METHOD DEVELOPMENT AND VALIDATION

FOR ANTI DIABETIC DRUG BY RP-HPLC

A Thesis submitted to
Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal (M.P.) In the
partial fulfillment of the requirement for the Degree of

In
Pharmaceutical Chemistry

Submitted By Supervised By

Mr. Romit Jain Mr. Prashant Vinode

(Asso. Professor)

(Enrolment No. 0602PY22MP10)

SAGAR INSTITUTE OF PHARMACEUTICAL

SCIENCES

NH-26, NARSINGHPUR ROAD, SIRONJA, SAGAR (M.P)

470228

2023-2024
 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE,PCI New Delhi, DTE Bhopal and Govt. of
M.P.(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

EXAMINER CERTIFICATE

This is to certified that the M. Pharm. Research Work and Colloquium final thesis

viva of Mr./Ms enrollment no.

of M. Pharm. IV Sem. MPC403P is held on . The external examiner

appointed by RGPV, Bhopal for thesis viva is examine the candidate.

Name and Signature Name and Signature

Internal examiner External examiner

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The sample injector, which is close to the column inlet, is used to inject the sample. The
components of the injected sample migrate through the column, moving between the stationary
phase and mobile phase, as it enters the column with mobile phase. The first stage in
developing a successful separation using the RP-HPLC technology is selecting the stationary
and mobile phases. It is crucial to pick a stationary phase that works well with the mobile phase
and can successfully trap the target analyte. It's critical to choose a mobile phase that elutes the
target analyte from the column and is compatible with the stationary phase. To get the best
separation conditions, it is possible to adjust a number of parameters, including column
temperature, mobile phase pH, flow rate, and gradient elution. The optimization process aims to
achieve maximum resolution, minimal analytical time, and optimal solvent consumption. The
most common side effects of sitagliptin phosphate are abdominal pain, nausea diarrhea,
vomiting and hypoglycemia. The symptoms of lactic acidosis are weakness, trouble breathing,
abnormal heartbeats, unusual muscle pain stomach discomfort, lightheadedness and feeling
cold. Patients at risk for lactic acidosis include those with reduced function of the kidneys or
liver, congestive heart failure severe acute illnesses, and dehydration. Specificity is the ability to
measure accurately and specifically the analyte of interest in the presence of other components
that may be expected to be present in the sample matrix. The other component may include
excipients, impurities, degradation product etc. The separation method was carried out by using
a mobile phase consisting of 0.02M dipotassium hydrogen phosphate and acetonitrile in the
ratio 55:45. The retention time of Sitagliptin phosphate was found to be 7.485 respectively. The
asymmetry factor or tailing 1.011 respectively, which indicates symmetrical nature of the peak.
The number of theoretical plates of Sitagliptin phosphate was found to be 12044 respectively,
which indicates the efficiency performance of the column. The peak of Sitagliptin phosphate are
found well separated at 7.485 respectively. The developed method was validated for various
parameters as per ICH guidelines like Accuracy, Precision, Linearity, Specificity, Ruggedness,
Robustness, LOQ and LOD. This method exhibited an excellent performance in terms of
sensitivity and speed. The major advantage of this technique is that it is less time consuming
and also eco-friendly because of its low consumption of organic solvents as compared to other
analytical techniques. The foundations of reversed-phase high-performance liquid
chromatography (RP-HPLC), including the separation principle, alternatives for stationary and
mobile phases, and variables affecting RP-HPLC separation, are discussed. We will also discuss
the many steps involved in the development and validation of RP-HPLC methods and highlight
the many applications of RP- HPLC, including pharmaceutical analysis, food analysis, and
environmental analysis. Along with other recent advancements in RP-HPLC, new stationary
and mobile phases, RP-HPLC downsizing, and hyphenated techniques like LC-MS will also be
addressed.
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 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE,PCI New Delhi, DTE Bhopal and Govt. of
M.P.(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

PLAGIARISM CERTIFICATE

This is to certified that Thesis entitled_____________________________

___________________________________________________________________________________________________
___________________________________________________________________________________________________
_ ____________________________________were plagiarized by student through software and
the matched result was found to be ____%. The thesis was healthy and does not
contain any copyright material. The thesis was prepared and submitted under
my supervision.

Student Name Internal Examiner

Mr. Romit Jain Mr. Prashant Vinode

(Asso. Professor)
 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE,PCI New Delhi, DTE Bhopal and Govt. of M.P.
(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

Certificate
This is to certify that the thesis entitled “METHOD
DEVELOPMENT AND VALIDATION FOR ANTI DIABETIC DRUG
BY RP-HPLC” submitted in partial fulfillment of the requirement for
the degree of Master of Pharmacy in Pharmaceutical Chemistry to
Sagar Institute of Pharmaceutical Sciences, Sagar M.P., affiliated to
Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal (M.P) and wor k
carried out by Mr. Romit Jain, Enrollment No. 0602PY22MP10 during
the academic session 2023-2024. His work is satisfactory and not
submitted anywhere for the award of any other degree.
I hereby forwarded his Thesis..

SUPERVISED BY

Mr. Prashant Vinode

(Asso. Professor)

Sagar Institute of Pharmaceutical Sciences, Sagar M.P.

 SAGAR INSTITUTE OF PHARMACEUTICALSCIENCES,
SAGAR (M.P.)
Approved by AICTE, PCI New Delhi, DTE Bhopal and Govt. of M.P.
(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

Forwarding Letter
This is to certify that the thesis entitled “METHOD
DEVELOPMENT AND VALIDATION FOR ANTI DIABETIC DRUG
BY RP-HPLC” submitted in partial fulfillment of the requirement for
the degree of Master of Pharmacy in Pharmaceutical Chemistry from
Sagar Institute of Pharmaceutical Sciences, Sagar and work carried
out by Mr. Romit Jain, Enrollment No. 0602PY22MP10 under the
guidance and supervision of Mr. Prashant Vinode during the academic
session 2023-2024.
I hereby forwarded his thesis..

Forwarded by
Mr. Prashant Vinode
(Asso. Professor)

Sagar Institute of Pharmaceutical Sciences, Sagar M.P.

 SAGAR INSTITUTE OF PHARMACEUTICAL
SCIENCES, SAGAR (M.P.)
Approved by AICTE, PCI New Delhi, DTE Bhopal and Govt. of M.P.
(Affiliated to Rajiv Gandhi Proudyogiki Vishwavidyalaya Bhopal)

Declaration

I hereby declare that the work incorporated in the thesis is

entitled “METHOD DEVELOPMENT AND VALIDATION FOR
ANTI DIABETIC DRUG BY RP-HPLC” embodies my own work
except the guidance and suggestions received from my supervisor Mr.
Prashant Vinode, Sagar Institute of Pharmaceutical Sciences, Sagar
(M.P.).
I further declare that to the best of my knowledge the thesis does
not contain any part of any work which neither has been submitted for
the award of degree in any University not has been published anywhere
without proper citation.
I also declare that a check for plagiarism has been carried out on
this dissertation and is found within the acceptable limit and report of
which is enclosed herewith.

Supervised By Submitted By

Mr. Prashant Vinode Mr. Romit Jain

(Asso. Professor) (0602PY22MP10)

Forwarded by
Dr. Yuvraj Singh Dangi
(Principal)
 ACKNOWLEDGEMENT

To acknowledge one’s own dept is not an easy task. I feel deeply

obliged to express my profound gratitude and indebtedness to my diligent
project guide Mr. Prashant vinode (Associate Professor, SIPS, Sagar), his
ever inspiring guidance, keen interest; patient supervision and optimism
helped me to solve various problems encountered during the course of this
work. Her earnest desire for my every success will always be my pride and
my devotion and respect for her will be forever.

I express my feelings for Dr. Y. S. Dangi (Principal, SIPS, Sagar)

bottom from my heart, for his support, guidance and encouragements during
the journey for this path.

I express my regards to my parents for their love, care and whole

hearted support all through.

I heartly express my sincere thanks to my all teachers of SIPS, for their

support including Dr. Varsha Kashaw, Mr. Mukesh Patel, Mr. Akash Sharma,
and thanks to my seniors Mr. Lavkush Vishwakarma Mr. Ram Kishan
Mishra, and my friend Mr. Subodh Kumar forever encouragement.

Above all, I am greatly indebted to the almighty, for showering with

his/her infinite bounties graces and mercies upon me and for being my
constant companion.

Date: Mr. Romit Jain

Place: - SAGAR 0602PY22MP10
 CONTENT
Page
S. No. Title
Profile
1
INTRODUCTION 1-16
2
REVIEW OF 17-20
LITERATURE
3
DRUG PROFILE 21-22
4 RESEARCH ENVISAGED AND
23-24
PLAN OFWORK
5 MATERIALS AND METHODS 25-36
6
RESULT AND DISCUSSION 37

7
SUMMARY AND CONCLUSION 38

8
BIBLOGRAPHY 39-42
CHAPTER- 1
INTRODUCTION
Chapter-1 Introduction

1. INTRODUCTION
The science and practice of figuring out a material's composition based on the
elements it contains is known as analytical chemistry. The field of pharmaceutical analysis
is concerned with the analytical techniques used to assess the quality, safety, and purity of
medications and chemicals. It includes methods for figuring out a new compound's identity,
strength, quality, and purity. It also entails steps for sorting, classifying, and calculating the
proportions of each component in a sample of matter. Determining the safety and efficacy
of medications is largely dependent on Chemistry. Its analytical techniques, which are
extremely sensitive and specific, are used in the design, development, standardization, and
Chemistry of pharmaceuticals. For investigations on drug metabolism and
pharmacokinetics, they are equally crucial both of which are critical for evaluating clinical
response and bioavailability.
Contemporary physical methods of analysis are incredibly sensitive, even with small sample
sizes. It can be automated easily and is quickly applicable. As a result, it is extensively
utilized in the creation of new products as well as in the formulation, manufacturing, and
usage of medications.
Pharmaceutical analysis includes both quantitative and qualitative evaluations of
pharmaceuticals and pharmaceutical materials, ranging from unfinished dosage forms to
bulk drugs. As a result, it is employed as a diagnostic tool in contemporary medicine by
analyzing chemical components of the human body that may change when a patient is ill.
The pharmacist must take the required actions to ascertain whether the medication product
is faulty if a doctor has concerns about its quality.
The drug manufacturer can be contacted regarding the issue with the product, laboratory
analysis of the preparation, loaning necessary equipment from a clinical laboratory if
needed, sending the sample to a private laboratory for analysis, or a combination of all these
methods can be used to achieve this. In any event, though, analysts are still in charge of
finding solutions to issues with medication quality.
When referring to a drug product, "quality" is defined as the totality of the elements that
either directly or indirectly support the product's dependability, safety, and effectiveness.

1
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

Significance of quality control:

The pharmaceutical industry is still an essential part of the health care cycle because it
conducts research and produces goods that both maintain and restore life. Human-use
modern medications must adhere to strict guidelines pertaining to their efficacy, safety, and
quality. The existence of suitable procedures for product quality control is a prerequisite for
the practical assessment of safety, efficacy, and maintenance. The sample's species' chemical
identities are revealed through quantitative analysis. Through quantitative analysis, the
relative abundance of one or more of these species or analytes is determined numerically.
Analytical Techniques
A pharmaceutical product's safety and effectiveness can only be guaranteed by analytical
quality control. As a result, a medication's overall purity needs to be evaluated during
storage, distribution, and use. If the specifications to be applied are based on a validated
procedure that can show the quality relationship between the substance under examination
and that which was initially subjected to pharmaceutical, toxicological, and pharmacological
evaluation, then it may be possible to achieve the goal.
Optical methods
Some of the optical methods are
 X-ray spectroscopy
 UV-Visible spectroscopy
 Infrared spectroscopy
 Atomic absorption spectroscopy
 Flame photometry
 Nuclear magnetic resonance spectroscopy
 Nephlo-turbidimetry
 Electron spin spectroscopy
Electro analytical methods:
Some of the electro analytical methods are
 Amperometry
 Voltametry
 Potentiometry
 Conductometry

2
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

Separation methods/chromatography
Some of chromatographic methods are
 Gas-liquid chromatography
 Gas-solid chromatography
 Liquid-liquid chromatography
 Liquid-solid chromatography
 Thin-layer chromatography
 Paper chromatography
 Gel permeation chromatography
Selecting the best procedure for a particular analysis is one of the most important decisions
an analyst must make. To do this, he must be knowledgeable about the theoretical
underpinnings of the analysis as well as the practical aspects. He must also understand the
conditions under which each method is reliable, be aware of potential interferences that may
occur, and be able to devise workarounds for such issues.
The instrumental separative techniques are divided into two categories
1. Chromatography
2. Electrophoresis
3. Mass spectroscopy
Chromatography
Chromatography is a technique by which a mixture is separated into its components as a
result of the relative ability of each component to be eluted along or through the stationary
phase by mobile phase. The sample is placed on edge of the stationary phase (a solid or
liquid) and a mobile phase is allowed to flow over the stationary phase to sweep the sample
along the length of the stationary phase.
Component which are strongly adsorbed to the stationary phase are swept less rapidly along
the length of the stationary phase than those components that are less strongly adsorbed to
stationary phase.
Chromatography is a technique that separates a mixture into its constituent
parts based on how well each part can be eluted by the mobile phase along or through the
stationary phase. The sample is positioned at the edge of the stationary phase, which can be
either liquid or solid, and it is swept along the stationary phase's length by allowing a mobile
phase to flow over it More components that are strongly adsorbed to the stationary phase

3
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

sweep along its length more slowly than less strongly adsorbed components. that are strongly
adsorbed to the stationary phase sweep along its length more slowly than less strongly
adsorbed components.
The Greek letters chromos, which means color, and graphy, which means
color writing are the roots of the word chromatography. The terms were first used by Tswett,
who used a chromatography column made up of an adsorbent powder and a liquid solvent
known as the mobile phase to separate color bands of plant pigments. This travels the entire
length of the tube, which is home to a stationary phase—an immobile phase made of liquid
or solid.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
The theoretical underpinnings of earlier chromatographic techniques, particularly column
chromatography, were known when the high-performance liquid chromatography (HPLC)
technique was developed in the late 1960s and early 1970s. The method is predicated on the
same separation modes as traditional columns. For example, gel permeation, ion exchange,
adsorption, and partitioning (including reverse phase partitioning). Column chromatography
and HPLC are not the same because HPLC uses a high-pressure pump to force the mobile
phase through the packed column. Improved separation resolution, quicker separation times,
and increased sensitivity, accuracy, and precision in quantifying the separated substances
are the main benefits of HPLC over traditional (gravity feed) Improved separation
resolution, accelerated separation times, and enhanced sensitivity, accuracy, and precision
in quantifying the separated substances are all benefits of column chromatography.
Basic principle of HPLC
The separation method known as high performance liquid chromatography (HPLC) makes
use of variations in the distribution of substances in phases known as the stationary phase
and the mobile phase. The liquid that is flowing over the fine particles is referred to as the
mobile phase, and the thin layer that is formed on their surface is known as the stationary
phase. Depending on the molecular size and solubility in the phases, each component in a
sample has a distinct distribution equilibrium under a given dynamic solution. The
component move over the stationary phase at varying speeds as a result, eventually
separating from one another. The column consists of a tube made of stainless steel (or resin)
that is filled with solid, spherical particles. A liquid pump continuously feeds the mobile
phase into the column inlet at a constant rate. The sample injector, which is close to the
column inlet, is used to inject the sample.

4
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

The components of the injected sample migrate through the column, moving between the
stationary phase and mobile phase, as it enters the column with mobile phase.
Compounds move through the column more quickly when they are in the mobile
phase as opposed to when they are more evenly distributed in the stationary phase. The
component is thus separated on the column and elutes from the outlet in a stepwise manner.
Every compound that elutes from the column is detected by a detector that is attached to the
column's outlet.
Beginning at the moment the sample is injected, the recorder tracks the separation process
and generates a graph. We refer to this graph as a chromatogram. The properties of the
compound determine the retention time—the amount of time needed for the compound to
elute—and the correlation between the compound's concentration and peak area.
Selectivity of HPLC
Most of the drugs can be analysed by HPLC method because of several
advantages.
 Speed (analysis can be accomplished in 20 minutes or less).
 Greater sensitivity (various detectors can be employed).
 Improved resolution (wide variety of stationary phase).
 Reliable columns (wide variety of stationary phase).
 Ideal for substances of low volatility.
 Easy sample recovery, handling and maintenance.
 Easy programming of the numerous functions in each module.
 Time programmable operation sequence, such as initiating operation of
detector lamp and pump to obtain stable baseline and equilibrated column before the work
day begins.
 Excellent reproducibility of retention time.
Different Modes of Separation of HPLC
- Normal phase mode - Reverse phase mode
- Reverse phase ion pair chromatography
- Ion exchange chromatography
- Affinity chromatography
- Size exclusion chromatography (gel permeation and gel filtration chromatography)

5
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

Instrumentation for HPLC

 A solvent reservoir that allows the column to receive the mobile phase at a variety of
pressures and flow rates. To extract dissolved air and other materials from the solvents, a
degasser is required.
 A pump that feeds the column with the mobile phase. There must be no pulses in the
pumping system. A pump ought to have a minimum operating pressure of 100 atm (1500
psm), which is appropriate for less costly chromatography. However, a more ideal pressure
limit is 400 atm (600 psi). It was necessary to generate a moderate flow rate of 0.5 to 2
milliliters per minute for numerous analytical columns.
 To stop tiny particles from contaminating the main column, there might be an inline
filter or a guard column at the head of the separation column.
 The packing required to achieve the intended HPLC separation is present in the
separation column.
 These could be gels with a certain porosity for exclusion chromatography, silicas for
adsorption chromatography, bonded phases for liquid-liquid chromatography, ion exchange
functional groups bound to stationary support for ion exchange chromatography, or some
special packing for a specific separation technique.
 Short, fast columns are 3 to 8 cm long with an internal diameter of 4 to 5 mm. The
majority of column lengths are between 10 and 30 cm. Particle diameters for preparative
chromatography typically range from 3 to 5 µm, but they can also occasionally reach 10 µm
or greater.
A detector with some type of data handling device, completes the basic instrumentation.
The various detectors are
 UV visible photometers
 Refractive index detector
 Flourimetric detector
 Conductivity detector
 Amperometric detector
 PDA
In HPLC, detector electronic integrators and computing integrators are commonly utilized
for peak area measurement. These gadgets identify peaks on their own and print the areas as
numbers. Peak width can be computed (thinking of the peak as a triangle) and the number
of theoretical plates can be computed with the aid of peak areas and height values.

6
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

Quantitation methods in HPLC

Peak heights or peak area measurements only provide a response in terms of detector signal.
This response must be related to the concentration or mass of the compound of interest. To
accomplish this, some type of calibration must be performed.
The four primary techniques for quantization are
a. Normalized peak area method.
b. External standard method.
c. Internal standard method.
d. Method of standard addition.
a) Normalized peak area method
Normalized peak area is the area percentage of each individual peak. This method is widely
used to determine the response factor for each component and to estimate the relative
amounts of small impurities or degradation compounds in a purified material.
b) External Standard method
Both standard and unknown are injected using this method; the unknown is identified either
numerically using response factors or graphically from a calibration plot.
A response factor (RF) can be determined for each standard as follows.
RF = Standard area (peak height) / Standard Concentration
The external standard approach for most samples in HPLC that do not require extensive
sample preparation.
c) Internal Standard method
Although it is distinct from the analyte, the internal standard is well-resolved during the
separation process. It is best to select an internal standard that closely resembles the sample
component's behavior. When samples need extensive pre-treatment or preparation, one of
the key justifications for employing an internal standard is that situation.
d) Method of standard addition
A calibration plot for quantitative analysis can be produced using the standard addition
method. It is employed in trace analysis more frequently.
The response prior to spiking additional analytes should be high enough to provide a
reasonable S/N ratio (<10); otherwise, the result will have poor precision. This is an
important aspect of the standard addition method.(Vogel’s 2003)
The foundations of reversed-phase high-performance liquid chromatography (RP-HPLC),
including the separation principle, alternatives for stationary and mobile phases, and

7
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

variables affecting RP-HPLC separation, are discussed. We will also discuss the many steps
involved in the development and validation of RP-HPLC methods and highlight the many
applications of RP-HPLC, including pharmaceutical analysis, food analysis, and
environmental analysis. Along with other recent advancements in RP-HPLC, new stationary
and mobile phases, RP-HPLC downsizing, and hyphenated techniques like LC-MS will also
be addressed.
The pharmaceutical sector has found RP-HPLC to be crucial since it can separate and
quantify impurities and active pharmaceutical ingredients (APIs). The effectiveness and
selectivity of RP-HPLC have improved recently as a result of developments in both
stationary and mobile phases. Miniaturization advances have made it possible to analyze
smaller sample quantities with RP-HPLC at a lower cost and in less time. The creation of
hybrid techniques like LC-MS has led to increased sensitivity and selectivity in RP-HPLC
analysis.
FUNDAMENTALS OF RP-HPLC
A number of chromatographic parameters are also used to gauge the effectiveness of RP-
HPLC separations. The retention time is the length of time it takes for a molecule to elute
from the column after injection, whereas the selectivity factor is the ratio of the distance
between two peaks. Resolution measures the space between two successive peaks, while
capacity factor measures the compound's relative affinity for the stationary phase.
The first stage in developing a successful separation using the RP-HPLC technology is
selecting the stationary and mobile phases. It is crucial to pick a stationary phase that works
well with the mobile phase and can successfully trap the target analyte. It's critical to choose
a mobile phase that elutes the target analyte from the column and is compatible with the
stationary phase. To get the best separation conditions, it is possible to adjust a number of
parameters, including column temperature, mobile phase pH, flow rate, and gradient elution.
The optimization process aims to achieve maximum resolution, minimal analytical time, and
optimal solvent consumption.
To guarantee that RP-HPLC methods are reliable, reproducible, and precise, validation is
crucial. Some of the factors that are investigated during validation include accuracy,
precision, linearity, resilience, and limitations of detection and quantification.
Recent advancements in RP-HPLC include its downsizing, the introduction of hyphenated
methodologies like LC-MS, and the creation of new stationary and mobile phases. More

8
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

precise separation and analysis of complex compounds may be possible with newly
constructed stationary and mobile phases with increased selectivity and efficiency.
Miniaturized RP-HPLC can analyze smaller sample volumes, reducing run durations and
solvent consumption. By combining the advantages of RP-HPLC with mass spectrometry,
hybrid techniques provide more accurate and sensitive analyte detection. In conclusion, RP-
HPLC is a vital analytical chemistry technique, particularly for the investigation of
medicines. The creation of accurate and dependable RP-HPLC techniques is necessary for
determining the quality, safety, and efficacy of drug products.
The stationary and mobile phases must be carefully considered, the separation parameters
must be fine-tuned, and RP-HPLC methodologies must be validated in order to achieve the
perfect separation. One way that RP-HPLC is changing to become more adaptable and
potent in the analytical environment is through the addition of new stationary and mobile
phases, downsizing, and hyphenated techniques.
APPLICATIONS OF RP-HPLC IN PHARMACEUTICAL ANALYSIS
RP-HPLC is a widely used technique in the pharmaceutical industry for drug analysis due
to its high sensitivity, selectivity, and reproducibility. The application of RP-HPLC in
pharmaceutical analysis involves the separation and quantification of active pharmaceutical
ingredients (APIs), impurities, degradation products, and other related substances.
One of the primary applications of RP-HPLC in pharmaceutical analysis is the determination
of drug purity. This involves the separation of the API from other components in the sample
and quantifying the amount of the API present. The analysis of drug impurities is also an
essential application of RP-HPLC.
Impurities can arise from various sources, such as starting materials, intermediates, and
degradation products. RP-HPLC is capable of separating these impurities from the API and
determining their concentration in the drug substance.
RP-HPLC is also used in the analysis of drug-drug interactions. This involves the separation
and quantification of the drug and its metabolites in biological matrices, such as plasma or
urine. The analysis of drug-drug interactions is essential to evaluate the safety and efficacy
of drugs and to determine the pharmacokinetic parameters of drugs . In addition to the above
applications, RP-HPLC is also used in the analysis of drug stability, bioavailability, and
pharmacokinetics. The stability of drugs is determined by subjecting the drug substance to
various stress conditions and analyzing the resulting degradation products. RP-HPLC is used
to separate and quantify the degradation products formed during the stability studies.

9
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

The bioavailability and pharmacokinetics of drugs are also determined by RP-HPLC

analysis of biological matrices such as plasma or urine.
In conclusion, RP-HPLC is an essential technique in pharmaceutical analysis due to its high
sensitivity, selectivity, and reproducibility. The applications of RP-HPLC in pharmaceutical
analysis include drug purity determination, impurity analysis, analysis of drug formulations,
analysis of drug degradation products, analysis of drug-drug interactions, drug stability
studies, and determination of bioavailability and pharmacokinetics of drugs.
VALIDATION
A crucial step in efficient Chemistry is validation. "Validation is established documented
evidence that offers a high degree of assurance that a device or process will reliably yield a
result that satisfies its predefined quality attributes and specifications." Definition
USFDA defines validation as “established documented evidence which provides a high
degree of assurance that a specific process will consistently produce a product of
predetermined specifications and quality attributes.”
EUGMP defines validation as “action of proving in accordance with the principle of Good
manufacturing practice (GMP), that any material activity or system actually lead to expected
result”.
AUSTRALIAN GMP defines validation as “the action of proving that any material,
process, procedure, system, equipment or mechanism used in manufacture or control can
and will be reliable and achieve the desire and intended result.
Importance of validation
1. Because routine quality control tests a statistically insignificant number of samples, the

product's quality cannot always be guaranteed.

2. A high degree of confidence that the same level of quality is consistently built into each

finished product from batch to batch should be provided by the validation, which should
demonstrate the system's or product's sufficiency and reliability in meeting the specified
criteria or attributes.
Objectives of validation
Validation serves as the foundation for written procedures for production and process
control, which are intended to ensure that pharmaceutical products are who they say they
are and are purported to be in terms of identity, quality, and purity.

10
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

1. Assurance of quality
2. Government regulation.

Types of validation
The following are frequently required to be validated on a pharmaceutical process
1. Equipment validation
2. Process validation
3. Cleaning validation
4. Analytical method validation
5. Facility validation including utilities.
ANALYTICAL METHOD VALIDATION
The process of verifying that the analytical technique used for a particular test is appropriate
for its intended use is known as method validation. To guarantee a pharmaceutical product's
purity, stability, safety, and efficacy, analytical testing is required. Validation of analytical
methods is a crucial component of the quality control system.
Analyzing a sufficient number of aliquots from a homogeneous sample to compute
statistically valid estimates of standard deviation or relative standard deviation is how one
assesses the precision of an analytical method.
PARAMETERS USED FOR ASSAY VALIDATION
The validations of the assay procedure are carried out using following parameters.
SPECIFICITY:
The capacity to clearly assess analyte in the presence of contaminants, degradants, matrix,
etc. that may be anticipated to be present is known as specificity. Other supporting analytical
procedures may make up for a particular analytical procedure's lack of specificity.
PRECISION:
Definition
The degree to which multiple measurements taken from the same homogeneous sample
under specified conditions agree with one another is expressed as the precision of an
analytical procedure. The variance, standard deviation, or coefficient of variation of a series
measurement are typically used to express the precision of an analytical process.
System precision A system precision is evaluated by measuring the peak response for the
six replicable injection of the same standard solution prepared as per the proposed method.
The %RSD is calculated and it should not be more than 2%.

11
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

Method precision
A method precision is evaluated by measuring the peak response for six replicate injection
of six different weigh of sample solution prepared as per proposed method. The %RSD is
calculated and it should not be more than 2%.
Determination
Analyzing a sufficient number of aliquots from a homogeneous sample to compute
statistically valid estimates of standard deviation or relative standard deviation is how one
assesses the precision of an analytical method.
ICH Requirements
The International Committee of Harmonization (ICH) recommended that in order to
evaluate repeatability, a minimum of nine determinations should be made within the
procedure's specified range (that is, three concentrations and three replicates of each
concentration, or a minimum of six determinations at 100% of the test concentration).
ACCURACY
Definition
The accuracy of an analytical procedure express the closeness of the agreement between the
values which is acceptable either as conventional true value or an accepted reference value
and the value found.
Determination
When determining the accuracy of a drug assay in a formulated product, the analytical
method can be applied to synthetic mixtures of the drug product's constituent parts to which
a known quantity of analyte has been added within the method's tolerances. If obtaining
every component of the product is not feasible, it might be acceptable to either add known
amounts of the analyte to the drug product or compare the outcomes with a second, well-
characterized method whose accuracy has been declared or defined.
The guidelines for submitting samples and analytical data for method validation state that
accuracy studies should be conducted at the 80,100, and 120% level of label claim for drug
substances and drug products. Replica samples are assessed at each suggested level of study.
The analysis variation or test method precision can be obtained from the replicates' RSD.
The accuracy of the test procedure is indicated by the mean of the replicates, which is
represented as %label claim.

12
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

ICH Requirements
The ICH documents recommended that accuracy should be assessed using a minimum of
nine determinations over a minimum of three concentration levels, covering the specified
range (i.e., three concentration and three replicates of each concentration).
LINEARITY
Definition
The linearity of an analytical procedure is its ability (with in a given range) to obtain the test
results which are directly proportional to the concentration (amount) of analyte in the
sample.
Determination
A minimum of five concentrations are required to establish the linearity of an analytical
procedure. First, it is determined visually by analyzing the plot of signals as a function of
the content's analyte concentration. Test results are established using the proper statistical
techniques if a linear relationship appears (i.e., by calculation of the regression line by the
method of least squares)
LIMIT OF DETECTION (LOD)
Definition
LOD is the lower concentration of the substance that the method can detect but not
necessarily quantify. LOD simply indicates that the sample below or above a certain level.
Determination
For non-instrumental methods, the detection limit is generally determined by the analysis of
samples with known concentration of analyte and by establishing the minimum level at
which the analyte can be reliably detected.
ICH Requirements
The ICH describes a common approach, which is to compare measured signal from samples
with known concentrations of analyte with those of blank samples. The minimum
concentration at which the analyte can reliably be detected is established. Typically,
acceptable signal-to-noise ratios are 2:1 or 3:1.
LIMIT OF QUANTITATION (LOQ)
Definition
The lower limit of quantification (LOQ) is the substance's concentration at which the
suggested method can estimate it quantitatively with a sufficient level of precision, accuracy,
and reliability. The lowest level at which an acceptable degree of accuracy and precision is

13
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

attained is known as the limit of quantification, or LOQ, and is established by analyzing

samples with progressively decreasing known quantities of the substance.
Determination
For non-instrumental methods, quantization limit is generally determined by the analysis of
samples with known concentration of analyte and by establishing the minimum level at
which the analyte can be determined with acceptable accuracy and precision.
RANGE
Definition
An analytical procedure's range is the range of concentrations (amounts) of analyte in the
sample, including these concentrations, for which the analytical procedure has been shown
to have an appropriate degree of linearity, accuracy, and precision.
Determination
The range of the method is validated by verifying that the analytical method provides
acceptable precision, accuracy and linearity when applied to samples containing analyte at
the extremes of the range as well as within the range.
ROBUSTNESS
Definition
An analytical procedure's resilience to small, purposeful changes in method parameters is
measured by its robustness, which also indicates how reliable it is under typical operating
conditions.
Determination
The method's robustness was evaluated by conducting an assay with purposeful parameter
changes (flow rate ±10%, mobile phase ratio ±2, mobile phase pH ±0.2, wave length
detection ±5 nm, temperature ±1 to 50), and confirming that the changes did not affect the
results.
RUGGEDNESS
Definition
The degree of repeatability of test results from the analysis of samples under various
conditions—different laborites, different analysts, different instruments, different lots of
reagents, varied elapsed assay times, varied assay temperatures, varied days, etc.—defines
the ruggedness of an analytical method.

14
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

Determination
The ruggedness of analytical method is determined by the analysis of aliquots from
homogenous lots in different laboratories, by different analysis, using operational and
environmental condition that may differ but are still within the specified parameters of the
assay. The degree of reproducibility of the results is that determined as a function of assay
variables. This reproducibility may be compared to the precision of assay under normal
condition to obtain a measure of the ruggedness of the analytical method.
SAMPLE SOLUTION STABILITY
It is important to assess the drug substance's or drug product's solution stability following
preparation in accordance with the test protocol. The majority of labs use auto samples that
are run overnight; the sample must spend hours in solution in the lab before the remaining
steps are finished.
This is particularly concerning for medications that can degrade via photolysis, hydrolysis,
or adhesion to glass surfaces.
SYSTEM SUITABILITY SPECIFICATION AND TESTS
The accuracy and precision of HPLC data collected begin with a well-behaved
chromatographic system. The system suitability specifications and tests are parameters that
provide assistance in achieving this purpose.
It consists of following factors:
1. Capacity factor
2. Precision\Injection repeatability
3. Relative retention
4. Resolution
5. Tailing factor
6. Theoretical plate number
1. Capacity factor (K’)
K’ = (tR-tO/tf)
The capacity factor is a measure of where the peak of interest is located with respect to the
void volume i.e., elution time of the non-retained components.
2. Precision/Injection repeatability (RSD)
The performance of the HPLC at the time the samples are analyzed, including the pumping,
column, and environmental conditions, is indicated by the injection precision, which is

15
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-1 Introduction

expressed as RSD (relative standard deviation). Note that variations in manufacturing and
sample preparation are not taken into account.
3. Relative retention (α)
α = K’1/K’2
Relative retention is a measure of the relative location of two peaks. This is not an essential
parameter as long as the resolution (Rs) is stated.
4. Resolution (Rs)
Rs = (tR2-tR1)/ (1/2) (tw1+tw2)
The distance between two peaks is measured in terms of Rs. Well-separated peaks are
necessary for accurate quantification. If you are concerned about possible inference peaks
(s), this parameter is quite helpful.
5. Tailing factor
T = Wx/2f
Because the integrator finds it difficult to determine when and where the peak ends, and
therefore to calculate the area under the peak, the accuracy of quantitation decreases as peak
tailing increases. The analyst uses the integrator variables to calculate the area for the peak
of interest as accurately as possible. Accuracy decreases if the integrator cannot pinpoint the
precise moment at which a downslope for an upslope occurs.
6. Theoretical plate number (N)

N = 16(tR/tw) 2 = L/H

Theoretical plate number is a measure of column efficiency, that is, how many peaks can be
located per unit run-time of the chromatograph.
N- Constant for each peak on the chromatogram with a fixed set of operating conditions.
H- Height equivalent of a theoretical plate.
L- Length of column.

16
Method Development and Validation for Anti Diabetic Drug By-HPLC
CHAPTER- 2
REVIEW
OF LITERATURE
Chapter-2 Review of Literature

2. REVIEW OF LITERATURE

Peram, M. R.,et,al.(2021): Reverse-phase liquid chromatography (RP-HPLC) is the

preferred separation mode for high-performance liquid chromatography (HPLC) due to its
adaptability and higher selectivity for hydrophobic compounds. This review article discusses
the fundamentals of reversed-phase high-performance liquid chromatography (RP-HPLC).
This covers the separation principle, various stationary and mobile phase types, and
separation-affecting variables. This article highlights the need of developing and testing
such methods in addition to outlining the advantages of using RP-HPLC in industries like
pharmaceutical, food, and environmental analysis. As examples of more recent
advancements in RP-HPLC, new stationary and mobile phases, RP-HPLC downsizing, and
hyphenated methods are also discussed. This review article provides a comprehensive tool
for designing, refining, and validating RP-HPLC processes.
Sharma S.,et,al.(2018): Analytical method development and validation play important roles
in the drug discovery, drug development and manufacture of pharmaceuticals. It involves detection
of the purity and toxicity of a drug substance. The present study focuses on the various steps,
parameters involved in HPLC condition. It can be adopted apparently for routine quality control
study of research and formulation tests. This article mainly focuses on the optimization of HPLC
conditions and other important aspects during method of process development and validation of
drug substances.
G.Mubeen et al., (2010) have been developed a simple Spectrophotometric method has
been developed and validated for the estimation of Metformin hydrochloride in bulk and in
tablet formulation. The primary amino group of Metformin hydrochloride was oxidized
using hydrogen peroxide to form a yellow chromogen, which is determined
spectrophotometrically at 400 nm. It obeyed Beer’s law in the range of 4- 26mcg/ml.The
percentage recovery of the drug for the proposed method ranged from 99-101.3% indicating
no interference of the tablet excipients. The proposed method was found to be accurate and
precise for routine estimation of Metformin hydrochloride in bulk and in tablet dosage
forms.
G. Mubeen, et.,al.(2010): Have been developed for determination of Metformin
hydrochloride in bulk and in tablets.The proposed method was validated as per the standard
analytical procedures.The precision studies of the method and system were carried out and
the % RSD was found to be less than 2% indicating that the method is precise.

17
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-2 Review of Literature

S.Havele et al., (2010) have developed a simple, rapid, and precise reversed-phase high-
performance liquid chromatographic method for simultaneous analysis of metformin
hydrochloride, gliclazide, and pioglitazone hydrochloride in a tablet dosage form has been
developed and validated. Chromatography was performed on a 25 cm × 4.6 mm i.d., 5-
column with 85:15 (v/v) methanol: 20 mM potassium dihydrogen phosphate buffer as
mobile phase at a flow rate of 1.2 ml/min. UV detection at 227 nm; metformin
hydrochloride, gliclazide, and pioglitazone hydrochloride were eluted with retention times
of 2.15, 3.787, and 4.57 min, respectively. The method was validated in accordance with
ICH guidelines. Validation revealed the method is specific, rapid, accurate, precise, reliable,
and reproducible. Calibration plots were linear over the concentration ranges 50– metformin
hydrochloride, 3.0 – r gliclazide, and 2– pioglitazone hydrochloride. Limits of detection
were 0.20, 0.04, and 0.10 metformin hydrochloride, gliclazide, and pioglitazone
hydrochloride, respectively. The high recovery and low coefficients of variation confirm the
suitability of the method for simultaneous analysis of the three drugs in tablets. Statistical
analysis proves that the method is suitable for the analysis of metformin hydrochloride,
gliclazide, and pioglitazone hydrochloride as a bulk drug and in pharmaceutical formulation
without any interference from the excipients. It may be extended to study the degradation
kinetics of three drugs and also, for its estimation in plasma and other biological fluids.

K.S. Lakshmi et al., (2009) have been developed a simple, sensitive and rapid reverse phase
high performance liquid chromatographic method was developed for the estimation of
Metformin Hcl (MET) and Pioglitazone (PIO) in pure and in pharmaceutical dosage forms.
A Gemini C18 column (150x4.6mm, 5μ) was used with a mobile phase containing a mixture
of Acetonitrile and Ammonium Acetate buffer (pH-3) in the ratio of 42: 58. The rate was
0.3ml/min and effluents were monitored at 255nm and eluted at 5.17min (MET) and 8.1min
(PIO). Calibration curve was plotted with a range from 0.5-50 μg/ml for MET and 0.3-30
μg/ml for PIO. The assay was validated for the parameters like accuracy, precision,
robustness and system suitability parameters. The proposed method can be useful in the
routine analysis for the determination on metformin and pioglitazone in pharmaceutical
dosage forms.
Maria-Cristina Ranetti et al., (2009) have been developed a simple HPLC method for the
simultaneous determination of metformin (MTF) and gliclazide (GCZ) in the presence of
glibenclamide, in human plasma, for the clinical monitoring of MTF and GCZ after oral
18
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-2 Review of Literature

administration or for bioequivalence studies. Ion-pair separation followed by UV detection

performed on deproteinised plasma samples was chosen for the determination of metformin
and gliclazide.The mobile phase was acetonitrile: methanol (1:1v/v) and sodium
dodecylsulphate 5mM, pH=3.5 with H3PO4 85% and gradient elution. The eluent was
monitored at 236 nm. The calibration curve was linear within the range of 0.05-5.00 μg/mL
(r2=0.99, n=6). The lowest limit of quantification (LLOQ) was 50 ng/mL for metformin and
49 ng/mL for gliclazide.The proposed method was validated and proved to be adequate for
metformin and gliclazide clinical monitoring, bioavailability and bioequivalence studies.
Bhusari KP et al., (2009): Simple, selective and sensitive spectrophotometric method has
been developed for the determination of Sitagliptin phosphate pharmaceutical
formulations. The method was completely validated and proven to be rugged. The
interferences of the other ingredients and excipients were not observed. The repeatability
and the performance of the proven method were conventional by point and interior
proposition and through recovery studies.
K.S. Lakshmi, et,al.(2009): The proposed method was found to be simple, precise, accurate
and rapid for simultaneous determination of Metformin and Pioglitazone from pure and in
pharmaceutical dosage forms. The mobile phase is simple to prepare and economical. The
sample recoveries in all formulations were in good agreement with their respective label
claims and they suggested non-interference of formulation excipients in the estimation.
Hence, the method can be easily and conveniently adopted for routine analysis of Metformin
and Pioglitazone in combined dosage forms and can also be used for dissolution.
Jitendra D et al. (2009): developed a simple, sensitive and accurate reversed phase high
performance liquid chromatographic method for Enoxaparin sodium and Sitaglipitin drugs.
Reversed phase chromatographic separation the above two drugs was performed a C18
column is used with different mobile phases of water, orthophosphoric acid,
tetrahydrofuranan methanol, acetonitrile, sodiumdihydrogen phosphate, orthophosphoric
acid respectively. The detection of wave length is 230 nm for Enoxaparin sodium and 270
nm for Sitagliptin phosphate.The percentage of recovery 99.5% for enoxaparin sodium and
97.6% for sitaglipitin. The proposed method is validated for linearity, accuracy, and
precision, limit of detection (LOD) and limit of quantification (LOQ) as per the guide lines
of Internationational Conference on Harmonization (ICH).
Denney, R.C.,et,al.(2003):developed a simple, sensitive and rapid reverse phase high
performance liquid chromatographic method for simultaneous estimation of Sitagliptin

19
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-2 Review of Literature

phosphate and Metformin. A BDS hypersil C18 column (250x4.0mm,5μ) was used with a
mobile phase containing a mixture of phosphate buffer (Ph-4) and Acetonitrile and in the
ratio of 60:40. The flow rate was 1.0ml/min and effluents were monitored at 260nm and
eluted at 2.8min and 2.0min respectively. Calibration curve was plotted with a range from
2-12μg/ml for Sitagliptin phosphate and 20-120 μg/ml for Metformin.
Beckett, A.H et.al, (1997): The method was validated for accuracy, precision, linearity,
LOD, LOQ, Robustness, Ruggedness, specificity, and sensitivity in accordance with ICH
guide lines. Validation revealed the method is specific, rapid, accurate,The high recovery
and low coefficients of variation confirm the suitability of the method for metaformin HCL
in tablets. The validated method was successfully used for quantitative analysis of
metaformin HCL tablets. Find out the impurities of given metaformin HCL by using the
validated method with help of HPLC.
Hanssen et.al,(1991): Pharmaceutical analysis is a bench of science that deals with the
analytical procedures used to determine the purity, safety and quality of drugs and chemicals.
It contains procedures to determine the identity, strength, quality and purity of new
compounds. It also involves procedures for separating, identifying, and determining the
relative amount of the components in sample of matter. Chemistry plays a key role in
finding the safety and efficiency of medicines. It has highly specific and sensitive
analytical methods for the design, development, standardization and quality control of
medicinal products.

20
Method Development and Validation for Anti Diabetic Drug By-HPLC
CHAPTER- 3
DRUG PROFILE
 3. DRUG PROFILE

SITAGLIPTIN PHOSPHATE

Molecular formulae: C16H20F6N5O6P

Molecular Weight: 523.32

IUPAC Name: (R)-4-oxo-4-[3-(trifluoromethyl)-5,6 dihydro

[1,2,4] triazolo[4,3-a] pyrazin 7(8H)-yl]-1-

(2,4,5-) butan-2-amine Phosphate (1:1)

monohydrate.

Category: Antidiabetic

Dose: 25 to 100 mg.

Solubility: Soluble in water and N, N Dimethyl formamide;

Melting Point: 216-219 OC

Storage: Store in well-closed containers.

21
Method Development and Validation for Anti Diabetic Drug By-HPLC
PHARMACOLOGY:
Mechanism of action:
Sitagliptin phosphate works to inhibit the enzyme dipeptidyl peptidase 4
(DPP-4). This enzyme breaks down the incretins GLP-land GIP,
gastrointestinal hormones released in response to a measure. By preventing
GLP-1 and GIP inactivation, they are able to potentiate the secretion of
insulin and suppress the release of glucagon by the pancreas. This drives blood
glucose levels towards normal. As the blood glucose level approaches normal,
the amounts of insulin released and glucagon suppressed diminishes, thus
tending to prevent an "overshoot" and subsequent low blood sugar
(hypoglycemia) which is seen with some other oral hypoglycemic agents

Sitagliptin phosphate is an oral anti hyperglycemic of the dipeptidyl

peptidase-4 (DPP-4) inhibitor class. This enzyme-inhibiting drug is used
either alone or in combination with other oral anti hyperglycemic agents
(such as metformin or a thiazolidinedione) for treatment of diabetes
mellitus type 2. Sitagliptin phosphate works to competitively inhibit the
enzyme DPP-4. This enzyme breaks down the incretins GLP-1 and GIP,
gastrointestinal hormones released in response to a meal. By preventing
GLP-1and GIP inactivation, they are able to increase the secretion of
insulin and suppress the release of glucagon by the pancreas. This drives
blood glucose levels towards normal.
Side effects:
The most common side effects of sitagliptin phosphate are abdominal pain,
nausea diarrhea, vomiting and hypoglycemia. The symptoms of lactic acidosis
are weakness, trouble breathing, abnormal heartbeats, unusual muscle pain
stomach discomfort, lightheadedness and feeling cold. Patients at risk for lactic
acidosis include those with reduced function of the kidneys or liver, congestive
heart failure severe acute illnesses, and dehydration.

22
Method Development and Validation for Anti Diabetic Drug By-HPLC
 CHAPTER- 4
RESEARCH ENVISAGED
AND PLAN OF WORK
Chapter-4 Research Envisaged and plan of work

4. RESEARCH ENVISAGED AND PLAN OF WORK

The drug analysis is playing an important role in the development of drugs, their
manufacture and therapeutic use. For the simultaneous estimation of drugs present in dosage
forms, lot of suitable methods are adopted like uv – spectrophotometer, HPLC, HPTLC etc.
These methods are powerful and rugged method. They are also extremely precise, specific,
accurate, linear and rapid.
A pharmaceutical industry depends upon quantitative chemical analysis to ensure that the
raw material used and the final product obtained meets the required specification. The drugs
will occur as a single component or multi component dosage forms. The later proves to be
effective due to its combined mode of action on the body.
The number of drugs or drug formulations introduced into the market is increasing at a fast
rate. These may be either new entries in the market or structural modification of the existing
drugs or novel dosage forms or multicomponent dosage forms. The complexity in the dosage
forms, including that of the multi component dosage forms creates considerable challenges
to the analytical chemist during the development of assay procedure for its accurate
estimation. The estimation of individual drugs in these multi component dosage forms
becomes difficult due to tedious extraction or isolation procedure.
The Sitagliptin phosphate was selected for the present study.
According to the literature survey conducted, it was observed that no method was reported
in RP-HPLC for the estimation of individual drug carried out. Hence present study aims to
develop an accurate, precise, specific, linear, simple, rapid, validated and cost-effective
analytical method for Sitagliptin phosphate in tablet dosage form by RP-HPLC method.
The scope of our work extends to validate for the developed method as per ICH guidelines.
RP-HPLC method development was obtained as
 Selection and optimization of mobile phase and stationary phase.
 Selection of detector wavelength.
 Selection of extraction procedure.
 Optimization of chromatographic condition.
 Estimation of Sitagliptin phosphate
 Method Validation

23
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-4 Research Envisaged and plan of work

PLAN OF WORK:

The purpose of project work will be performed as per below mention plan-:

1. Literature survey
2. Procurement of drug and chemicals
3. Analytical method development by RP-HPLC.
4. Validation of methodology
(a) Linearity
(b) Range
(c) Accuracy
(d) Precision
(e) Specificity
(f) Robustness
5. Statistical analysis of the results.
6. Compilation of data & thesis submission.

24
Method Development and Validation for Anti Diabetic Drug By-HPLC
CHAPTER- 5
MATERIALS AND
METHODS
Chapter-5 Materials and Methods

5. MATERIALS AND METHODS

Sitagliptin phosphate (API Form) was received as a gift sample form Biotech solutions and
pharmaceuticals Pvt. Ltd. Madhura nagar Hyderabad India. Marketed formulation of is
Sitagliptin phosphate Accept SR in 100 mg tablet for Adult. these registered trademark of
vivid biotek inc. Delhi etc. All other chemicals used were of HPLC grade.
Instrumentation:

S.No. Name of instrument Model Make

1. Semi micro balance CPA225D Sartorius
2. pH meter Metler Toledo Thermo Orion
3. HPLC LC-20 AT Shimadzu
4. C 18 Column Phenomenex Gemini
5. Sonicator USB Spectro lab
6. UV 1700 series Shimadzu

Initial separation condition

The following chromatographic conditions were fixed initially to improve

the separation of drugs.
Instrument : Shimadzu prominence
Column : Phenomenex Gemini C18 (250 × 4.6mm), 5µ.
Column oven temperature : Ambient
Wavelength : 260nm
Flow rate : 1.2ml/min
Injection volume : 20µl
Run time : 10 min
Mobile phase : Solvent A – Phosphate Buffer
Solvent B – Methanol
Solvent Ratio : 50:50% V/V

25
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

METHOD DEVELOPMENT GUIDE

Information on sample, define separation goals.

Need for special procedure, sample treatment.

Choose detector and detector settings.

Choose the method: preliminary run: estimate the best separation condition

Optimize separation conditions.

Validate the method.

Selection of chromatographic method

Proper selection of the method depends on the nature of the sample (ionic or ionizable or
neutral molecules), its molecular weight, pka value and stability. The drugs selected in the
present study are polar and so reversed phase or ion exchange chromatography can be
used.The reverse phase HPLC was selected for the initial separation because of its
simplicity and suitability.For the literature survey and with knowledge of properties of the
selected drugs, Phenominex Gemini C18 (250 × 4.6mm) 5µ column was chosen as
stationary phase and mobile phase with different compositions such as Acetonitrile was
used. The separations were not observed so use of buffer was finalized.

26
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

Sitagliptin phosphate standard solution of 100 ppm was scanned at 200-400 nm and
UV Spectrum was recorded. By observing the spectrum of standard solution, λmax of
267 nm was taken for trails to develop the proposed method. UV-spectra of sitagliptin
phosphate phosphate is shown in figure.1.

Fig.1 UV spectrum of sitagliptin phosphate in water (100 µg/ml)

Preparation of buffer

Phosphate buffer was prepared by dissolving 0.68 gm of potassium dihydrogen

orthophosphate in 500 mL of double distilled water. PH was adjusted to 2.5 with
0.2% ortho phosphoric acid and solution was filtered through 0.45 μ Millipore
Nylon filter.
Mobile phase preparation

0.01M Phosphate buffer adjusted to PH 2.5: methanol in a ratio 50:50 % v/v was
taken, sonicated for 15 minutes and filtered through 0.45µ Millipore Nylon filter
under Vacuum filtration. The prepared solution was used as Mobile phase.
27
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

Diluent Preparation Diluent 1: Water Diluent 2: Mobile phase

Preparation of solutions Standard stock solution:
10 mg of Sitagliptin phosphate was accurately weighed and dissolved in diluent1 in
a 100 ml volumetric flask and the solution was made up to 100 mL with diluent 1to
obtained concentration of 100 µg/ml.
Working Standard solution:
1mL of standard stock solution was pipetted into 10 mL volumetric flask and
diluted up to the mark with diluent 2 and filtered through 0.45μ Millipore Nylon
filter to obtained concentration of 10 µg/ml.
Sample stock solution:

20 tablets were weighed and average weight of tablet was calculated. The tablets
were crushed into a fine powder using mortar and pestle. 42 mg of tablet powder
equivalent to 10 mg of Sitagliptin phosphate monohydrate was weighed accurately
and transferred into a 100 mL clean and dry volumetric flask. Then 70 ml of diluent
1 was added, sonicated for 15 min. and then volume was made up to the mark with
the diluent 1 to obtained concentration of 100 µg/ml. Further1 mL of above sample
stock solution was pipetted into a 10 mL volumetric flask and diluted up to the
mark with diluent 2, filtered through 0.45 μ Millipore Nylon filter to obtained final
concentration of 10 µg/ml.
Calculation:

Determine the % amount of Sitagliptin phosphate in tablets according to the following

formula.
AT ×W R × 3 × 100 × 50 × PR × Average Weight
%Assay = × 100
AR × 100 × 50 × WT × 3 × 100 × LA

Where, AT = Area in the test solution

AR = Area in the standard solution

WR = Weight of standard solution (mg)

WT = Weight of sample in test preparation (mg)

PR = Purity of working standard (%)

LA = Labeled amount of Sitagliptin phosphate per Tablets.

28
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

METHOD VALIDATION
The method was validated in terms of the following parameters; linearity,
specificity, accuracy, precision, and system suitability parameters as per the ICH
guidelines.
Specificity
To determine specificity, a volume of 10µl of working standard, sample and blank
solution were injected separately and the chromatograms were recorded and are
shown in fig. 2 and 3.

Fig.2 Chromatogram of sitagliptin phosphate in standard solution

Fig.3: Chromatogram of sitagliptin phosphate in sample solution

29
Method Development and Validation for Anti Diabetic Drug By-HPLC
 Chapter-5 Materials and Methods

Linearity

A series of working standard solutions of Sitagliptin phosphate monohydrate

were prepared in the concentration range from 5 to 30(μg/mL) and injected
into the chromatographic system. A calibration graph is plotted between
concentration of Sitagliptin phosphate monohydrate (μg/mL) and
chromatographic peak area (mV). The results are tabulated in Table no.1 and
linearity graph was shown in Fig 4.
Table 1: Data of linearity study

S. No. Concentration Peak Area Statistical analysis

(µg/mL)

1. 5 32500
2. 10 66306
Slope=6326 Intercept=945.7
3. 15 99201 Correlation coefficient= 0.999

4. 20 122467
5. 25 156734
6. 30 193693

Fig.4: Calibration curve of sitagliptin phosphate (5-30 µg/ml)

30
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

Accuracy studies

A known amount of working standard at three different levels i.e. 50%, 100%,
and 150% were added to pre analyzed sample solution of 100% concentration
and injected each three times in to the chromatographic system. From this %
recovery was calculated. Results of the recovery studies are shown in Table
no 2.

Table: 2 Data of Accuracy (Recovery

studies)

Statistical Analysis
S. Spiked Amount of Amount added amount %
No level drug from (µg/ml) found Recovery
Mean± %RSD
Formulatio (μg/mL) SD(n=3)
n (μg/mL)

1. 15.10 100.7 100.6 ± 0.37

50% 10 5 0.374
15.02 100.1

15.15 101.0

2. 19.91 99.5 99.84 ± 0.25

100% 10 10 0.251
20.02 100.1
19.98 99.92
25.22 100.8
3. 150% 10 15 25.34 101.3 100.4 0.97
24.78 99.14 ±0.923

Precision Studies
PRECISION
Precision is the measure of the degree of repeatability of an analyte method under
normal operation and is normally expressed as percent relative standard deviation for
a significant number of the samples.
According to the ICH precision should be performed at three different levels:
Repeatability, Intermediate precision, Reproducibility.
System precision:
The system precision was established by injecting six replicate injections of working
standard solution into the chromatographic system and the results are shown in table no.3
31
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

Table 3: Data of system precision

Injection Retention time (min) Peak area

Injection-1 3.073 66287

Injection-2 3.100 67736

Injection-3 3.173 66286

Injection-4 3.113 66262

Injection-5 3.200 67585

Injection-6 3.173 67591

Mean ± S.D 3.138 ± 0.045 66957 ± 681.3

%RSD 1.43 1.01

Method precision:
The method precision was established by injecting six freshly prepared sample
solutions into the chromatographic and the results are shown in table no.4

Table 4: Data of method precision

Injection Retention time (min) Peak area

Injection-1 3.120 71511

Injection-2 3.113 72575
Injection-3 3.113 71676
Injection-4 3.120 71946
Injection-5 3.160 71106
Injection-6 3.120 71851
Mean ± S.D 3.124 ± 0.016 488.1
%RSD 0.512 0.679

32
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

Sensitivity

The sensitivity of Sitagliptin phosphate by the use of proposed method was estimated
in terms of the limit of quantitation (LOQ) and the limit of detection (LOD). The LOQ
and LOD were found to be 0.6 µg/ml and 1.9 µg/ml.
LIMIT OF QUANTITATION (LOQ)
The limit of Quantitation (LOQ) is defined as the lowest concentration of the analyte
in a sample that can be determined with acceptable precision and accuracy under the
stated operational conditions of the method. Limit of Quantitation (LOQ) is also
based on standard deviation of the response and theslope of calibration curve.

LOQ = 10s
S
s = Standard deviation of the response

S = Slope of calibration curve

Limit of Quantitation study

Table-5

LOQ Sitagliptin
phosphate
(µg/ml)

1. 0.6

LIMIT OF DETECTION (LOD)

The limit of detection (LOD) is defined as the lowest concentration of an analyte in a

sample that can be detected, though not necessarily quantitated. It is a limit test that
specifies whether or not an analyte is above or below a certain value.
ICH has recommended some method for determining the limit of detection. The
method maybe either instrumental or non-instrumental. They are
 Visual Evaluation
 Signal – to – Noise ratio convention

33
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

Based on Standard deviation of the response and the slope of calibration

curve Limit of detection (LOD) based on standard deviation of the response and
the slope of calibration curve.

LOD = 3.3S

s = Standard deviation of the

responseS = Slope of
calibration curve

Limit of detection study

Table-6

LOD Sitagliptin
phosphate
(µg/ml)

1. 1.9

Acceptance Criteria:

 The % RSD for the individual recoveries of each level and mean recovery
should not be more than 2 %.
 The % recovery at each level and mean recovery should be in between
98.0% to 102%.

ROBUSTNESS

Robustness is the capacity of a method to remain unaffected by small deliberate

variations in method parameters. The robustness of a method is evaluated by varying
method parameters such as percent organic solvent, pH, ionic strength or temperature
and determining the effect on the results of the method.
Robustness tests were generally introduced to avoid problems in linear laboratory
studies and to identify the potentially responsible factors.

34
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

Determination of robustness
Robustness was performed by varying the
-PH

- Flow rate

Robustness study for Sitagliptin phosphate

Table – 7

Robustness Criteria RT of
Sitagliptin phosphate
Change in flow +0.2 3.629

Change in flow -0.2 3.260

Change in wavelength by -PH 3.340

Change in wavelength by + PH 3.390

Acceptance Criteria:
 Shall comply the system suitability parameters.

 Measured variation to be reported with appropriate recommendations.

RUGGEDNESS
Ruggedness of analytical method is the degree of reproducibility of the results
obtained by the analysis of the same samples under a variety of test conditions such
as different laboratories, analysts, instruments, temperature, different days etc.
Determination of Ruggedness
The Ruggedness of an analytical method was determined by the analysis of aliquots
from homogenous lots in different laboratories by different analysts using operational
and environmental condition that may differ but are still within the specified
parameters of the assay. The degree of reproducibility of the results is then
determined as a function of assay variables.
This reproducibility may be compared to the precision of assay under normal
condition to obtain a measure of the ruggedness of the analytical method.

35
Method Development and Validation for Anti Diabetic Drug By-HPLC
Chapter-5 Materials and Methods

To determine the degree of reproducibility of the results by this method involved the
studies of the analyst to analyst and day to day; that is to carry out precision study in
six replicate of an assay of a single batch sample by two different analysts on two
different days.

Ruggedness Interday Analysis Study

Table –8
Sample. No % Assay of
Sitagliptin
phosphate
1 101.0
2 99.8
3 98.4
4 98.1

Acceptance Criteria:

 The % recovery at each level and mean recovery should be in between 98.0% to 102%.

36
Method Development and Validation for Anti Diabetic Drug By-HPLC
CHAPTER- 6
RESULT AND
DISCUSSION
Chapter-6 Result and Discussion

6. RESULT AND DISCUSSION

The working condition for the RP-HPLC method was established for Sitagliptin
phosphate was applied on pharmaceutical dosage forms. A simple reverse phase
liquid chromatographic method has been developed and subsequently validated.
Mobile Phase 0.01M Phosphate buffer adjusted to P H 2.5: methanol in a ratio
50:50 % v/v was taken, sonicated for 15 minutes and filtered through 0.45µ
Millipore Nylon filter under Vacuum filtration. The prepared solution was used
as Mobile phase.
The proposed method was developed and validated as per the ICH guidelines.
Linearity was observed over a concentration range of 5 to 30 μg/ml. System
suitability parameters were satisfactory and the theoretical plates were obtained
above 2000. Tailing factor was found below 2. %RSD also found below 2%.
The assay of Sitagliptin was found to be 99.89% and the low % RSD value
confirms the robustness of the method.
The calibration was linear in concentration range of 5-30 µg/ml for Sitagliptin
phosphate p respectively. The sensitivity for the drugs has been calculated and the
LOQ and LOD of the Sitagliptin phosphate was found to be 0.6µg/ml and 1.9µg/ml.
The low values of % R.S.D. indicate the method is precise and accurate. Ruggedness
of the proposed methods was determined by analysis of aliquots from homogeneous
slot by different analysts, using similar operational and environmental conditions;
the % R.S.D. reported was found to be less than 2 %.
From the linearity studies, specified concentration levels were determined. The
linearity range of 5-30 µg/ml for Sitagliptin phosphate were found. The validation
of proposed method was verified by recovery studies. The percentage recovery
range was found to be satisfied which represent in results. The robustness studies
were performed by changing the pH and wavelength. The ruggedness study was
also performed.
The analytical method validation was carried as per ICH guidelines and given below
are the tables are the summary of the result.

37
Method Development and Validation for Anti Diabetic Drug By-HPLC
CHAPTER- 7
SUMMARY AND
CONCLUSION
Chapter-7 Summary and Conclusion

7. SUMMARY AND CONCLUSION

Summary of Validation Parameters

S. No. Parameter Results

1 Limit of Linearity 5-30 µg/ml
2 Regression equation Y= 6326x+945.7
Slope 6326
Intercept 945.7
Correlation coefficient (r2) 0.999
3 Accuracy (%) 99.8-100.6
4 Precision(%cv)
System precision 1.22
Method precision 0.59
5 LOD (µg/ml) 0.6
6 LOQ (µg/ml) 1.9

CONCLUSION
The proposed RP-HPLC method was found to be simple, accurate,
precise, linear, robust and specific for quantitative estimation of
Sitagliptin phosphate in bulk and its formulation. The proposed RP-HPLC
method was cost effective and less time consuming. The values for system
suitability parameters showed feasibility of this method for routine
pharmaceutical application. Hence, the present RP-HPLC method is
suitable for routine assay of Sitagliptin phosphate in bulk and tablet
dosage form in the quality control laboratories.

38
Method Development and Validation for Anti Diabetic Drug By-HPLC
CHAPTER- 8
BIBLIOGRAPHY
Chapter-8

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Method Development and Validation for Anti Diabetic Drug By-HPLC
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Method Development and Validation for Anti Diabetic Drug By-HPLC