Here are the key IR frequencies identified in the sample that match the reference standard of propafenone: - C-C stretch at 1186 cm-1 - C=C stretch at 1651 cm-1 - C-H stretch (symmetric) at 2939 cm-1 - C-H bend at 1328 cm-1 - CH2 stretch (symmetric) at 1369 cm-1 - CH2 bend at 1485 cm-1 - CH3 bend at 1398 cm-1 - C-O stretch at 1100 cm-1 - C=O stretch at 1695 cm-1 - N-H stretch at 3417 cm-1 The IR spectrum

1. THESIS PRESENTED ON
Stability Indicating Method Development and
Validation for Assay of Propafenone in Extended
Release Capsule Dosage Form
Presented by:
Vaibhav S.Bhatt
M. Pharm(Sem IV)
Quality Assurance
122080904015
Industrial Guide:
Mr. Hemang Parikh
Group leader , ADL-US
Zydus Cadila
Guided by:
Mr. Hiren kadikar
M.pharm
Asst. Professor (Q.A)
ARIHANT SCHOOL OF PHARMACY & BRI
Uvarsad Crossroad, S.G. Highway, Adalaj,
Dist. Gandhinagar-382421, Gujarat

2. INTRODUCTION[1-39]

3. Arrhythmia is an abnormality of rate, origin or conduction or spread of
cardiac impulses.
The rhythm of the heart is determined by the generation of an impulse by
some pacemaker cell, and the conduction of that impulse to the rest of the
heart.
Disturbances in cardiac rhythm are a result of abnormalities in impulse
initiation, or conduction, or both.
Arrhythmias are of various types :-
• Ventricular arrhythmia
• Supraventricular arrhythmia
• Paroxysmal tachycardia
• Atrial flutter
• Atrial fibrilation
• Ventricular flutter
• Ventricular fibrilation
Propafenone is mainly useful in Supraventricular Arrhythmias.
ARRHYTHMIA

4. 1. Class I agents sodium channel blockers:-
A. Intermediate channel binding and recovery:- Quinidine,
procainamide, Disopyramide
B. Rapid channel binding and recovery :- lidocaine, Mexiletine,
Tocainide
C. Slow channel binding and recovery:- Propafenone, flecainide
2. Class II beta blockers:- Propranolol, Esmolol, Acebutolol, sotalol
3. Class III potassium channel blockers:- Amiodarone, Bretylium,
Ibutilide, Dofetilide
4. Class IV calcium channel blockers:- verapamil , diltiazem
CLASSIFICATION

5. HPLC
Performance Liquid Chromatography (HPLC) is one of the most used
analytical techniques due to its simplicity, high specificity and wide range
of sensitivity.
Most of the drugs in multi component dosage forms can be analyzed by
HPLC method because of the several advantages like rapidity, specificity,
accuracy, precision and ease of automation in this method.
The separation of compounds is due to their relative differences in travel
through column on application of pressure exerted through mobile phase
for carrying liquid.
The compounds of the mixture travel with different rates due to their
relative affinities with the solvent and stationary phase. Compound with
higher affinity towards stationary phase of the column travels slowly and
vice-versa.

6. VALIDATION
Validation of analytical method is defined as the process by which it is
established, by laboratory studies, that the performance characteristics of
the method meet the requirements for the intended analytical application.
It is the documented evidence that provides high degree of assurance that
the given method , process or procedure will consistently produce the
product which will meet the pre- determined specification.
The objective of validation of an analytical procedure is to demonstrate
that it is suitable for its intended purpose.

7. VALIDATION PARAMETERS
 Accuracy
 Precision
 Specificity
 Linearity
 Range
 Robustness
 Limit of detection
 Limit of quantification

9. Stability Indicating Assay Method (SIAM)
ICH harmonized Tripartite Guideline described Stability Testing of New
Drug Substances and Products [ICH Q1AR2].
According to ICH Q1AR2, SIAM defined as “Method which is reliable,
meaningful and specific means the content of active ingredients, degradation
products and other contents of interest in a drug product can be accurately
measured without interference.”
According to US-FDA stability guideline of 1998, SIAM defined as,
“Validated quantitative analytical methods that can detect the changes with
time in the chemical, physical, or microbiological properties of the drug
substance and drug product, and that are specific so that the contents of
active ingredient, degradation products, and other components of interest
can be accurately measured without interference”

10. OBJECTIVES OF STABILITY TESTING
To provide evidence on how the quality of a drug substance or drug
product varies with time under the influence of a variety of condition.
To determine structural transformation of the drug substance and drug
product.
To determine lowest possible conc. of potential degradation products.
To establish the retest period for the drug substance.
To determine the shelf life of the drug product.
To propose the recommended storage conditions.
To provide assurance to the patients.

11. STRESS TESTING CONDITIONS
• Acid stress testing
• Base stress testing
• Peroxide stress testing
• Thermal stress testing
• Humidity stress testing
• Photo stability

12. DRUG PROFILE[40-43]

13. Name of drug Propafenone
Structure
Mol. formula C21H27NO3.HCL
Mol. Wt. 377.92
CAS No. 54063-53-5
IUPAC name 1-{2-[2-hydroxy-3(propylamino)-propoxy]phenyl}-3-
phenylpropan-1-one
Description Propafenone hydrochloride is a colourless crystals to white
fine crystalline powder with a bitter taste.
Solubility It is slightly soluble in alcohol and chloroform. It is very
slightly soluble in acetone. It is soluble in methanol and hot
water. It is insoluble in diethyl ether and toluene.

14. Melting point 173°C - 176oC
pKa 8.91
Dosage forms Tablet, Capsule, Injection and Suspension.
Dispensing and
Storage
Dispense in a tight light-resistant container.
Store at 20°–25°C (68°–77°F).
Mechanism of
action
Propafenone HCL is a sodium channel protein inhibitor.
Propafenone works by slowing the influx of sodium ions into
the cardiac muscle cells, causing a decrease in excitability of
the cells Propafenone differs from the prototypical class Ic
antiarrhythmic in that it has additional activity as a beta-
adrenergic blocker which can cause bradycardia and
bronchospasm . Mainly used for supraventricular
arrhythmias.

15. Pharmacokinetics Absorption: From G. I. Tract
Distribution: 97% drug bound to plasma protein.
Metabolism: Metabolized primarily in the liver where it is
rapidly and extensively metabolized to two active
metabolites, 5-hydroxypropafenone and N-
depropylpropafenone. These metabolites have
antiarrythmic activity comparable to propafenone but are
present in concentrations less than 25% of propafenone.
Excretion: Approximately 50% of propafenone metabolites
are excreted in the urine following administration of
immediate release tablets.
Volume of distribution : 252 Lit.

16. LITERATURE REVIEW[44-54]

17. Sr.
No.
Source Method Description Ref
No.
1 EP 2008 Assay by
potentiometric
titration
Dissolve 0.3 g in 2 ml of formic acid,
add 50 ml of acetic anhydride , titrate
with 0.1M perchloric acid
potentiometrically, perfom blank.
Each ml of 0.1M perchloric acid is
equivalent to 377.92 mg of
propafenone HCL.
44
2 EP 2008 Liquid
chromatography
M.P: Phosphate buffer pH 2.5:
ACN (65:35)
S.P: Octyl silyl silica gel column
Flowrate: 1ml/min
Detection: Uv detector at 222 nm
44
Official Methods for Propafenone

18. 3 USP30
NF-25
Assay by
potentiometric
titration
Dissolve 0.250 g in 30 ml of
methanol, add 15 ml of mercuric
acetate, titrate with 0.1M
perchloric acid potentiometrically.
Each ml of 0.1M perchloric acid is
equivalent to 377.92 mg of
propafenone HCL
45
4 USP30
NF-25
Liquid
chromatography
S.P: Octyl silyl silica gel column
M.P: Ammonium sulphate buffer:
ACN (16:9)
Flowrate: 1ml/min
Detection: UV detector at 222 nm
45
5 USP30
NF-25
Gas
chromatography
Career gas: Helium
S.P: Silica coated with
polymethylsiloxane
Detection: Flame ionisation
detactor
45

19. 6 BP
2012
Assay by
potentiometric
titration
Dissolve 0.3 g in 2 ml of formic acid,
add 50 ml of acetic anhydride, titrate
with 0.1M perchloric acid
potentiometrically, perfom blank.
Each ml of 0.1M perchloric acid is
equivalent to 377.92 mg of propafenone
HCL
46
7 BP
2012
Liquid
chromatography
M.P : Phosphate buffer PH 2.5:
ACN(65:35)
S.P : Octyl silyl silica gel column
Flowrate : 1ml/min.
Detection : Uv detector at 222 nm
46

20. Sr.
No.
Drug/ Dosage Form/
Matrix
Description Ref
No.
1 Propafenone and 5-
hydroxy Propafenone
in pharmaceutical
formulation
S.P : 25×0.4 cm 5 μm ODS column.
M.P: 0.1 M di potassium hydrogen
phosphate (K2HPO4) buffer (pH 2.5) and
acetonitrile (63:37)
Detection: UV detection at 249 nm.
Rt: Retention times of the internal
standard, propafenone, and 5-OH
propafenone are 4.3, 6.0 and 2.9 minutes
respectively.
47
2 Propafenone and 5-
hydroxypropafenone
enantiomers in
human plasma
S.P : Toluene : diethyl ether phase (9:1).
M.P : silica gel column using N-tert-Boc-
(L)-proline as the chiral additive to the
mobile phase.
Detection : UV detection at 249nm.
48
Reported methods for Propafenone

21. 3 Propafenone HCL
in presence of its
Degradation
product in
Pharmaceutical
Dosage form
S.P: ODS (250 X 4.6 mm, 5μm) column
M.P: 0.01 M potassium dihydrogen phosphate
(pH 3.0) : ACN 65:35 (v/v)
Flowate: 1.0 mL/ min
Detection: UV detection at 249 nm.
Rt: 7.41 min
49
4 propafenone in
bulk drug and it’s
pharmaceutical
formulation.
Method A: Extractive Spectrophotometric
estimation , propafenone was estimated at 301 nm
using ethanol as a solvent & linearity was
observed in the conc. range of 20 - 200 μg /ml.
Method B: Hydrotropic method , propafenone
was estimated at 301 nm using diphenhydramine
citrate as a solvent & linearity was observed in
the conc. range of 15 - 150 μg /ml.
50
5 Propafenone
Hydrochloride in
tablet dosage form
S.P : Aluminum plate
10 × 10 & 20 × 10 cm) with 250 μm thickness.
M.P: Chloroform : methanol : ammonia (8 : 2 :
0.2 v/v).
Rf value: 0.49
51

22. 6 Propafenone
HCL in tablet
dosage form.
HPTLC method
S.P: precoated aluminium backed TLC plate (10
cm x 10 cm).
M.P: methanol : ethyl acetate : triethylamine
(1.5 : 3.5 : 0.4 v/v/v)
Scaning time: 10 min.
Detecion: UV at 250 nm.
52
7 Propafenone
and Its Major
Metabolites in
Human Serum
S.P: C18 column (25 × 0.46 cm i.d., 5 µm)
M.P: ACN : phosphate buffer gradient at a flow
rate of 1.7 ml min-1.
Detection: UV at 210 nm.
53
8 propafenone in
biological
fluids
Gas-liquid chromatographic method.
S.P : 25 m × 0.31 mm cross linked , phenyl
methyl silicone coated fused-silica capillary
column was employed for all analyses.
Conc.range : 10-100 ng/ml
54

23. RATIONALE

24. • The review of literature reveals that Stability Indicating HPLC and
HPTLC method is developed for estimation of Propafenone HCL in bulk
and solid dosage form.
• Generally, majority of analysis in industries are done on HPLC.
• Propafenone HCL is available in the market with Brand name
RHYTHMOL.
• Propafenone mainly used for Supraventricular tachycardia.
• My goal is to developed more accurate, precise and efficient stability
indicating HPLC method than the available one.
• The Stability of Capsule formulation in acid/base, thermal/humidity,
oxidation, light, etc. is more important.
• So area of interest is to Develop and Validate Simple, Accurate, Precise
and Economic stability indicating RP-HPLC method for estimation of
propafenone in capsule formulations as per ICH guideline.
• To perform Forced degradation study as per ICH guidelines.

25. SUMMARY OF PSAR REPORT
Sr.
No.
Patent Application
number
Title of Patent
1 IB2009/007126 Propafenone extended release composition
2 11/146006 Sustained release propafenone
hydrochloride capsules
3 PCT/CA2005/000863 Propafenone hydrochloride capsules
containing microtablets
4 12/419790 Novel Antiarrhythmic Drug Formulations
5 07/189544 Long lasting composition of propafenone
and quinidine for treatment of cardiac
conditions
Novelty of Project: Looking at above 5 patents the dissertation
title is 50-75% novel.

26. •Propafenone HCL is available in the market with Brand name
RHYTHMOL & mainly used for Supraventricular tachycardia.
•The Stability of Capsule formulation in acid/base,
thermal/humidity, oxidation, light, etc. is more important.
•Stability Indicating HPLC and HPTLC method is developed for
estimation of Propafenone HCL in bulk and solid dosage form.
•So area of interest is to Develop and Validate better , different and
precise stability indicating method for estimation of propafenone
in capsule formulations as per ICH guideline.

27. AIM OF WORK

28. • Day to day numbers of newer drugs and their formulations either in single or
in combined dosage forms are marketed. Drug analysis plays a vital role in
drug product design, development, and manufacturing. This analysis
provides assurance that the product meets the established quality
requirement.
• Though Stability indicating HPLC and HPTLC methods are available for
estimation of Propafenone, my goal is to developed more accurate, precise
and efficient stability indicating HPLC method than the available one.
• To develop a simple and accurate HPLC method for the determination of
Propafenone HCL in Propafenone HCL Extended release capsules.
• Validation of the developed method.
• To carry out forced degradation study of Propafenone HCL according to
ICH guideline Q1A(R2).

29. EXPERIMENTAL WORK

30. Melting Point Standard Observed
Propafenone 173oC - 176oC 176.78oC
IDENTIFICATION OF SAMPLE BY MELTING POINT (44)
DSC of Propafenone

31. IDENTIFICATION OF SAMPLE BY IR SPECTRUM
Reference IR spectrum of propafenone
IR spectrum of sample
IDENTIFICATION OF SAMPLE BY IR SPECTRUM

32. PROCEDURE FOR RECORDING IR SPECTRUM
•Mix 3 mg of sample with 900 mg Kbr, grind the mixture and record IR
absorption spectrum between 3800 cm-1 to 650 cm-1 using air as a
reference.
•The IR absorption spectra of sample exhibit maxima, which are only at
the same wavelength in the spectrum of propafenone USP working
standard prepared in the same manner.
•So it is identified that the given sample is propafenone.

33. Functional Group IR Frequency (Cm-1)
C-C 1186
C=C 1651
C-H (S) 2939
C-H (B) 1328
CH2 (S) 1369
CH2 (B) 1485
CH3 (B) 1398
C-O 1100
C=O 1695
N-H 3417
O-H 3319
Aromatic Ring 910
Interpretation of IR Spectra

34. INSTRUMENTS

35. Sr. No Instrument Make Model
1. HPLC Waters 2862
2. ANALYTICAL BALANCE Mettler Toledo XR205SM-DR
3. PH METER Lab India 420 A+
4. MILLI Q WATWR SOURCE Thermofisher
scientific
Branstead D3750
5. INJECTOR Rhenodyne valve
with 20μl fixed
loop
-
6. PUMP Quaternary pump -
7. DETECTOR UV detector
PDA detector
-
8. HOT AIR OVEN Lab line Cintex-906
9. SONICATOR Roop telesonic Branson 2510

36. MATERIALS

37. No. Name Grade Source/Supplier
1 Water HPLC Nanopure Diamond
water Purification
2 Di potassium hydrogen
phosphate
AR Merck
3 Orthophosphoric acid HPLC Spectrochem
4 Acetonitrile HPLC Rankem
5 Hydrochloric acid AR Merck
6 Sodium hydroxide AR Merck
7 Hydrogen peroxide AR Merck

38. Buffer
preparation
• Dissolve 5.2 gm of K2HPO4 anhydrous in 2000 ml of Water,
and adjust the pH 2.5 with Orthophosphoric acid (OPA).
• Filter the solution through 0.45 μ nylon filter and degas
before use.
Mobile
phase
• Prepared a mixture of 600 ml of Buffer and 400 ml of
Acetonitrile.
• Adjust pH 2.5 with diluted Orthophosphoric acid (OPA).
Diluent
• Milli-Q water : Acetonitrile (60:40)
PREPARATION OF SOLUTIONS

39. Standard preparation: (100 μg/ml):
100 mg of Propafenone hydrochloride standard to
100 ml volumetric flask. Add 70ml of diluent and
sonicate to dissolve.
Dilute 5ml of this solution to 50ml with
diluent.
Make volume up to mark
with diluents and mix.
Equivalent to 500 mg of Propafenone
hydrochloride powder to 250 ml volumetric flask.
Add 150ml of diluent and sonicate to dissolve
for 30 min.
Dilute 5ml of this solution to 100ml with diluent.
Make volume up to mark
with diluents and mix.
Sample Preparation: (100 μg/ml):

40. HPLC METHOD DEVELOPEMENT

41. Selection of Detection Wavelength
UV spectra of Propafenone HCl
•The standard Propafenone solution in diluent (100 μg/ml) was analyzed in the
UV region of 200-400 nm and the spectrum was recorded. The detection
wavelength for propafenone HCl was found to be 249 nm.

42. TRIALS FOR MOBILE PHASE OPTIMIZATION

43. Trial
No.
Mobile Phase Retention
Time (min)
Remarks
1. Water : Methanol (60:40) 0.824 Early elution, Poor peak symmetry
2. Water : Methanol (70:30) 0.881 Early elution, Poor peak symmetry
3. Water : ACN (60:40) 1.250 Early elution
4. Water : ACN (70:30) 1.925 Early elution
5. Buffer (KH2PO4, pH 6.8) :
ACN (60:40)
10.625 Late elution
6. Buffer (KH2PO4, pH 4.5) :
ACN(60:40)
5.167 Good Rt, Proper peak symmetry
7. Buffer (KH2PO4, pH 2.5) :
ACN(60:40)
3.36 Effective Rt, Proper peak
symmetry

44. OPTIMIZED CHROMATOGRAPHIC CONDITION
PARAMETERS SPECIFICATION
Stationary Phase Inert ODS 3V C18 (250 X 4.6
mm), 5μ
Flow rate 1.5 ml/min
Wavelength 249 nm
Injection volume 10 μl
Coloumn oven temperature 45 °C
Mobile phase Buffer (KH2PO4, pH 2.5) : ACN (60:40)
Diluent Milli - Q water : ACN (60:40)
Retention time 3.368 min
Run time 10 min

45. Chromatogram Of Propafenone HCl Standard

46. FORCED DEGRDATION

47. Degradation Condition
Acid Hydrolysis 15 ml 5N HCL Water Bath 90 0C 1 Hr
Alkali Hydrolysis 15 ml 5N NAOH Water Bath 90 0C 1 Hr
Peroxide Oxidation 15 ml 30% H2O2 Water Bath 90 0C 30 min
Thermal Degradation 7 Days at 100 0C
Photolytic Degradation 7 Days under UV light
Humidity Degradation 7 Days at 40 0C / 75% RH
DEGRADATION CONDITION

48. Acid Degradation
Chromatogram of Acid Degradation (Placebo)
Chromatogram of Acid Degradation (Standard)
Chromatogram of Acid Degradation (Sample)

49. Base Degradation
Chromatogram of Base Degradation (Placebo)
Chromatogram of Base Degradation (Standard)
Chromatogram of Base Degradation (Sample)

50. Peroxide Degradation
Chromatogram of Peroxide Degradation (Placebo)
Chromatogram of Peroxide Degradation (Standard)
Chromatogram of Peroxide Degradation (Sample)

51. Thermal Degradation
Chromatogram of Thermal Degradation (Placebo)
Chromatogram of Thermal Degradation (Standard)
Chromatogram of Thermal Degradation (Sample)

52. Photolytic Degradation
Chromatogram of Photolytic Degradation (Standard)
Chromatogram of Photolytic Degradation (Sample)
Chromatogram of Photolytic Degradation (Placebo)

53. Humidity Degradation
Chromatogram of Humidity Degradation (Placebo)
Chromatogram of Humidity Degradation (Standard)
Chromatogram of Humidity Degradation (Sample)

54. DEGRADATION SUMMARY
Degradation
Condition
Peak Area % Degradation
Standard 796308 -
Sample as such 762441 -
Standard Sample Standard Sample
Acid Hydrolysis 677983 641219 14.8 15.9
Base Hydrolysis 693882 681100 12.8 10.7
Peroxide Oxidation 633524 614778 20.4 19.3
Thermal Degradation 793506 757998 0.35 0.58
Photolytic Degradation 794228 759526 0.27 0.38
Humidity Degradation 795974 760939 0.05 0.20

55. PEAK PURITY MATCH
Degradation Condition Peak purity factor
Acid Hydrolysis 999.953
Base Hydrolysis 999.925
Peroxide Oxidation 999.945
Thermal Degradation 999.916
Photolytic Degradation 999.958
Humidity Degradation 999.478

56. •Based on the results, obtained from the analysis of forced degraded
samples using described method, it shows that there was no other co-
eluting peaks of interference from recipients and degradation products
due to variable stress components with the main peaks and the method
was specific for the estimation of Propafenone HCl in presence of
various degradants.
•So previously described method can be used as stability indicating
method for Assay of Propafenone HCl from its ER Capsule dosage
forms.
DISCUSSION

57. METHOD VALIDATION

58. 100 mg of Propafenone standard Dilute to 100 ml with diluent
(1000 μg/ml) (LSS)
Further dilute LSS up to
100 ml with diluent
25 μg/ml
2.5 ml
to 100
ml
50 μg/ml 75 μg/ml 100 μg/ml 125 μg/ml 150 μg/ml
5 ml to
100 ml
7.5 ml
to 100
ml
10 ml to
100 ml
12.5 ml
to 100
ml
15 ml
to 100
ml
Preparation of solutions for linearity study

59. Result of Linearity – Range study
SR.
NO
CONCENTRATION
(µg/ml)
AREA (VOLTS)
± SD
% RSD
1 25 189311 ± 750.798 0.39
2 50 396392 ± 2053.373 0.51
3 75 594183 ± 3739.276 0.62
4 100 787092 ± 5439.013 0.69
5 125 977668 ± 4363.375 0.44
6 150 1194025 ±8770.450 0.73

60. Concentration (µg/ml)
Area
(Volts)
Calibration curve of Propafenone HCl

61. PARAMETERS VALUE
Linearity range 25-150 µg/ml
Regression equation Y= 7954.6 x – 6252.2
Correlation Co-efficient (R2) 0.999
Slope of curve 7954.6
Overlain Chromatogram of Propafenone HCl (25 - 150 µg/ml)
Summary of linearity - range of Propafenone Hydrochloride

62. Limit of Detection (LOD) – Limit of Quantification (LOQ)
PARAMETERS VALUE
LOD 0.04 µg/ml
LOQ 0.12 µg/ml

63. Preparation of solutions for recovery study
Accuracy
Level
Weight
of API
(mg)
Weight
of
Placebo
(mg)
Dilute to
Volume
With
Diluent
(ml)
Accuracy
Stock
Solution
(ml)
Further
Dilute to
Volume
with
Diluent
(ml)
Final
Conc.
(μg/ml)
50% 250 20 250 5 100 50
100% 500 20 250 5 100 100
150% 750 20 250 5 100 150

64. Set No. Accuracy
level
(%)
Expected
amount of
Propafenone
(μg/ml)
Recovered
amount of
Propafenone
(μg/ml)
%
Recovery
Mean
± SD
%
RSD
Set – 1
50
50 50.27 100.54 50.57
±
0.31
0.61Set – 2 50 50.89 101.78
Set – 3 50 50.54 100.08
Set – 1
100
100 100.73 100.73 100.82
±
0.59
0.59Set – 2 100 101.28 101.28
Set – 3 100 101.46 101.46
Set – 1
150
150 148.28 99.85 148.92
±
0.79
0.53Set – 2 150 148.67 99.28
Set – 3 150 149.81 99.87
Result of Recovery study (n=3)

65. Sr.
No.
Concentration (µg/ml) Area (Volts)
1. 100 788016
2. 100 796675
3. 100 785323
4. 100 789448
5. 100 782619
6. 100 792897
Mean AUC (n=6) 789163
SD (n=6) 5086.045
% RSD 0.64
Result of repeatability study (n=6)

66. Sr. No. Concentration
(µg/ml)
Mean Area ± SD Area (Volts)
1 50 398136 ± 1837.267 0.46
2 100 789546 ± 4883.726 0.52
3 150 1197845 ± 8474.793 0.70
Result of Intra-day precision (n=3)

67. Sr. No. Concentration (µg/ml) Mean Area
± SD
Area (Volts)
1 50 395698 ± 2274.792 0.57
2 100 781392 ± 5363.155 0.68
3 150 1199025 ± 9364.180 0.78
Result of Inter-day precision (n=3)

68. Specificity
Chromatogram of blank (Diluent)
Chromatogram of Placebo

69. Chromatogram of Standard for Propafenone Hydrochloride
Chromatogram of Sample for Propafenone Hydrochloride

70. •In the specificity study, Blank, placebo and standard and sample were
injected into the system.
•The chromatograms of blank and placebo do not show any interferences
at the retention time of Propafenone Hydrochloride as it can be seen
from respective chromatograms.
•By computing peak purity of main peak using PDA detector, Standard
and sample solution was analyzed as per proposed method. Analysis was
replicated for six times. Peak purity factor was found to be 999.888 for
Propafenone Hydrochloride. Result of specificity study by peak purity is
depicted in table .

71. Sr.
No.
Concentration
(µg/ ml)
Peak Purity Mean Peak Purity
1. 100 999.888
999.888
2. 100 998.783
3. 100 999.574
4. 100 998.462
5. 100 998.812
6. 100 999.894
Result of specificity study

72. Robustness
Robustness of method was studies by small but deliberate change in
chromatographic condition as below:
i. Change in Flow rate : 1.4 ml/min, 1.6 ml/min
ii. Change in mobile phase composition : Acetonitrile : Phosphate
buffer (pH 2.5) (62:38, 58:42 %v/v)
iii. Change in pH : 2.3, 2.7
iv. Change in wavelength : 247 nm, 251 nm
v. Change in temperature : 40ºC, 50ºC

73. Result of Change in Flow Rate
Sr.
No.
Flow Rate: 1.4 ml/ min
Area (Volts)
Flow Rate: 1.6 ml/ min
Area (Volts)
1. 931558 701947
2. 938876 712698
3. 933569 704589
4. 933548 705489
5. 939958 708268
6. 934498 713589
Mean AUC
(n=6)
935334 707764
SD (n=6) 3322.325 4639.455
% RSD 0.35 0.65

74. Result of Change in Mobile Phase composition
Sr.
No.
Buffer (KH2PO4, pH 2.5) :
ACN (62 : 38 % v/v)
Area (Volts)
Buffer (KH2PO4, pH 2.5) : ACN
(58 : 42 % v/v)
Area (Volts)
1. 811161 800139
2. 815487 808698
3. 816984 813257
4. 814581 801936
5. 813259 807854
6. 819589 809359
Mean AUC
(n=6)
815178.166 806873.833
SD (n=6) 2931.887 4918.320
% RSD 0.35 0.61

75. Result of Change in pH
Sr.
No.
pH : 2.3
Area (Volts)
pH : 2.7
Area (Volts)
1. 812937 830698
2. 814589 838579
3. 813957 836987
4. 812698 843597
5. 817598 831458
6. 818589 842985
Mean AUC
(n=6)
815061.333 837384
SD (n=6) 2466.572 5502.331
% RSD 0.30 0.65

76. Result of Change in Wavelength
Sr.
No.
ƛmax: 247 nm
Area (Volts)
ƛmax: 251 nm
Area (Volts)
1. 775896 728589
2. 773698 726699
3. 774589 726486
4. 772689 726458
5. 771893 724589
6. 776983 711364
Mean AUC (n=6) 774291.666 724030.833
SD (n=6) 1928.259 4442.863
% RSD 0.25 0.61

77. Result of Change in Temperature
Sr.
No.
Temperature : 40 °C
Area (Volts)
Temperature : 50 °C
Area (Volts)
1. 774404 771226
2. 771268 776751
3. 772940 774638
4. 770291 780947
5. 774853 772120
6. 776944 783905
Mean AUC
(n=6)
773449.333 776598.833
SD (n=6) 2451.902 5001.638
% RSD 0.31 0.64

78. Hours Area (Volts)
4 817192
8 815665
12 814968
16 812879
20 809587
24 807998
Mean AUC (n=6) 803048.166
SD (n=6) 3611.576
% RSD 0.44
Solution stability study

79. Sr.
No.
Volume of
sample
discarded ( ml)
PVDF
(0.45 µm)
Nylon
(0.45 µm)
Area (Volts) Area (Volts)
1. Unfiltered 807689 818965
2. 1 805578 818457
3. 3 804547 806357
4. 5 803658 814798
5. 7 801946 812658
6. 10 800589 811457
Mean AUC (n=6) 804002 813782
SD (n=6) 2544.358 4723.367
% RSD 0.32 0.58
Filter compatibility study

80. Sr. no. Parameters Value Obtained Standard Value
1. Retention time (Rt) 3.36 -
2. % RSD of area of six
replicate injections
0.64 NMT 2.00
3. Mean Theoretical plates
(N) or Column efficiency
4256 NLT 2000
4. Mean Tailing factor (As)
or Symmetry factor
0.9 NMT 2.00
System suitability parameters

81. Label Claim (mg) % Assay
425
101.1
100.5
101.4
101.5
101.1
Mean ± SD 101.1 ± 3255.256
% RSD 0.48
Analysis of marketed formulation
Assay of Propafenone Hydrochloride (n=5)

82. Sr. No. Parameters Results
1 Linearity (μg/ml) 25-150
2 Correlation Coefficient 0.999
3 Accuracy (% Recovery) 99.28 – 101.78
4 LOD (μg/ml) 0.04
5 LOQ (μg/ml) 0.12
6 Repeatability (% RSD) 0.64
7 Intra-day Precision (%RSD) 0.46 – 0.70
8 Inter-day Precision (%RSD) 0.57 – 0.78
9 Specificity Specific: No co-elution
with main peak
10 Robustness
Complies with the acceptance
criteria
11 Solution Stability Study
12 Filter Compatibility Study
13 System Suitability study
Validation parameters summary

83. SUMMARY
•For the Propafenone hydrochloride, isocratic method was developed by
taking trials for the ratio of Water and methanol, water and acetonitrile,
and then the ratio of buffer and acetonitrle. The ratio of buffer and
acetonitrle were selected on the basis of good retention time and peak
symmetry, it was finalized as Mobile Phase in the ratio of 60:40%v/v.
•For the linearity and range, correlation coefficient value of the linearity
curve should not be less than 0.995. Correlation coefficient value for the
Propafenone hydrochloride was found to be 0.999, which is greater than
0.995. Hence, the method is linear within the range.
•Accuracy was determined over the range from lowest sample concentration
to highest concentration (i.e. from 50% - 150%). For each level, for the
drug, % R.S.D was found in the range of 0.53 – 0.61 %, which is well
within the acceptance criterion. Hence, the method is found to be accurate.

84. •In order to show the precision of the method, repeatability (System & Method
Precision), intra-day precision and inter-day precision were checked. For the
repeatability, % R.S.D of the six replicates of sample should not be more than
2%. The obtained % R.S.D was found to be 0.64 % for Propafenone
hydrochloride, which is well within the limit of acceptance criteria. While for
the intra-day precision and inter-day precision of the method, the % R.S.D was
found in the range of 0.46 – 0.70 % and 0.57 – 0.78 % respectively. Hence, the
method is found to be precise.
•In order to show the stability of the method, specificity was carried out. After
the specificity, it was observed that there was no interference of degradation
products, placebo, with the main peak. Peak purity index was found to be
greater than 990. All the results obtained were found within the acceptance
criteria. Hence, the method can be termed as specific.

85. •Robustness is performed to prove the efficiency of the method despite
deliberate changes in the normal method conditions i.e. column oven
temperature, flow rate, mobile phase pH, organic solvent composition,
wavelength etc. According to the acceptance criteria, the % R.S.D should not
be more than 2. The obtained % R.S.D was found to be in the range of 0.25 –
0.65 %. Therefore, it is within the acceptance criteria. Hence, the method can
be termed as robust.
•From the results obtained, it is obvious that the proposed method is applicable
for the determination of Propafenone hydrochloride without interference and
with good sensitivity. The results obtained indicate that the proposed method
for the estimation of Propafenone hydrochloride is specific, rapid, linear,
accurate, precise, and suitable for intended use. These merits suggest the use
of the proposed method in routine and quality control analysis without
interference from commonly encountered excipient.

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