RP-HPLC method development & validation for estimation of Flecainide acetate in bulk and tablet dosage form

Musthafa et al / Int. J. of Farmacia, 2015; Vol-(1) 2: 70-78
70
International Journal of Farmacia
Journal Home page: www.ijfjournal.com
RP-HPLC method development & validation for estimation of Flecainide
acetate in bulk and tablet dosage form
Md.Musthafa1
*, Ravi Kumar Vejendla2
, J.Srawanthi2
, Arshiya Jabeen2
, K.Sindhu2
1*
Nizam Institute of Pharmacy, Deshmukhi, Nalgonda, Telangana
2
Sri Indu Institute of Pharmacy, Sheriguda, Ibrahimpatnam, Hyderabad, Telangana
*
Email id: mustafa2529@gmail.com
ABSTRACT
A novel stability indicating liquid chromatographic assay method was developed and validated as per ICH guide
lines for the quantitative estimation of Flecainide in tablet formulation. An isocratic reverse phase LC-method was
developed using C18 ODS 250cm x 4mm x 5µm particle size column and a mobile phase comprising of a mixture of
buffer: acetonitrile (40:60), pH adjusted to 3.0 with Ammonium di-hydrogen phosphate. The detector set at 299nm
with flow rate of 1.0ml min-1
. The method is linear between 5µg ml-1
to 25µg ml-1
and the limit of detection (LOD)
is 0.5 µg ml-1
. The Accuracy of the method was found to be in the range of 99.70% to 100.26%. The mean Inter and
Intraday assay Relative Standard deviation (% RSD) were less than 0.69%. The Proposed method was found to be
Linear, precise and accurate for the quantitative estimation of Flecainide in tablet formulations and can be used for
commercial purposes.
Keywords: Flecainide, Liquid Chromatography, Stress degradation and Method validation.
INTRODUCTION
Flecainide acetate is a class Ic antiarrhythmic agent
used to prevent and treat tachy-arrhythmias
(abnormal fast rhythms of the heart). It is used to
treat a variety of cardiac arrhythmias including
paroxysmal atrial fibrillation (episodic irregular
heartbeat originating in the upper chamber of the
heart), paroxysmal supraventricular tachycardia
(episodic rapid but regular heartbeat originating in
the atrium), and ventricular tachycardia (rapid
rhythms of the lower chambers of the heart).
Flecainide works by regulating the flow of sodium in
the heart, causing prolongation of the cardiac action
potential. Flecainide acetate is an anti-arrhythmic
drug. Molecular formulae is C17H20F6N2O3 molecular
weight is 414.343 g/mol. Flecainide acetate is a white
crystalline substance it has an aqueous solubility of
48.4 mg/ml at 37°C. Chemical name: Flecainide
acetate is benzamide, N-(2-piperidinylmethyl)-2,5-bis
(2,2,2trifluoroethoxy)-mono-acetate. A through
literature survey has revealed the following reported
methods for the estimation of Flecainide in bulk,
formulation and biological fluids using different
analytical techniques. The reported methods include
estimation of Flecainide in biological samples using

71
LC-UV and LC-MS, HPLC, and Spectrophotometry
for the estimation of Flecainide in bulk and
pharmaceutical formulations [3-16]. In the present
study attempts were made to develop a rapid,
economical, precise and accurate liquid
chromatographic method for the estimation of
Flecainide in tablet formulations.
MATERIALS AND METHODS
Materials
All the reagents were of analytical and HPLC grade
unless stated otherwise. Milli-Q-Water was used
throughout the study. Hydrochloric acid, Sodium
Hydroxide, Acetonitrile, sodium di-hydrogen
phosphate, potassium di-hydrogen phosphate,
ammonium di-hydrogen phosphate etc.(Merck,
Mumbai, India) were used. Flecainide Standard was
obtained as a gift sample and Flecainide tablets were
purchased from Local Pharmacy.
Instrument employed
Shimadzu-1700 double beam – UV – Visible
spectrophotometer with pair of 10mm matched quartz
cells, Shimadzu HPLC, C18 ODS 250cmx4mmx5µm
particle size column, HPLC detector is PDA, HPLC
Injecting Syringe (25 ml) (HAMILTON), PH
analyzer, Ultra Sonicator etc. specification of the
instruments are mentioned the data give for
Electronic
Selection of solvents
The solubility of flecainide was determined in a
variety of solvents as per Indian Pharmacopoeia
standards. Solubility test for Flecainide was carried
out in different polar and non-polar solvents. From
the solubility studies, methanol, acetonitrile was
selected solvent for proposed method.
Preparation of standard Stock Solution
100mg of Flecainide raw material was accurately
weighed and transferred into the 25 ml volumetric
flask and dissolved in minimum quantity of mobile
phase and made up to 25 ml. from this dilution 20,
40, 60, 80,100 & 120 µg/mL were made in 100 mL
volumetric flasks & make up with phosphate buffer
of pH 3.0.
Selection of λ max
The solution was scanned between 200 and 400 nm
range mobile phase as blank. From the UV Spectra
299nm was selected as ‫ג‬max for analysis of Flecainide.
Stability of the Flecainide in mobile phase was
studied by measuring the same solution at this ‫ג‬max in
different time intervals. It was observed that
Flecainide in mobile phase was stable for more than 2
hours.
Method development
Selection of chromatographic method
Proper selection of the method depends upon the
nature of the sample, molecular weight, and
solubility. The drug selected for the present study
was polar in nature. So reversed phase
chromatography can be used, this reverse phase
HPLC was selected for the initial separation from the
knowledge of properties, C18 column was chosen as
stationary phase.
Preparation of mobile phase
0.01M solution of buffer was prepared by using water
as solvent to this organic solvent acetonitrile was
added. While conducting trials various buffers was
used by changing the proportional of buffer and
acetonitrile along with altering the PH
to decide the
final development method. Then it is subjected to
vacuum filtration and then proper sonication should
be done which is for 15 min
Sample injection
After preparation of mobile phase, sample was
prepared by taking little a quantity of mobile phase of
subsequent trials in a test tube ,to this a little quantity
of sample was added and then it is sonicated. Before
injecting the sample, pump is subjected to purging
and then wavelength of 299nm was adjusted then the
blank was injected initially in order to get stable base
line after attaining stable baseline sample was
injected with syringe.
Table 1: Optimized chromatographic conditions
.Parameter Optimized condition
Chromatograph HPLC (Shimadzu with 2487 PDA)
Column C18 ODS 250cmx4mmx5µm particle size
Mobile Phase* (0.01M Ammonium di-hydrogen Phosphate - pH 3.0)
acetonitrile: buffer ( 40 : 60 % v/v)
Flow rate 1 ml/min

72
Detection at UV wave length 299 nm
Injection volume 20µl
Column Temperature Ambient
Retention time 2.773 min.
.
Figure 1: Optimized spectra of Flecainide
Method validation
The proposed RP-HPLC method was validated as per
ICH guidelines.
Linearity
The calibration curve was plotted with five
concentrations of the standard drug solution 5.0-100
µg/ml solution and chromatography was repeated
thrice for each dilution. The linearity was evaluated
by linear regression analysis, before injecting
solutions; the column was equilibrated for at least 30
min with the mobile phase flowing through the
system five determinations were carried out for each
solution, peak area ratios were recorded for all the
solutions. The correlation graph was constructed by
plotting the peak area ratios obtained at the optimum
wave length of detection versus the injected amounts
of the respective concentrations.
Specificity
The specificity of the RP-HPLC method was
determined by comparison of the chromatogram of
standard and sample solution. The parameters like
retention time (Rt), resolution (Rs) and tailing factor
(Tf) were calculated.
Precision
Precision study was performed to find out intra-day
and inter-day variation. It was carried out by
estimating the corresponding responses 3 times on
the same day and on 3 different day (first, second and
fifth day) for particular concentration of flecainide
(100µg/ml) and the results are reported in terms of
relative standard deviation (RSD). The repeatability
studies were carried out by estimating response of
particular concentration of flecainide (100µg/ml) for
5times.

73
Stability
In order to demonstrate the stability of the standard
solution of MS during analysis, the solution was
stored over a period of 24 hr at room temperature and
then analyzed.
Robustness
As defined by the ICH, the robustness of an
analytical procedure describes to its capability to
remain unaffected by small and deliberate variations
in method parameters. Robustness was performed by
small variation in the chromatographic conditions
and found to be unaffected by small variations like
±2% variation in volume of mobile phase
composition, ±0.1 ml/min in flow rate of mobile
phase, ±0.1 variation in pH.
Accuracy
To study the reliability, suitability and accuracy of
the method recovery experiments were carried out. A
known quantity of the pure drug of 10 mcg i.e.,
from spiking standard( prepared by taking 100mcg of
drug in 100ml of mobile phase so concentration is
1mg/ml that is for 10mcg 10ml is taken) was added
to the pre-analyzed sample of 80 mcg,100,120 mcg
respectively. After this each sample was injected for
3 times. The contents were determined from the
respective chromatograms. The concentration of the
drug product in the solution was determined using
assay method and % RSD was calculated by using
the following formula; the lower values of %RSD of
assay indicate the method is accurate. The mean
recoveries were in range of 99.8-101.20 % which
shows that there is no interference from excipients.
% recovery = (b-a) / c (100)
Where,
a- The amount of drug found before the addition of
standard drug
b- The amount of drug found after the addition of
standard drug
c-The amount of standard drug added
Assay of Flecainide in tablets
Twenty tablets were weighed and finely powdered.
An accurately weighed portion of the powder
equivalent to 100 mg of flecainide was transferred to
100 ml volumetric flask containing 10ml of buffer
solution and the contents of the flask were sonicated
for 15 min, to ensure the complete solubility of the
drug .The mixture was then made up to 100 ml with
mobile phase. The resulting solution was thoroughly
mixed and filtered through vacuum filter. 5 ml of this
solution was added to 100 ml volumetric flask and
made up to the mark with phosphate buffer of pH
(3.0). This solution (20 µl) was injected three times
into the column. The mean values of peak areas of
five such determinations were calculated and the drug
content in the tablets was quantified using the
regression equation.
Ruggedness
The ruggedness of the method was determined by
carrying out the experiment on different instruments
like Shimadzu HPLC and Agilent HPLC by different
operators using different columns of similar types.
The %RSD values with two different instruments
Shimadzu HPLC and Agilent HPLC, analysts and
columns were 0.5- 0.5, 0.6- 0.5 and 0.4- 0.3%
respectively.
RESULTS AND DISCUSSION
From the optical characteristics of these proposed
methods, it was found that trail method IV for
Flecainide obey Accuracy, Precision and Linearity
within the concentration respectively. From the
results, it was found that the percentage recovery
values of pure drugs from the pre-analyzed solution
of formulation were 99.40 % for Flecainide, which
indicates that the proposed method is accurate and
also reveals that the commonly used excipients and
additives in the pharmaceutical formulations were not
interfering in the proposed method. Chromatograms
are attached for the trials, linearity, accuracy,
precession specificity, robustness and ruggedness.
Table 2: Linearity
Linearity
Concentration mcg Area of the peak

74
Figure 2: Linearity curve of Flecainide
Table 3: Precision
Flecainide Precision (100 mcg)
S. No RT Area
1 2.753 571.164
2 2.75 570.437
3 2.757 570.945
4 2.76 570.295
5 2.757 571.338
Average 2.7554 570.8358
St.dev. 0.003911521 0.453693399
% RSD 0.141958389 0.079478792
Table 4: Accuracy
20 114.714
40 232.316
60 326.214
80 427.313
100 550.506
120 641.868
Spikiking Standard Concentration 100mcg
Spiking Standard Areas
Standard 1 553.617
Standard 2 553.452
Standard 3 553.494
y = 5.2735x + 13.011
R² = 0.9986
0
100
200
300
400
500
600
700
0 50 100 150
Area
Conc. mcg
Linearity

75
Flecainide 130mcg
Sample 120+10 130 mcg
Spiking Standard Area 553.521
Sample 1 712.762
Sample 2 715.771
Sample 3 716.803
Average 715.112
Mcg Recovery 129.1932917
% Recovery 99.38%
Flecainide 110mcg
Sample 100+10= 110 mcg
Sample 1 603.352
Sample 2 605.729
Sample 3 606.318
Average 605.133
% Recovery 99.39%
Average 553.521
Standard value 0.070014284
% RSD 0.015492
Flecainide 90mcg
Sample 80+10= 90 mcg
Sample 1 496.127
Sample 2 498.198
Sample 3 495.608
Average 496.6443333
% Recovery 99.69%

76
Assay
Separately inject equal volumes of diluents, standard
preparation and sample preparation into
chromatograph, record the chromatograms, and
measure the pea area response for the major peaks
calculate the amount content of Flecainide in the
portion of Flecainide tablets by the formula.
Sample peak area x Dilution factor of standard x
Average weight of tablets
Standard peak area x dilution factor of sample
Table 5: Assay
Flecainide Assay
Standard Areas
Standard 1 594.328
Standard 2 595.446
Standard 3 596.406
Average 595.926
Sample Areas
Sample 1 571.164
Sample 2 570.16
Average 570.662
Standard Area 595.926
Sample Area
570.662
Standard Weight
25.2mg
Sample Weight
72.8mg
Label Claim 50mg
Standard purity
99.83%
Average Weight 150.2mg
Content of Flecainide 99.40%
CONCLUSION
An attempt has been made to estimate Flecainide by
RP-HPLC. Even though a number of RP-HPLC
methods have been reported earlier for Flecainide
individually and with other combinations, an effort
has been made to identify a common mobile phase to
come up with the isocratic elution of drug
individually. The present drug of Flecainide was
marketed as formulation. Flecainide – 150mg /tablet.
The formulation was diluted in the linearity range
and peak areas were determined, the concentrations
Flecainide were then determined by comparing the
peak areas of sample with that of standard peak areas
of that can be identified by their retention times for
Flecainide. The results obtained from HPLC method
were reproducible and encouraging. The values
percentage deviation was satisfactorily low and
recovery close to 100 % indicating reproducibility
and accuracy of method. The proposed method were
proved to be superior to most of the reported method
and this can be used as alternative method for the
routine determination of selected drugs under the
study in bulk and pharmaceutical dosage forms. Thus
the purpose of the present investigation was
successfully achieved.

77
REFERENCES
[1]. Corkeron M, van Heerden P, Newman S, Dusci L (1999). "Extracorporeal circulatory support in near-fatal
flecainide overdose". Anaesth Intensive Care 27 (4): 405–8. PMID 10470398.
[2]. Echt D, Liebson P, Mitchell L, Peters R, Obias-Manno D, Barker A, Arensberg D, Baker A, Friedman L,
Greene H (1991). "Mortality and morbidity in patients receiving encainide, flecainide, or placebo. The Cardiac
Arrhythmia Suppression Trial". N Engl J Med 324 (12): 781–8. doi:10.1056/NEJM199103213241201.
PMID 1900101.
[3]. Fornieles-Pérez H, Montoya-García M, Levine P, Sanz O (2002). "Documentation of acute rise in ventricular
capture thresholds associated with flecainide acetate". Pacing Clin Electrophysiol 25 (5): 871–2.
doi:10.1046/j.1460-9592.2002.00871.x. PMID 12049386.
[4]. Gasparini M, Priori S, Mantica M, Napolitano C, Galimberti P, Ceriotti C, Simonini S (2003). "Flecainide test
in Brugada syndrome: a reproducible but risky tool". Pacing Clin Electrophysiol 26 (1 Pt 2):338–41.
doi:10.1046/j.1460-9592.2003.00045.x. PMID 12687841.
[5]. Gill J, Mehta D, Ward D, Camm A (1992). "Efficacy of flecainide, sotalol, and verapamil in the treatment of
right ventricular tachycardia in patients without overt cardiac abnormality". Br Heart J 68 (4): 392–7.
doi:10.1136/hrt.68.10.392. PMC 1025139. PMID 1449923.
[6]. Morganroth J (1992). "Early and late proarrhythmia from antiarrhythmic drug therapy". Cardiovasc Drugs
Ther 6 (1): 11–4. doi:10.1007/BF00050910. PMID 1533532.
[7]. Greenberg H, Dwyer E, Hochman J, Steinberg J, Echt D, Peters R (1995). "Interaction of ischaemia and
encainide/flecainide treatment: a proposed mechanism for the increased mortality in CAST I". Br Heart J 74
(6): 631–5. doi:10.1136/hrt.74.6.631. PMC 484119. PMID 8541168.
[8]. Guetens G., Prenen H., De Boeck G., Van Dongen W., Esmans E, Lemiere F., Van Oosterom A. T., Schöffski
P., De Bruijn E. A.Journal of chromatography, 2005, vol 1082[1], pp 2-5.
[9]. Haefeli W, Bargetzi M, Follath F, Meyer U (1990). "Potent inhibition of cytochrome P450IID6 (debrisoquin 4-
hydroxylase) by flecainide in vitro and in vivo". J Cardiovasc Pharmacol 15 (5): 776–9.
doi:10.1097/00005344-199005000-00013. PMID 1692938.
[10]. Katristis D, Rowland E, O'Nunain S, Shakespeare C, Poloniecki J, Camm A (1995). "Effect of flecainide on
atrial and ventricular refractoriness and conduction in patients with normal left ventricle Implications for
possible antiarrhythmic and proarrhythmic mechanisms". Eu Heart J 16 (1): 1930–1935.
[11]. L. R. Snyder, J.J. Kirkland, and J. L. Glajch,John Wiley & Sons,Practical HPLC Method Development, New
York, 1997.
[12]. L. R. Snyder, J.J. Kirkland, and J. W. Dolan,John Wiley & Sons.Introduction to Modern Liquid
Chromatography, New York, 2009.
[13]. Ming Zhao ; Hartke Carol ; Jimeno Antonio ; Jing Li ; Ping He ; Zabelina Yelena ; Journal of chromatography.
B, 2005, Hidalgo Manuel (1) ; Baker Sharyn D. vol. 819[1], pp. 73-80.
[14]. N Engl J Med. Cardiac Arrhythmia Suppression Trial (CAST) Investigators (1989). "Preliminary report: effect
of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial
infarction." 321 (6): 406–412. doi:10.1056/NEJM198908103210629. PMID 2473403.
[15]. Ohki R, Takahashi M, Mizuno O, Fujikawa H, Mitsuhashi T, Katsuki T, Ikeda U, Shimada K (2001).
"Torsades de pointes ventricular tachycardia induced by mosapride and flecainide in the presence of
hypokalemia". Pacing Clin Electrophysiol 24 (1): 119–21. doi:10.1046/j.1460-9592.2001.00119.x.
PMID 11227957.
[16]. Padrini R, Piovan D, Busa M, al-Bunni M, Maiolino P, Ferrari M (1993). "Pharmacodynamic variability of
flecainide assessed by QRS changes". Clin Pharmacol Ther 53 (1): 59–64. doi:10.1038/clpt.1993.9.
PMID 8422742.
[17]. Ramos E, O'leary M (2004). "State-dependent trapping of flecainide in the cardiac sodium channel". J Physiol
560 (Pt 1): 37–49. doi:10.1113/jphysiol.2004.065003. PMC 1665201. PMID 15272045.
[18]. Santinelli V, Arnese M, Oppo I, Matarazzi C, Maione S, Palma M, Giunta A (1993). "Effects of flecainide and
propafenone on systolic performance in subjects with normal cardiac function". Chest 103 (4): 1068–73.
doi:10.1378/chest.103.4.1068. PMID 8131440.

78
[19]. S. Ahuja and H. T. Rasmussen (ed),HPLC Method Development for Pharmaceuticals, Academic Press, 2007.
[20]. Sakurada H, Hiyoshi Y, Tejima T, Yanase O, Tokuyasu Y, Watanabe K, Motomiya T, Sugiura M, Hiraoka M
(1990). "[Effects of oral flecainide treatment of refractory tachyarrhythmias]". Kokyu to Junkan 38 (5): 471–6.
PMID 2115193.
[21]. Smith G (1985). "Flecainide: a new class Ic antidysrhythmic". Drug Intell Clin Pharm 19 (10): 703–7.
PMID 3902429.
[22]. Winkelmann B, Leinberger H (1987). "Life-threatening flecainide toxicity. A pharmacodynamic approach".
Ann Intern Med 106 (6): 807–14. PMID 3107447.
[23]. Wang Z, Fermini B, Nattel S (1993). "Mechanism of flecainide's rate-dependent actions on action potential
duration in canine atrial tissue". J Pharmacol Exp Ther 267 (2): 575–81. PMID 8246130.
[24]. Watanabe, Hiroshi; Nagesh Chopra, Derek Laver, Hyun Seok Hwang, Sean S. Davies, Daniel E. Roach, Henry
J. Duff, Dan M. Roden, Arthur A. M. Wilde, Björn C. Knollmann (2009-04-01). "Flecainide prevents
catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans.". Nature Medicine 15
(4): 380–383. doi:10.1038/nm.1942. PMC 2904954. PMID 19330009.. Retrieved 2009-05-04.
[25]. Wiley, 2006. M.W. Dong .Modern HPLC for practicing scientists.
[26]. Yasui R, Culclasure T, Kaufman D, Freed C (1997). "Flecainide overdose: is cardiopulmonary support the
treatment?". Ann Emerg Med 29 (5): 680–2. doi:10.1016/S0196-0644(97)70257-9. PMID 9140253.

RP-HPLC method development & validation for estimation of Flecainide acetate in bulk and tablet dosage form

More Related Content

Similar to RP-HPLC method development & validation for estimation of Flecainide acetate in bulk and tablet dosage form (20)

More from SriramNagarajan18 (20)

Recently uploaded (20)

RP-HPLC method development & validation for estimation of Flecainide acetate in bulk and tablet dosage form