Advanced Method Development and

Validation for Propafenone Assay in

Extended Release Capsule Dosage
Forms
 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.
 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.
 VALIDATION PARAMETERS

 Accuracy
 Precision
 Specificity
 Linearity
 Range
 Robustness
 Limit of detection
 Limit of quantification
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”
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.
 STRESS TESTING CONDITIONS

• Acid stress testing

• Base stress testing
• Peroxide stress testing
• Thermal stress testing
• Humidity stress testing
• Photo stability
DRUG PROFILE[40-43]
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.
Melting point 173°C - 176oC
pKa 8.91

Dosage forms Tablet, Capsule, Injection and Suspension.

Dispensing and Dispense in a tight light-resistant container.

Storage Store at 20°–25°C (68°–77°F).

Mechanism of Propafenone HCL is a sodium channel protein inhibitor.

action 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.
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.

LITERATURE REVIEW[44-54]
 Official Methods for Propafenone
Sr. Source Method Description Ref
No. No.
1 EP 2008 Assay by Dissolve 0.3 g in 2 ml of formic acid, 44
potentiometric add 50 ml of acetic anhydride , titrate
titration with 0.1M perchloric acid
potentiometrically, perfom blank.
Each ml of 0.1M perchloric acid is
equivalent to 377.92 mg of
propafenone HCL.

2 EP 2008 Liquid M.P: Phosphate buffer pH 2.5: 44

chromatography ACN (65:35)
S.P: Octyl silyl silica gel column
Flowrate: 1ml/min
Detection: Uv detector at 222 nm
3 USP30 Assay by Dissolve 0.250 g in 30 ml of 45
NF-25 potentiometric methanol, add 15 ml of mercuric
titration acetate, titrate with 0.1M
perchloric acid potentiometrically.
Each ml of 0.1M perchloric acid is
equivalent to 377.92 mg of
propafenone HCL

4 USP30 Liquid S.P: Octyl silyl silica gel column 45

NF-25 chromatography M.P: Ammonium sulphate buffer:
ACN (16:9)
Flowrate: 1ml/min
Detection: UV detector at 222 nm

5 USP30 Gas Career gas: Helium 45

NF-25 chromatography S.P: Silica coated with
polymethylsiloxane
Detection: Flame ionisation
detactor
6 BP Assay by Dissolve 0.3 g in 2 ml of formic acid, 46
2012 potentiometric add 50 ml of acetic anhydride, titrate
titration with 0.1M perchloric acid
potentiometrically, perfom blank.
Each ml of 0.1M perchloric acid is
equivalent to 377.92 mg of propafenone
HCL
7 BP Liquid M.P : Phosphate buffer PH 2.5: 46
2012 chromatography ACN(65:35)
S.P : Octyl silyl silica gel column
Flowrate : 1ml/min.
Detection : Uv detector at 222 nm
 Reported methods for Propafenone
Sr. Drug/ Dosage Form/ Description Ref
No. Matrix No.
1 Propafenone and 5- S.P : 25×0.4 cm 5 μm ODS column. 47
hydroxy PropafenoneM.P: 0.1 M di potassium hydrogen
in pharmaceutical phosphate (K2HPO4) buffer (pH 2.5) and
formulation 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.
2 Propafenone and 5- S.P : Toluene : diethyl ether phase (9:1). 48
hydroxypropafenone M.P : silica gel column using N-tert-Boc-
enantiomers in (L)-proline as the chiral additive to the
human plasma mobile phase.
Detection : UV detection at 249nm.
3 Propafenone HCL S.P: ODS (250 X 4.6 mm, 5μm) column 49
in presence of its M.P: 0.01 M potassium dihydrogen phosphate
Degradation (pH 3.0) : ACN 65:35 (v/v)
product in Flowate: 1.0 mL/ min
Pharmaceutical Detection: UV detection at 249 nm.
Dosage form Rt: 7.41 min
4 propafenone in Method A: Extractive Spectrophotometric 50
bulk drug and it’s estimation , propafenone was estimated at 301 nm
pharmaceutical using ethanol as a solvent & linearity was
formulation. 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.
5 Propafenone S.P : Aluminum plate 51
Hydrochloride in 10 × 10 & 20 × 10 cm) with 250 μm thickness.
tablet dosage form M.P: Chloroform : methanol : ammonia (8 : 2 :
0.2 v/v).
Rf value: 0.49
6 Propafenone HPTLC method 52
HCL in tablet S.P: precoated aluminium backed TLC plate (10
dosage form. 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.

7 Propafenone S.P: C18 column (25 × 0.46 cm i.d., 5 µm) 53

and Its Major M.P: ACN : phosphate buffer gradient at a flow
Metabolites in rate of 1.7 ml min-1.
Human Serum Detection: UV at 210 nm.

8 propafenone in Gas-liquid chromatographic method. 54

biological S.P : 25 m × 0.31 mm cross linked , phenyl
fluids methyl silicone coated fused-silica capillary
column was employed for all analyses.
Conc.range : 10-100 ng/ml
RATIONALE
• 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.
 SUMMARY OF PSAR REPORT
Sr. Patent Application Title of Patent
No. number
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.
•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.
AIM OF WORK
• 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).
EXPERIMENTAL WORK
IDENTIFICATION OF SAMPLE BY MELTING POINT (44)

Melting Point Standard Observed

Propafenone 173oC - 176oC 176.78oC

DSC of Propafenone
IDENTIFICATION OF SAMPLE BY IR SPECTRUM
Reference IR spectrum of propafenone

IR spectrum of sample
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.
 Interpretation of IR Spectra
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
INSTRUMENTS
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 Branstead D3750
scientific

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
MATERIALS
No. Name Grade Source/Supplier

1 Water HPLC Nanopure Diamond

water Purification

2 Di potassium hydrogen AR Merck

phosphate

3 Orthophosphoric acid HPLC Spectrochem

4 Acetonitrile HPLC Rankem

5 Hydrochloric acid AR Merck

6 Sodium hydroxide AR Merck

7 Hydrogen peroxide AR Merck

 PREPARATION OF SOLUTIONS
• Dissolve 5.2 gm of K2HPO4 anhydrous in 2000 ml of Water,
and adjust the pH 2.5 with Orthophosphoric acid (OPA).
Buffer • Filter the solution through 0.45 μ nylon filter and degas
preparation before use.

• Prepared a mixture of 600 ml of Buffer and 400 ml of

Acetonitrile.
Mobile • Adjust pH 2.5 with diluted Orthophosphoric acid (OPA).
phase

• Milli-Q water : Acetonitrile (60:40)

Diluent
Standard preparation: (100 μg/ml):
100 mg of Propafenone hydrochloride standard to
100 ml volumetric flask. Add 70ml of diluent and
sonicate to dissolve.

Make volume up to mark

with diluents and mix.

Dilute 5ml of this solution to 50ml with

diluent.

Sample Preparation: (100 μg/ml):

Equivalent to 500 mg of Propafenone
hydrochloride powder to 250 ml volumetric flask.
Add 150ml of diluent and sonicate to dissolve
for 30 min.
Make volume up to mark
with diluents and mix.

Dilute 5ml of this solution to 100ml with diluent.

HPLC METHOD DEVELOPEMENT
 Selection of Detection Wavelength

•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.

UV spectra of Propafenone HCl

TRIALS FOR MOBILE PHASE OPTIMIZATION
Trial Mobile Phase Retention Remarks
No. Time (min)
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) : 10.625 Late elution

ACN (60:40)
6. Buffer (KH2PO4, pH 4.5) : 5.167 Good Rt, Proper peak symmetry
ACN(60:40)

7. Buffer (KH2PO4, pH 2.5) : 3.36 Effective Rt, Proper peak

ACN(60:40) symmetry
 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
Chromatogram Of Propafenone HCl Standard
FORCED DEGRDATION
 DEGRADATION CONDITION
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

 Acid Degradation

Chromatogram of Acid Degradation (Placebo)

Chromatogram of Acid Degradation (Standard)

Chromatogram of Acid Degradation (Sample)

 Base Degradation

Chromatogram of Base Degradation (Placebo)

Chromatogram of Base Degradation (Standard)

Chromatogram of Base Degradation (Sample)

 Peroxide Degradation

Chromatogram of Peroxide Degradation (Placebo)

Chromatogram of Peroxide Degradation (Standard)

Chromatogram of Peroxide Degradation (Sample)

 Thermal Degradation

Chromatogram of Thermal Degradation (Placebo)

Chromatogram of Thermal Degradation (Standard)

Chromatogram of Thermal Degradation (Sample)

 Photolytic Degradation

Chromatogram of Photolytic Degradation (Placebo)

Chromatogram of Photolytic Degradation (Standard)

Chromatogram of Photolytic Degradation (Sample)

 Humidity Degradation

Chromatogram of Humidity Degradation (Placebo)

Chromatogram of Humidity Degradation (Standard)

Chromatogram of Humidity Degradation (Sample)

 DEGRADATION SUMMARY
Degradation Peak Area % Degradation
Condition

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
 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

 DISCUSSION
•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.
METHOD VALIDATION
 Preparation of solutions for linearity study
100 mg of Propafenone standard Dilute to 100 ml with diluent
(1000 μg/ml) (LSS)

Further dilute LSS up to

100 ml with diluent

2.5 ml 5 ml to 7.5 ml 10 ml to 12.5 ml 15 ml

to 100 100 ml to 100 100 ml to 100 to 100
ml ml ml ml

25 μg/ml 50 μg/ml 75 μg/ml 100 μg/ml 125 μg/ml 150 μg/ml

 Result of Linearity – Range study
SR. CONCENTRATION AREA (VOLTS) % RSD
NO (µg/ml) ± SD

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

 Calibration curve of Propafenone HCl

Area
(Volts)

Concentration (µg/ml)
 Overlain Chromatogram of Propafenone HCl (25 - 150 µg/ml)

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

Summary of linearity - range of Propafenone Hydrochloride

Limit of Detection (LOD) – Limit of Quantification (LOQ)
PARAMETERS VALUE

LOD 0.04 µg/ml

LOQ 0.12 µg/ml

 Preparation of solutions for recovery study
Accuracy Weight Weight Dilute to Accuracy Further Final
Level of API of Volume Stock Dilute to Conc.
(mg) Placebo With Solution Volume (μg/ml)
(mg) Diluent (ml) with
(ml) Diluent
(ml)
50% 250 20 250 5 100 50
100% 500 20 250 5 100 100
150% 750 20 250 5 100 150
 Result of Recovery study (n=3)
Set No. Accuracy Expected Recovered % Mean %
level amount of amount of Recovery ± SD RSD
(%) Propafenone Propafenone
(μg/ml) (μg/ml)
Set – 1 50 50.27 100.54 50.57
Set – 2 50 50 50.89 101.78 ± 0.61
Set – 3 50 50.54 100.08 0.31
Set – 1 100 100.73 100.73 100.82
Set – 2 100 100 101.28 101.28 ± 0.59
Set – 3 100 101.46 101.46 0.59
Set – 1 150 148.28 99.85 148.92
Set – 2 150 150 148.67 99.28 ± 0.53
Set – 3 150 149.81 99.87 0.79
 Result of repeatability study (n=6)

Sr. Concentration (µg/ml) Area (Volts)

No.

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 Intra-day precision (n=3)

Sr. No. Concentration Mean Area ± SD Area (Volts)

(µg/ml)

1 50 398136 ± 1837.267 0.46

2 100 789546 ± 4883.726 0.52

3 150 1197845 ± 8474.793 0.70

 Result of Inter-day precision (n=3)

Sr. No. Concentration (µg/ml) Mean Area Area (Volts)

± SD

1 50 395698 ± 2274.792 0.57

2 100 781392 ± 5363.155 0.68

3 150 1199025 ± 9364.180 0.78

 Specificity

Chromatogram of blank (Diluent)

Chromatogram of Placebo
Chromatogram of Standard for Propafenone Hydrochloride

Chromatogram of Sample for Propafenone Hydrochloride

•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 .
 Result of specificity study
Sr. Concentration Peak Purity Mean Peak Purity
(µg/ ml)
No.

1. 100 999.888

2. 100 998.783

3. 100 999.574

4. 100 998.462 999.888

5. 100 998.812

6. 100 999.894
 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
 Result of Change in Flow Rate
Sr. Flow Rate: 1.4 ml/ min Flow Rate: 1.6 ml/ min

No. Area (Volts) Area (Volts)

1. 931558 701947
2. 938876 712698
3. 933569 704589
4. 933548 705489
5. 939958 708268
6. 934498 713589
Mean AUC 935334 707764
(n=6)
SD (n=6) 3322.325 4639.455
% RSD 0.35 0.65
 Result of Change in Mobile Phase composition
Sr. Buffer (KH2PO4, pH 2.5) : Buffer (KH2PO4, pH 2.5) : ACN
ACN (62 : 38 % v/v) (58 : 42 % v/v)
No.
Area (Volts) Area (Volts)
1. 811161 800139
2. 815487 808698
3. 816984 813257
4. 814581 801936
5. 813259 807854
6. 819589 809359
Mean AUC 815178.166 806873.833
(n=6)
SD (n=6) 2931.887 4918.320
% RSD 0.35 0.61
 Result of Change in pH
Sr. pH : 2.3 pH : 2.7

No. Area (Volts) Area (Volts)

1. 812937 830698
2. 814589 838579
3. 813957 836987
4. 812698 843597
5. 817598 831458
6. 818589 842985
Mean AUC 815061.333 837384
(n=6)
SD (n=6) 2466.572 5502.331
% RSD 0.30 0.65
 Result of Change in Wavelength
Sr.
ƛmax: 247 nm ƛmax: 251 nm
No.
Area (Volts) 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
 Result of Change in Temperature
Sr. Temperature : 40 °C Temperature : 50 °C

No. Area (Volts) Area (Volts)

1. 774404 771226
2. 771268 776751
3. 772940 774638
4. 770291 780947
5. 774853 772120
6. 776944 783905
Mean AUC 773449.333 776598.833
(n=6)
SD (n=6) 2451.902 5001.638
% RSD 0.31 0.64
 Solution stability study
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
 Filter compatibility study
Sr. Volume of PVDF Nylon
sample
No. (0.45 µm) (0.45 µm)
discarded ( ml)
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
 System suitability parameters
Sr. no. Parameters Value Obtained Standard Value

1. Retention time (Rt) 3.36 -

2. % RSD of area of six 0.64 NMT 2.00

replicate injections

3. Mean Theoretical plates 4256 NLT 2000

(N) or Column efficiency

4. Mean Tailing factor (As) 0.9 NMT 2.00

or Symmetry factor
 Analysis of marketed formulation
Assay of Propafenone Hydrochloride (n=5)
Label Claim (mg) % Assay

101.1
425
100.5

101.4

101.5

101.1

Mean ± SD 101.1 ± 3255.256

% RSD 0.48
 Validation parameters summary
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
11 Solution Stability Study Complies with the acceptance
12 Filter Compatibility Study criteria
13 System Suitability study
 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.
•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.
•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.