ANALYTICAL METHOD DEVELOPMENT AND

VALIDATION FOR DETERMINATION OF GLIPIZIDE

AND METFORMIN IN BULK FORM AND MARKETED
PHARMACEUTICAL DOSAGE FORM BY RP-HPLC
Presented 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)
 CONTENTS

 Introduction
 Literature review
 Aim and Objective
 Drug profile
 Plan of work
 Experimental Methods
 Results and Discussions
 Summary & Conclusion
 References
 Introduction

 Pharmaceutical analysis plays a major role today, 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.
 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.
 TYPES OF HPLC TECHNIQUES

 Normal phase chromatography

 Reverse phase chromatography
 Reverse phase chromatography

 the polar nature of the silanol group by chemically reacting silicon with organic
silanes. The object was to make silica less polar or nonpolar 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.
 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.
 INSTRUMENTATION OF HPLC
 Mobile phase and reservoir
 Solvent
 Pump
 Injector
 HPLC Column
 Detector
 waste
 Data system
 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.
 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.
 Steps in HPLC method development

1. Information on sample, define separation Goals

2. Need special HPLC procedure, sample pre-treatment, etc..
3. Choose detector and detector setting
4. Choose LC method; preliminary run, estimate best separation
5. Optimize separation conditions
6. Check for problems or requirement for special procedures
7. Recover Purified material
8. Quantitative calibration
9. Qualitative method
10. Validate method for release to routine laboratory
ANALYTICAL METHOD VALIDATION

 FDA defines validation as “Establishing documented evidence, which Provides a

high degree of assurance that a specific process will consistently produce a
product of predetermined specifications and quality attributes.
 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.
 VALIDATION PARAMETERS (ICH)

I. Accuracy
II. Precision
III. Specificity
IV. Linearity
V. Detection limit
VI. Quantification limit
VII. Range
VIII. Robustness
 LITERATURE REVIEW

 Sri Lakshmi D, et al., (2015)

 Bagadane Snehal Bapusaheb, et al., (2019)
 D. Sireesha, et al., (2016)
 Suresh Kumar GV, et al. (2012)
 Crispin R. Dass, et al. (2019)
 AIM

 Aim of this project is 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.
 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.
 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 PROFILE
GLIPIZIDE DRUG PROFILE

 Name : Glipizide
 Chemical formula : C21H27N5O4S
 Molecular mass : 445.535 g/mol
 Physical appearance : Glipizide is a whitish, odorless powder.
 Category : oral hypoglycemic agent.
 Protein binding : 98-99%
 Volume of distribution : 10L
 Half-life : 2 to 5 hours after single or multiple doses
STRUCTURE OF GLIPIZIDE
 METFORMIN DRUG PROFILE

 Name : Metformin
 Chemical formula : C4H11N5
 Category : Anti-Diabetic Agent
 Protein binding : Negligibly bound to plasma proteins.
 Volume of distribution : 63-276 L
 Half-life : 6.2 hours. Duration of action is 8-12 hours.
STRUCTURE OF METFORMIN
COMBINED DRUG FORMULATION

 Drug name : Glipizide and Metformin

 Label Claim : 5mg/500mg
 Brand name: Glipimet Forte Tab
 Company : Sun Pharma
 PLAN OF WORK

 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
 EXPERIMENTAL METHODS
INSTRUMENTS USED
SL.No Instrument Model

WATERS, software: Empower 2, 2695

1 HPLC
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

 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

 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.
 PREPARATION 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.
 PREPARATION OF STOCK 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 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.
 PROCEDURE
Inject each level into the chromatographic system and measure the peak area.

 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):
 0.4 ml 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):
 0.5 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with diluent.
 Preparation of Level – IV (60 ppm of Glipizide & 60ppm of Metformin):
 0.6 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with diluent.
 Preparation of Level – V (70 ppm of Olanzapine & 70ppm of Metformin):
 0.7 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with diluent.
 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.
 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.
 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

S.No Peak name Rt Area Height USP Resolution USP Tailing USP plate count

1 Glipizide 3.315 3524121 478954 2.8 1.02 986

 Trial 2: Single drug trails-Metformin

 Mobile phase : Water; Methanol (60:40%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 : 10 µl

S.No Peak name Rt Area Height USP Resolution USP Tailing USP plate count

1 Metformin 2.064 521414 524154 3.8 1.63 2145

 Trial 3:
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
 Column temp : Ambient
 Sample Temp : Ambient
 Injection Volume : 20 µl

S.No Peak name Rt Area Height USP Resolution USP Tailing USP plate count

1 Glipizide 4.276 214541 12541   0.78 2314

2 Metformin 5.152 19865 12044 2.9 1.19 985
 Trial 8
Optimized Chromatographic Condition (Glipizide + Metformin)

 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 Make: Waters) or equivalent
 Flow rate : 1 ml/min
 Wavelength : 268 nm
 Column temp : Ambient
 Sample Temp : Ambient
 Injection Volume : 10 µl

S.No Peak name Rt Area Height USP Resolution USP Tailing USP plate count
1 Glipizide 2.242 4256351 565842   0.68 6584
2 Metformin 3.678 265284 285441 3.6 1.47 4857
 VALIDATION PARAMETERS
Peak Results for assay standard of Glipizide + Metformin

S.No Peak name Rt Area Height USP Resolution USP Tailing USP plate count
1 Glipizide 2.241 4256587 545145   0.86 6352
2 Metformin 3.68 268547 27547 3.6 1.48 4652
3 Glipizide 2.245 4268541 545241   0.69 6541
4 Metformin 3.683 265412 26854 3.6 1.49 4684
5 Glipizide 2.245 4258417 552415   0.75 7684
6 Metformin 3.683 269854 26859 3.6 1.47 4365
 Linearity Results: For Glipizide

S.No. Linearity Level Concentration Area

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

Results for Linearity for Glipizide

Acceptance Criteria: Correlation coefficient should be not less than 0.99.
 Linearity Results: For Metformin

S.No. Linearity Level Concentration Area

1 I 30ppm 158547

2 II 40ppm 215475

3 III 50ppm 265284

4 IV 60ppm 319866

5 V 70ppm 365214

Correlation Coefficient 0.999

Results for Linearity for Metformin

Acceptance Criteria: Correlation coefficient should be not less than 0.99.
 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
 Metformin:2.19µg/ml
 SUMMARY AND CONCLUSION

 A new method was established for simultaneous estimation of Glipizide and

Metformin by RP-HPLC method.
 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 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.
 The suggested RP-HPLC method can be used for routine analysis of Glipizide and
Metformin in API and Pharmaceutical dosage form.
 REFERENCES

 ICH Q2A, “validation of analytical methods, definitions and terminology”, ICH Harmonized
tripartite guideline, (1999).
 https://www.drugbank.ca/drugs/DB01067
 https://pubchem.ncbi.nlm.nih.gov/compound/glipizide
 https://en.wikipedia.org/wiki/Glipizide
 https://www.drugbank.ca/drugs/DB00331
 https://pubchem.ncbi.nlm.nih.gov/compound/4091
 https://en.wikipedia.org/wiki/Metformin
 Sri Lakshmi D*, Jane T Jacob, Srinivas D, Satyanarayana D, Bagadane Snehal Bapusaheb*, Jadhav
Prerana B, K. Ganesh1, G. Nikitha1, D. Sireesha2*, B. Vasudha3, Suresh Kumar Gv, D.Triveni, S.B.
Puranik, N.Sateesh Kumar4 K.A.SR, Ahmed Gedawy a, Hani Al-Salami a,b, Crispin R. Dass a,b.
 ETC……
THANK YOU