
Knowledge Space.

ControlStrategyisdefinedas“aplannedsetofcontrols,derivedfromcurrentproductandprocessunde
rstandingthatensuresprocessperformanceandproductquality.Thecontrolscanincludeparametersa
ndattributesrelatedtodrugsubstanceanddrug productmaterialsand components, facility and
equipment operating conditions, in-process controls, finished product specifications, and the
associated methods and frequency of monitoring and control.”

AControlStrategyisdesignedtoensurethataproductofrequiredqualitywillbeproducedconsistently
.Theelementsofthecontrolstrategyshoulddescribeandjustifyhowin-
processcontrolsandthecontrolsofinputmaterials(drugsubstanceandexcipients),intermediates(in
-
processmaterials),containerclosuresystem,anddrugproductscontributetothefinalproductquality.
Thesecontrolsshouldbebasedonproduct,formulationandprocessunderstandingandshould
include,at a minimum,control oftheCPPsandCMAs.In
aQbDapproach,pharmaceuticaldevelopmentwillgenerateprocessandproductunderstandingandi
dentifysourcesofvariability.Thissourcesofvariabilitymayimpactonproductqualityandtherefores
houldbeidentified,understood,andsubsequentlycontrolled.Productandprocessunderstanding,inc
ombinationwithqualityriskmanagement,willsupportthecontroloftheprocesssuchthatthevariabili
tycanbecompensatedforinanadaptablemannertodeliverconsistentproductquality

Scale-
up,technologytransferandmanufacturingexperiencecanleadtorefinementsofthecontrolstrategy.

1.6.8 Continuousimprovement throughoutproductlifecycle

QbDfocusesonbuildingqualityintotheproductandmanufacturingprocesses,aswellascontinuousp
rocessimprovement.Continuousimprovementofa
productandprocessshouldbeemployedthroughoutthelifecycleofaproduct.

ICHQ10describesamodelfortheestablishmentofaneffectivePharmaceuticalQualitySystem(PQS
) that can be used by manufacturers implementing QbD systems and can evaluate and
improve product quality throughout the product lifecycle. In fact, PQS facilitate continual
improvement,helpingthe identificationandimplementationof
appropriateproductandprocessqualityimprovements,reducingthevariability,andidentifyingandp
rioritizingareasforcontinualimprovement.Itisimportanttosharetheknowledgegainedduringdevel
 CHAPTER I INTRODUCATION

opmentandimplementationthatisrelevantforutilizationofthatDesignSpaceonthemanufacturingfl
oorandunderthePQS.Thisknowledgecanincluderesultsofriskassessments,assumptionsbasedonp
riorknowledge,andstatisticaldesignconsiderations.LinkagesamongtheDesignSpace,ControlStr
ategy,CQAandQTPPareanimportantpartofthissharedknowledge.
Inthecaseofchangestoanapproved designspace,appropriatefilingsshouldbemade
tomeetregulatoryrequirements.Movementwithintheapproveddesignspace,asdefinedintheICHQ
8(R2)glossary,doesnotcallforaregulatoryfiling.Formovementoutsidethedesignspace,
theuseofriskassessmentcouldbehelpfulindeterminingtheimpactofthechange on
quality,safetyand efficacy and theappropriate regulatory filing strategy.
 CHAPTER II DRUG PROFILE

CHAPTER 2

DRUG PROFILE
 CHAPTER II DRUG PROFILE

Stability-indicating High performance liquid chromatography method development and

validation of Trabectedin in bulk drug and pharmaceutical dosage forms.

1.1 Highlights of the Developed Method

 Developed the Method for Routine analysis of Trabectedin

 The organic phase of mobile phase and pH of the buffer are minimized with high
theoretical plates.
 Estimation of stability for Trabectedin by RP-HPLC technique.
 Acid, Basic Degradent is calculated
 Thermal, Peroxide and Photolytic Degradent is calculated
 The Retention time and peak asymmetry of analysis is minimized.
 The developed method is simple, precise, validated and optimized method for
estimation of Trabectedin in bulk and pharmaceutical dosage form.
 Optimized Stability data
1.2 Physical, chemical and biological properties of Trabectedin

Name Trabectedin

Structure

Structure of Trabectedin

CAS NO 114899-77-3

Molucular Formula C39H43N3O11S

Molucular Weight 761.84 g·mol−1

Monoisotopic Mass 761.84 g·mol−1

 CHAPTER II DRUG PROFILE

 (1'R,6R,6aR,7R,13S,14S,16R)-6',8,14-trihydroxy-7',9-
dimethoxy-4,10,23-trimethyl-19-oxo-
IUPAC Name 3',4',6,7,12,13,14,16-octahydrospiro[6,16-
(epithiopropano-oxymethano)-7,13-imino-6aH-1,3-
dioxolo[7,8]isoquino[3,2-b][3]benzazocine-20,1'(2'H)-
isoquinolin]-5-yl acetate
 DNA Blocker in Myxoid liposarcoma
 Metastatic leiomyosarcoma
Category & Medical use  Metastatic liposarcoma
 Relapsed platinum-sensitive ovarian cancer
 Unresectable leiomyosarcoma

pKa 9.26

Water Solubility 0.328 mg/mL

Storage Store in a refrigerator at 2°C to 8°C (36°F to 46°F).

Trabectedin blocks DNA binding of the oncogenic

transcription factor FUS-CHOP and reverses the
transcriptional program in myxoid liposarcoma. By
Mechanism of Action
reversing the genetic program created by this transcription
factor, trabectedin promotes differentiation and reverses
the oncogenic phenotype in these cells.
CHAPTER III LITERATURE REVIEW

CHAPTER 3

LITERATURE
REVIEW
 CHAPTER III LITERATURE REVIEW

Comparison of the Present work with available Literature methods

 Monique Zangarini et al, Quantification of trabectedin in human plasma: Validation

of a high-performance liquid chromatography–mass spectrometry method and its
application in a clinical pharmacokinetic study, 2014

Abstract
A rapid, sensitive and specific HPLC–MS/MS method was developed and validated for the
quantification of trabectedin in human plasma after using deuterated trabectedin as Internal
Standard (IS). After the addition of ammonium sulphate, the analyte was extracted from
human plasma with acidified methanol (0.1 M HCl). Chromatographic separation was done
on an Accucore XL C18 column (4 μm; 50 mm × 2.1 mm) using a Mobile Phase (MP)
consisting of CH3COONH4 10 mM, pH 6.8 (MP A) and CH3OH (MP B). The mass
spectrometer worked with electrospray ionization in positive ion mode and Selected Reaction
Monitoring (SRM), using target ions at [M−H2O+H]+ m/z 744.4 for trabectedin and
[M−H2O+H]+ m/z 747.5 for the IS. The standard curve was linear (R2 ≥ 0.9955) over the
concentration range 0.025–1.0 ng/ml and had good back-calculated accuracy and precision.
The intra- and inter-day precision and accuracy determined on three quality control samples
(0.04, 0.08 and 0.80 ng/ml) were <9.9% and between 98.3% and 105.3%, respectively. The
extraction recovery was >81% and the lower limit of quantification 0.025 ng/ml. The method
was successfully applied to study trabectedin pharmacokinetics in a patient with a
liposarcoma who received 1.3 mg/m2 as a 24 h continuous i.v. infusion.

 Laura Ceriani et al, Hplc–MS/MS Method to Measure Trabectedin in Tumors:

Preliminary PK Study in A Mesothelioma Xenograft Model, 2015
Abstract
Background: Trabectedin is an anticancer agent registered for the second-line treatment of
soft tissue sarcoma and ovarian cancer. No preclinical data are available on its tumor
distribution, so a method for quantification in neoplastic tissues is required. Methods/results:
We validated an LC–MS/MS assay determining the recovery, sensitivity, linearity, precision
and accuracy in mouse tumor and liver samples. The limit of quantification was 0.10 ng/ml
with a curve range of 0.10–3.00 ng/ml (accuracy 96.1–102.1%). Inter-day precision and
accuracy of QCs were 6.0–8.2 and 97.0–102.6% respectively. The method was applied in
mesothelioma xenografts treated with therapeutic doses. Conclusion: The method was
 CHAPTER III LITERATURE REVIEW

validated for measuring trabectedin in tissues. In a mesothelioma xenograft model,

trabectedin distributed preferentially in tumor compared with liver.
Emanuela Di Gregorio et al, Novel method for fast trabectedin quantification using
hydrophilic interaction liquid chromatography and tandem mass spectrometry for
human pharmacokinetic studies, 2020
Abstract
Few time-consuming bioanalytical methods are currently available for trabectedin
quantification in clinical investigations. Here we present a novel, fast and sensitive method
for trabectedin determination in human plasma based on hydrophilic interaction liquid
chromatography and tandem mass spectrometry (HILIC-MS/MS). Plasma samples are treated
with acetonitrile–0.1 % formic acid and the solvent extract is directly injected into an Acquity
BEH Amide column (2.1 × 100 mm, 1.7 μm) operating in HILIC mode at 0.2 mL/min with
80:20 acetonitrile–0.1 % formic acid in water. The analyte is separated by an organic solvent
gradient and quantified by an Agilent Ultivo triple quadrupole mass spectrometer operating in
multiple reaction monitoring (MRM) mode. The quantitative MRM transitions were m/z
762→234 and m/z 765→234 for trabectedin and its d3-labeled derivative, respectively. The
lower limit of quantification (LLOQ) was 0.01 ng/mL and the assay was linear up to
2.5 ng/mL. The intra- and inter-day relative error ranged from 1.19 % to 8.52 %, while the
relative standard deviation was less than 12.35 %. The method was used to determine the
pharmacokinetic profiles of trabectedin in 26 patients with soft tissue sarcoma, showing that
this new HILIC-MS/MS method is suitable for use in clinical research.
E. Stokvis et al, Simple and sensitive liquid chromatographic quantitative analysis of the
novel marine anticancer drug Yondelis™ (ET-743, trabectedin) in human plasma using
column switching and tandem mass spectrometric detection, 2004
Abstract
The development of a simple and sensitive assay for the quantitative analysis of the marine
anticancer agent Yondelis (ET-743, trabectedin) in human plasma using liquid
chromatography (LC) with column switching and tandem mass spectrometric (MS/MS)
detection is described. After protein precipitation with methanol, diluted extracts were
injected on to a small LC column (10 × 3.0 mm i.d.) for on-line concentration and further
clean-up of the sample. Next, the analyte and deuterated internal standard were back-flushed
on to an analytical column for separation and subsequent detection in an API 2000 triple-
quadrupole mass spectrometer. The lower limit of quantitation was 0.05 ng mL−1 using 100
µl of plasma with a linear dynamic range up to 2.5 ng ml−1. Validation of the method was
 CHAPTER III LITERATURE REVIEW

performed according to the most recent FDA guidelines for bioanalytical method validation.
The time needed for off-line sample preparation has been reduced 10-fold compared with an
existing LC/MS/MS method for ET-743 in human plasma, employing a labor-intensive solid-
phase extraction procedure for sample pretreatment. The proposed column switching method
was successfully applied in phase II clinical trials with Yondelis and pharmacokinetic
monitoring. Copyright © 2004 John Wiley & Sons, Ltd.

G Sirisha et al, Analytical Method Development And Validation For The Estimation Of
Trabectedin In Bulk And Parenteral Dosage Form By RP-HPLC, 2018
Abstract: A new RP-HPLC method for the quantitative determination of Trabectedin was
developed and validated as per ICH guidelines. The drugs were injected into Zorbax SB,
C18,(150x4.6mm); 3.5µm column maintained at ambient temperature and effluent
monitored at 215nm. The mobile phase consisted of phosphate buffer (pH 3.0) and
Acetonitrile in the ratio of 70:30 v/v. The flow rate was maintained at 0.8 ml/min. The
calibration curve for Trabectedin was linear from 50-175µg/ml (r2 for Trabectedin = 1). The
proposed method was adequate, sensitive, reproducible, accurate and precise for the
determination of Trabectedin in bulk and pharmaceutical dosage forms.

Review of research and development in the subject

(a) International status
1. Jane T.Jacob et al, (2015) Gliptins represent a dipeptidyl peptidase-4 inhibitors that improve
beta cell health and suppress glucagon, leading to improved post-prandial and fasting
hyperglycemia used for the treatment of type 2 diabetes mellitus. A simple and quick
approach of stability indicating RP-HPLC technique was developed for the determination of
Metformin, (Met), Saxagliptin (Saxa) and Sitagliptin (Sita) in bulk and pharmaceutical
dosage forms. This projected methodology is apt for the multicomponent estimation of 2
totally different commercially existing combinations in pharmaceutical market used for the
treatment of type II diabetes mellitus viz. Sitagliptin and metformin, saxagliptin and
metformin in 8 min. A chromatographic separation of the three drugs was attained with a
Inertsil C18 (4.6 × 250 mm, 5 μm) analytical column using a buffer potassium dihydrogen
phosphate adjusted pH 4 with orthophosphoric acid: methanol:acetonitrile (70:10:20%v/v) in
isocratic mode at a flow rate of mL/min, column at ambient temperature and detection of
each 3 drugs were monitored at 215 nm using a DAD detector. These established techniques
were subjected for forced degradation studies in different stress conditions. This suggested
methodology was found to be specific and stability indicating as no interfering peaks of
 CHAPTER III LITERATURE REVIEW

degradation compounds in forced degradation study and excipients was noticed. The
Robustness study and percentage of the assay of the formulations was established within the
limit of ICH guidelines
2.
(b) National status

1. Blessy M et al, (2013) Forced degradation is a degradation of new drug substance and
drug product at conditions more severe than accelerated conditions. It is required to
demonstrate specificity of stability indicating methods and also provides an insight into
degradation pathways and degradation products of the drug substance and helps in
elucidation of the structure of the degradation products. Forced degradation studies show the
chemical behavior of the molecule which in turn helps in the development of formulation and
package. In addition, the regulatory guidance is very general and does not explain about the
performance of forced degradation studies. Thus, this review discusses the current trends in
performance of forced degradation studies by providing a strategy for conducting studies on
degradation mechanisms and also describes the analytical methods helpful for development
of stability indicating method.

2. S. V. Mulgund et al, A simple, specific, accurate and stability-indicating reversed phase high
performance liquid chromatographic method was developed for the simultaneous
determination of mephenesin and diclofenac diethylamine, using a Spheri-5-RP-18 column
and a mobile phase composed of methanol: water (70:30, v/v), pH 3.0 adjusted with o-
phosphoric acid. The retention times of mephenesin and diclofenac diethylamine were found
to be 3.9 min and 14.5 min, respectively. Linearity was established for mephenesin and
diclofenac diethylamine in the range of 50-300 μg/ml and 10-60 μg/ml, respectively. The
percentage recoveries of mephenesin and diclofenac diethylamine were found to be in the
range of 99.06-100.60% and 98.95-99.98%, respectively. Both the drugs were subjected to
acid, alkali and neutral hydrolysis, oxidation, dry heat, photolytic and UV degradation. The
degradation studies indicated, mephenesin to be susceptible to neutral hydrolysis, while
diclofenac diethylamine showed degradation in acid, H2O2, photolytic and in presence of UV
radiation. The degradation products of diclofenac diethylamine in acidic and photolytic
conditions were well resolved from the pure drug with significant differences in their
retention time values. This method can be successfully employed for simultaneous
quantitative analysis of mephenesin and diclofenac diethylamine in bulk drugs and
 CHAPTER III LITERATURE REVIEW

formulations
3.M. Vamsi Krishna et al, Quality by Design (QbD) approach to develop HPLC method for
berconazole nitrate:Application to hydrolytic, thermal, oxidative and photolytic degradation
kinetics, Stability of eberconazole nitrate (EBZ) was investigated using a stability indicating
HPLC method. Quality by Design (QbD) approach was used to facilitate method
development. EBZ was exposed to different stress conditions, including hydrolytic (acid,
base, neutral), oxidative, thermal and photolytic. Relevant degradation was found to take
place in all the conditions. The degradation of EBZ followed (pseudo) first-order kinetics
under experimental conditions. The kinetic parameters (rate constant,t1/2, andt90) of the
degradation of EBZ were calculated

 Importance of the study in the context of current status

Forced degradation studies by QbD approach provides knowledge about possible degradation
pathways and degradation products of the active ingredients and help elucidate the structure
of the degradants. Degradation products generated from forced degradation studies are
potential degradation products that may or may not be formed under relevant storage
conditions but they assist in the developing stability indicating method. It is better to start
degradation studies earlier in the drug development process to have sufficient time to gain
more information about the stability of the molecule. This information will in turn help
improve the formulation manufacturing process and determine the storage conditions. As no
specific set of conditions is applicable to all drug products and drug substances and the
regulatory guidance does not specify about the conditions to be used, this study requires the
experimenter to use common sense. The aim of any strategy used for forced degradation is to
produce the desired amount of degradation i.e., 5–20%. A properly designed and executed
forced degradation study would generate an appropriate sample for development of stability
indicating method.

 Expected outcome from the project

Stress tests for developing a stability indicating method should always be designed and
evaluated with common sense and chemical knowledge, keeping in mind the manufacturing
process and the nature of the final drug product.
The stability profile will established for drug product to assure safety, efficacy and quality.
The results coming from these studies can support formulation and packaging. The know-
how coming from stress tests are useful to reduce time and money, related to the drug and
final product development, giving the possibility of forecasting analytical problems. A
 CHAPTER III LITERATURE REVIEW

generic approach can be used as a starting point to set up a stress testing study, but a case-by-
case approach for stress testing is essential to allow flexibility. This is also recognized by the
regulatory agencies because very detailed instructions about how to perform stress testing are
not given in the available guidance documents. In first instance, a SIM should be applied to
quantification of the main active in a precise and accurately, even in the presence of
interferences (degradation products, impurities, and placebo). In this manner, some
evaluation of mass balance can be done, but the applicant must have in mind, the limitation
about the obtained values, since %area is a useful but not precise/accurate tool to determine
concentrations. If during long-term stability studies or quality control determinations, the
main active concentration decreases, mass balance investigations may start, evaluating
%peak, DAD spectra and inparallel, different/contributing analytical techniques.

 Importance of the study in the context of current status

Forced degradation studies by QbD approach provides knowledge about possible degradation
pathways and degradation products of the active ingredients and help elucidate the structure
of the degradants. Degradation products generated from forced degradation studies are
potential degradation products that may or may not be formed under relevant storage
conditions but they assist in the developing stability indicating method. It is better to start
degradation studies earlier in the drug development process to have sufficient time to gain
more information about the stability of the molecule. This information will in turn help
improve the formulation manufacturing process and determine the storage conditions. As no
specific set of conditions is applicable to all drug products and drug substances and the
regulatory guidance does not specify about the conditions to be used, this study requires the
experimenter to use common sense. The aim of any strategy used for forced degradation is to
produce the desired amount of degradation i.e., 5–20%. A properly designed and executed
forced degradation study would generate an appropriate sample for development of stability
indicating method.

 Expected outcome from the project

Stress tests for developing a stability indicating method should always be designed and
evaluated with common sense and chemical knowledge, keeping in mind the manufacturing
process and the nature of the final drug product.
The stability profile will established for drug product to assure safety, efficacy and quality.
The results coming from these studies can support formulation and packaging. The know-
how coming from stress tests are useful to reduce time and money, related to the drug and
 CHAPTER III LITERATURE REVIEW

final product development, giving the possibility of forecasting analytical problems. A

generic approach can be used as a starting point to set up a stress testing study, but a case-by-
case approach for stress testing is essential to allow flexibility. This is also recognized by the
regulatory agencies because very detailed instructions about how to perform stress testing are
not given in the available guidance documents. In first instance, a SIM should be applied to
quantification of the main active in a precise and accurately, even in the presence of
interferences (degradation products, impurities, and placebo). In this manner, some
evaluation of mass balance can be done, but the applicant must have in mind, the limitation
about the obtained values, since %area is a useful but not precise/accurate tool to determine
concentrations. If during long-term stability studies or quality control determinations, the
main active concentration decreases, mass balance investigations may start, evaluating
%peak, DAD spectra and inparallel, different/contributing analytical techniques.

Conclusion:
As per literature survey, No one has done the force degradation study for Trabectedin by
HPLC method also nobody did the quality by design approach any method development and
validation.
It was best opportunity to develop the stability indicating method for the estimation of
Trabectedin by Quality by Design Approach.
CHAPTER III LITERATURE REVIEW
CHAPTER IV AIM AND OBJECTIVES

CHAPTER 4

AIM AND
oBJECTIVES
 CHAPTER IV AIM AND OBJECTIVES

2.1 Justification for the selection of the Topic

Analyt icalmethodsarerequiredtocharacterizedrugsubstancesanddrugproductsCompositionduri
ngallphasesofpharmaceuticaldevelopment.Developmentofmethodstoachievethefinalgoalofens
uringthequalityofdrugsubstancesanddrugproductsmustbeimplementedinconjunctionwithanund
erstandingofthechemicalbehaviourandphysicochemicalpropertiesofthedrugsubstance.Thisdete
rminationrequireshighlysophisticatedinstrumentsandmethodslikeHPLC and
Spectrophotometeretc.
Extensiveliteraturesurveyrevealsthatseveralanalyticalmethodshavebeenreportedfortheestimati
onofAlectinib inpharmaceuticaldosageformwhichincludes Spectrophotometricmethods UV-
Vis Spectrophotometer, HPLC, LC-MS and
HPTLCApartfromabovenootherspectroscopicandchromatographicmethodslike Force
Degradation Study available for the sameRP-HPLCmethodswerereportedforthis
compound.Hence therewasaneedforthedevelopmentofnewer,simple,sensitive,
rapid,accurateand reproducibleanalyticalmethodsfortheroutineestimationofabove four
drugsinbulkand pharmaceuticaldosageform and apply this method to Force degradation study.
Inviewoftheneedforasuitablemethodforroutineanalysisofsaid drugsinformulations,
attemptswerebeingmade todevelopsimple,preciseandaccurateanalyt icalmethods
forestimationofsaid drugsandextenditfortheirdeterminationinstability indicating Method.

2.2 Followings were objectives according to Drug were selected,

Aim: Stability-indicating High performance liquid chromatography method development
and validation of Trabectedin in bulk drug and pharmaceutical dosage forms.

The objective of the work was

 To develop HPLC method for Alectinib
 To validate the developed methods as per ICH guidelines.
 To Select Methodology of Force Degradation Study.
 To evaluate the degradent of Acid hydrolysis
 To Evaluate the degradent of Base Hydrolysis
 To Evaluate the degradent of oxidation
 To Evaluate the degradent of thermal stability
 To Evaluate the degradent of Base Photolytic study
 Statistical analysis of the recovery data obtained from Force Degradation Study.
 CHAPTER IV PLAN OF WORK

CHAPTER 5

PLAN OF WORK
CHAPTER IV PLAN OF WORK
 CHAPTER IV PLAN OF WORK

1.4Experimental

1.4.1 Materials

Table 1.2: Active Pharmaceutical Ingredient

Sr.
Name Description
No.
1. Trabectedin White, Crystalline Solid
2. 1 mg per Vial

Table 1.3: List of Chemicals

Sr.
Name ofChemicals Grade Manufacturer
No
1 Methanol HPLC Grade Merck Lie Sciences Pvt. Ltd, Mumbai.
2 Acetonitrile HPLC Grade Merck Lie Sciences Pvt. Ltd, Mumbai.
3 Ethanol HPLC Grade Merck Lie Sciences Pvt. Ltd, Mumbai.
4 Water HPLC Grade Merck Lie Sciences Pvt. Ltd, Mumbai.
Potassium dihydrogen
5 LR Grade Research Fine Chem. Indu.
phosphate
6 Sodium hydroxide LR Grade Research Fine Chem. Indu.
7 Triethylamine LR Grade Research Fine Chem. Indu.
 CHAPTER IV PLAN OF WORK

1.4.2 Instrumentation and Chromatographic Conditions

Table 1.4: List of Instruments

Sr. Name of Equipment/

Model/Specification Manufacturer
No. Instruments
HPLC Series2000
1.Pump PU2080
2. Sample Injection port Rheodyne Injector Jasco
1 3. UV/Visible Detector UV 2075 plus
4.Software Borwin
Double beam UV-
2 VisibleSpectrophotomet UV-1800 240V SHIMADZU
er
3 pH meter EQ-636 Equip-Tronics
4 Balance BL-220H Shimadzu
5 Sonicator 1.5L 50H Rolex

Method
Method Development
The best peak parameter plays a significant role to determine a suitable solvent system and
column. (ACN: Buffer, ACN: Water, Methanol: Water) these three mobile phases were
selected for the factorial design. We performed trails on C18 columns at pH 4.0 and 6.0.
These mobile phases were screened by varying the organic phase composition from 70 to 90
% v/v and flow rate from 1.00 mL/ min flow rate consider standard. After performing the
trials, we optimized chromatographic conditions according to Peak properties by scaling it
with satisfactory, partially satisfactory, extremely satisfactory, dissatisfactory

1.4.4 Preparation of Phosphate Buffer Solution

6.8 gm of Potassium dihydrogen orthophosphate was dissolved in sufficient water(HPLC
grade) with aid of sonicator. Then add triethylamine or orthophosphoric acid wasused to
adjust the pH to 5.
 CHAPTER IV PLAN OF WORK

1.4.5 Preparation of stock solutions

10 mg of Trabectedin diluted with 10mL Acetonitrile in volumetric flask to get concentration
of 1000 µg/ml. From the resulting solution 0.1 ml was diluted to 10 ml with Acetonitrile to
obtain concentration of 10 µg/ml of Trabectedin.

1.4.6 Chromatographic procedure

Chromatographic separations were carried out on Analytical column: C18 column Waters X
Bridge (4.6× 250mm id. particle size 5µm), UV detection: 215 nm, Injection volume: 20 µL,
Flow rate: 1.00 mL min -1, Temperature: Ambient, Run time: 10 min
A mixture of acetonitrile, Ammonium Format Buffer (pH-5) and methanol (90:10) was used
as the mobile phase. The pH of the buffer solution was adjusted by phosphoric acid (H3PO4)
and triethylamine (TEA). The wavelength of 215 nm was used as detection at which both
drugs gave good response.

5.4.8 Selection of detection wavelength

From the standard stock solution further dilutions were done using Acetonitrile and scanned
over the range of 200-400 nm and the spectra were overlain. It was observed that drug
showed considerable absorbance at 215 nm.

5.4.9 Preliminary Analysis of Drug

Color of Trabectedin was compared with reported characters mentioned in drug bank and
Melting Point was found to be 244 °C. It shows poor solubility in aqueous phase, organic
phase like methanol, acetonitrile and freely soluble in dimethyl sulfoxide. Wavelength of
maximum absorbance (λmax) in methanol showed at 265 nm.
CHAPTER V RESULT AND DISCUSSION

RESULT AND
DISCUSSION
 CHAPTER V RESULT AND DISCUSSION

1.5Results and Discussion

 Hydrolysis
 Acid Degradation Studies
Approximately 1 mL of solution of stock Trabectedin was added, followed by 1 mL of 1 N
hydrochloric acid, which was then refluxed for 30 minutes at 60 0C/400C for 1 to 5 sampling
Days. A 400 ppm and a 50 ppm solution were prepared from the resulting solution, and 0.30
μL solutions were injected into the system, and the chromatograms were recorded in order to
determine the stability of the sample.
Table 1. Software has given Conditions for forced degradation studies.

Storage Sampling
Condition in time Retention Retention
Sr. Peak Are of Peak Are of Percentage
time of time of
No Trabectedin Degradent Degradation
Temp in Trabectedin Degradent
0 Days
C
1 60 1 5.689 5708920.514 3.23 608353.4862 9.63
2 40 5 5.687 5909809.827 3.231 407464.173 6.45
3 60 5 5.689 5251549.876 3.231 1065724.124 16.87
4 40 1 5.689 6077217.588 3.23 240056.412 3.80
Standard Peak Area of Trabectedin 30ug/mL: 6317274

Day 01
Results of Hydrolysis by 1 N HCl at 400C
The percentage degradation of Trabectedin was found to be 3.80 % in acidic condition. The
percentage degradation was within acceptable criteria (NMT 10%). The extra peaks were
eluted at retention times of 3.23min and the chromatogram was shown in Figure
 CHAPTER V
RESULT AND DISCUSSION

Day 05
Results of Hydrolysis by 1 N HCl at 400C
The percentage degradation of Trabectedin was found to be 6.45% in acidic condition. The
percentage degradation was within acceptable criteria (NMT 10%). The extra peaks were
eluted at retention times of 3.231min and the chromatogram was shown in Figure

Day 01
Results of Hydrolysis by 1 N HCl at 600C
The percentage degradation of Trabectedin was found to be 9.63% in acidic condition. The
percentage degradation was within acceptable criteria (NMT 10%). The extra peaks were
eluted at retention times of 3.23min and the chromatogram was shown in Figure
 CHAPTER V RESULT AND DISCUSSION

Day 05
Results of Hydrolysis by 1 N HCl at 600C
The percentage degradation of Trabectedin was found to be 16.87% in acidic condition. The
extra peaks were eluted at retention times of 3.23min and the chromatogram was shown in
Figure

Conclusion: Less degradation will exist 400C and Day 1st also all runs shown percentage
degradationis less than 10%. The percentage degradation was within acceptable criteria
(NMT 10%) and for 60 degree it shows more degradation.

 Alkali Degradation Studies

Approximately 1 mL of solution of stock Trabectedin was added, along with 1 mL of 1 N
sodium hydroxide, and the mixture was refluxed for 30 minutes at 600C. It was necessary to
dilute the resulting solution to create 400 parts per million and 50 parts per million solutions,
which were then injected into the system and the chromatograms were recorded to determine
the stability of the sample.

Storage Sampling
Retention Retention
Sr. Condition in time Peak Are of Peak Are of Percentage
time of time of
No Trabectedin Degradent Degradation
Temp in Trabectedin Degradent
0 Days
C
1 60 1 5.689 4909153.625 3.231 1408120.375 22.29
2 40 5 5.688 5081615.206 3.231 1235658.794 19.56
3 60 5 5.689 4239522.581 3.231 2077751.419 32.89
4 40 1 5.689 5482762.105 3.231 834511.8954 13.21
Standard Peak Area of Trabectidin 30ug/mL: 6317274
 CHAPTER V RESULT AND DISCUSSION

Day 01
Results of Hydrolysis by 1 N NaOH at 400C
The percentage degradation of Trabectedin was found to be 13.21 % in basic condition. The
extra peaks were eluted at retention times of 3.231 min and the chromatogram was shown in
Figure

Day 05
Results of Hydrolysis by 1 N NaOH at 400C
The percentage degradation of Trabectedin was found to be 19.56 % in basic condition. The
extra peaks were eluted at retention times of 3.231 min and the chromatogram was shown in
Figure
 CHAPTER V
RESULT AND DISCUSSION

Day 01
Results of Hydrolysis by 1 N NaOH at 600C
The percentage degradation of Trabectedin was found to be 22.29 % in basic condition. The
extra peaks were eluted at retention times of 3.231 min and the chromatogram was shown in
Figure

Day 05
Results of Hydrolysis by 1 N NaOH at 600C
The percentage degradation of Trabectedin was found to be 32.89% in basic condition. The
extra peaks were eluted at retention times of 3.231 min and the chromatogram was shown in
Figure
 CHAPTER V
RESULT AND DISCUSSION

Conclusion: Less degradation will exist 400C and Day 1stand its percentage is 13.21% . The
percentage degradation was not within acceptable criteria (NMT 10%) and for 60 degree it
shows more degradation so that method says that drug is basic degradable.

 Peroxide Degradation Studies

1 mL of Trabectedin solution of stocks, as well as 1 mL of hydrogen peroxide (H 2O2) at a
concentration of 20 Percentage, was added individually. The solutions were maintained at 25
to 60 degree Celsius for 30 minutes.
As part of the HPLC investigation, the resulting solution was diluted to create 400 ppm and
50 ppm solutions, and 0.30 μL was put into the system, with chromatograms recorded to
determine sample stability.TwoProminent degradation peaks was observed.
 CHAPTER V RESULT AND DISCUSSION

Storage Sampling
Condition in time Retention Peak Area Peak Area
r. Peak Are of Rt of 1st Rt of 2nd Percentag
time of of 1st of 2nd
o Trabectedin Degradent Degradent Degradatio
Temp in Trabectedin Degradent Degradent
0 Days
C
60 1 5.689 4279953 7.214 2037321 9.31 679107 32.25
25 5 5.687 4360183 7.214 1957091 9.31 652364 30.98
60 5 5.689 3655806 7.214 2661468 9.31 887156 42.13
25 1 5.689 4891465 7.214 1425809 9.31 475270 22.57
Standard Peak Area of Trabectedin 30ug/mL: 6317274

Day 01
Results of Oxidation by 20 PercentH2O2 at 600C
The percentage degradation of Trabectedin was found to be 32.25% in Oxidative condition.
The extra peaks were eluted at retention times of 7.214min and9.31 min, the chromatogram
was shown in Figure

Day 05
Results of Oxidation by 20 PercentH2O2 at 600C
The percentage degradation of Trabectedin was found to be 42.13% in Oxidative condition.
The extra peaks were eluted at retention times of 7.214min and9.31 min, the chromatogram
was shown in Figure
 CHAPTER V RESULT AND DISCUSSION

Day 01
Results of Oxidised by 20 PercentH2O2 at 250C
The percentage degradation of Trabectedin was found to be 22.57% in Oxidative condition.
The extra peaks were eluted at retention times of 7.214min and9.31 min, the chromatogram
was shown in Figure

Day 05
Results of Oxidised by 20 PercentH2O2 at 250C
The percentage degradation of Trabectedin was found to be 30.98% in Oxidative condition.
The extra peaks were eluted at retention times of 7.214min and9.31 min, the chromatogram
was shown in Figure
 CHAPTER V RESULT AND DISCUSSION

Conclusion: Less degradation will exist on 250C and 1st Day with 22.57% but all runs shown
percentage degradationis more. Method concluded that the drug is oxidative.

Thermal degradation studies

The standard medication solution was baked in an oven at 30°C to 105°C for 6 hours to
investigate dry heat degradation from 1st day to 5th Day. HPLC analysis was carried out by
 CHAPTER V
RESULT AND DISCUSSION

diluting the resulting solution to 400 parts per million (ppm) and 50 parts per million (ppm)
solutions, injecting 0.30 μL of the solution into the system and recording the chromatograms
to determine the stability of the sample.No major degradation wasobserved.

Storage Sampling
Condition in time Retention Retention Peak Are
Peak Are of Percentage
Sr. No time of time of of
Trabectedin Degradation
Temp in Trabectedin Degradent Degradent
0 Days
C
1 105 1 5.689 1756833.899 8.324 4560440.1 72.19%
2 30 5 5.687 2991229.239 8.324 3326044.76 52.65%
3 105 5 5.689 765653.6088 8.324 5551620.39 87.88%
4 30 1 5.689 3643803.643 8.324 2673470.36 42.32%
Standard Peak Area of Trabectedin 30ug/mL: 6317274

Day 01
Results of Thermolysis at 300C
The percentage degradation of Trabectedin was found to be 42.32% in thermolytic condition.
The extra peaks were eluted at retention times of 8.324min; the chromatogram was shown in
Figure

Day 05
Results of Thermolysis at 300C
 CHAPTER V RESULT AND DISCUSSION

The percentage degradation of Trabectedin was found to be 52.65% in thermolytic condition.

The extra peaks were eluted at retention times of 8.324min; the chromatogram was shown in
Figure

Day 01
Results of Thermolysis at 1050C
The percentage degradation of Trabectedin was found to be 72.19%in thermolytic condition.
The extra peaks were eluted at retention times of 8.324min; the chromatogram was shown in
Figure

Day 05
 CHAPTER V
RESULT AND DISCUSSION

Results of Thermolysis at 1050C

The percentage degradation of Trabectedin was found to be 87.88%in thermolytic condition.
The extra peaks were eluted at retention times of 8.324min; the chromatogram was shown in
Figure

Conclusion: The drug is thermo labile because it shown the more degradation in all
conditions. It is not within prescribed limits.

1.5.2 Optimization Result

Results of various trials performed on C1 Column at different Mobile phase with different pH
of aqueous phase.
Table 1.5: Trials performed at mobile phase (70:30 v/v) with aqueous phase pH 4
 CHAPTER V
RESULT AND DISCUSSION

Sr.
Composition Observation Remarks
no.
Peak asymmetry is good but Partly
1 ACN: Water
less theoretical plates satisfactory
Greater peak asymmetry and
2 Methanol: Water Not satisfactory
lower theoretical plates
Good Retention Time, Less
Extremely
3 ACN: buffer Asymmetry & Remarkable
Satisfactory
theoretical plates

Table 1.6: Trials performed at mobile phase (90:10 v/v) with aqueous phase pH 6
Sr.
Composition Observation Remarks
no.
Less peak asymmetry with More than
1 ACN: Water
more theoretical plates Satisfied
Less peak asymmetry but less
2 Methanol: Water Satisfied
theoretical plates
Very
3 ACN: buffer No peak appearence
Dissatisfied

Table 1.7: Trials performed at mobile phase (70:30 v/v) with aqueous phase pH 6
Sr.
Composition Observation Remarks
no.
More retention time Not
1 ACN: Water
and less peak height satisfactory
Not at all
2 Methanol: Water More retention time
satisfactory
Very
3 ACN: buffer No peak appearence
Dissatisfied

Table 1.8: Trials performed at mobile phase (90:10 v/v) with aqueous phase pH 4
Sr.
Composition Observation Remarks
no.
1 ACN: Water Good peak properties Satisfactory
Not
2 Methanol: Water More retention time
satisfactory
Greater peak Not
3 ACN: buffer
asymmetry satisfactory

1.5.4 Optimized chromatographic conditions

Mobile phase: Phosphate buffer: Acetonitrile(30.00:70.00v/v), pH of buffer: 4, Analytical
column: C18 column Waters XBridge (4.6× 250mm id. particle size 5µm), UV detection:
-1
215nm, Injection volume: 10 µL, Flow rate: 1.00 mL min , Temperature: Ambient, Run
 CHAPTER V
RESULT AND DISCUSSION

time: 10 min.

 Analytical Method Development

Different mobile phases were investigated to develop the suitable HPLC method for the
analysis of Trabectedin in formulations. For the selection of media the criteria employed was
sensitivity of the method, ease of sample preparation, miscibility of the drug, cost of solvents
and applicability of method to various purposes. Retention time and peak area of Trabectedin
in the selected medium at respective wavelengths were determined and compared with the
reference standards and formulation also.

Figure 1.4: A typical chromatogram of Trabectedin

The applied chromatographic conditions permitted a good separation of Trabectedin in a
short retention time of 5.689 min. (Fig.1.4), no drug decomposition was observed during the
analysis. The LC method was validated for the parameters reported below.
 System suitability study
According to USP, system suitability parameters are an essential part of analytical methods.
Therefore, Retention time, peak area, and theoretical plates were stated for standard solutions.
A sample of 25µg/ml was run at 265 nm and the achieved data was compared with the
prescribed limit.Results were shown in the table. 5.27.
Table.1.13: System suitability parameters
Sr. No. Parameters Observation
1 Retention time 5.689 min
2 Peak area 952879
3 Theoretical plates 9895
 CHAPTER V RESULT AND DISCUSSION

 Preparation of Calibration Curve

The stock solution was prepared using 10mg of Trabectedin in 10ml of Acetonitrile and
further solutions were diluted with the mobile phase. Furthermore, the Final solutions were
sonicated about 5 min. The linearity range was taken from 5 to 25µg/ml and plotted
calibration curve was found to be linear with 0.9978. A calibration curve is shown in fig. 5.5
and details are in table 5.28.
Table 1.14: Result of linearity
Sr. Concentration Retention Asymmetric Theoretical
Peak Area
No (µg) Time Factor Plates
1 5 1052879 5.688 1.212 9895
2 10 2105758 Peak Properties 5.689 1.211 10201
3 15 3108637 5.688 1.211 9921
4 20 4211516 5.689 1.211 9952
5 25 5264395 5.689 1.212 9719
6 30 6317274 5.689 1.213 9981
Slope 210861.51 Six Replicates used
Standard Error 22625.31

Calibration Curve
7000000
6000000 y = 210862x - 13333
R² = 0.9999
5000000
Peak Area

4000000
3000000
2000000
1000000
0
0 5 10 15 20 25 30 35
Concentration

Figure 5.5:Linearity graph for Trabectedin

 CHAPTER V RESULT AND DISCUSSION

 Linearity

Figure 1.6: Chromatogram of Trabectedin (5 µg/ml)

Figure 1.7: Chromatogram of Trabectedin (10 µg/ml)

CHAPTER V RESULT AND DISCUSSION

Figure 1.8: Chromatogram of Trabectedin (15 µg/ml)

Figure 1.9: Chromatogram of Trabectedin (20 µg/ml)

CHAPTER V RESULT AND DISCUSSION

Figure 1.10: Chromatogram of Trabectedin (25µg/ml)

Figure 1.11: Chromatogram of Trabectedin (30µg/ml)

 CHAPTER V RESULT AND DISCUSSION

 Selectivity
Brand name of Trabectedin Formulation is Yondelis1mg.Vial Injectable of Trabectedin
formulation in volumetric flask and 1ml (1 mg of pure drug) including other excipients was
shifted in 10 ml volumetric flask and volume were adjusted with Acetonitrile. Retention time
and another peak parameter of the formulation were compared with the API of the drug. A
chromatogram is shown in figure 5.11. Retention time, Area, Asymmetric factor, and
Theoretical Plates were found to be 5.689min, 1052879, 1.212 and 9895 respectively. These
results concluded that the excipient of the capsule does not interfere with the separation of the
drug in the selected mobile phase.

Figure
1.11: A typical chromatogram of Trabectedin [Concentration 25ug/ml]

 Sensitivity
The sensitivity of the method can be determined by the limit of detection and the limit of
quantitation. Signal to noise ratio is of paramount importance in the calculation. Six
replicates of the blank sample were run and calculate noise level which is three times of
LOD and ten times of LOQ respectively.

Table 1.16: Result of Sensitivity

1 LOD (μg/mL) 0.3541
 CHAPTER V RESULT AND DISCUSSION

2 LOQ (μg/mL) 1.0730

 Accuracy
To develop a routine analytical method, accuracy has great importance invalidation. It is
nothing but a comparative study of the reference value and found value. Accuracy existed
between specified ranges of 99.92 to 100.36%. It was conducted by preparing the five
replicates of three different concentrations and estimated the found concentration and %
Recovery. The results obtained are shown in table 4.
Table 1.17: AccuracyresultsofTrabectedinbyRP-HPLC

Found
Concentration
Sr. No Peak area Concentration % Recovery
(μg/mL)
(μg/mL)
1 16 3394816.8 15.99 99.92
2 20 4243521 19.99 99.95
3 24 5092225.2 24.09 100.36

 Precision
Precision is the series of measurements in different environments of all day. We performed
intraday precision and Interday precision. Six replicates of 20 µg/ml solution were prepared
and took 6 readings in a day right from morning to evening. Intraday precision proved that the
method remains stable in different climatic conditions of the day. Relative standard deviation
% was found to be 1.5652likewise Interday precision is the sample analysis for three days
and relative standard deviation % was found to be 1.730. Analytical precision yields reliable
results and complies with the ICH guideline. Repeatability was performed by preparing the
six replicates and RSD% was 0.6940. The results obtained are shown in table 5.30.
Table 1.18:SystemPrecisionresultsforTrabectedinbyRP-HPLC
Sr. Concentration
Intraday Interday
No (μg/mL)
1 20 4235992.5 4224355
2 20 4261017 4265265.333
3 20 4087630 4350678
4 20 4242997 4130308
5 20 4274752 4211218
6 20 4248203.25 4176939
Average 4225098.6 4226460.6
Standard Deviation 62750.91 69366.48
RSD% 1.4852 1.641
 CHAPTER V RESULT AND DISCUSSION

Table 1.18 (a): Results of Intraday Precision

Sr. No Concentration 0 hr 2hr 4hr 6hr Average

1 20 4216755 4217442 4303229 4206544 4235992.5
2 20 4306742 4196221 4388818 4152287 4261017

3 20 4302634 3401628 4390970 4255288 4087630

4 20 4315269 4138568 4311543 4206608 4242997

5 20 4284172 4222911 4310507 4281418 4274752
6 20 4216852 4252183 4218488 4305290 4248203.25
Average 4225099
Standard Deviation 62750.9
RSD 1.4852

Table 1.18 (b): Results of Inter day Precision

Sr. No Concentration 1st Day 2nd Day 3rd Day Average
1 20 4242402 4339549 4091114 4224355
2 20 4256963 4415530 4123303 4265265
3 20 4341273 4554326 4156435 4350678
4 20 3985991 4359696 4045237 4130308
5 20 4095125 4438674 4099855 4211218
6 20 4251923 4343203 3935691 4176939
Average 4226460.56

Standard Deviation 69366.48

RSD 1.641

 Specificity
Chromatogram of Trabectedin Esilate in capsule formulation showed a peak at a retention time
of 4.217min. The mobile phase designed for the method resolved the drug very efficiently and
The Retention time of Trabectedin was 4.217± 0.0018 min. The detection of wavelength was
265 nm. The peak properties of a formulation containing the drug were compared with
standard and it was observed that the peak was adequately resolved without the interference of
the excipient. Recovery was achieved at 99.94%. The result was shown in table 5.31.
Table 1.15: Results of Specificity
Label Claim
Sample Amount Found Recovery Retention Time
(mg)
Vial 1 0.98 98% 5.689
 CHAPTER V RESULT AND DISCUSSION

 Repeatability
Demonstrationofprecisionwas done undertwocategories.Theinjectionrepeatabilit y
(SystemPrecision)wasassessedbyusingsixinjectionsofthestandardsolutiono fTrabectedinandthe
%RSDofthereplicateinjectionswascalculated. Results obtained are shown in table 100.
Table 1.16:Repeatability resultsforTrabectedinbyRP-HPLC
Concentration
Sr. No Peak Area
(μg/mL)
1 20 4216734
2 20 4196734
3 20 4216747
4 20 4209860
5 20 4266734
6 20 4214522
Average 4220221.8
Standard Deviation 21905.7

RSD% 0.519

 Robustness
Robustnessisameasureofcapacityofamethodtoremainunaffectedbysmall,but
deliberatevariationsinthemethodconditions,andisindicationsofthereliabilityofthemethod.Ameth
odisrobust,ifitisunaffectedbysmallchangesinoperating
conditions.Todeterminetherobustnessofthismethod,theexperimentalconditionsweredeliberately
alteredatthreedifferentlevelsandretentiontimeand chromatographicresponsewereevaluated.
Onefactoratatimewaschangedtostudy theeffect.Variationofmobilephasecompositio n
(70:30v/v), flowrateby1ml/min(0.9 and1.1ml/min), Variation of wavelength by 265 nm (263
nm and 267 nm), Variations in pH by 5 (4.8 and 5.2)hadnosignificanteffectonthe retention
time and chromatographic response of 10 μg/ml solution, indicating that the method was
robust, The result was shown in table

Table 1.17: Result of Robustness

Sr. No Parameter Response Parameter Response

Detection
Acetonitrile : Buffer Retention Wavelength Peak Area
Time (min)
(V/V) (nm)
CHAPTER V RESULT AND DISCUSSION

1 69 31 5.592 213 4153389

2 70 30 5.689 215 4211704
3 71 29 5.788 217 4244968
Average 5.690 Average 4203354
Standard
Standard Deviation 0.080
Deviation 37850.36
RSD% 1.406 RSD% 0.900
Flow Rate Retention pH of Buffer
Peak Area
(mL/min) Time (min) (mmol/L)
1 0.9 5.82 3.8 4239776
2 1 5.689 4 4211877

3 1.1 5.575 4.2 4136256

Average 5.695 Average 4195970

Standard
Standard Deviation 0.1001 43733.13
Deviation

RSD% 1.758 RSD% 1.0423

 Recovery
The The recovery of the method was evaluated by the addition of standard at three levels 80,
100, and 120 % to the mixture. Six replicates were run and calculated the mean amount of
drug recovered and percentage recovery. It found that results were observed within the
prescribed limit. Results were shown in table 8.

Table 1.18:Recovery resultsof TrabectedinbyRP-HPLC

Amount Amount of
Amount of
of Drug Drug
Sample
Sr. No Added Recovered Recovery %

(µg/ml) (µg/ml) (µg/ml)

1 20 10 9.989 99.89
2 20 20 19.99 99.96
3 20 30 30.01 100.03
 CHAPTER V RESULT AND DISCUSSION

Part-II

Development and Validation of Stability Indicating Liquid Chromatographic Methods

for Trabectedin and Its Formulation

According to the above prescribed, the RP-HPLC method was developed and validated as per
ICH guideline. Optimized mobile phase by design expert software was used to estimate drug
in human plasma. Details are as follows.

1.6 Optimized chromatographic conditions

Mobile phase: Ammonium format buffer: Acetonitrile(30.00:70.00v/v), pH of buffer: 4,
Analytical column: C18 column Waters XBridge (4.6× 250mm id. particle size 5µm), UV
-1
detection: 215 nm, Injection volume: 10 µL, Flow rate: 1.00 mL min , Temperature:
Ambient, Run time: 10 min.

 Introduction of research:
 Stability requirements during development of a drug
Chemical stability of pharmaceutical molecules is a matter of great concern as it affects the
safety and efficacy of the drug product. The FDA and ICH guidance’s state the requirement
of stability testing data to understand how the quality of a drug substance and drug product
changes with time under the influence of various environmental factors. Knowledge of the
stability of molecule helps in selecting proper formulation and package as well as providing
proper storage conditions and shelf life, which is essential for regulatory documentation.
Forced degradation is a process that involves degradation of drug products and drug
substances at conditions more severe than accelerated conditions and thus generates
degradation products that can be studied to determine the stability of the molecule. The ICH
guideline states that stress testing is intended to identify the likely degradation products
which further helps in determination of the intrinsic stability of the molecule and establishing
degradation pathways, and to validate the stability indicating procedures used. But these
guidelines are very general in conduct of forced degradation and do not provide details about
the practical approach towards stress testing. Although forced degradation studies are a
regulatory requirement and scientific necessity during drug development, it is not considered
as a requirement for formal stability program.

It has become mandatory to perform stability studies of new drug moiety before filing in
registration dossier. The stability studies include long term studies (12 months) and
 CHAPTER V RESULT AND DISCUSSION

accelerated stability studies (6 months). But intermediate studies (6 months) can be

performed at conditions milder than that used in accelerated studies. So the study of
degradation products like separation, identification and quantitation would take even more
time. As compared to stability studies, forced degradation studies help in generating
degradants in much shorter span of time, mostly a few weeks. The samples generated from
forced degradation can be used to develop the stability indicating method which can be
applied latter for the analysis of samples generated from accelerated and long term stability
studies. This review provides a proposal on the practical performance of forced degradation
and its application for the development of stability indicating method.

 Origin of research problem

Stability indicating methods are developed to supervise the stability of drug substance and
pharmaceutical dosage forms for the duration of the early phase of medicine development,
and once the medicine is entered into the marketed, for the continuing product stability
studies which must be performed as per ICH or regulatory guidelines. The reason of stability
studies testing to give evidence on how the quality of drug differs with moment under the
influence of a multiplicity of ecological factors such as humidity, temperature and light,
enables suggested storage conditions, re-analysis intervals and shelf life (live) to be
recognized. The development of these methods for pharmaceutical dosage forms and API can
be come near from quite a few possibilities. Methods can be developed which determine the
impurities of drug left over.
The ICH guideline primarily addresses the in sequence necessary in Registration
Applications for new drug products. This instruction does not at present look for to wrap the
information necessary for abbreviated, variations, clinical trial new applications, etc. The
preference of test conditions distinct in the guideline is based on the real time analysis of the
belongings of climatic circumstances in the three zones of the Japan, EC and USA. The
represent or average kinetic temperature in any zone or region of the globe can be
consequential from climatic information.
In order to the stability of the drug is an essential part of the organized approach to stability
assessment as shelf life. The goal of stability program depends on the stage of development
of the drug product. At very beginning of product development, it is essential to recognize the
inherent firmness of the medicine and it’s interaction with the proposed excipients. At this
stage the effect of pH, moisture, air (oxygen), and light on the stability of the drug substance
 CHAPTER V RESULT AND DISCUSSION

also storage produced. The accelerated testing on drug substance and drug product provides
the in sequence to the inherent stability of the bulk drug molecule formation and may
establish the likely degradation pathways. The formulation group also has the responsibility
for recommending to the toxicology group about stability of drug substance in the medium

 Objectives
 To Apply Stability indicating method to the Trabectedin
 To establish degradation pathways of drug substances and drug products.
 To determine the intrinsic stability of a drug substance in formulation.
 To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis or
photolysis of the drug substance and drug product
 To establish stability indicating nature of a developed method.
 To understand the chemical properties of drug molecules.
 To produce a degradation profile similar to that of what would be observed in a formal
stability study under ICH conditions.
CHAPTER VI CONCLUSION

CONCLUSION
 CHAPTER VI CONCLUSION

.
❖ Method I

The method is economical, reliable and reproducible.

Optimized Mobile phase: Phosphate buffer: Acetonitrile (30.00:70.00v/v), pH of buffer: 4,
Analytical column: C18 column Waters XBridge (4.6× 250mm id. particle size 5µm), UV
detection: 215nm, Injection volume: 10 µL, Flow rate: 1.00 mL min -1, Temperature:
Ambient, Run time: 10 min.
Validation is done as per ICH guidelines
❖ Method II
 Acid Degradation Studies
Less degradation will exist 400C and Day 1st also all runs shown percentage
degradationis less than 10%. The percentage degradation was within acceptable
criteria (NMT 10%) and for 60 degree it shows more degradation.
 Alkali Degradation Studies
Less degradation will exist 400C and Day 1stand its percentage is 13.21% . The
percentage degradation was not within acceptable criteria (NMT 10%) and for 60
degree it shows more degradation so that method says that drug is basic degradable.
 Peroxide Degradation Studies
Less degradation will exist on 250C and 1st Day with 22.57% but all runs shown
percentage degradationis more. Method concluded that the drug is oxidative.
 Thermal degradation studies
The drug is thermo labile because it shown the more degradation in all conditions. It
is not within prescribed limits.
CHAPTER VII REFERENCE

REFERENCE
CHAPTER VII REFERENCE

Chapter

References

1. ChatwalGR,AnandSK.InstrumentalMethodsofChemicalAnalysis:Himalaya Publishing
House; 1979.

2. VA.ATextbookofQuantitativeAnalysis:ForeignLanguagesPublishingHouse.
3. Skoog DA, Holler FJ, Crouch SR. Principles of Instrumental Analysis:
ThomsonBrooks/Cole; 2007.
4. WillardHH,MerrittJrLL,DeanJA,SettleJrFA.Instrumentalmethodsofanalysis. 1988.
5. Lawrence XY. Pharmaceutical quality by design: product and process
development,understanding, and control. Pharmaceutical research. 2008;25(4):781-91.
6. MartensH,MartensM.Multivariateanalysisofquality.Anintroduction.IOPPublishing; 2001.
7. Guidance for industry [electronic resource] : Q8(R2) pharmaceutical
development.CenterforDrugE,Research,CenterforBiologicsE,Research,InternationalConf
erenceonH,editors.Rockville,MD:U.S.Dept.ofHealthandHumanServices,FoodandDrugA
dministration,CenterforDrugEvaluationandResearch : Center for Biologics Evaluation
and Research; 2009.
8. Guidance for industry [electronic resource] : Q9 quality risk management. Centerfor
Drug E, Research, Center for Biologics E, Research, International Conferenceon H,
editors. Rockville, MD: U.S. Dept. of Health and Human Services, Food
andDrugAdministration,CenterforDrugEvaluationandResearch:CenterforBiologics
Evaluation and Research; 2006.
9. Dudhe, P.; Shelke, P., Chavare, P. Determination of Apixaban From Bulk and Tablet
Dosage Form by Area Under Curve and First Order Derivative Spectrophotometric
Methods. Int J PharmTech Res., 2017,10(5), 703-711.
10. Katta, R.; Nagaraju, C.; Ramasrinivas.; Rao G. Two Novel Validated RP-HPLC and Uv
Spectrophotometric Methods for Estimation of Apixaban In Bulk and Pharmaceutical
Dosage Form. Am. j. PharmTech Res., 2015, 5(4), 451-460.
11. Kashid, A.; Vidhate, M.; Ingale, M. Analytical Method Development and Validation for
Estimation of Apixaban by RP-HPLC. Indian Drugs., 2017, 54(4), 76-79.
12. Landge, S.; Jadhav, S.; Dahale, S.; Solanki, P.; Bembalkar, S.; Mathad, V. Development
and Validation of Stability Indicating RP-HPLC Method on Core Shell Column for
Determination of Degradation and Process Related Impurities of Apixaban—An
CHAPTER VII REFERENCE

Anticoagulant Drug, Am J Analyt Chem., 2015, 6, 539-550.

13. Soundharya, R. Method Development and Validation of Apixaban using RP-HPLC
Method and Its Stress Stability Studies. Int.j. chem. pharm. anal., 2017, 5(1), 1-11.
14. Prabhune, S.; Jaguste, R.; Pradhan, N. Stability-Indicating High-Performance Liquid
Chromatographic Determination of Apixaban In the Presence of Degradation Products.
Scipharm., 2014, 82(4), 777-785.
15. Kumar, S.; Ajitha, M.; Awate, M.; Harika, K. Stability Indicating Assay Method
Development and Validation of Apixaban Tablets by RP-HPLC. Int. J. Pharm. Sci.,
2017, 6(1), 71-75.
16. Zaky, M.F., Megahed, M.A., Hammady, T.M., Gad, S., Ghorab, M.M. and El-Say, K.M.,
2023. Tailoring Apixaban in Nanostructured Lipid Carrier Enhancing Its Oral
Bioavailability and Anticoagulant Activity. Pharmaceutics, 15(1), p.80.
17. Kumar, N., Sangeetha, D., Kalyanraman, L. and Sainath, K., 2018. Stability-indicating
HPLC method for simultaneous determination of degradation products and process-
related impurities of avanafil in avanafil tablets. Acta Chromatographica, 30(3), pp.158-
163.
18. Prabhune, S.S., Jaguste, R.S., Kondalkar, P.L. and Pradhan, N.S., 2014. Stability-
indicating high-performance liquid chromatographic determination of Apixaban in the
presence of degradation products. Scientia pharmaceutica, 82(4), pp.777-786.
19. Reddy, V. Method Development and Validation of Apixaban In Bulk and Tablet Dosage
Form by RP-HPLC. J. Pharma Creations., 2018, 5(1). 703-711.
20. Jain, H.; Nikam, V. Formulation Development and Stability Indicating HPLC Assay of
Tablets of Apixaban, Int J Pharm Pharm Sci., 2017, 9(10), 78-82.
21. Sangshetti, J.N.; Deshpande, M.; Zaheer, Z.; Shinde, D.; Arote, R. Quality by Design:
Regulatory Need. Arab. J. Chem., 2017, 10, S3412–S3425.
22. Bhatt, A.; Rane, A. QbD Approach to Analytical RP-HPLC Method Development and its
Validation. Int J Pharm Pharm Sci., 2011, 3(1), 179- 187.
23. Chavan, S.D.; Pimpodkar, N. V., Kadam, A. M., Gaikwad, P. S., 2015. Quality by
Design. Research and Reviews: Journal of Pharmaceutical Quality Assurance. 1(2), 18-
24
24. Bindhu, R. Quality by Design: A Brief Introduction. Pharmacovigilance., 2015,3(4),
e142.
25. Shanmugasundaram, P. and Kamarapu, S.K., 2018. Reverse-phase high-performance
liquid chromatographic method development and validation for the simultaneous
 CHAPTER VII REFERENCE

estimation of Hydrochlorothiazide and Propranolol in bulk and Pharmaceutical dosage

form in biorelevant dissolution media. Media, 10, pp.202-211.
26. Izat, N.; Yerlikaya, F.; Capan, Y. A glance on the history of pharmaceutical quality by
design. Drug Design & Delivery., 2014, 2(1), 1-8.
27. Purnachand, D., Veerareddy, A., Ramadevi, B., Kameswarrao, C.V.S.L., Reddy, G.S.
and Madhusudhanreddy, B., 2015. Development and validation of a simple and sensitive
stability indicating RP-HPLC assay method for determination of Nintedanib and stress
degradation studies. J Chem Pharm Res, 78, pp.774-782.
28. International Conference on Harmonization (ICH) Harmonized Tripartite Guideline,
Validation of Analytical Procedure: Text and Methodology Q2 (B), 2005.
29. International Conference on Harmonization (ICH) of Technical Requirements for
Registration of Pharmaceuticals for Human Use: Harmonized Tripartite Guideline
Validation of Analytical Procedure: Methodology, recommended for adoption at step 4
of the ICH Process on November 1996 by the ICH steering Committee, IFPMA,
Switzerland.
30. Patve RS, Shaikh AR, Inamdar N, Bhise K. Method development and validation of
lenvatinib by HPLC and UV-Spectroscopy, Indian Drugs, 2018; 55: 39-7.
31. Prashanthi Y, Ahmed MA, Vijaya K, Riyazuddin. Method Development And Validation
of Lenvatinib Drug By RP-HPLC In Pharmaceutical Drug Dosage Form, Indo Am. j.
pharm. sci, 2016; 3: 1078-85.
32. Panigrahy UP and Reddy Ak, A novel validated RP-HPLC-DAD method for the
estimation of Lenvatinib Mesylate in bulk and pharmaceutical dosage form, J. Chem.
Pharm. Res, 2015; 7:872-81.
33. Waghmare SA and Sumithra M, Full factorial experimental design for development and
validation of RP-HPLC method for estimation of Apixaban in Bulk and Pharmaceutical
Formulations, J of Seybold Rep, 2020; 15: 3428-42.
34. Waghmare SA, Kale PB, Desai RS Takale S, Jadhav M, Bayas JP. Analytical Method
Development and Validation for Determination of Telmisartan in Bulk and
Pharmaceutical Formulation by QbD Approach, IJAEMA, 2020; 12: 1567-71.
35. Pukale AK, Giri SS, Waghmare SA, Qbd Approach to UV-Visible Spectroscopic
Method Development and Validation for The Estimation of Ranolazine in Bulk and
Pharmaceutical Formulation, European j. biomed. Pharm. Sci, 2018; 5: 740-45.
36. Waghmare SA, Kashid AM, Reverse Phase-High Performance Liqui Chromatography
method development and validation for estimation of efavirenz by Quality by Design
 CHAPTER VII REFERENCE

approach, J. drug deliv. ther, 2019; 9: 319-30.

37. Sanap A, Choudhary PS, Kale PB, Waghmare SA, Analytical Method Development and
Validation for Estimation of Oseltamivir Phosphate in Bulk and Pharmaceutical
Formulation by QbD Approach, IJAEMA, 2020; 12: 463-70.
38. Giri SS, Pukale AK, Waghmare SA, Spectroscopic Method Development and Validation
for Estimation of Apixaban in Bulk and Pharmaceutical Formulation By QbD Approach,
European j. biomed. Pharm. Sci, 2018; 5: 637-42.
39. Rajkotwala S, Shaikh SS, Dedania ZR, Dedania RR, Vijendraswamy SM. QbD
Approach to Analytical Method Development and Validation of Piracetam By HPLC,
World J Pharm Pharm Sci, 2016; 5: 1771-84.
40. Garg NK, Sharma G, Singh B, Nirbhavane P, Katare OP. Quality by design (QbD)-based
development and optimization of a simple, robust RP-HPLC method for the estimation
of methotrexate. J Liq Chrom Relat Tech. 2015; 38: 1629-37.
41. Mandlik SK, Agrawal PP, Dandgavhal HP, Implementation of Quality by Design (Qbd)
Approach In Formulation And Development of Ritonavir Pellets Using Extrusion
Spheronization Method, Int. J. Appl. Pharm, 2020; 12: 139-46.
42. Srujani CH, Annpurna P, Nataraj KS, Pawar KM, Analytical Quality by Design
Approach in RP-HPLC Method Development and Validation for the Estimation of
Duvelisib, Asian J. Pharm. Clin. Res, 2021; 14:99-08.
43. Reis, Rafael, Laurence Labat, Marie Allard, Pascaline Boudou-Rouquette, Jeanne
Chapron, Audrey Bellesoeur, Audrey Thomas-Schoemann et al. "Liquid
chromatography-tandem mass spectrometric assay for therapeutic drug monitoring of the
EGFR inhibitors afatinib, erlotinib and osimertinib, the ALK inhibitor crizotinib and the
VEGFR inhibitor nintedanib in human plasma from non-small cell lung cancer patients."
Journal of pharmaceutical and biomedical analysis 158 (2018): 174-183.
44. Vaidya, Bhuvaneshwar, Snehal K. Shukla, Srikanth Kolluru, Melanie Huen, Nihal
Mulla, Neelesh Mehra, Dipti Kanabar et al. "Nintedanib-cyclodextrin complex to
improve bio-activity and intestinal permeability." Carbohydrate polymers 204 (2019):
68-77.
45. Alam, Parvez, Ali S. Abdelhameed, Ravi Kant Rajpoot, and Rizwan Hasan Khan.
"Interplay of multiple interaction forces: Binding of tyrosine kinase inhibitor nintedanib
with human serum albumin." Journal of Photochemistry and Photobiology B: Biology
157 (2016): 70-76.
46. Kumar, Rohit, Vijaya Kumar Munipalli, Raman Mohan Singh, and Sayali Warde.
 CHAPTER VII REFERENCE

"Validated RP-HPLC Method for Determination and Quantification of Nintedanib in

Pharmaceutical Formulation." Journal of Advancement in Pharmacology 1, no. 1.
47. Purnachand, Dasari, Arava Veerareddy, Bhoomireddy Ramadevi, and Ch VSL.
"Development and validation of a simple and sensitive stability-indicating RP-HPLC
assay method for determination of Nintedanib and stress degradation studies." J Chem
Pharm Res 78 (2015): 774-782.
48. Wind, Sven, Ulrike Schmid, Matthias Freiwald, Kristell Marzin, Ralf Lotz, Thomas
Ebner, Peter Stopfer, and Claudia Dallinger. "Clinical pharmacokinetics and
pharmacodynamics of nintedanib." Clinical pharmacokinetics (2019): 1-17.
49. Bhole, R., Chadar, K., Zambare, Y. and Bonde, C.G., 2020. Development and validation
of HPTLC method for estimation of dofetilide in pharmaceutical dosage form and
determination of its degradation profile by MS-MS method. İstanbul Journal of
Pharmacy, 50(2), pp.71-78.
50. Dutta, D., Das, S., Seijas, J.A. and Ghosh, M., 2019. Validated Stability-Indicating Hptlc
Method For Nintedanib & Characterization Of Degradants By Lc-Msn.
51. Sieger, P., Y. Cui, and S. Scheuerer. "pH-dependent solubility and permeability profiles:
A useful tool for prediction of oral bioavailability." European Journal of Pharmaceutical
Sciences 105 (2017): 82-90.
52. Pasquini, Benedetta, Serena Orlandini, Sandra Furlanetto, Roberto Gotti, Massimo Del
Bubba, Francesca Boscaro, Bruno Bertaccini, Michal Douša, and Giuseppe Pieraccini.
"Quality by Design as a risk-based strategy in pharmaceutical analysis: Development of
a Liquid chromatography-Tandem mass spectrometry method for the determination of
Nintedanib and its impurities." Journal of Chromatography A 1611 (2020): 460615.
53. Jayagopal, Balaji, and Shivashankar Murugesh. "QbD-mediated RP-UPLC method
development invoking an FMEA-based risk assessment to estimate nintedanib
degradation products and their pathways." Arabian Journal of Chemistry 13, no. 9
(2020): 7087-7103.
54. Shah, Purvi, Tosha Pandya, Mukesh Gohel, and Vaishali Thakkar. "Development and
Validation of HPLC method for simultaneous estimation of Rifampicin and Ofloxacin
using experimental design." Journal of Taibah University for Science 13, no. 1 (2019):
146-154.
55. Sahu, Prafulla Kumar, Nageswara Rao Ramisetti, Teresa Cecchi, Suryakanta Swain,
Chandra Sekhar Patro, and Jagadeesh Panda. "An overview of experimental designs in
HPLC method development and validation." Journal of pharmaceutical and biomedical
 CHAPTER VII REFERENCE

analysis 147 (2018): 590-611.

56. Subramanian, Velusamy B., Naresh Kumar Katari, Thirupathi Dongala, and Sreekantha
B. Jonnalagadda. "Stability‐indicating RP‐HPLC method development and validation for
determination of nine impurities in apixaban tablet dosage forms. Robustness study by
quality by design approach." Biomedical Chromatography 34, no. 1 (2020): e4719.
57. Garg, Neeraj K., Gajanand Sharma, Bhupinder Singh, Pradip Nirbhavane, and Om
Prakash Katare. "Quality by design (QbD)-based development and optimization of a
simple, robust RP-HPLC method for the estimation of methotrexate." Journal of Liquid
Chromatography & Related Technologies 38, no. 17 (2015): 1629-1637.
58. Liu, Hongfei, Kunyu Du, Dongli Li, Yi Du, Jumei Xi, Ying Xu, Yan Shen, Tao Jiang,
and Thomas J. Webster. "A high bioavailability and sustained-release nano-delivery
system for nintedanib based on electrospray technology." International Journal of
Nanomedicine 13 (2018): 8379.
59. Rathod, Ravsaheb H., Suraj R. Chaudhari, Amod S. Patil, and Atul A. Shirkhedkar.
"Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) in practice:
analysis of drugs and pharmaceutical formulations." Future Journal of Pharmaceutical
Sciences 5, no. 1 (2019): 6.
60. Kirthi, A., R. Shanmugam, M. Shanti Prathyusha, and D. Jamal Basha. "A review on
bioanalytical method development and validation by RP-HPLC." Journal of global trends
in pharmaceutical sciences 5, no. 4 (2014): 2265-2271.
61. Dubala, Anil, Rizwanbasha Khatwal, Jayasankar Kosaraju, Venkat Meda, and M.
Samanta. "Bioanalytical method development and validation of sitagliptin phosphate by
RP-HPLC and its application to pharmacokinetic study." Int J Pharm Pharm Sci 4, no. 2
(2012): 691-694.
62. Pharne, A. B., B. Santhakumari, A. S. Ghemud, H. K. Jain, and M. J. Kulkarni.
"Bioanalytical method development and validation of vildagliptin a novel dipeptidyl
peptidase IV inhibitor by RP-HPLC method." International Journal of Pharmacy and
Pharmaceutical Sciences 4, no. 3 (2012): 119-123.
63. Jayaseelan, S., S. Suresh, G. Sathishkumar, V. Sekar, and P. Perumal. "Bioanalytical
method development and validation of Lamivudine by RP-HPLC method." Int J Chem
Tech Res 2 (2010): 163-7.
64. D’cruz, Delma, Anu Babu, Eena Joshy, and T. P. Aneesh. "Bioanalytical method
development and validation of ticagrelor by RP-HPLC." International Journal of Applied
Pharmaceutics, Innovare Academics Sciences Pvt. Ltd 9, no. 3 (2017): 51-54.
CHAPTER VII REFERENCE

65. Bhinge, Somnath D., Sharangouda M. Malipatil, and Lalit V. Sonawane. "Bioanalytical
method development and validation for simultaneous estimation of cefixime and
dicloxacillin by RP-HPLC in human plasma." Acta Chimica Slovenica 61, no. 3 (2014):
580-586.
66. Jayaseelan, S., S. Suresh, G. Sathishkumar, V. Sekar, and P. Perumal. "Bioanalytical
method development and validation of Lamivudine by RP-HPLC method." Int J Chem
Tech Res 2 (2010): 163-7.
67. Lin, Dan, Li-man Qiao, Yu-niao Zhang, Yuan Liu, and Xin-she Liu. "Simultaneous
determination of nintedanib and its metabolite by UPLC–MS/MS in rat plasma and its
application to a pharmacokinetic study." Journal of pharmaceutical and biomedical
analysis 117 (2016): 173-177.
68. Ameeduzzafar, Javed and Asgar A, "Development and validation of UPLC/ESI-Q-TOF-
MS for carteolol in aqueous humor: Stability, stress degradation, and application in the
pharmacokinetics of nanoformulation"Arabian Journal of Chemistry 10, no. 3 (2017):
S2969-S2978.
69. Ameeduzzafar, Ibrahim E, Nabil K., Imam S, Ahmed F, Mohammed H, Ahmad N, and
Elmowafy M, Quality by design (QbD) based development and validation of
bioanalytical RP-HPLC method for dapagliflozin: Forced degradation and preclinical
pharmacokinetic stud, Journal of Liquid Chromatography & Related Technologies 43, no
1-2, (2020): 01-12.
CHAPTER VIII PRESENTATION AND PUBLICATION

PRESENTATION
&
PUBLICATION
CHAPTER VIII PRESENTATION AND PUBLICATION

1. Coumarin sulfonamid: A Highly versatile scaffold in drug discovery (WJPR

Publication).

2. Menorrhagia –Understanding &Managing Menstrual Bleeding (IJPS

Publication)
ERRATA