MPHARAM PROJECT DOC Rajesh PDF

I take pleasure A Project Report On
ANALYTICAL METHOD DEVELOPMENT AND VALIDATION

FOR DETERMINATION OF GLIPIZIDE AND METFORMIN IN
BULK FORM AND MARKETED PHARMACEUTICAL
DOSAGE FORM BY RP-HPLC
Submitted in partial fulfillment of the
Requirement for the award of the degree
of
Master of Pharmacy
in
PHARMACEUTICAL ANALYSIS
BY
DOMMETI PUJITHA
(Reg. No:19JQ1S1603)
Under the esteemed guidance of
Mrs. G.MANGADEVI (M.Pharm)
Assistant Professor
Department of Pharmacy
KAKINADA INSTITUTE OF TECHNOLOGY AND SCIENCE
(Approved by A.I.C.T.E., New Delhi Affiliated to JNT University, Kakinada)
TIIRUPATHI (V), DIVILI, PEDDAPURAM 533 433, (Estd-2008)
EAST GODAVARI, A.P
Accredited by NAAC with B+ Grade
TIIRUPATHI (V), DIVILI, PEDDAPURAM 533 433, (Estd-2008) EAST
GODAVARI, A.P.
CERTIFICATE
This is to certify that the dissertation entitled “ANALYTICAL METHOD

DEVELOPMENT AND VALIDATION FOR DETERMINATION OF GLIPIZIDE
AND METFORMIN IN BULK FORM AND MARKETED PHARMACEUTICAL
DOSAGE FORM BY RP-HPLC” being submitted by DOMMETI PUJITHA,
Roll No:19JQ1S1603 in partial fulfillment of the requirements for the award of in the
PHARMACEUTICAL ANALYSIS to the Jawaharlal Nehru Technological University
-Kakinada is a record of bonafide work carried out by her under my guidance and
supervision during 2019-2021. The results embodied in this thesis have not been
submitted to any other University for the award of any Degree or Diploma.
Guide’s Signature: Signature of the Head of theDept.

Mrs.G.MANGADEVI M.Pharm Mrs.G.MANGADEVI M.Pharm
Assitant Professor. Assitant Professor.
Department of M.Pharmacy
KITS/ Tirupathi-533 433.
External Examiner
TIIRUPATHI (V), DIVILI, PEDDAPURAM 533 433, (Estd-2008)
EAST GODAVARI, A.P.
DECLARATION
The work presented in “ANALYTICAL METHOD DEVELOPMENT AND

VALIDATION FOR DETERMINATION OF GLIPIZIDE AND METFORMIN IN
BULK FORM AND MARKETED PHARMACEUTICAL DOSAGE FORM BY RP-
HPLC” being submitted by DOMMETI PUJITHA. The thesis is carried out by the SURA
LABS, Hyderabad and Laboratories of Kakinada Institute of Technology and
Science,Divili under the guidance of Mrs.G.MANGADEVI. The extent of information
derived from the existing literature has been indicated in the body of thesis at
appropriate places giving the source confirmation. The work is original and has not
been submitted in Part or Full for any Diploma or Degree of this University or any
other University.
DOMMETI PUJITHA
(Reg. No: 19JQ1S1603)
PLACE:
DATE:
ACKNOWLEDGEMENT
A single flower cannot make a garland or a single star cannot make the beautiful shiny
sky in the night. A research work can never be the outcome of a single individual’s talent or
efforts. During my journey from objective to goal, I have experienced a shower of blessings,
guidance and inspiration from my Great God, Family, Teachers and all my well wishers.
“Words are tools for expressing the feelings but they might fail miserably when it comes
to thanks giving.”
I take this moment to thank numerous people who lent their constant support and
Encouragement for the successful completion of this project.
I express my warmest gratitude to my esteemed guide, Mrs.G.MANGADEVI

M.Pharm., Madam for his constructive criticism, perpetual encouragement, timely advice and
meticulous attention were the real driving forces. His keen interest in my project encouraged
me a lot.
I thank our honorable Chairman Sri B.SRINIVASARAO garu

Correspondent,Kakinada Institute Of Technology and Science for providing us with good
faculty support throughout the course.Involuntarily,
I am sincerely thankful to my principal Dr.E.SARVARAMESWARUDU.,Ph.D sir for
providing me an opportunity to carry out my dissertation work in the college.
My heartful gratitude to Mrs.G.MANGADEVI M.Pharm, Madam Head of the

Department of PHARMACEUTICAL ANALYSIS for the encouragement and assistance to
me, which contribute to the successfulCompletion of this project.
To express my sincere thanks to all the administrative staff, teaching and non teaching
staff, Kakinada Institute of Technology and Science, Divili and SURA LABS for helping
me throughout the project.
I especially thank all my friends and classmates from the core of my heart for their
continuous support.
By
DOMMETI PUJITHA
Date: I 2-07-2021.
This is to certify ihai Miss. DOMMETI PU.IITHA (HT. No. I9JQ1S1603) pursuing her
M,Pharmacy in KITS COLLEAGE OF PHARMACY has concerned out her project work in our
Organisation entitled “ANALYTCAL METHOD DEVELOPMENT AND VALIDATION
FOR DETERMINATION OF GLIPIZIDE AND METFORMIN IN BULK FORM AND
MARKETED PHARMACEUTICAL DOSAGE FORM BY RP-HPLC” In the department of
Pharmaceutical Analysis from 08-FEB-2021 to 12 JULY-2021.
During her tenure she was sincere, hard working and Punctual in her Project work,
We wish her to success in her future career.

DEPARTMENT OF PHARMACY
ABSTRACT
A rapid, precise, accurate, specific and simple RP-HPLC method was developed for the
simultaneous estimation of Glipizide and Metformin in bulk and its combined pharmaceutical
dosage form. A High performance liquid chromatography WATERS, software: Empower
2, 2695 separation module, 996 PDA detector, using Phenomenex Luna C18 (4.6mm x
250mm, 5µm, Make: Waters) or equivalent column, with mobile phase composition of
Acetonitrile: Phosphate Buffer (pH-6.8) [20:80 % (v/v)] was used. The flow rate of 1.0 ml
min-1 and effluent was detected at 268 nm. The retention time of Glipizide and Metformin
was found to be 2.242min and 3.678 minutes respectively. Linearity was observed over
concentration range of 30-70µg ml-1 for Glipizide and 30-70µg ml-1 for Metformin
respectively.
The accuracy of the proposed method was determined by recovery studies and the
Glipizide was found to be 100.41% and Metformin was found to be 99.58% respectively. The
proposed method is applicable to stability studies and routine analysis of Glipizide and
Metformin in bulk and pharmaceutical formulations. The proposed method was validated for
various ICH parameters like linearity, limit of detection, limits of quantification, accuracy,
precision, range and specificity.
Key Words: Glipizide, Metformin, RP-HPLC, Robustness and ICH Guidelines.

ABBREVIATIONS
HPLC - High Performance liquid chromatography
.UV - Ultra violet spectroscopy
TLC - Thin layer chromatography
LOD - Limit of Detection
LOQ - Limit of Quantitation
S.D - Standard Deviation
%RSD - Percentage Relative Standard Deviation
RS - Peak Resolution
M.P - Mobile Phase
mg - milligrams
µg - Micrograms
ml - Milliliters
% - Percentage
w/w - Weight/weight
v/v - volume/volume
µg/ml - micrograms per milliliter
nm - Nanometer
Rt - Retention Time
Min - Minutes
ICH - International conference on Harmonization
Ola - Olanzapaine
Flu - Fluoxetine
INDEX
S.NO. TITLE PAGE NO.
1. Introduction 15-44
2. Literature review 45-47
3. Aim and Objective 48
4. Drug profile 49-55
5. Plan of work 56
6. Experimental Methods 57-67
7. Results and Discussions 68-107
8. Summary & Conclusion 108
9. References 109-111
S.NO NAME OF THE TABLE PAGE.NO
1 Classification of analytical methods 17
2 Different types of chromatographic techniques 21
3 Physical properties of common HPLC solvents 25
4 Separation goals in HPLC method development 34
5 Sample and analyte information 35
6 System suitability parameters and recommendation 40
7 METFORMIN DRUG PROFILE 52
8 COMBINED DRUG FORMULATION 55
9 Instruments used 57
10 Chemicals used 57
11 Results of Trial-1 69
18 Results of Optimized Chromatographic Condition 77

(Glipizide +Metformin)
19 Results of system suitability parameters for Glipizide and 78

Metformin
20 Peak Results for assay standard of Glipizide + Metformin 80
21 Peak Results for assay Sample of Glipizide + Metformin 82
22 Results of method precision for Glipizide + Metformin 85
23 Results of Intermediate precision for Glipizide + Metformin: 87
24 Results of Intermediate precision for Glipizide + Metformin: 89
25 Results of Chromatogram Sample Concentration-50% 91
28 Accuracy (recovery) data for Glipizide: 96
29 Accuracy (recovery) data for Metformin 96
30 Results for Linearity for Glipizide 100
31 Results for Linearity for Metformin 100
32 Analytical performance parameters of Glipizide and Metformin 102
33 System suitability results for Glipizide: 105
34 System suitability results for Metformin: 105
35 System suitability results for Glipizide 107
36 System suitability results for Metformin 107

S.NO NAME OF THE FIGURE PAGE.NO
1 Schematic diagram of HPLC instrumentation 24
2 Steps in HPLC method development 33
3 GLIPIZIDE DRUG PROFILE 49
4 Chromatogram of Trial-1 69
11 Optimized Chromatographic Condition (Glipizide + 76

Metformin)
12 Chromatogram for Blank Solution 77
13 Chromatogram for system suitability 78
14 Chromatogram showing assay of sample injection-1 79

17 Chromatogram showing assay of standard injection -1 81
20 Chromatogram for standard injection-1 83
25 Chromatogram for sample injectiocn-1 86
31 Chromatogram for sample concentration-50% 90

40 Chromatogram for linearity concentration-30 µg/ml of 97

Glipizide & 30 µg/ml of Metformin

42 Chromatogram for linearity concentration-50ppm of 98

Glipizide & 50ppm of Metformin

44 Chromatogram for linearity concentration-70µg/ml of 99

45 Calibration graph for Glipizide at 268 nm 101
46 Calibration graph for Metformin at 268 nm 101
47 Chromatogram showing less flow of 0.8ml/min 104

48 Chromatogram snowing more flow of 1.2ml/min 104
49 Chromatogram showing less organic composition 106
50 Chromatogram showing more organic composition 106

1. INTRODUCTION
Analytical methods development and validation play important roles in the discovery,
development, and manufacture of pharmaceuticals. The current good manufacturing practice
(CGMP) and food drug administration (FDA) guidelines insist for adoption of sound methods
of analysis with greater sensitivity and reproducibility. Development of a method of analysis
is usually based on prior art (or) existing literature, using the same (or) quite similar
instrumentation. It is rare today that an HPLC-based method is developed that does not in
same way relate (or) compare to existing, literature based approaches. Today HPLC (high
performance liquid chromatography) is the method of choice used by the pharmaceutical
industry to assay the intact drug and degradation products. The appropriate selection and
chromatographic conditions ensure that the HPLC method will have the desired specificity.
UV spectroscopy is also a simple analytical tool widely used for routine assay of drugs.
Hence for the assay of the selected drugs HPLC and UV spectroscopy has been chosen for
these proposed methods.
The developed chromatographic methods further validated as per ICH or

USFDA guidelines for all the critical parameters. To access the precision and to evaluate the
results of analysis the analyst must use statistical methods. These methods include confidence
limit, regression analysis to establish calibration curves. In each analysis the critical response
parameters must be optimized and recognized if possible.
Pharmaceutical analysis plays a major role today, and it can be considered as an

interdisciplinary subject. Pharmaceutical analysis derives its principles from various branches
like chemistry, physics and microbiology etc. Pharmaceutical analytical techniques are
applied mainly in two areas, quantitative analysis and qualitative analysis, although there are
several other applications.
Drugs and pharmaceuticals are chemicals or like substances, which or of organic inorganic or
other origin. Whatever may be the origin, we some property of the medicinal agent to
measure them quantitatively or qualitatively.
In recent years, several analytical techniques have been evolved that combine two or more
methods into one called “hyphenated” technique e.g. GC/MS, LC/MS etc. The complete
analysis of a substance consists of four main steps.
The concept of analytical chemistry lies in the simple, precise and accurate measurements.
These determinations require highly sophisticated instruments and methods like mass
spectroscopy, gas chromatography, high performance thin layer chromatography, high
performance liquid chromatography etc. The HPLC method is sensitive, accurate, precise and
desirable for routine estimation of drugs in formulations.
Thereby it is advantageous than volumetric methods. Many HPLC methods has been
developed and validated for the quantitative determination of various marketed drugs.
Analytical method development and validation places an important role in drug discovery
and manufacture of pharmaceuticals. These methods are used to ensure the identity, purity,
potency and performance of drug products majority of analytical development effort goes
into validating a stability indicating method. So it is a quantitative analytical method based on
the structure and chemical properties of each active ingredient of the drug formulation.
Most of the drugs can be analyzed by HPLC method because of several advantages like
rapidity, specificity, accuracy, precision, reproducibility, ease of automation and eliminates
tedious extraction and isolation procedures.
On the literature survey, it was found that most of the analytical method available for the
above mentioned drug is applicable for quantification in plasma samples, the most widely
used method being liquid chromatography-mass chromatography. So it is felt that there is a
need to develop accurate, precise analytical methods for the estimation of the drug in solid
dosage formulation.
Newer analytical methods are developed for these drugs or drug combinations of the
below reasons:
● There may not be suitable method for a particular analyte in the specific matrix.
● Existing method may be too error prone or unreliable (have poor accuracy and
precision).
● Existing method may be expensive, time consuming, energy intensive and may not be
provide sensitive or analyte selectivity, and not easy for automation.
● Newer instrumentation and techniques may have evolved that provide opportunities
for improved methods.
● There may be need for an alternate method to confirm, for legal and scientific reasons.
The newly developed analytical methods having their importance in different fields that
include, research and development centre (R&D), quality control department (QC), approved
testing laboratories, chemical analysis laboratories etc. For analysis of these drugs different
analytical methods are routinely being used.
The analytical methods are classified as classical and instrumental. These methods signal
measured in those methods was mentioned in following table.
Table-1: Classification of analytical methods
Measurement signal Analytical method
Chromatographic techniques
Electrical Gas chromatography (Thermal conductivity detector)
Increase in electrical current Gas chromatography (Flame ionization detector)
Decrease in electrical current Gas chromatography (Flame capture detector)
Electromagnetic radiation Liquid chromatography (Ultraviolet light detector, diode

absorbed array detector)
Electrical Ion chromatography
Spectrophotometric method
Emission radiation Emission spectroscopy (X-ray, UV, Visible), Fluorescence

and phosphorescence (X-ray, UV, Visible), radiochemistry.
Absorption of radiation Spectrophotometry (X-ray, UV, Visible, IR) NMR and

electron spin resonance spectroscopy.
Scattering of radiation Turbidimetry, nephelometry, raman spectroscopy
Refraction of radiation Refractometry, interferometry
Diffraction of light X-ray and electron diffraction
Rotation of radiation Polarimetry, optical rotatory dispersion
Mass to charge ratio Mass spectroscopy
Electro chemical techniques
Electrical potential Potentiometry
Electrical current Polarography, amperometry
Electrical resistance Conductometry
Miscellaneous techniques
Rate of reaction Kinetic method
Thermal properties DTA, DSC
Classical methods
Mass Gravimetric analysis
Volume Volumetric analysis

2. CHROMATOGRAPHY
Techniques related to chromatography have been used for centuries to separate materials such
as dyes extracted from plants. Russian botanist Tswett is credited with the discovery of
chromatography. In 1903 he succeeded in separating leaf pigments using a solid polar
stationary phase, It was not until 1930s that this technique was followed by Kuhn and Lederer
as well as Reichstein and van Euw for the separation of natural products. Martin and Synge
were awarded the Nobile prize for their work in 1941 in which they described liquid-liquid
chromatography. Martin and Synge applied the concept of theoretical plates as a measure of
chromatographic efficiency. The term “chromatography” (Color-writing derived from the
Greek for Color-chroma and Write-Graphing).
CHROMATOGRAPHY IN THE PHARMACEUTICAL WORLD
In the modern pharmaceutical industry, chromatography is the major and integral analytical
tool applied in all stages of drug discovery, development, and production. The development
of new chemical entities (NCEs) is comprised of two major activities drug discovery and
development. The goal of the drug discovered is to investigate a plethora of compounds
employing fast screening approaches, leading to generation of lead compounds and then
narrowing the selection through targeted synthesis and selective screening (lead
optimization). The main functions of drug development are to completely characterize
candidate compounds by performing drug metabolism, preclinical and clinical screening, and
clinical trials. Throughout this drug discovery and development paradigm, rugged analytical
HPLC separation methods are developed, at each phase of development to analyses of a
myriad of samples are performed to adequately control and monitor the quality of the
prospective drug candidates, excipients and final products. Effective and fast method
development is of paramount importance throughout this drug development life cycle. This
requires a thorough understanding of HPLC principles and theory which have solid
foundation for appreciating the many variables that are optimized during fast and effective
HPLC method development and optimization.
3. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
3.1 Brief Historical prospective of chromatography:
The historical development of liquid chromatography has been extensively reviewed and can
be traced as far back as they early 1900, where the Russian botanist Zwett used a variant of
liquid chromatography to separate some colored plant substances.
The focus was on modern development in HPLC, a term that was coined in late 1960s with
the advent of more sophisticated instrumentation, better engineered separation columns, and
reliable and highly efficient stationary phases and packaging materials.
These technological advances have been, In part, fuelled, by the need to separate an
increasingly large variety of differing compounds classes encountered as API s, e.g.
Antibiotic, sulphonamides nucleosides, fat soluble vitamins neutral and non polar
compounds. Additional challenges include developing faster and more consistent HPLC
methods requiring higher flow rates, while maintaining peak shape, peak symmetry and
efficiencies. Another important analytical challenge is the desire to detect and accurately
quantify low levels of impurities at level present in API materials.
High-pressure liquid chromatography quickly improved with the development of column

packing materials. Additional convenience of on-line detectors became rapidly a powerful
separation technique and is today called as high-performance liquid chromatography (HPLC)
⮚ One of the early problems with liquid state chromatography was the slow rate at
which analysis took place. Early methods use gravity feed, and it was not uncommon
diffusion and soon.
⮚ This problem was largely overcome by the advent high-performance liquid
chromatography (HPLC). In this system the pressure is applied to the column forcing
the mobile phase through at much higher rate.
⮚ For an analysis to take several days to complete. This led not only to great delay but
also the excessive time on the column and thus inevitably led to loss of resolution.
Table-2: Different types of chromatographic techniques
S. no Basic principle involved Type of chromatography
1 Techniques by chromatographic Column chromatography

bed shape
Paper chromatography
Thin layer chromatography
2 Techniques by physical state of Gas chromatography

mobile phase
Liquid chromatography
3 Affinity chromatography Super critical fluid chromatography
4 Techniques by separation Ion exchange chromatography

mechanism
Size exclusion chromatography
5 Special techniques Reversed phase chromatography
Two-dimensional chromatography
HPLC
In high performance liquid chromatography, mobile as well as the stationary phase compete
for the distribution of the sample components. In case of HPLC, separation is based on
adsorption and partition. Adsorption chromatography employs high-surface area particles that
adsorb the solute molecules. Usually a polar solid such as silica gel, alumina or porous glass
beads and a non-polar mobile phase such as heptanes, octane or chloroform are used in
adsorption chromatography.
In partition chromatography, the solid support is coated with a liquid stationary phase. The
relative distribution of solutes between the two liquid phases determines the separation..
The stationary phase can either polar or non-polar. If the stationary phase is non-polar, it is
called normal phase partition chromatography.
If the opposite case holds, it is called reversed-phase partition chromatography. In normal

phase mode, the polar molecule partition preferentially in to the stationary phase and are
retained longer than non-polar compounds.
In reverse phase partition chromatography, the opposite behavior is observed
3.2 TYPES OF HPLC TECHNIQUES:

Based on modes of chromatography:
● Normal phase chromatography
● Reverse phase
chromatography Based on
principle of separation:
❖ Adsorption chromatography
❖ Ion exchange chromatography
❖ Size exclusion chromatography
❖ Affinity
chromatography Based on elution
technique:
o Isocratic separation
o Gradient
separation Based on the scale
of operation:
⚪ Analytical HPLC
⚪ Preparative HPLC
Ion exchange chromatography: Due to differences in the affinity of ions for the in exchange.
Size exclusion chromatography: Due to differences in molecular weight and size of the
molecules to be separated.
Affinity chromatography: Separation is based on a chemical interaction specific to the

target species. The more popular revered phase mode uses a buffer and added counter-ion of
opposite.
charge to the sample with separation being influenced by pH, ionic strength, temperature,
concentration and type of organic co-solvents.
Chiral chromatography: Separation of the enantiomers can be achieved on chiral stationary

phases by the formation of diastereomers.
Analytical HPLC: Only analysis of the samples is done. Recovery of the

samples for reusing is normally not done
3.3 MOST COMMONLY USED METHODS IN
HPLC Normal phase chromatography:
For a polar stationary bed like silica we need to choose a relatively non-polar Mobile phase.
This mode of operation is termed as normal phase chromatography. Here the least polar
component elutes first, and increasing the mobile phase polarity leads to decrease in elution
time. Non-polar solvents like pentane, Hexane, isooctane, cyclohexane etc. are more popular.
It is mainly used for separation of nonionic, non-polar to medium polar substances.
Reverse phase chromatography:
In 1960s, chromatographers started modifying the polar nature of the silanol group by
chemically reacting silicon with organic silanes. The object was to make silica less polar or
non- polar so that polar solvents can be used to separate water-soluble polar compounds.
Since the ionic nature of the reverted, the chromatographic separation carried out with such
silica is preferred to as reverse- phase chromatography. Here the most post components elutes
first. Increasing mobile phase polarity leads to decrease in elution time. Common solvents
used in this mode include methanol /Acetonitrile /isopropanal etc. Mostly used for separation
of ionic and polar substances. The parameters that govern the retention in reversed phase
system are the following:
a. The chemical nature of the stationary phase surface.

b. The type of solvents that compose the mobile phase.
c. pH and ionic strength of the mobile phase.
Isocratic elution: A separation in which the mobile phase composition remains constant
throughout the procedure is termed as a isocratic (meaning constant composition).
Gradient elution: The mobile phase composition does not have to remain constant. A
separation in which the mobile phase composition is changed during the separation process is
described as a gradient elution.
3.4 INSTRUMENTATION OF HPLC:
The mobile phase components HPLC instrument and their working functions are described
below.
● Mobile phase and reservoir

● Solvent degassing system
● Pump
● Injector
● Colum
● Detector
● Data system
Figure-1: Schematic diagram of HPLC instrumentation

I. MOBILE PHASE AND RESERVIOR:
The most common type of solvent reservoir is a glass bottle. The mobile phase is pumped
under pressure from one of several reservoirs and flows through the column at a constant
rate. With micro particulate packing, there is a high-pressure drop across a chromatography
column. Mobile phase used for HPLC are typically mixtures of organic solvents and water or
aqueous buffers. The following points should also be considered when choosing a mobile
phase:
● The essential to establish that the drug is stable in the mobile phase for
at least the duration of the analysis.
● Excessive salt concentrations should be avoided. High salt
concentrations can result in precipitation, which can damage HPLC equipment.
● The mobile phase should have a pH 2.5 and pH 7.0 to maximize the
lifetime of the column.
● Reduce cost and toxicity of the mobile phase by using methanol instead
of acetonitrile when possible minimizes the absorbance of buffer.
● Use volatile mobile phase when possible, to facilitate collection of
products and LC-MS analysis. Volatile mobile phases include ammonium, acetate,
ammonium phosphate, formic acid, and trifluoroacetic acid. Some caution is needed
as these buffers absorb below 220nm.
Mobile phase used for HPLC are typically mixtures of organic solvents and water or aqueous
buffers. Physical properties of some HPLC solvents were summarized in table: 3.
Table-3: Physical properties of common HPLC solvents
Solvent MW BP RI UV Density Viscosity Dielectric
(25OC) λ Cut- g/ml(25Oc) CP(25OC) Constant

off(nm)
Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8

Dioxane 88.1 101 1.420 215 1.034 1.26 2.21
Ethanol 46.1 78 1.359 205 0.789 1.19 24.5
Ethylacetate 88.1 77 1.372 256 0.901 0.450 6.02
Methanol 32.0 65 1.326 205 0.792 0.584 32.7
CH2CI2 84.9 40 1.424 233 1.326 0.44 8.93
Isopropanol 60.1 82 1.375 205 0.785 2.39 19.9
N-propanol 60.1 97 1.383 205 0.804 2.20 20.3
THF 72.1 66 1.404 210 0.889 0.51 7.58
a :The wavelength at which the absorbance of 1cm is 1.0
II. SOLVENT DEGASSING SYSTEM:
The constituents of the mobile phase should be degassed and filtered before use. Several
methods are employed to remove the dissolved gases in the mobile phase. They include
heating and stirring, vacuum degassing with an aspirator, filtration through 0.45µ filters,
vacuum degassing with an air-soluble membrane, helium purging ultra signification or
purging or combination of these methods. HPLC systems are also provided an online
degassing system, which continuously removes the dissolved gases from the mobile phase.
III. PUMP:
High pressure pumps are needed to force solvents through packed stationary phase beds.
Smaller bed particles require higher pressures. There are many advantages to using smaller
particles, but they may not be essential for all separations.
The degree of flow of control also varies with pump expense. More expensive pumps include
such state of the art technology as electronic feedback and multithreaded configurations. It is
desirable to have an integrated degassing system, either helium purging, or membrane
filtering.
IV. INJECTOR:
Sample introduction can be accomplished in various ways. The simplest method is to use an
injection value in more sophisticated LC systems, automatic sampling devices are
incorporated where the sample is introduced with the help of auto samplers and
microprocessors in liquid chromatography, liquid samples may be injected directly and solid
samples need only be dissolved in an appropriate solvent. Sample introduction techniques can
be used with a syringe an injection valve.
V. COLUMN:
The heart of the system is the column. Many different reverse phase columns will provide
excellent specificity for any particular separation. It is therefore best to routinely attempt
separations with a standard C8 or C18 column and determine if it provides good separations.
Reverse phase columns differ by the carbon chain length, degree of end capping and percent
carbon loading. Diol, cyano and amino groups can also be used for reverse phase
chromatography. Typical HPLC columns are 5, 10, 15, and 25 cm in length and are filled
with small diameter (3, 5 or 10µm) particles. The internal diameter of the columns is usually
4.6 mm; this is considered the best compromise for sample capacity, mobile phase
consumption, speed and resolution. However, if pure substances are to be collected
(preparative scale), then larger diameter columns may be needed.
VI. DETECTOR:
The detection of UV light absorbance offers both convenience and sensitivity for molecules.
When a chromophore is present, the wavelength of detection for a drug should be based on its
UV spectrum in the mobile phase and not in pure solvents. The most selective wavelength for
detecting a drug is frequently the longest wavelength maximum to avoid interference from
solvents, buffers and excipient. Other method of detection can be useful are required in some
instances.
1. Solute specific detectors (UV-Vis, fluorescence, electrochemical, infra-red, radio

activity).
2. Bulk property detectors (Refractive index, viscometer, conductivity).

3. Desolvation detector (Flame ionization etc).
4. LC-MS detectors.
5. Reaction detectors.
VII. DATA SYSTEM:
Since the detector signal is electronic, using modern data collection techniques can aid the
signal analysis. In addition, some systems can store data in a form for highly sophisticated
computer analysis at a later time. The main goal in using electronic data systems is to
increase analysis accuracy and precision, while reducing operator attention.
PERFORMANCE CALCULATIONS:
Calculating the following values (which can be included in a custom report) used to access
overall system performance.
1. Relative retention
2. Theoretical plates
3. Capacity factor
4. Resolution
5. Peak asymmetry
6. Plates per meter
The following information furnishes the parameters used to calculate these system
performance values for the separation of two chromatographic components. (Note: where the
terms w and t both appear in the same equation they must be expressed the same units).
System suitability parameters:
The theory of chromatography has been used as the basis for system-suitability tests,
which are set of quantitative criteria that test the suitability of the chromatographic system to
identify and quantify drug related samples by HPLC at any step of the pharmaceutical
analysis.
1. Relative retention: The time elapsed between the injection of the sample
components in to the column and their detection is known as the retention time (Rt).
α = (t2-ta) / (t1-ta)
Where,
α =Relative retention.
t1= Retention time of the peak measured from point of injection.
t2 = Retention time of the second measured from point of injection.
ta = Retention time of an inert peak not retained by the column, measured from point of injection.
2. Theoretical plates:
n =16 (tR / w) 2
Where,
n =Theoretical plates.
tR = Retention time of the component.
W = Width of the base of the component peak using tangent method.
3. Capacity factor: The capacity factor describes the thermodynamic basis of the
separation and its definition is the ratio of the amounts of the solute at the stationary
and mobile phases within the analyte band inside the chromatographic column.
K1 = (t2/t a)-1
Where,
K1 = Capacity factor.
ta = Retention time of an inert peak not retained by the column, measured from point of injection.
4. Resolution: the gap between two peaks

R =2 (t2- t1) / (w2-w1)
Where,
R =Resolution between a peak of interest (peak 2) and the peak preceding it
(peak 1). W2 = Width of the base of component peak 2.
W1 = Width of the base of component peak 1.
5. Peak asymmetry
T =W0.05/ 2f
Where,
T = Peak asymmetry, or tailing factor.
W0.05 = Distance from the leading edge to the tailing edge of the peak, measured at a point 5
% of the peak height from the baseline.
f= Distance from the peak maximum to the leading edge of the peak.
6. Plate Per Meter:
N =n/L
Where,
N = plates per meter.
L = column length in meters.
Advantages:
● HPLC separations can be accomplished in a minutes, in some cases even in seconds.

● High resolution of complex sample mixture into individual components.
● Rapid growth of HPLC is also because of its ability to analyze substances that are
unsuitable for gas liquid chromatographic (GLC) analysis due to non-volatility or
thermal-instability.
2. Need special HPLC

procedure, sample pre-
treatment, etc?
● Quantitative analysis is easily and accurately performed and errors of less than 1 %
are common to most HPLC methods.
● Depending on sample type and detector used, it is frequently possible to measure 10-9
g or 1 ng of sample. With special detectors, analysis down to 10-12 pg has been
reported.
● As HPLC is versatile, it can be applied to wide variety of samples like organic,
inorganic, high molecular weight liquids, solids, and ionic-nonionic compounds.
Disadvantages:
● HPLC instrumentation is expensive and represents a major investment for many

laboratories.
● HPLC cannot handle gas samples.
● HPLC is poor identifier. It provides superior resolution but it does not provide the
information that identifies each peak.
● Only one sample can be analyzed at a time.
Finally, at present there is no universal and sensitive detector.
4. ANALYTICAL METHOD DEVELOPMENT
Methods are developed for new products when no official methods are available. Alternate
methods for existing (non-pharmacopoeia) products are developed to reduce the cost and
time for better precision and ruggedness. Trail runs are conducted, method is optimized and
validated.
When alternate method proposed is intended to replace the existing procedure, comparative
laboratory data includes merits /demerits should be made available.
The important factors, which to be taken into account to obtain reliable quantitative analysis, are
1. Careful sample and sample preparation.

2. Appropriate choice of the column.
3. Selection flow rate.
4. Selection of detector wavelength.

5. Selection of column temperature.
Documentation starts at the very beginning of the development process. A system for full
documentation of development studies must be established. Analyte standard characterization.
a) All known information about the analyte and its structure is collected i.e., physical and
chemical properties.
b) The literature for all type of information related to the analyte is surveyed.
c) Using the information in the literatures and prints, methodology is adapted. The methods
are modified where ever necessary.
d) The required instrumentation is setup. Installation, operational and performance
qualification of instrumentation using laboratory SOPs are verified.
HPLC method development is based on few basic steps which include:
1.Information on sample, define separation goals
2.Need special HPCL procedure, sample pre-treatment etc
3.Choose detector and detector setting

4. Choose LC method; preliminary run estimate best seperation
↓
5. Optimize separation conditions
6.Check for problems or requirement for special procedures
7b.Quantitative 7c Qualitative
8.Validate method
calibration method
for release to routine
laboratory
7a.Recover
purified
material
8.Validate methods for release to routine labouratry

Figure-2: Steps in HPLC method development
Method goals:
Analytical method goals are often defined as method acceptance criteria for peak resolution,
precision, specificity, sensitivity. For instance, pharmaceutical methods for potency assays of
an API typically require the following:
● Minimal sample work-up (extra and inject if possible).

● Robust method that doesn’t require extensive execution.
● Low cost per analysis.
Table 4: Separation goals in HPLC method development
Goals Comment
Resolution Precise and rugged quantitative analysis requires that Resolution

be greater than 1.5.
%RSD Precision of retention time and peak area, ‹1-2%RSD.
Range Linearity in the range of 50-150% of the lab label claim.
Analysis time ῀5-30min ( ῀60min for complex samples).
Separation time ‹5-10min is desirable for routine procedures.
Quantification ≤2% (%RSD) for assays, ≤5% for less demanding analyses,
≤15% for trace analyses.
Pressure ‹150 bar is desirable, ‹200bar is usually essential(new column

assumed).
Peak height Narrow peaks are desirable for large signal/ noise ratios.
Sample analyte information:
The information is useful for the selection of appropriate sample preparation procedures as
well as the initial detection and chromatographic modes. If data not available (e.g., PKa,
solubility) separate studies should be initiated as soon as possible. The sample related
information is summarized in Table 5.
Table 5: Sample and analyte information
Sample/analyte Information
Sample Number of components
concentration range of analytes
Analyte (s) Chemical structure, molecular weight and functional groups
PKa, Solubility, Chrpmophores, wavelength (max), Chiral

centers, isomers, spectral data (MS,NMR, IR, and UV), Stability
and toxicity.
Others Purity of reference standard materials.
1. Careful sampling and sample preparation:
Before beginning method development, it is need review what is known about the sample in
to define the goals of separation. The sample related information that is important to
summarized in table 5. The chemical composition of the sample can be provide valuable
clues for the best choice of initial conditions for an HPLC separation.
▪ Number of compounds present

▪ Molecular weight of compounds
▪ PKa values of compounds
▪ UV spectra of compounds
▪ Concentration range of compounds in samples of interest
▪ Sample solubility
2. Separation goals
The goals of HPLC separation need to be specified clearly, which include
● The use of HPLC to isolate purified sample components for spectral identification Or
quantitative analysis.
● It may necessary to separate all degradants or impurities from a product for reliable
content assay or not.
● In quantitative analysis, the required levels of accuracy and precision should be known.
● Whether a single HPLC procedure is sufficient for raw materials or one or more
different procedures are desired for formulations.
● When the number of samples for analysis at one time is greater than 10, a run time of
less than 20 minutes often will be important. Knowledge on the desired HPLC
equipment.
● HPLC equipment, HPLC experience and academic training do to operators
have Sample preparation: Samples come in various forms
o Solutions ready for injection.

o solutions that require dilution, buffering, addition of an
internal standard or other volumetric manipulation.
o solids must be dissolved or extracted.
o Samples that require pretreatment to remove interference and / or to protect
the column or equipment from damage.
3. Appropriate choice of the column:
The selection of the column in HPLC is somewhat similar to the selection of column in G.C,
in the sense that, in the adsorption and partition modes, the separation mechanism is based on
inductive forces, dipole-dipole interaction and hydrogen bond information.
Column plays the important role in achieving the chromatographic separation. The following
parameters should be considered while selecting a column
i. Length and diameter of the column.

ii. Packing material.
iii. Size and shape of the particles.
iv. Pore size, surface area and end capping.
v. Percentage of carbon loading.
Columns with silica as a packing material used widely in normal phase chromatography,
where the eluent (mobile phase) is non-polar consisting of various organic solvents and the
stationary phase is polar. The silanol groups on the surface of the silica give it a polar
character.
In reverse phase chromatography a wide variety of columns is available covering a wide

range of polarity by cross linking the silanol groups with alkyl chains like C6, C8, C18 and
nitrile groups (-CN), phenyl groups (-C6H6) and amino groups (-NH2)
ORDER OF THE SILICA BASED COLUMNS

I------Non polar------Moderately polar------Polar I
C18<C8<C6‹ Phenyl ‹ Amino ‹ Cyano ‹ Silica
4. Selection of flow rate.
Flow rate is selected based on the follows
● Retention time
● Column composition
● Separation impurities
● Peak symmetry
Preferably flow rate shall not be more than 2.5 mL/min a flow rate that gives least retention
times, good peak symmetries, least back pressure and better separation of impurities from
API peak shell be selected.
5. Selection of detector wavelength:
Selection of detector wavelength is a critical step in finalization of the analytical method. To

determine the exact wavelength standard API is injected into chromatographic system with
photo diode array detector and the wavelength, which gives higher response for the
compound
6. Selection of column temperature:
Ambient temperature is always preferred as a column temperature. However if the peak

Symmetry could not be achieved then the column temperature can be varied between 30º C to
80º C if a column temperature above 80º C is found necessary, packing material which can
withstand to that temperature shall be chosen. The increase in column temperature generally
will result in reduction in peak asymmetry and peak retentions.
5. ANALYTICAL METHOD VALIDATION:
Method validation can be defined as (ICH) “Establishing documented evidence, which

provides a high degree of assurance that a specific activity will consistently produce a desired
result or product meeting is predetermined specifications and quality characteristics”.
Method validation study include system suitability, linearity, precision, accuracy, specificity,
ruggedness, robustness, limit of detection, limit of quantification and stability of samples,
reagents, instruments.
VALIDATION DEFINITION:
FDA defines validation as “Establishing documented evidence, which Provides a high degree
of degree of assurance that a specific process will consistently produce a product of
predetermined specifications and quality attributes.
OBJECTIVE OF METHOD VALIDATION:
The objective of validation is to form a basis for written procedure for production and
control, which are designed to assure that the drug products have the identity, quality, and
purity.
TYPES OF ANALYTICAL PROCEDURES:
i. Identification tests.
ii. Quantitative tests for impurities content.
iii. Limit test for control of impurities.
iv. Quantitative tests of the active moiety in samples of drug substances or drug product
or other selected components(s) in the drug product.
v. Dissolution testing for drug products.
vi. Particle size determination for drug substances.
6. VALIDATION PARAMETERS (ICH)
Typical validation study include system suitability
I. Accuracy
II. Precision
III. Specificity
IV. Linearity
V. Detection limit
VI. Quantification limit
VII. Range
VIII. Robustness
1. System suitability
Prior to the analysis of samples of each day, the operator that the HPLC system and
procedure are capable of providing data of acceptable quality. This is accomplished with
system suitability experiments, which can be defined as tests to ensure that the method can
generate results of acceptable accuracy and precision. The requirements for system suitability
are usually developed after method development and validation has been completed.
Table-6: System suitability parameters and recommendation
Parameter Recommendation
Capacity factor The peak should be well-resolved from

other peaks and the void volume.
Generally K> 2.0
Repeatability RSD ≤1% N ≥ 5 is desirable.
Relative retention Not essential as long as the resolution is

stated.
Resolution Resolution is > 2 between the peak

interest and the closes to eluting
potential interferent (impurity, excipient,
degradation product, internal standard,
etc)
Tailing factor T of ≤ 2
Theoretical plates N> 2000
Non-interference of placebo:
The portion of specificity evaluation applies to the finished drug product only. Excipients
present in the formulation should be evaluated and must not interfere with the detection of the
analyte.
2. Linearity
The linearity of a method is a measure of how well a calibration plot of response vs.
concentration approximates a straight line. Linearity can be assessed by performing single
measurement at several analyte concentrations. The data is then processed using a linear
least- squares regression. The resulting plot slope, intercept and correlation coefficient
provide the desired information on linearity.
3. Precision
Precision can be defined as “The degree of agreement among individual test results when the
procedure is applied repeatedly to multiple samplings of a homogenous sample”. A More
comprehensive definition proposed by the international conference on harmonization (ICH)
divides precision into three types.
1. Repeatability
2. Intermediate precision
3. Reproducibility
Repeatability: is the precision of a method under the same operating conditions over a short
time period.
Intermediate precision: is the agreement of complete measurements (including standards)

when the same method is applied many times within the same laboratory.
Reproducibility: examine the precision between laboratories and is often determined in

collaborative studies or method transfer experiments.
4. Accuracy:
The accuracy of a measurement is defined as the closeness of the measured value to the true
value.
In a method with high accuracy, a sample (whose “true value” is known) is analyzed and the
measured value is identical to the true value. Typically, accuracy is represented and
determined by recovery studies. There are three ways to determine accuracy:
1. Comparison to a reference standard

2. Recovery of the analyte spiked into black matrix or
3. Standard addition of the analyte.
It should be clear how the individual or total impurities are to be determined. e.g., weight/
weight or area percent in all cases with respect to the major analyte.
5. Specificity/ selectivity
The terms specificity are often used interchangeably. According to ICH, the term specific
generally refers to a method that produces a response for a single analyte only while the term
selective refers to a method which provides responses for a number of chemical entities that
may or may not be distinguished from each other. If the response is distinguished from all
other responses, the method said to be selected. Since there are very few methods that
respond to only one analyte, the term selectivity is more appropriate. The analyte should have
no interference from other extraneous components and be well resolved from them. A
representative chromatogram or profile should be generated and submitted to show that the
extraneous peak either by addition of known compounds or samples from stress testing are
baseline resolved from the parent analyte.
6. Ruggedness:
The ruggedness of an analytical method is the degree of reproducibility of test results

obtained by the analysis of the same samples under a variety of normal test conditions such
as different
laboratories, different analysts, using operational and environmental conditions that may
differ but are still within the specified parameters of the assay. The testing of the ruggedness
is normally suggested when the method is to be used in more than one laboratory.
Ruggedness normally expressed as the lack of the influence on the test results of operational
and environmental variables of the analytical method.
For the determination or ruggedness, the degree of reproducibility of test result is determined
as a function of the assay variable. This reproducibility may be compared to the precision of
the assay under normal conditions to obtain a measure of the ruggedness of the analytical
method.
7. Robustness:
The concept of robustness of an analytical procedure has been defined by the ICH as “A
measure of its capacity to remain unaffected by small, but deliberate variations in method
parameters”. A good practice is to vary important parameters in the method systematically
and measure their effect on separation. The variable method parameters in HPLC technique
may involves flow rate, column temperature, sample temperature, pH and mobile phase
composition.
8. Stability:
To generate reproducible and reliable results, the samples, standards, and reagents used for
the HPLC method must be stable for a reasonable time (e.g., one day, one week, and one
month depending on need). Therefore, a few hours of standard and sample solution suitability
can required even for short (10 min) separation. When more than one sample is analyzed
(multiple lots of one sample or samples from different storage conditions from a single lot),
automated, overnight runs often are performed for better lab efficiency such practices add
requirements for greater solution stability.
9. Limit of detection:
Limit of detection (LOD) is the lowest concentration of analyte in a sample that can be
detected, but the necessarily quantitated under the stated experimental conditions.
● Based on visual evaluation.

● Based on the standard deviation of the blank.
● Based on the calibration curve.

● Based on signal-to-noise: A signal-to-noise ratio of 3 or
2:1 is acceptable.
LOD may be expressed as
LOD
=3.3σ/s Where, σ = the standard deviation of the
response
S= the slope of the calibration curve
The slope S may be estimated from the calibration of the analyte.
10. Limit of quantification:
Limit of quantification is the lowest concentration of analyte in a sample that can be

determined with acceptable precision and accuracy under the stated experimental conditions.
Several approaches for determining the quantification limit are possible.
● Based on visual evaluation.

● Based on standard deviation of the blink.
● Based on the calibration curve.
● Based on the signal-to-noise approach: A typical signal-to-
Noise is 10:1
LOQ may be
expressed as LOQ =
10σ /s
Where = Standard deviation of the response.
S= the slope of the calibration curve.

LITERATURE REVIEW27-31
Sri Lakshmi D, et al., (2015): A simple, accurate, economical and precise reverse phase
high performance liquid chromatographic (RP-HPLC) method has been developed for the
simultaneous determination of Metformin and Glipizide. The separation was achieved on
Inertsil C 18 column (250 x 4.6 mm, 5 µm) as stationary phase with a mobile phase
comprising of Phosphate buffer p H (8.0): Acetonitrile (50:50) in an isocratic mode, at a flow
rate of 2 ml/min. The detection was monitored at 257 nm. The retention time of Metformin
and Glipizide were
2.41 min and 4.21 min respectively. The linearity was found to be in the range of 60-140
µg/ml and 3.6-8.4 µg/ml for Metformin and Glipizide respectively with correlation
coefficient of 0.999. The proposed method was validated according to ICH guidelines for
parameters like linearity, accuracy, precision and specificity. All validation parameters were
within the acceptable range. The developed method was successfully applied for the
estimation of Metformin and Glipizide in pure and pharmaceutical dosage form.
Bagadane Snehal Bapusaheb, et al., (2019): A simple, accurate, economical and precise
reverse phase high performance liquid chromatographic (RP-HPLC) method has been
developed for simultaneous estimation of Metformin hydrochloride and Glipizide in bulk and
pharmaceutical dosage form. The separation of Metformin hydrochloride and Glipizide was
achieved by using Cosmosil C 18 (250 mm × 4.6 ID, particle size-5 micron) column as
stationary phase with the mobile phase comprising of methanol and water (60:40 ,pH 3
adjusted with ortho-phosphoric acid) in an isocratic mode and flow rate of 0.8 ml/min with
UV detection at 226 nm. The retention time of Metformin hydrochloride and Glipizide were
found to be 4.159 min and 5.571 min respectively. The proposed method was validated
according to ICH guidelines for the parameters like linearity, accuracy, precision, percent
recovery, robustness, ruggedness, limit of detection and limit of quantitation. The % RSD is
found to be less than 2 % and the tailing factor for both the drugs are found to be less than 2.
The number of theoretical plates for Metformin hydrochloride and Glipizide were found to be
more than 2000.All validation parameters were within the acceptable range. The developed
method was successfully applied for the estimation of Metformin hydrochloride and
Glipizide in bulk and pharmaceutical dosage forms.
D. Sireesha, et al., (2016): A simple, rapid and precise Spectrophotometric method has
been developed for simultaneous estimation of Metformin and Glipizide. The method
involved estimation of Metformin and Glipizide by simultaneous equation at 272nm and
232nm respectively in their solution in water. This method was validated with respect to
linearity, accuracy, precision, LOD and LOQ. Beer’s law obeyed in the concentration range
of 5-25μg/ml and 20-50μg/ml for Metformin and Glipizide respectively with the correlation
coefficient of above 0.99. Limit of detection and quantification values were determined to be
0.214μg/ml and 0.649μg/ml for Metformin and 0.608μg/ml and 1.854μg/ml for Glipizide
respectively. Mean recovery of Metformin and Glipizide were found to be in the range of 98-
102% signifies the accuracy of the method. The method was found to be precise as %RSD
was less than 2.
Suresh Kumar GV, et al. (2012): The present work describes development and validation of
simple, precise and accurate reversed-phase liquid chromatographic method for simultaneous
estimation of Glipizide and Metformin hydrochloride in both bulk drugs and pharmaceutical
dosage forms. The chromatographic separation was achieved on (Enable, symmetry C18,
250mm x 4.6mm, 5μ) analytical column. A mobile phase consisting mixture of potassium
dihydrogen phosphate (0.2M, pH 5.8 adjusted with dilute sodium hydroxide) and Acetonitrile
in ratio (60:40 v/v) at flow rate of 1.0ml/min and UV detector wavelength 258 nm. The
retention time of Glipizide and Metformin Hcl was found to be 7.9 and 2.5 minutes
respectively. The method was successfully validated in accordance to ICH guidelines for
accuracy, precision, specificity, linearity, ruggedness and robustness. The linear regression
analysis data for calibration plots showed good linear relationship in the concentration range
60-140μg/mL for both Glipizide and Metformin hydrochloride.
Crispin R. Dass, et al. (2019): An efficient and simple HPLC method has been developed
and validated for the simultaneous determination of Gliclazide and Metformin hydrochloride
in bulk and was applied on marketed Metformin and Gliclazide products. The mobile phase
used for the chromatographic runs consisted of 20mM ammonium formate buffer (pH 3.5)
and Acetonitrile (45:55, v/v) The separation was achieved on an Alltima CN (250 mm 4.6
mm x5m) column using isocratic mode. Drug peaks were well separated and were detected
by a UV detector at 227 nm. The method was linear at the concentration range 1.25-150
mg/ml for Gliclazide and 2.5-
150 mg/ml for Metformin respectively. The method has been validated according to ICH
guidelines with respect to system suitability, specificity, precision, accuracy and robustness.
Metformin limit of detection (LOD) and limit of quantification (LOQ) were 0.8 mg/ml and
2.45 mg/ml respectively while LOD and LOQ for Gliclazide were 0.97 mg/ml and 2.95
mg/ml respectively.
4. AIM & OBJECTIVE
Existing literature reveals that Glipizide and Metformin can be analyzed by HPLC using UV
detection, TLC, HPTLC, HPLC in bulk and pharmaceutical forms.
A comprehensive, validated and simple analytical method development and validation of

Glipizide and Metformin tablets is, therefore, crucial. HPLC with PDA detector is a good
selection as PDA detector is available in most laboratories.
Therefore, in proposed project a successful attempt has been made to develop, simple,
accurate, and economic methods for analysis of Glipizide and Metformin tablets validated.
OBJECTIVE
The objective of the present work is to development and validates a HPLC method with
PDA detector for the development and validation Glipizide and Metformin of tablets.
To be employed in routine and stability tests. In the method development of Glipizide and
Metformin we have decided to carry out our project work by incorporating the reverse
phase high performance liquid chromatography (HPLC).
Then the developed method will be validated according to ICH guidelines for its various
parameters.
DRUG PROFILE21-23
GLIPIZIDE DRUG PROFILE
Name : Glipizide
Description : Glipizide is an oral hypoglycemic agent in the second-

generation sulfonylurea drug class that is used to control blood sugar levels in patients with
type 2 diabetes mellitus. It was first introduced in 1984 3 and is available in various countries
including Canada and the U.S. According to the 2018 Clinical Practice Guidelines by
Diabetes Canada, sulfonylurea drugs are considered a second-line glucose-lowering therapy
following Metformin.
Structure :
IUPAC Name : N-[2-(4-{[(cyclo hexyl carbamoyl) amino] sulfonyl}

phenyl) ethyl]-5-methylpyrazine-2-carboxamide.
Chemical formula : C21H27N5O4S
Molecular mass : 445.535 g/mol
Physical appearance : Glipizide is a whitish, odorless powder.
Solubility : It is insoluble in water and alcohols, but soluble in 0.1 N

NaOH; it is freely soluble in dimethyl formamide, sparingly soluble in acetone and soluble
in methylene chloride (Dichloromethane).
Category : Glipizide is an oral hypoglycemic agent.

Mechanism of action : Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder

with increasing prevalence worldwide. Characterized by higher-than-normal levels of blood
glucose, T2DM is a complex disorder that arises from the interaction between genetic,
environmental and behavioral risk factors. Insulin is a peptide hormone that plays a critical
role in regulating blood glucose levels. In response to high blood glucose levels, insulin
promotes the uptake of glucose into the liver, muscle cells, and fat cells for storage. Although
there are multiple events occurring that lead to the pathophysiology of T2DM, the disorder
mainly involves insulin insensitivity as a result of insulin resistance, declining insulin
production, and eventual failure of beta cells of pancreatic islets that normally produce
insulin.5 Early management with lifestyle intervention, such as controlled diet and exercise,
is critical in reducing the risk of long-term secondary complications, such as cardiovascular
mortality.
Pharmacodynamics: Glipizide is a blood glucose-lowering agent. The initial onset of blood

glucose-lowering effect occurs around 30 minutes post-administration with the duration of
action lasting for about 12 to 24 hours.8 While the chronic use of Glipizide does not result in
elevations in the fasting insulin levels over time, the postprandial insulin response, or insulin
response to a meal, is observed to be enhanced, even after 6 months of treatment. The main
therapeutic actions of Glipizide primarily occur at the pancreas where the insulin release is
stimulated, but Glipizide also mediates some extra pancreatic effects, such as the promotion
of insulin signaling effects on the muscles, fat, or liver cells.9 Due to its action on the
endogenous cells, sulfonylureas including Glipizide is associated with a risk for developing
hypoglycemia and weight gain in patients receiving the drug.5,6 Chronic administration of
Glipizide may result in down- regulation of the sulfonylurea receptors on pancreatic beta
cells, which are molecular targets of the drug, leading to a reduced effect on insulin secretion.
Absorption: Gastrointestinal absorption of Glipizide is uniform, rapid, and essentially

complete. The absolute bioavailability of Glipizide in patients with type 2 diabetes receiving
a single oral dose was 100%. The maximum plasma concentrations are expected to be
reached within 6 to 12 hours following initial dosing. The steady-state plasma concentrations
of Glipizide from extended-release oral formulations are maintained over the 24-hour dosing
interval. In healthy volunteers, the absorption of Glipizide was delayed by the presence of
food but the total absorption was unaffected.
Volume of distribution: The mean volume of distribution was approximately 10 L following

administration of single intravenous doses in patients with type 2 diabetes mellitus. In mice
and rat studies, the presence of the drug and its metabolites was none to minimal in the fetus
of pregnant female animals.9 Other sulfonylurea drugs were shown to cross the placenta and
enter breast milk 6 thus the potential risk of Glipizide in fetus or infants cannot be excluded.
Protein binding: Glipizide is about 98-99% bound to serum proteins, with albumin being the
main plasma protein.
Metabolism: Glipizide is subject to hepatic metabolism, in which its major metabolites are
formed from aromatic hydroxylation. These major metabolites are Glipizide are reported to
be pharmacologically inactive. In contrast, an acetylaminoethyl benzine derivative is formed
as a minor metabolite which accounts for less than 2% of the initial dose and is reported to
have one- tenth to one-third as much hypoglycemic activity as the parent compound.
Route of elimination: Glipizide is mainly eliminated by hepatic biotransformation, where

less than 10% of the initial dose of the drug can be detected in the urine and feces as
unchanged Glipizide. About 80% of the metabolites of Glipizide are excreted in the urine
while 10% is excreted in the feces.
Half-life: The mean terminal elimination half-life of Glipizide ranged from 2 to 5 hours after
single or multiple doses in patients with type 2 diabetes mellitus.
Side Effects: Side effects of Glucotrol including easy bruising or bleeding (nosebleeds,
bleeding gums), tiredness, shortness of breath, upper stomach pain, itching, dark urine, clay-
colored stools, jaundice (yellowing of the skin or eyes); pale skin, fever, confusion; or
throbbing headache.
INTERACTIONS:
Drug Interactions:
5- androstenedione: The metabolism of 5-androstenedione can be decreased when combined

with Glipizide.
6-O-benzylguanine: The metabolism of 6-O-benzylguanine can be decreased when

combined with Glipizide.
7,8-Dichloro-1,2,3,4-tetrahydroisoquinoline: 7,8-Dichloro-1,2,3,4-tetrahydroisoquinoline
may increase the hypoglycemic activities of Glipizide.
9- aminocamptothecin: The metabolism of 9-aminocamptothecin can be decreased when

combined with Glipizide.
Medical Uses: Glipizide is an oral diabetes medicine that helps control blood sugar levels by
helping your pancreas produce insulin. Glipizide is used together with diet and exercise to
improve blood sugar control in adults with type 2 diabetes mellitus. Glipizide is not for
treating type 1 diabetes.
METFORMIN DRUG PROFILE24-26
Name Metformin
Description This compound belongs to the class of organic compounds known as

biguanides. These are organic compounds containing two N-linked
guanidines.
Structure
Categories Anti-Diabetic Agent
Weight Average: 129.1636
Chemical Formula C4H11N5
IUPAC Name 1-carbamimidamido-N,N-dimethylmethanimidamide

Classes Guanidines
Solubility Metformin was found to be freely soluble in water; slightly soluble in

alcohol; practically insoluble in acetone and in methylene chloride.
Pharmacology
Indication For use as an adjunct to diet and exercise in adult patients (18 years and
older) with NIDDM. May also be used for the management of metabolic
and reproductive abnormalities associated with polycystic ovary syndrome
(PCOS). Jentadueto is for the treatment of patients when both Linagliptin
and Metformin is appropriate.
Pharmacodynamics Metformin is an oral antihyperglycemic agent that improves glucose

tolerance in patients with NIDDM, lowering both basal and postprandial
plasma glucose. Metformin is not chemically or pharmacologically related
to any other class of oral antihyperglycemic agents. Unlike sulfonylureas,
Metformin does not produce hypoglycemia in either patients with NIDDM
or healthy subjects and does not cause hyperinsulinemia. Metformin does
not affect insulin secretion.
Mechanism of Metformin mechanisms of action differ from other classes of oral

action antihyperglycemic agents. Metformin decreases blood glucose levels by
decreasing hepatic glucose production, decreasing intestinal absorption of
glucose, and improving insulin sensitivity by increasing peripheral glucose
uptake and utilization. These effects are mediated by the initial activation
by Metformin of AMP-activated protein kinase (AMPK), a liver enzyme
that plays an important role in insulin signaling, whole body energy
balance, and the metabolism of glucose and fats. Activation of AMPK is
required for Metformin’s inhibitory effect on the production of glucose by
liver cells. Increased peripheral utilization of glucose may be due to
improved insulin binding to insulin receptors. Metformin administration
also increases AMPK activity in skeletal muscle. AMPK is known to cause
GLUT4 deployment to the plasma membrane, resulting in insulin-
independent glucose uptake. The rare side effect, lactic acidosis, is thought
to be caused by decreased liver uptake of serum lactate, one of the
substrates of gluconeogenesis. In those with healthy renal function, the
slight excess is simply cleared. However, those with severe renal
impairment may accumulate clinically significant serum lactic acid levels.
Other conditions that may precipitate lactic acidosis include severe hepatic
disease and acute/decompensated heart failure.
Absorption Absorbed over 6 hours, bioavailability is 50 to 60% under fasting

conditions. Administration with food decreases and delays absorption.
Some evidence indicates that the level of absorption is not dose-related,
suggesting that absorption occurs through a saturable process. Limited data
from animal and human cell cultures indicate that absorption occurs
through a passive, non-saturable process, possibly involving a paracellular
route. Peak action occurs 3 hours after oral administration.
Volume of 654 L for Metformin 850 mg administered as a single dose. The volume of
distribution distribution following IV administration is 63-276 L, likely due to less
binding in the GI tract and/or different methods used to determine volume
of distribution.
Protein binding Metformin is negligibly bound to plasma proteins.
Metabolism Metformin is not metabolized.
Route of Intravenous single-dose studies in normal subjects demonstrate that

elimination Metformin is excreted unchanged in the urine and does not undergo
hepatic metabolism (no metabolites have been identified in humans) nor
biliary excretion. Approximately 90% of the drug is eliminated in 24 hours
in those with healthy renal function. Renal clearance of Metformin is
approximately 3.5 times that of creatinine clearance, indicating the tubular
secretion is the primary mode of Metformin elimination.
Half life 6.2 hours. Duration of action is 8-12 hours.

Affected organisms ● Humans and other mammals.
Side Effects Side effects of Metformin include: physical weakness (asthenia), diarrhea,
gas (flatulence), symptoms of weakness, muscle pain (myalgia), upper
respiratory tract infection, low blood sugar (hypoglycemia), abdominal
pain (GI complaints), and lactic acidosis (rare), low blood levels of vitamin
B-12.
Drug Interactions Abaloparatide: The therapeutic efficacy of Metformin can be decreased
when used in combination with Abaloparatide.
Abemaciclib: The excretion of Abemaciclib can be decreased when

combined with Metformin.
Acarbose: The risk or severity of hypoglycemia can be increased when

Acarbose is combined with Metformin.
Aceclofenac: Aceclofenac may decrease the excretion rate of Metformin

which could result in a higher serum level.
Food Interactions Avoid alcohol.
Take with food to reduce gastric irritation.
COMBINED DRUG FORMULATION:
S.No. Drug name Label Claim Brand name Company
1 Glipizide and 5mg/500mg Glipimet Forte Sun Pharma

Metformin Tab
5.PLAN OF WORK
In order to develop a simple, reliable and an accurate method development and validation of
Glipizide and Metformin in bulk and pharmaceutical dosage form by reverse phase HPLC and
validate the method for its repeatability and reproducibility.
Plan of the proposed work includes the following steps:
⮚ Selection of drug and literature survey.
⮚ Solubility studies and optimization of conditions.
⮚ Analytical method(s) development using HPLC etc.
⮚ Assay of the drugs(s) in marketed formulations using the proposed method(s).
⮚ Procurement of raw materials.
⮚ Establishment of system suitability parameters.
⮚ Trails for the method development of Glipizide and Metformin.
⮚ Setting of the optimized method.
⮚ Validation of the optimized method for Glipizide and Metformin.
Validation parameters include:
❖ System suitability
❖ Specificity
❖ Method precision
❖ Linearity
❖ Accuracy
❖ Range
❖ Robustness
6. EXPERIMENTAL METHODS
6: INSTRUMENTS USED
Table-7: Instruments used
SL.No Instrument Model
1 HPLC WATERS, software: Empower 2, 2695

separation module. 996 PDA detector.
2 UV/VIS spectrophotometer LABINDIA UV
3 pH meter Lab India
4 Weighing machine Sartorius
5 Pipettes and Burettes Borosil
6 Beakers Borosil
7 Digital ultra sonicator Ener tech
6.1: CHEMICALS USED:
Table-8: Chemicals used
SL.No Chemical Brand
1 Glipizide Sura Labs
2 Metformin Sura labs
3 KH2PO4 FINER chemical LTD
4 Water and Methanol for HPLC LICHROSOLV (MERCK)

5 Acetonitrile for HPLC Merck
6. Triethyl amine Sura labs
7 Orthophosphoric acid Sura labs
6.1: HPLC METHOD DEVELOPMENT:
6.1.1 Mbile Phase Optimization:
Initially the mobile phase tried was Acetonitrile: Water and Acetonitrile:
Sodium dihydrogen phosphate buffer with varying proportions. Finally, the mobile
phase was optimized to Acetonitrile with Sodium dihydrogen phosphate buffer (pH
6.8), in proportion 20:80 v/v respectively.
6.3.3. Optimization of Column:
The method was performed with various columns like C18 column, X- bridge
column, Xterra, and C8 column. Phenomenex Luna C18 (4.6mm x 250mm, 5μm,
Make: Waters) was found to be ideal as it gave good peak shape and resolution at
1ml/min flow.
6.4. OPTIMIZED CHROMATOGRAPHIC CONDITIONS:
Instrument used : Waters HPLC with auto sampler and PDA
detector 996 model.
Temperature : Ambient
Column : Phenomenex Luna C18 (4.6mm x 250mm, 5μm,
Make: Waters) or equivalent
Buffer : 1.1998gm of Sodium dihydrogen phosphate in 1000ml

HPLC water pH (6.8) adjusted with ortho

phosph
oric acid.
pH : 6.8
Mobile phase : 80% buffer 20%
Acetonitrile Flow rate : 1 ml per min
Wavelength : 268
nm Injection volume :
10 μl Run
time :
6min.
Optimized chromatogram is shown in the figure and blank is shown in the figure. System
suitability parameters are shown in figure. and the results are shown in Table.
6.5: PREPARATION OF BUFFER AND MOBILE PHASE:
6.5.1: Preparation of Phosphate buffer:
Accurately 1.1998 gm of Sodium dihydrogen phosphate was taken in a 1000 ml

volumetric flask, dissolved in 150 mL of HPLC water and adjusted to pH 6.8 with
Orthophosphoric acid diluted to 1000ml with HPLC water and Filtered by using 0.45µ
filter paper and sonicated.
6.5.2Preparation of mobile phase:
Accurately measured 800 ml (80%) of above buffer and 200 ml of HPLC grade
acetonitrile (20%) were mixed and degassed in a digital ultra sonicater for 10 minutes
and then filtered through 0.45 µ filter under vacuum filtration.
6.6.1 Diluent Preparation:
The Mobile phase was used as the diluent.
: VALIDATION PARAMETERS:
6.6.1: Method Precision:
6.6.1.1: Preparation of Standard Solution:
Accurately weigh and transfer 10 mg of Glipizide and 10mg of Metformin working

standard into a 10mL and 100ml of clean dry volumetric flasks add about 7mL and
70ml of Diluents and sonicated to dissolve it completely and make volume up to the
mark with the same solvent. (Stock solution)
Further pipette 0.5 ml and 0.5 ml of the above Glipizide stock solution into a 10ml
volumetric flask and dilute up to the mark with diluents.
6.6.1.2Preparation of Sample Solution:
Accurately weigh 10 tablets crush in mortor and pestle and transfer equivalent to 10
mg of Glipizide and Metformin (marketed formulation) sample into a 10mL clean dry
volumetric flask add about 7mL of Diluents and sonicate to dissolve it completely and
make volume up to the mark with the same solvent. (Stock solution)
Further pipette 0.5 ml of above stock solution into a 10ml volumetric flask and dilute
up to the mark with diluent.
The standard and sample solutions of 50 µg/ml of Glipizide and 50 µg/ml of

Metformin were injected for five times and the peak areas were recorded as shown in
Figure.
The mean and percentage relative standard deviation were calculated from the
peak areas and shown in the Table.
5.5.1 : Intermediate Precision/Ruggedness:
To evaluate the intermediate precision (also known as Ruggedness) of the method,

Precision was performed on different day by using different make column of same
dimensions.
Preparation of stock solution:
mg of Glipizide and Metformin (marketed formulation) sample into a 10mL clean dry
volumetric flask add about 7mL of Diluents and sonicate to dissolve it completely and
Further pipette 0.5 ml of Glipizide and Metformin of the above stock solution into a
10ml volumetric flask and dilute up to the mark with diluent
Procedure:
The standard solution was injected for five times and measured the area for all five
injections in HPLC. The %RSD for the area of five replicate injections was found to
be within the specified limits.
Chromatograms were recorded as shown in Figure and results are shown in
Table. 5.6.3: Accuracy:
Preparation of Standard stock solution:
Accurately weigh and transfer 10 mg of Glipizide and 10mg of

Metformin working standard into a 10mL and 100ml of clean dry volumetric flasks
add about 7mL and 70ml of Diluents and sonicate to dissolve it completely and make
volume up to the mark with the same solvent. (Stock solution)
Further pipette 0.5ml and 0.5ml of the above Glipizide and Metformin stock solution
into a 10ml volumetric flask and dilute up to the mark with diluents
Preparation Sample solutions:
For preparation of 50% solution (With respect to target Assay concentration):
Accurately weigh and transfer 10mg of Glipizide and 10mg of Metformin working
standard into a 10mL and 100ml of clean dry volumetric flask add about 7mL of
Diluents
and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock Solution).
Further pipette 0.25ml and 0.25ml of the above Glipizide and Metformin stock
solution into a 10ml volumetric flask and dilute up to the mark with diluent.
Accurately weigh and transfer 10 mg of Glipizide and 10mg of Metformin working

70ml of Diluents and sonicate to dissolve it completely and make volume up to the
Further pipette 0.5 ml and 0.5ml of the above Glipizide stock solution into a 10ml
volumetric flask and dilute up to the mark with diluents
Accurately weigh and transfer 10 mg of Glipizide and 10 mg of Metformin working

70ml of Diluents and sonicate to dissolve it completely and make volume up to the
Further pipette 0.75 ml and 0.75 ml of the above Glipizide and Metformin stock
solution into a 10ml volumetric flask and dilute up to the mark with diluents.
Procedure:
Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150%
solutions.
Calculate the Amount found and Amount added for Glipizide & Metformin and
calculate the individual recovery and mean recovery values. These solutions were
filtered through 0.45µ membrane and then each concentration; three replicate
injections were made under
the optimized conditions. Recorded the chromatograms and measured the peak
responses. The chromatograms were shown in figure. Results are shown in the table.
5.6.4 : LINEARITY:
5.6.4.1 : Preparation of stock solution:
mg of Olanzapine and Metformin (marketed formulation) sample into a 100mL clean
dry volumetric flask add about 70mL of Diluents and sonicate to dissolve it
completely and make volume up to the mark with the same solvent. (Stock solution)
Preparation of Level – I (30 ppm of Glipizide & 30pm of Metformin):

0.3 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark
with diluent.
Preparation of Level – II (40 ppm of Glipizide & 40ppm of Metformin):
0.4ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark
with diluent.
Preparation of Level – III (50 ppm of Glipizide & 50ppm of Metformin):
with diluent.
Preparation of Level – IV (60 ppm of Glipizide & 60ppm of Metformin):
with diluent.
Preparation of Level – V (70 ppm of Olanzapine & 70ppm of Metformin):
with diluent.
Procedure:
Inject each level into the chromatographic system and measure the peak area.
Plot a graph of peak area versus concentration (on X-axis concentration and on Y-
axis Peak area) and calculate the correlation coefficient.
The chromatograms were recorded as show in Figure and results are shown in
Table. 5.6.5: LIMIT OF DETECTION (for Glipizide):
Preparation of 50 µg/ml solution:
Accurately weigh and transfer 10 mg of Glipizide and into a 10mL clean dry
volumetric flask add about 7mL of Diluent and sonicate to dissolve it completely and
Further pipette 0.5 ml of the above stock solution into a 10 ml volumetric flask and
dilute up to the mark with diluent.
Preparation of 0.01µg/ml solution):
Further pipette 0.1ml of the above stock solution into a 10ml volumetric flask and
Pipette 0.1mL of solution into a 10 ml of volumetric flask and dilute up to the mark with
diluent.
Chromatograms were shown in the figure Results are shown in the
table. 5.6.6: LIMIT OF QUANTIFICATION:
Preparation of 50 µg/ml solution:
Accurately weigh and transfer 10 mg of Glipizide working standard into a 10mL clean
dry volumetric flask add about 7mL of Diluent and sonicate to dissolve it completely
and make volume up to the mark with the same solvent. (Stock solution)
Pipette 0.3mL of above solution into a 10 ml of volumetric flask and dilute up to the
mark with diluent.
The chromatograms were recorded as show in Figure and results are shown in
Table.
5.6.7 : LIMIT OF DETECTION: (for Metformin)
Preparation of 0.8µg/ml solution:
Accurately weigh and transfer 10mg of Metformin working standard into a

100mL clean dry volumetric flask add about 70mL of Diluent and sonicate to dissolve
it completely and make volume up to the mark with the same solvent. (Stock solution)
Further pipette 0.008ml of the above Metformin stock solution into a 10ml
volumetric flask and dilute up to the mark with diluent.
Chromatograms were shown in the figure Results are shown in the table.
5.6.8 : LIMIT OF QUANTIFICATION:

Preparation of 2.4µg/ml solution:
Accurately weigh and transfer 10mg of Metformin working standard into a 10mL
clean dry volumetric flask add about 7 mL of Diluent and sonicate to dissolve it
completely and make volume up to the mark with the same solvent. (Stock solution)
Further pipette 0.0219ml of the above stock solution into a 100ml volumetric flask
and dilute up to the mark with diluent.
The chromatograms were recorded as show in Figure and results are shown in Table
5.6.9 : ROBUSTNESS:
The analysis was performed in different conditions to find the variability of test
results. The following conditions are checked for variation of results. .
Preparation of sample solution (50µg/ml of Glipizide 50 µg/ml of Metformin)
About 10mg of Glipizide and 10 mg of Metformin were weighed and transferred to

100ml volumetric flask, it was dissolved with diluent and the volume was made up to
the mark with same solvent. Further 0.5 ml of above solution was diluted to 10ml with
the diluent to get 50µg/ml of Glipizide 50µg/ml of Metformin
Effect of Variation of flow:
The sample was analyzed at 0.8 ml/min and 1.1 ml/min instead of 1ml/min, remaining
conditions are same. 10µl of the above sample was injected twice and chromatograms
were recorded.
Effect of Variation of mobile phase organic composition:
The sample was analyzed by variation of mobile phase i.e. Phosphate buffer:
Acetonitrile was taken in the ratio and 85:15, 75:25 instead of 80:20, remaining
conditions are same. 10µl of the above sample was injected twice and chromatograms
were recorded.
The chromatograms were recorded as shown in Figure and results are shown in Table.
5.8 formulae
AT WS DT P Avg. Wt
Assay% = x x x x X
100 AS DS WT 100 Label Claim
Where:
AT = average area counts of sample

preparation. AS = average area counts of
standard preparation. WS = Weight of
working standard taken in mg.
P = Percentage purity of working
standard LC = Label claim.
7. RESULTS AND DISCUSSION
The present investigation reported in the thesis was aimed to develop a new
method development and validation for the simultaneous estimation of Glipizide and
Metformin by RP-HPLC method.
Literature reveals that there are no analytical methods reported for the
simultaneous estimation of Glipizide and Metformin by RP-HPLC method. Hence, it
was felt that, there is a need of new analytical method development for the
simultaneous estimation of Glipizide and Metformin in pharmaceutical dosage form.
Method Development
The detection wavelength was selected by dissolving the drug in mobile phase
to get a concentration of 50µg/ml for individual and mixed standards. The resulting
solution was scanned in U.V range from 200-400nm.
The overlay spectrum of Glipizide and Metformin was obtained and the
isobestic point of Glipizide and Metformin showed absorbance’s maxima at 268nm.
The chromatographic method development for the simultaneous estimation of
Glipizide and Metformin were optimized by several trials for various parameters as
different column, flow rate and mobile phase, finally the following chromatographic
method was selected for the separation and quantification of Glipizide and Metformin
in API and pharmaceutical dosage form by RP-HPLC method.

7.1. Optimized chromatogram is obtained by following
conditions Trial 1: (Single drug trails-Glipizide)
Mobile phase: Acetonitrile: Methanol (70:30% v/v)
Column: Make; waters, symmetry C18 (4.6*250mm) 5µm particle
size Flow rate: 1.0 ml/min
Wavelength: 268 nm
Column temp: Ambient
Sample Temp: Ambient
Injection Volume: 10µl
Fig-3: Chromatogram of Trial-1
Table-9: Results of Trial-1
USP
S. USP USP plate
Peak name Rt Area Height Resolutio
No Tailing count
n
1 Glipizide 3.315 3524121 478954 1.02

2.8 986
Trial 2: (Single drug trails-Glipizide)
Mobile phase: Water: Methanol (20:80% v/v)
Column: Make; waters C18 (4.6*150mm) 5µm particle
Wavelength: 268 nm
Injection Volume: 10 µl
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.206 6352415 469551 1.59

3.7 2541
Trial 3: (Single drug trails-Metformin)
Mobile phase: Water: Methanol (60:40%v/v)
Column: Make; waters Symmetry C18 (4.6*250mm) 5µm Particle
Wavelength: 268 nm
USP
S. USP USP plate
No Tailing count
n
1 Metformin 2.064 521414 524154 1.63

3.8 2145
Trial 4: (Single drug trails-Metformin)
Mobile phase: Phosphate buffer (0.02M) pH 4.8: Methanol
(70:30%v/v) Column: Symmetry C18 5µm (4.6*250mm) Make;
waters make Flow rate: 1ml/min
Wavelength: 268 nm
USP
S. USP USP plate
No Tailing count
n
1 Metformin 3.686 8564754 562487 1.51

3.6 2658
Trial 5: (Combination drug trails-Glipizide + Metformin)
Mobile phase: Phosphate buffer (0.05M) pH 5.6: Methanol
(60:40%v/v) Column: Symmetry C18 5µm (4.6*150mm) Make;
waters
Flow rate: 1ml/min
Wavelength: 268 nm
Fig-7: Chromatogram of Trial-5 (Glipizide + Metformin)
Table-13: Results of Trial-5 (Glipizide + Metformin)
S. USP USP USP plate

Peak name Rt Area Height
No Resolution Tailing count
1 Glipizide 4.276 214541 12541 0.78 2314
2 Metformin 5.152 19865 12044 1.19

2.90 985
From the above chromatogram it was observed that the Glipizide, Metformin peaks were
separated but resolution was not passed.
Trial 6:
Mobile phase: Phosphate buffer (0.03M) pH 5.6: Acetonitrile
(50:50%v/v) Column: X bridge C18 5µm (4.6*150mm) 5µm particle size
Flow rate: 1 ml/min
Wavelength: 268 nm
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.696 265894 465844 0.71 985

2 Metformin 3.271 3521454 214541 1.09

3.0 2104
From the above chromatogram it was observed that the Glipizide and Metformin
peaks are splitted.
Trial 7:
Mobile phase:Phosphate buffer (0.02M) pH 6.2: Acetonitrile (60:40%v/v)
Column:Phenomenex Luna C18 (4.6mm x 250mm, 5µm Particle size
Flow rate: 1.0 ml/min
Wavelength: 268 nm

S. USP USP USP plate

Peak name Rt Area Height
No Resolution Tailing count
1 Glipizide 2.584 3562845 521415 0.69 5621
2 Metformin 3.413 275842 225416 1.21

3.1 2514
Trial 8: (Optimized Chromatographic Condition)
Mobile phase: Phosphate buffer (0.01M) pH 6.8: Acetonitrile (80:20%v/v)
Column: Phenomenex Luna C18 (4.6mm x 250mm, 5µm Particle size
Flow rate: 1 ml/min
Wavelength: 268 nm
Fig-10: Optimized Chromatographic Condition (Glipizide + Metformin)

Table-16: Results of Optimized Chromatographic Condition

(Glipizide + Metformin)
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.242 4256351 565842 0.68 6584
2 Metformin 3.678 265284 285441 1.47

3.6 4857
From the above chromatogram it was observed that the Glipizide and Metformin
peaks are well separated
Retention time of Glipizide – 2.242
min Retention time of Metformin –
3.678 min
Figure-11: Chromatogram for Blank Solution
From the above chromatogram it was observed that there are no interferences
7.2 : SYSTEM SUITABILITY:
Figure-12: Chromatogram for system suitability
Table-17: Results of system suitability parameters for Glipizide and

Metformin
USP USP USP plate

S. No Peak name Rt Area Height
Resolution Tailing count
1 Glipizide 2.242 4263524 545145 0.85 7568
2 Metformin 3.679 267412 27582 1.26

3.9 4214
Acceptance criteria:
● Resolution between two drugs must be not less than 2
● Theoretical plates must be not less than 2000
● Tailing factor must be not less than 0.9 and not more than 2.
● It was found from above data that all the system suitability parameters for
developed method were within the limit.
6.3 : VALIDATION PARAMETERS:
Assay calculation for Glipizide and Metformin

The assay study was performed for the Glipizide and Metformin. Each three
injections of sample and standard were injected into chromatographic system. The
chromatograms are shown in Fig. and results are tabulated in Table.
Fig-13: Chromatogram showing assay of sample injection-1

Table-18: Peak Results for assay standard of Glipizide +
Metformin
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.241
4256587 545145 0.86 6352
3.680 268547 27547 1.48
2 Metformin 3.6 4652
2.245
3 Glipizide 4268541 545241 0.69 6541
3.683 265412 26854 1.49

2.245
5 Glipizide 4258417 552415 0.75 7684
3.683 269854 26859 1.47

Fig-16: Chromatogram showing assay of standard injection -1
Fig-17: Chromatogram showing assay of standard injection -2

Fig-18: Chromatogram showing assay of standard injection -
3 Table-19: Peak Results for assay Sample of Glipizide +
Metformin
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.241
4236521 545847 0.62 6522
3.680 275874 27521 1.44
2.245
3 Glipizide 4298542 546541 0.68 6632
3.683 278547 27452 1.45

2.245
5 Glipizide 4265844 556352 0.63 6851
3.683 278798 26425 1.66

%ASSAY =
Sample area Weight of standard Dilution of sample Purity Weight of tablet

× × × × ×100
Standard area Dilution of standard Weight of sample 100 Label claim
= 4266969 / 4261181.667 × 10/50 × 50/15.87 × 99.86/100 × 47.62/30 × 100
= 100.016%
The % purity of Olanzapine and Metformin in pharmaceutical dosage form was found to be
100.016%.
6.3.1 : Precision:
Precision of the method was carried out for both sample and standard solutions as
described under experimental work. The corresponding chromatograms and results are
shown below.
Fig-19: Chromatogram for standard injection-1


Table-21: Results of method precision for Glipizide + Metformin
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.255
4263582 565842 0.67 6579
3.690 266521 285441 1.42
2.252
3 Glipizide 4265851 565842 0.69 6524
3.688 268542 285441 1.42

2.253
5 Glipizide 4285422 565842 0.66 6635
3.687 268542 285441 1.42

2.258
7 Glipizide 4268594 565842 0.68 6589
3.691 265648 285441 1.46

2.258
9 Glipizide 4275845 565842 0.65 6854
3.688 265845 285441 1.43

● %RSD for sample should be NMT 2
● The %RSD for the standard solution is below 1, which is within the limits
hence method is precise.
6.3.2. INTERMEDIATE PRECESSION (ruggedness)
There was no significant change in assay content and system suitability parameters at
different conditions of ruggedness like day to day and system to system variation.
Day1, Analyst1:
Fig-24: Chromatogram for sample injectiocn-1
Fig-25: Chromatogram for sample injection-2

Table-22: Results of Intermediate precision for Glipizide + Metformin:
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.255
4275248 562524 0.69 6553
3.685 267854 285362 1.41
2.262
3 Glipizide 4258641 562415 0.65 6865
3.687 269658 286321 1.43

2.257
5 Glipizide 4278474 564142 0.66 6682
3.687 268451 283625 1.47

● %RSD of five different sample solutions should not more than 2.
● The %RSD obtained is within the limit, hence the method is rugged.
Day2, Analyst2:

Table-23: Results of Intermediate precision for Glipizide + Metformin:
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.266 4254784

563251 0.62 6585
3.687 284521 1.40
2 Metformin 265874 3.6 4765
2.275
3 Glipizide 4268947 566325 0.68 6784
3.684 285241 1.45

4 Metformin 268797 3.6 4954
2.266
5 Glipizide 4289354 565847 0.69 6481
3.686 285621 1.46

6 Metformin 267895 3.6 4698
● %RSD of five different sample solutions should not more than 2.
● The %RSD obtained is within the limit, hence the method is rugged.
6.3.3 : ACCURACY:
Sample solutions at different concentrations (50%, 100%, and 150%) were prepared
and the % recovery was calculated.
Chromatogram for sample concentration-
50% Injection-1
Fig-30: Chromatogram for sample concentration-50%
Injection-2

Injection-3
Table-24: Results of Chromatogram Sample Concentration-
50%
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.203 3363851

562541 0.63 6586
3.680 283625 1.39
2 Metformin 209895 3.6 4785
2.206
3 Glipizide 3398843 564685 0.69 6763
3.677 285412 1.48

4 Metformin 209878 3.6 4952
2.205
5 Glipizide 3356245 566352 0.63 6652
3.678 284784 1.47

6 Metformin 209948 3.6 4745
100% Injection-1
Injection-2

Injection-3
100%
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.283 4278554

563652 0.69 6685
3.682 285421 1.42
2 Metformin 261828 3.6 4568
2.282
3 Glipizide 4269887 568547 0.68 6659
3.678 286395 1.46

4 Metformin 262587 3.6 4658
2.283
5 Glipizide 4287846 567481 0.68 6854
3.676 288654 1.46

6 Metformin 261876 3.6 4639
150% Injection-1
Injection-2

Injection-3
150%
USP
S. USP USP plate
No Tailing count
n
1 Glipizide 2.247 5086545

568545 0.65 6854
3.682 286859 1.41
2 Metformin 318951 3.6 4223
2.245
3 Glipizide 5089654 567847 0.69 6476
3.683 286854 1.47

4 Metformin 318547 3.6 4698
2.243
5 Glipizide 5078977 565898 0.69 6487
3.685 287847 1.43

6 Metformin 319124 3.6 4895
Accuracy Std:
Table-27: Accuracy (recovery) data for Glipizide:
%Concentrati
Amount Amount
on % Mean
Area Added Found
(at specification Recovery Recovery
(mg) (mg)
Level)
50% 3372980 40 39.893 99.732%
100% 4285059 50 50.617 101.234% 100.41%
150% 5085059 60 60.163 100.271%
Acceptance Criteria:
● The % Recovery for each level should be between 98.0 to 102.0%.
Table-28: Accuracy (recovery) data for Metformin
%Concentrati
Amount Amount
on % Mean
Area Added Found
(at specification Recovery Recovery
(mg) (mg)
Level)
50% 209948 40 39.612 99.03%
100% 262097 50 49.538 99.076% 99.58%
150% 318874 60 60.383 100.638%

● The percentage recovery was found to be within the limit (97-103%).
The results obtained for recovery at 50%, 100%, 150% are within the limits. Hence
method is accurate.
6.3.4 LINEARITY:
The linearity range was found to lie from 10µg/ml to 50µg/ml of Glipizide,
1µg/ml to 5µg/ml 0f Metformin and chromatograms are shown below.
Fig-39: Chromatogram for linearity concentration-30 µg/ml of Glipizide & 30

µg/ml of Metformin
Fig-40: Chromatogram for linearity concentration-40 µg/ml of Glipizide &

40µg/ml of Metformin
Fig-41: Chromatogram for linearity concentration-50ppm of Glipizide &

50ppm of Metformin
Fig-42: Chromatogram for linearity concentration-60 µg/ml of Glipizide & 60

µg/ml of Metformin
Fig-43: Chromatogram for linearity concentration-70µg/ml of Glipizide & 70

µg/ml of Metformin
Linearity Results: (for Glipizide)
Table-29: Results for Linearity for Glipizide
Linearity
S.No. Concentration Area
Level
1 I 30 ppm 2544547
2 II 40 ppm 3358542
3 III 50 ppm 4231546
4 IV 60 ppm 5127547
5 V 70 ppm 5874451
Correlation Coefficient 0.999
Acceptance Criteria: Correlation coefficient should be not less than 0.99.
Linearity Results: (for Metformin)
Table-30: Results for Linearity for Metformin
Linearity
S.No. Concentration Area
Level
1 I 30ppm 158547
2 II 40ppm 215475
3 III 50ppm 265284
4 IV 60ppm 319866
5 V 70ppm 365214
Correlation Coefficient 0.999

● Correlation coefficient should be not less than 0.99.
Fig-44: Calibration graph for Glipizide at 268 nm
Fig-45: Calibration graph for Metformin at 268 nm

Table-31: Analytical performance parameters of Glipizide and Metformin
Parameters Glipizide Metformin
Slope (m) 84463 5258
Intercept (c) 3491 1625
Correlation coefficient (R2) 0.999 0.999
Correlation coefficient (R2) should not be less than 0.999.
● The correlation coefficient obtained was 0.999 which is in the acceptance limit.
The linearity was established in the range of 10% to 50% of Glipizide and 1%
to 5% of Metformin.
6.3.5 : LIMIT OF DETECTION FOR GLIPIZIDE AND METFORMIN
LIMIT OF DETECTION
The detection limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be detected but not necessarily quantitated as an exact
value.
LOD= 3.3 × σ / s
Where
σ = Standard deviation of the
response S = Slope of the
calibration curve
Result:
Glipizide: 0.8µg/ml
Metformin: 0.7µg/ml
LIMIT OF QUANTITATION
The quantitation limit of an individual analytical procedure is the lowest
amount of analyte in a sample which can be quantitatively determined.
LOQ=10×σ/S
Where
σ = Standard deviation of the
response S = Slope of the
calibration curve Result:
Glipizide:
2.4µg/ml
Metfor
min:
2. 19µg/ml
6.3.7 : ROBUSTNESS:
The standard and samples of Glipizide and Metformin were injected by changing the
conditions of chromatography. There was no significant change in the parameters like
resolution, tailing factor, asymmetric factor, and plate count.
6.3.7.1 : Variation in flow
Fig-46: Chromatogram showing less flow of 0.8ml/min
Fig-47: Chromatogram snowing more flow of 1.2ml/min

System suitability results for Glipizide:
Table-32: System suitability results for Glipizide:
System Suitability Results
Flow Rate
S.No USP Plate Count USP Tailing
(ml/min)
1 0.8 6686 0.69
2 1.0 6584 0.68
3 1.2 6785 0.67
* Results for actual flow (1.0 ml/min) have been considered from Assay standard.
System suitability results for Metformin:
Table-33: System suitability results for Metformin:
System Suitability Results
Flow Rate
S.No USP Plate Count USP Tailing
(ml/min)
1 0.8 4986 1.49
2 1.0 4857 1.47
3 1.2 4998 1.53
* Results for actual flow (1.0ml/min) have been considered from Assay standard.
6.3.7.2 Variation of mobile phase organic composition:
Fig-48: Chromatogram showing less organic composition
Fig-49: Chromatogram showing more organic composition

System suitability results for Glipizide:
Table-34: System suitability results for Glipizide
Change in System Suitability Results

S.No Organic
Composition in USP Plate Count USP Tailing
the Mobile Phase

1 10% less 6087 0.59
2 *Actual 6584 0.68
3 10% more 6989 0.57
System suitability results for Metformin:
Table-35: System suitability results for Metformin
Change in System Suitability Results

Organic
S.No
Composition in USP Plate Count USP Tailing
the Mobile
Phase
1 10% less 4169 1.39
2 *Actual 4857 1.47
3 10% more 4468 1.38
* Results for actual mobile phase have been considered from Assay standard.
8. SUMMARY AND CONCLUSION
A new method was established for simultaneous estimation of Glipizide and
Metformin by RP-HPLC method. The chromatographic conditions were successfully
developed for the separation of Glipizide and Metformin by using Phenomenex Luna C18
(4.6mm x 250mm, 5µm, Make: Waters) or equivalent, flow rate was 1ml/min, mobile phase
ratio was (20:80 v/v) Acetonitrile: Phosphate buffer pH 6.8 (pH was adjusted with
orthophosphoric acid), detection wave length was 268nm. The instrument used was
WATERS HPLC Auto Sampler, Separation module 2695, photo diode array detector 996,
Empower-software version-2. The retention times were found to be 2.242mins and 3.678
mins. The % purity of Glipizide and Metformin was found to be 99.85% and 100.14%
respectively. The system suitability parameters for Glipizide and Metformin such as
theoretical plates and tailing factor were found to be within limits. The analytical method was
validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study on Glipizide and
Metformin was found in concentration range of 30µg-70µg and 30µg-70µg and correlation
coefficient (r2) was found to be 0.999 and 0.999, % recovery was found to be 100.41% and
99.83%, %RSD for repeatability was 0.207 and 0.534. The precision study was precise,
robust, and repeatable. LOD value was 0.8 and 0.7, and LOQ value was 2.4 and 2.19
respectively.
Hence the suggested RP-HPLC method can be used for routine analysis of Glipizide
and Metformin in API and Pharmaceutical dosage form.

9. REFERENCES
1. Sahajwalla CG a new drug development, vol 141, Marcel Dekker Inc., New York, (2004),
PP 421–426.
2. Introduction to Column.
(Online),URL:http://amitpatel745.topcities.com/index_files/study/co lumn care.pdf
3. Detectors used in HPLC (online
)URL:http://wiki.answers.com/Q/What_detectors_are_used_ in_HPLC
4.Draft ICH Guidelines on Validation of Analytical Procedures Definitions and terminology.
Federal Register, vol 60. IFPMA, Switzerland, (1995), PP 1126.
5.Code Q2B, Validation of Analytical Procedures; Methodology. ICH Harmonized
Tripa
rtite Guidelines, Geneva, Switzerland, (1996), PP 1- 8.
6.Introduction to analytical method validation (online), available from:
URL: http://www.standardbase.hu/tech/HPLC%20validation%20PE.pdf.
7. Data elements required for assay validation, (online) available from:
URL: http://www.labcompliance.com/tutorial/methods/default.aspx.
8.Snyder LR practical HPLC method development, 2nd edition. John Wiley and sons, New
York, (1997), PP 180-182.
9.Skoog D A, West D M, Holler FJ: Introduction of analytical chemistry. Sounder college of
publishing, Harcourt Brace college publishers. (1994), PP 1-5.
10. Dr. Kealey and P.J Haines, Analytical Chemistry, 1stedition, Bios Publisher, (2002), PP 1-7.
11. A.BraithWait and F.J.Smith, Chromatographic Methods, 5thedition, Kluwer
Academic Publisher, (1996), PP 1-2.
12. Andrea Weston and Phyllisr. Brown, HPLC Principle and Practice, 1st
edition, Academic press, (1997), PP 24-37.
13. Yuri Kazakevich and Rosario Lobrutto, HPLC for Pharmaceutical
Scientists, 1stedition, Wiley Interscience A JohnWiley & Sons, Inc., Publication, (2007),
PP 15-23.
14. Chromatography, (online). URL:http://en.wikipedia.org/wiki/Chromatography.
15. Meyer V.R. Practical High-Performance Liquid Chromatography, 4th Ed. England, John
Wiley & Sons Ltd, (2004), PP 7-8.
16. Sharma B K, Instrumental method of chemical analysis Meerut. (1999), PP 175-203.
17. Breaux J and Jones K: Understanding and implementing efficient analytical method
development and validation. Journal of Pharmaceutical Technology (2003), 5, PP 110-
114.
18. Willard, H. y. Merritt L.L, Dean J.A and Settle F.A “Instrumental methods of analysis”
7th edition CBS publisher and distributors, New Delhi, (1991), PP 436-439.
19. ICH Q2A, “validation of analytical methods, definitions and terminology”, ICH
Harmonized tripartite guideline, (1999).
20. https://www.drugbank.ca/drugs/DB01067
21. https://pubchem.ncbi.nlm.nih.gov/compound/glipizide
22. https://en.wikipedia.org/wiki/Glipizide
23. https://www.drugbank.ca/drugs/DB00331
24. https://pubchem.ncbi.nlm.nih.gov/compound/4091
25. https://en.wikipedia.org/wiki/Metformin
26. Sri Lakshmi D*, Jane T Jacob, Srinivas D, Satyanarayana D, Simultaneous Estimation Of
Metformin And Glipizide By RP-HPLC And Its Validation, World Journal of Pharmacy
and Pharmaceutical Sciences, Volume 4, Issue 09, 740-750.
27. Bagadane Snehal Bapusaheb*, Jadhav Prerana B, Development and validation of RP-
HPLC method for simultaneous estimation of Metformin hydrochloride and Glipizide in
bulk and pharmaceutical dosage form, Journal of Drug Delivery and Therapeutics, 2019;
9(3-s):146- 155.
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UV Spectrophotometric Method for Simultaneous Estimation of Metformin and Glipizide
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(2019) 315-322.

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I take pleasure A Project Report On

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION

Under the esteemed guidance of

Mrs. G.MANGADEVI (M.Pharm)

Accredited by NAAC with B+ Grade

This is to certify that the dissertation entitled “ANALYTICAL METHOD

Guide’s Signature: Signature of the Head of theDept.

Accredited by NAAC with B+ Grade

The work presented in “ANALYTICAL METHOD DEVELOPMENT AND

Encouragement for the successful completion of this project.

I express my warmest gratitude to my esteemed guide, Mrs.G.MANGADEVI

I thank our honorable Chairman Sri B.SRINIVASARAO garu

My heartful gratitude to Mrs.G.MANGADEVI M.Pharm, Madam Head of the

We wish her to success in her future career.

Key Words: Glipizide, Metformin, RP-HPLC, Robustness and ICH Guidelines.

HPLC - High Performance liquid chromatography

.UV - Ultra violet spectroscopy

TLC - Thin layer chromatography

LOD - Limit of Detection

LOQ - Limit of Quantitation

S.D - Standard Deviation

%RSD - Percentage Relative Standard Deviation

M.P - Mobile Phase

µg/ml - micrograms per milliliter

ICH - International conference on Harmonization

S.NO. TITLE PAGE NO.

2. Literature review 45-47

3. Aim and Objective 48

4. Drug profile 49-55

6. Experimental Methods 57-67

7. Results and Discussions 68-107

8. Summary & Conclusion 108

S.NO NAME OF THE TABLE PAGE.NO

1 Classification of analytical methods 17

2 Different types of chromatographic techniques 21

3 Physical properties of common HPLC solvents 25

4 Separation goals in HPLC method development 34

5 Sample and analyte information 35

6 System suitability parameters and recommendation 40

7 METFORMIN DRUG PROFILE 52

8 COMBINED DRUG FORMULATION 55

18 Results of Optimized Chromatographic Condition 77

19 Results of system suitability parameters for Glipizide and 78

20 Peak Results for assay standard of Glipizide + Metformin 80

21 Peak Results for assay Sample of Glipizide + Metformin 82

22 Results of method precision for Glipizide + Metformin 85

23 Results of Intermediate precision for Glipizide + Metformin: 87

24 Results of Intermediate precision for Glipizide + Metformin: 89

25 Results of Chromatogram Sample Concentration-50% 91

26 Results of Chromatogram Sample Concentration-100% 93

27 Results of Chromatogram Sample Concentration-150% 95

28 Accuracy (recovery) data for Glipizide: 96

29 Accuracy (recovery) data for Metformin 96

30 Results for Linearity for Glipizide 100

31 Results for Linearity for Metformin 100

32 Analytical performance parameters of Glipizide and Metformin 102

33 System suitability results for Glipizide: 105

34 System suitability results for Metformin: 105

35 System suitability results for Glipizide 107

36 System suitability results for Metformin 107

S.NO NAME OF THE FIGURE PAGE.NO

1 Schematic diagram of HPLC instrumentation 24