DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC AND CHROMATOGRAPHIC METHOD FOR DETERMINATION OF PIOGLITAZONE IN BULK DRUG AND DOSAGE FORMS

M. Pharmacy Research Project
DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC AND CHROMATOGRAPHIC
METHOD FOR DETERMINATION OF PIOGLITAZONE IN BULK DRUG AND DOSAGE
FORMS
Presented By:- Mr. Reddy D. D.
M Pharmacy, Second Year, Sem IV
Research Guide :- Prof. Gholve S. B.
M. Pharm., Ph.D. Scholar
Dept. of Qulity assurance.

CONTENTS
Channabasweshwar Pharmacy College (Degree), Latur2
75%
85%
Introduction
Need of study
Review of literature
Aim and objective
Plan of work
Materials and methods
Observation & Result
References

INTRODUCTION
 Analytical Method Development – It is the process of providing demostrating that
analytical procedures are suitable for their intended use & they support the identity , strength,
quality, purity , potency of the drug substances & drug products
Validation- The process of providing documented evidence, which provides a high degree of
assurance that specific process will produce a product meeting its predetermined specification
& quality attributes.
Analytical method development & validation play important role in discovery, development
& manufacturing of pharmaceuticals.
12/2/2020

INTRODUCTION
Channabasweshwar Pharmacy College (Degree), Latur
4
Spectroscopy:-
UV Spectroscopy:- The ultraviolet region is normally defined as 200–400 nm.
 Principle of Spectroscopy
• The principle is based on the measurement of spectrum of a sample containing atoms
/molecules.
• Spectrometer is an instrument design to measure the spectrum of a compound.
• Spectrum is a graph of intensity of absorbed or emitted radiation by sample various
frequency (ν) or wavelength (λ).
Fig. UV Spectroscopy
12/2/2020

INTRODUCTION
 Chromatography :-
 HPLC :- The technique is based on the pumping of mobile phase through the packed
column under high-pressure.
 Types HPLC
1. Mechanism
2. Polarity
 Principle:-When a mixture of a components are introduced into a HPLC column, they
travel according to their relative affinities to words the stationary phase.
• The components which has more affinity to words stationary phase travel slower.
• The components which has less affinity to words stationary phase travel faster.
12/2/2020

INTRODUCTION
 Pioglitazone is a diabetes drug (thiazolidinedione-type) used along with a proper diet and
exercise program to control high blood sugar in patients with type 2 diabetes. It works by
helping to restore your body's proper response to insulin, thereby lowering your blood sugar.
Pioglitazone activates peroxisome proliferator-activated receptor gamma (PPAR-gamma), a
ligand-activated transcription factor, thereby inducing cell differentiation and inhibiting cell
growth and angiogenesis.
 Controlling high blood sugar helps prevent kidney damage, blindness, nerve problems, loss
of limbs, and sexual function problems. Proper control of diabetes may also less your risk of
a heart attack or stroke.
5‐{p‐[2‐(5‐ethyl‐2‐pyridyl)ethoxy]benzyl}‐2,4‐thiazolidinedione hydrochloride
12/2/2020

NEED OF STUDY
 To introduce the simple, precise, accurate, robustness & economical method to the drug &
formulation.
 The method has found to be better than previously reported methods because of wide range
of linearity, use of economical & readily available mobile phase, UV detection , low flow rate
& retention time mainly use C18 column.
 Analytical method need to be validated & revalidated before their introduction into routine
use ,whenever the conditions change for which the method has been validated.
 It can therefore be concluded that the use of method can save much time & money & can be
used in small laboratories with high accuracy & wide linearity range.
 No. of drugs introduced into the market is increasing every year before introduction of new
validation method is must.
12/2/2020

PLAN
OF
WORK
Purpose
Characterization Of The Analyte
Requirement
Review Of Literature
Choosing And Analytical Method
Setting Up Of Instruments
Optimization Of The Method
Statistical Analysis & Calculation
Compilation Of Result & Interpretation

LITERATURE SURVEY
Sr.No. AUTHOR PERTICULARS
1. B.Siddarth.et al (2013) Pioglitazone was analyzed on a BDS
Thermohypersil C18 column (150x4.6mm, 5µ)
was used with a mobile phase containing a
mixture of Methanol and Ortho phosphoric acid
buffer (pH-3) in the ratio of 40:60 (v/v) . on
dosage form
2. Sharmila Begum Shaik et al
(2014)
Pioglitazone was analyzed Intersil ODS C18
(150 mm × 4.6 mm, 5µm) column with mobile
phase consisting of Ammonium acetate buffer
with Acetonitrile and Glacial acetic acid in the
ratio 50:50:1 (v/v) and quantified by UV
detection at 269 nm with flow rate of 0.7 ml/min
3. D.Shrinivas et al (2010) pioglitazone was analyzed using C18 column
(250x4.6mm), mixture of Buffer: acetonitrile
(55:45%v/v) as the mobile phase at a flow rate
1.0 ml/min. The analyte was monitored using
UV detector at 254 nm .
12/2/2020

LITERATURE SURVEY
4. Syed imran ali et al (2018) Pioglitazone was analyzed in human plasma .the
mobile phase comprises, 0.1 M ammonium acetate
and glacial acetic acid (25:25: v/v/v) at a flow rate
of 1.2 mL/min., using macherey-nagel column C18,
(dimension: 5 µm; 250 × 4.6 mm) with a guard
column.
5. Karthik A et al (2008) Pioglitazone and glimepride was analyzed using
intersil C18 ODS (250 × 4.6 mm, 5µ) column and
mobile phase comprised of acetonitrile and
ammonium acetate (pH 4.5 ; 20 mM) in proportion
of 60:40 (v/v) the flow rate was 1.0 ml/min and the
effluent was monitored at 230 nm.
6. Pattana sripalakit et al
(2006)
Pioglitazone was analyzed in human plasma a
reversed phase Apollo C18 colum and a mobile
phase of methanol-acetonitrile-mixed phosphate
buffer (pH 2.6; 10mM) (40:12:48,v/v/v) with a flow
rate of 1.2 ml/min
12/2/2020

PIOGLITAZONE DRUG PROFILE
IUPAC Name
(±)‐5‐{p‐[2‐(5‐ethyl‐2‐pyridyl)ethoxy]benzyl}‐2,4‐thiazoli
dinedione
Molecular weight 356.44 g/mol
Molecular formula C19H20N2O3 S
Melting point 183 to 184 °C (361 to 363 °F)
Half-life 3 to 7 hours
Excretion Urine and Feces
Solubility Soluble in Ethanol, Methanol, Acetonitrile,0.2 ,0.1N HCL.
12/2/2020

MATERIALS AND METHODS
12
Sr. No. Name of Reagent Used Make
1. Ortho phosphoric Acid (PH:3) Analytical and HPLC grade
2. Methanol Analytical and HPLC grade
Chemicals and reagents :-
Instruments :-
Sr. No. Name of Equipment Source
1. HPLC Agilent 1220 Infinity LC
2. UV Shimadzo, Model: UV-1800
3. Detector Variable wavelength detector
4. Electronic weighing balance Shimadzo BL- 220 H
5. Hot air oven Nisco company
6. Sonicator Ultrasonic’s PCi Analytics Sonicator
12/2/2020

MATERIALS AND METHODS
Used Drugs for the method development :-
Drug Standard Procured By
Pioglitazone Indian Pharmacopoeia FDC LIMITED,GOA
•Forced Degradation:
1) Acid degradation
2) Base degradation
3) Photo stability study
4) Hydrogen peroxide degradation
12/2/2020

VALIDATION
PARAMETER
SPECIFICITY
LINEARITY
RANGE
PRECISION
ACCURACY
LIMIT OF DETECTION
LIMIT OF QUANTITATION
ROBUSTNESS
14

OBSERVATIONS AND RESULTS
 Drug Identification and Authentication:-
• Drug identification and authentication is done by Melting point, solubility, UV
spectrum & FT-IR was the proof for its authenticity.
• Identity of drug was confirmed by comparing FT-IR spectra with Pioglitazone
(IP) Standard for presence of functional groups.
• Melting point- 184 ℃.
• Solubility- Soluble in Methanol, 0.1N HCL.
• UV spectrum- observed λ max of at 268 nm.
12/2/2020

FT-IR Graph of Pioglitazone :-
Chemical name Standard peak Reange (cm1) Observed Peak (cm1)
NH-C=O 1670-1820 1683
S=O 1510-1380 1509
C-CH3 3000-2800 2927
C=C 1680-1600 1620
cbpharmacy 220
Name
Sample 220 By cbpharmacy Date Thursday, January 23 2020
Description
4000 4503500 3000 2500 2000 1500 1000 500
98
52
55
60
65
70
75
80
85
90
95
cm-1
%T
1683.33cm-1, 52.75%T
849.60cm-1, 63.68%T
1242.45cm-1, 68.90%T
1231.18cm-1, 69.02%T
1148.17cm-1, 69.10%T
1038.11cm-1, 72.11%T 711.72cm-1, 72.21%T
584.93cm-1, 73.71%T
513.77cm-1, 75.78%T
1509.52cm-1, 76.61%T
1741.78cm-1, 77.55%T
659.33cm-1, 79.14%T
1313.02cm-1, 79.98%T 872.47cm-1, 80.32%T 550.33cm-1, 80.53%T
1 3 3 3 . 7 4 c m - 1 , 8 1 . 3 9 % T
1461.47cm-1, 82.75%T
644.99cm-1, 83.65%T
602.83cm-1, 83.91%T
1620.73cm-1, 85.02%T2615.83cm-1, 85.67%T
1293.77cm-1, 85.87%T 790.79cm-1, 85.99%T
1475.33cm-1, 86.02%T
1176.29cm-1, 86.66%T
1397.16cm-1, 87.61%T 738.58cm-1, 88.01%T2927.48cm-1, 88.01%T
9 9 9 . 7 1 c m - 1 , 8 8 . 3 1 % T
470.48cm-1, 90.22%T1552.77cm-1, 90.43%T
1065.39cm-1, 90.97%T
931.31cm-1, 91.76%T
12/2/2020

Selection of wavelength :-
Standard stock solution of 100 mg/ml of pioglitazone was prepared in 0.1N HCL diluents.
from the above stock solution, pipette out 10 ml in to 100ml volumetric flask and 1 ml finally
made up the volume with 10 ml volumetric flask 0.1N HCL to produce a concentration of
10µg/ml. The sample was then scanned in UV spectrophotometer from a range of 200-400 nm
against 0.1N HCL as blank and the wavelength corresponding to maximum absorbance in
0.1N HCL was found at 268 nm
12/2/2020

For UV Spectroscopic Method
Sr. No. Concentration µg/ml Absorbance
1. 2 0.210
2. 4 0.582
3. 6 0.868
4. 8 1.166
5. 10 1.393
6. 12 1.631
y = 0.0198x + 0.0514
R² = 0.980
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.5 1 1.5
Concentration µg/ml
12/2/2020

Linearity Parameters of UV :-
Sr. No. Parameters Data
1 λ max 268nm
2 Linearity 2-16 µg/ml
3 Regression equation Y=0.0194x + 0.0514
4 Correlation coefficient R2 = 0.980
5 Slop 0.0194
6 Intercept 0.084
7 LOD 1.5 µg/ml
8 LOQ 3.701 µg/ml
12/2/2020

20
UV Precision :-
Sr. No.
Absorbance
4 8 12
1 0.567 0.853 1.323
2 0.561 0.855 1.335
3 0.562 0.852 1.347
4 0.571 0.864 1.330
5 0.559 0.867 1.334
6 0.563 0.856 1.338
Average 0.5638 0.8678 1.3345
SD 0.0044 0.0061 0.0080
%RSD 0.7805 0.7202 0.0960
Sr. No.
Absorbance
4 8 12
1 0.441 0.712 1.235
2 0.451 0.719 1.237
3 0.459 0.713 1.239
4 0.453 0.710 1.228
5 0.455 0.708 1.233
6 0.457 0.721 1.231
Average 0.4526 0.7138 1.0238
SD 0.0063 0.0051 0.0040
%RSD 1.408 0.7166 0.3889
Intraday precision Interday precision
12/2/2020

21
Robustness :-
Sr. No.
Absorbance
4 8 12
1 0.415 0.671 1.143
2 0.409 0.679 1.136
3 0.417 0.665 1.129
4 0.425 0.662 1.130
5 0.416 0.673 1.133
6 0.417 0.671 1.136
Average 0.416 0.670 1.134
SD 0.005 0.006 0.005
%RSD 1.231 0.897 0.448
Change in the wavelength (230nm)
12/2/2020

22
Accuracy :-
Accuracy level % Recovery (AVG) Statistical analysis
Mean % RSD
80% 93 0.372 1.12
100% 95 0.570 1.19
120% 101 0.810 0.55
12/2/2020

23
Quantitation limit is the lowest amount of analytes in a sample which can be quantitatively
determined with suitable precision and accuracy.
The detection limit of an individual analytical procedure is the lowest amount of analytes in a
sample which can be detected but not necessarily quantities as an exact value .
where σ = the standard deviation of the response S =the slope of the calibration curve
 LOQ was found to be 3.0µg/ml
 LOD was found to be 1.54µg/ml
LIMIT OF QUANTITATION:-
LIMIT OF DETECTION:-
12/2/2020

24
Analysis of Marketed Formulation :-
Sr.
No.
Concentration
(µg/ml)
Absorbance Mean SD %RSD %Assay
1 10 0.361
0.364 0.0033 0.919
95
2 10 0.363
3 10 0.365
4 10 0.364
5 10 0.370
6 10 0.361
12/2/2020

25
Optimized chromatographic condition (HPLC) :-
SR. NO. PARAMETER OPTIMIZED CONDITION
1 Column Phenomena Gemini (C18, 4.6×250mm, 5μm.)
2 Mobile Phase Methanol : orthophosphoric acid (40: 60 )
3 Wavelength 268 nm
4 Flow rate 1.5 ml/min
5 Retention time 4.5
6 Run time 10-20 min
7 Injection volume 20 µl
12/2/2020

26
Sr. No
Concentration
(µg/ml)
Area
1 2 16267
2 4 24003
3 6 32353
4 8 40325
5 10 48435
6 12 56525
y = 4036.5x + 8062.2
R² = 0.980
0
10000
20000
30000
40000
50000
60000
0 2 4 6 8 10 12 14
Calibration Curve
Figure HPLC chromatogram of standard solution of pioglitazone
12/2/2020

27
Accuracy :-
Accuracy level
%Recovery(AVG)
Statistical Analysis
Mean SD %RSD
80 99.46 40265 93.88 0.2331
100 97.80 48242 443.78 0.9199
120 98.76 56285 596.04 1.058
12/2/2020

28
Precision:-
Intraday precision Interday precision
Sr No Conc. Area RT Area RT
1. 10 48435 4.36 48967 4.25
2. 10 47652 4.39 48853 4.36
3. 10 48566 4.41 47963 4.36
4. 10 47965 4.39 49631 4.37
5. 10 48861 4.40 48963 4.39
6. 10 47963 4.38 47896 4.38
Avg 48240.3 4.38 48711.5 4.35
SD 453.44 0.0172 665.92 0.051
%RSD 0.9399 0.392 1.36 1.175
12/2/2020

29
Robustness:-
Change in wavelength :-
Change in wavelength (250
nm)
Sr. no Concentration Area RT
1. 10 47236 4.19
2. 10 47650 4.25
3. 10 48013 4.25
4. 10 47950 4.29
5. 10 48001 4.27
6. 10 47925 4.28
Avg 47795 4.25
SD 304.75 0.035
%RSD 0.6376 0.837
Fig HPLC chromatogram of Change in the
wavelength (250nm)
12/2/2020

30
Change in flow rate:-
Change in flow rate (1.2)
Sr. no Concentration Area RT
1. 10 45692 4.15
2. 10 45675 4.25
3. 10 45563 4.18
4. 10 46251 4.29
5. 10 45701 4.27
Avg 45776 4.22
SD 271.05 0.060
%RSD 0.592 1.423
Fig HPLC chromatogram of Change in
Flow rate (1.2 ml/min)
12/2/2020

31
Analysis of Marketed Formulation :-
Conc.(µg/
ml)
Retention
time
Area % Assay
10 4.28 48257
96.87
10 4.26 48150
10 4.29 48123
10 4.35 48350
10 4.32 48263
Avg. 4.32 48228
SD 0.035 92.273
% RSD 0.822 0.191
Fig Chromatogram of Pioglitazone tab analysis
12/2/2020

32
Quantitation limit is the lowest amount of analytes in a sample which can be quantitatively
determined with suitable precision and accuracy
The detection limit of an individual analytical procedure is the lowest amount of analytes
in a sample which can be detected but not necessarily quantities as an exact value.
where σ = the standard deviation of the response S =the slope of the calibration curve
 LOQ was found to be 13.52µg/ml
 LOD was found to be 7.4537µg/ml
LIMIT OF QUANTITATION:-
LIMIT OF DETECTION :-
12/2/2020

DISCUSSION
33
• The selection of wavelength test was performed by using standard preparation for six
times.
• The maximum absorption found at the wavelength 268nm for Pioglitazone.
• The intraday and interday precision was performed by preparing three different
concentration using for six times for three days and calculating %RSD should be not more
than 2%.
• The linearity study was carried out by establishing calibration curve. Calibration curve was
linear over the concentration range of 2-16µg/mL for Pioglitazone with a correlation
coefficient (R2) of 0.980.
• The % assay of Pioglitazone were found in the 95% by UV spectroscopy and the % assay
found in tablet by HPLC 96.87%, which indicates the method is accurate.
• The force degradation study on UV spectroscopy completed successfully.
12/2/2020

SUMMARY & CONCLUSION
• A simple, precise & accurate UV spectroscopy & HPLC method was developed &
validated as per ICH guideline.
• In UV spectroscopy 0.1HCL used as diluent & in HPLC Methanol :ortho phosphoric acid
(40:60%v/v) used.
• Thus based on validation data it is concluded that present method is economical, less time
consuming, precise , accurate for estimation of Pioglitazone in bulk drug & formulations.
• This method can be used to determine the purity of the drug available from various
sources by detecting the related impurities.
12/2/2020

REFERENCES
35
 Validation of analytical procedures, Text and Methodology, ICH Harmonized Tripartite
Guideline Q2 (R1), Commission of the European Communities, 2005.
 B. K. Sharma, Instrumental Methods of Chemical Analysis, Introduction to Analytical
Chemistry, Goel publishing house, Meerat, 19th edition, page no. 1-4, 200-203, 2000.
 CDER. Reviewer Guidance. "Validation of Chromatographic Methods." 1994.
 Y. R. Sharma, (2002), Elementary organic spectroscopy principal and chemical analysis, S.
Chand publication Page No 9.
 B.Siddartha., Analytical method development and method validation for the estimation of
pioglitazone hydrochloride in tablet dosage form by RP-HPLC, international journal of
pharmacy and pharmaceutical sciences , vol5, issue 3, 2013
12/2/2020

REFERENCES
36
 Chatwal GR, Anand SK. Instrumental methods of chemical analysis.Himalaya
publishing House, Mumbai, 4th ed., 1994.
 Sharmila Begum Shaik., Analytical method development and validation of pioglitazone
hydrochloride by RP-HPLC, Journal of chemical of chemical and pharmaceutical Research,
2014, 6(6):16-21
 Syed Imran Ali., Development and validation of HPLC method for the determinationof
pioglitazone in human plasma, Researchgate, july 2018
12/2/2020

THANK
YOU
3712/2/2020
Channabasweshwar Pharmacy College
(Degree), Latur

DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC AND CHROMATOGRAPHIC METHOD FOR DETERMINATION OF PIOGLITAZONE IN BULK DRUG AND DOSAGE FORMS

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC AND CHROMATOGRAPHIC METHOD FOR DETERMINATION OF PIOGLITAZONE IN BULK DRUG AND DOSAGE FORMS

Similar to DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC AND CHROMATOGRAPHIC METHOD FOR DETERMINATION OF PIOGLITAZONE IN BULK DRUG AND DOSAGE FORMS (20)

Recently uploaded

Recently uploaded (20)

DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC AND CHROMATOGRAPHIC METHOD FOR DETERMINATION OF PIOGLITAZONE IN BULK DRUG AND DOSAGE FORMS

Editor's Notes