1. No category

International Journal of Pharma Sciences
Vol. 5, No. 3 (2015): 1030-1035
Research Article
Open Access
ISSN: 2320-6810
Analytical Method Development and Validation of RPHPLC Method for Simultaneous Estimation of
Ranolazine and Dronedarone in Bulk
Renuka Avvaru* and V. Prabakaran
Department of Pharmaceutical Analysis and Quality Assurance, Krishna Theja Pharmacy College, Renigunta road, Tirupathi, A.P, India.
* Corresponding author: Renuka Avvaru; email: [email protected]
Received: 22 February 2015
Accepted: 25 March 2015
Online: 01 May 2015
ABSTRACT
A simple, precise, accurate and rapid reverse phase high performance liquid chromatographic method was
developed for simultaneous estimation of Ranolazine and Dronedrone in bulk. The separation was achieved by
Xterra C18 column (150 mm × 4.6 mm; 5.0 μm). The estimation was carried out in isocratic program using buffer
and acetonitrile (50:50 v/v) as mobile phase, at a flow rate of 1mL/min. diluent was water and methanol(60:40)
and detection was carried out at 275nm. The retention time obtained for Ranolazine was 2.152min and Dronedrone
was 6.213 min. The calibration curves were linear in the concentration range of 15-90µg/ml for Ranolazine and 318µg/ml for dronedrone correspondingly with correlation coefficient (r2) 0.999.The mean recoveries were found to
be 99.97% for ranolazine and 100.02% for dronedarone. The proposed method has been validated as per ICH
guidelines and successfully applied to the estimation of ranalazine and dronedarone in their combined form.
Keywords: Ranolazine, Dronedarone, RP-HPLC simultaneous estimation.
1. INTRODUCTION
Ranolazine is anti-anginal drug and chemically it is a
piperazine derivative. IUPAC name of ranolazine is N(2,6-dimethylphenyl)-2-[4-[2-hydroxy-3-(2-methoxy
phenoxy) propyl] piperazin-1-yl]acetamide. Ranolazine
acts via altering the trans-cellular late sodium current.
It is by altering the intracellular sodium level that
ranolazine affects the sodium-dependent calcium
channels during myocardial ischemia. Thus, ranolazine
indirectly prevents the calcium overload the etiology of
cardiac ischemia. Ranolazine is indicated for the
treatment of chronic angina. Ranolazine may be used
with beta blockers, nitrates, and calcium channel
blockers, antiplatelet therapy, lipid-lowering therapy,
ACE inhibitors, and angiotensin receptor blockers.
Dronedarone, a benzofuran derivative, N-{2-butyl3
[4(3 di-butyl amino propoxy) benzoyl]-benzofuran-5yl} methanesulfonamide, is a potent drug mainly used
for the indication of cardiac arrhythmias. Dronedarone
is a multi-ion channel blocker, inhibiting the potassium
currents involved in cardiac re- polarisation including
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IKr, IKs, IKur, and IK (Ach) and has been shown to be
effective in the treatment of cardio- vascular
hospitalization in patients with paroxysmal or
persistent atrial fibrillation (AF) or atrial flutter (AFL).
There were some estimation methods for single and for
combination with other drugs but there is no method
found for simultaneous estimation of ranolazine and
dronidarone. [1-7] Hence the aim of present study is to
develop simple, fast, accurate, precise and specific
reversed
phase
high
performance
liquid
chromatographic
method
for
simultaneous
determination of ranolazine and dronedarone in bulk.
Figure-1a: Chemical structure of Ranolazine
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water bath for 5 minutes. Filter through 0.45 µ filter
under vacuum filtration.
Figure-1b: Chemical structures of Dronedarone
2. MATERIALS AND METHODS
2.1 Chemicals and solvents:
Pure samples of Raolazine and Dronedarone were
obtained respectively from Spectrum pharma research
solutions, Hyderabad, India. Ammonium acetate,
Methanol and water used are of HPLC grade.
2.2 Instrumentation:
The chromatographic separations were performed
using HPLC-Waters alliance (Model-2996) consisting of
an inbuilt auto sampler, a column oven and 2965 PDA
detector. The data was acquired through Empower-2software. The column used was Xterra C18
(150×4.6mm i.d, 5µm particle size) (make: Waters).
Meltronics sonicator was used for enhancing
dissolution of the compounds. Elico pH meter was used
for adjusting the pH of buffer solution. All weighing was
done on Sartorius balance (model AE-160).
2.3 Chromatographic conditions:
Detector
:
275nm
Injection volume :
10µl
Flow rate
:
1.0ml/min
Temperature
:
Ambient Temperature
Run time
:
9min
Mobile phase
: (pH 4.0) Ammonium acetate buffer and
Acetonitrile taken in the ratio 50B:50A
Diluent
:
Firstly dissolved in methanol and
then made up with water
2.4 Preparation of buffer solution (pH 4):
Accurately weighed and transferred 0.77gr of
Ammonium acetate in 1000ml of Volumetric flask add
about 900ml of milli-Q water added and degas to
sonicate and finally make up the volume with water
then PH adjusted to 4.0 with dil. Acetic acid solution.
2.5 Preparation of mobile phase:
Mix a mixture of above Buffer 500 mL (50%), 500 mL
of Acetonitrile HPLC (50%) and degased in ultrasonic
Concentration of
S.No
Ranolazine in µg/ml
1
15ppm
2
30ppm
3
45ppm
4
60ppm
5
75ppm
6
90ppm
Correlation Coefficient
2.6 Preparation of standard stock solutions:
(600µg/ml and120µg/ml)
Accurately Weighed and transferred 30mg of
Ranolazine and 6mg of Dronidarone working Standards
into 50ml clean dry volumetric flasks, add 3/4th volume
of diluent, sonicated for 5 minutes and make up to the
final volume with diluents.
(Stock solution)
1ml from the above two stock solutions was taken into
a 10ml volumetric flask and made up to 10ml.
(60µg/ml and12µg/ml)
2.7 Preparation of sample solution:
5 tablets were weighed and calculate the average
weight of each tablet. weight equivalent to
1tablet(1260mg)
was transferred into a 500mL
volumetric flask, 250mL of diluent was added and
sonicated for 25 min, further the volume was made up
with diluent and filtered.
(Stock solution)
From the filtered solution 0.4ml was pipette out into a
10 ml volumetric flask and made up to 10ml with
diluent. (60µg/ml and12µg/ml)
2.8 METHOD VALIDATION:
The developed method was validated as per the ICH
(International
Conference
on
Harmonization)
guidelines with respect to System suitability, Precision,
Specificity, Linearity, Accuracy, Limit of detection and
Limit of quantification.
2.8.1 Linearity:
Aliquots of 0.25, 0.5, 0.75, 0.1, 1.25 and 1.50 ml were
taken from stock solution of concentration 600µg of
ranolazine and 120µg of dronedarone, and then diluted
up to 10ml with diluents such that the final
concentrations were in the range 15-90µg for
ranolazine and 3-18µg for dronedarone. Volume of
10µl of each sample was injected in five times for each
concentration level and calibration curve was
constructed by plotting the peak area versus drug
concentration. The observations and calibration curve
were shown in Table 1 and Fig. 2, 3.
Table-1: Linearity data
Ranolazine Area
156864
324871
466249
634275
783151
952249
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Concentration of
Dronedarone in µg/ml
3ppm
6ppm
9ppm
12ppm
15ppm
18ppm
Dronedarone Area
169929
325400
479513
649881
826607
973167
0.999
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Figure-2: Ranolazine calibration curve
Figure-3: Dronedarone calibration curve
2.8.2 Assay:
Accurately weighed powder equivalent to 30mg of
Ranolazine and 6mg of Dronedarone was transferred
into a 50mL clean dry volumetric flask add diluent and
sonicate to dissolve it completely and make volume up
to the mark with the same solvent.
Drug
Ranolazine
Dronedarone
% linearity level
50
100
150
(Stock solution)
Further pipette 1ml of Ranolazine and Dronedarone of
the above stock solution into a 10ml volumetric flask
and diluted up to the mark with diluents it gives 60µg
of Ranolazine and 12µg of Dronedarone. The results
were shown in Table-2.
Table-2: Ranolazine and Dronedarone assay
Label claim
mg/tab
750
150
Amount found
mg/tab
751.95
150.195
Label
claim(% )
100.26
100.13
Table-3: Accuracy (Ranolazine)
% recovery
98.27
101.09
100.97
99.79
100.06
98.29
99.97
100.46
100.77
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S.D*
% R.S.D
0.73
0.602
0.73
0.60
%mean recovery ± S.D
% RSD
99.97%
1.06%
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% linearity level
50
% recovery
99.95
100.21
99.88
99.24
100.62
100.38
100.59
101.25
98.11
100
150
Accuracy (Dronedarone)
2.8.3 Accuracy:
Accuracy of the method was done by recovery study.
Sample solutions were prepared by spiking at about
50%, 100%, and 150% of specification limit to placebo
and analyzed by the proposed HPLC method. Results
are shown in Table-3.
2.8.4 Specificity:
The specificity of the method was performed by
injecting blank solution( without any sample) and then
a drug solution of 10µl injected into the column, under
optimized chromatographic conditions, to demonstrate
the separation of both Ranolazine and Dronedarone
from any of the impurities, if present. As there was no
interference of impurities and also no change in the
retention time, the method was found to be Specific.
INJECTIONS
1
2
3
4
5
6
AVG
S.D
%R.S.D
Drug
Ranolazine
Dronedarone
Injections
1
2
3
4
5
6
1
2
3
4
5
6
%mean recovery ± S.D
% RSD
100.02%
0.91%
2.8.5 Limit of detection (LOD) and Limit of
quantification (LOQ):
The parameters LOD and LOQ were determined on the
basis of response and slope of the regression equation.
The linearity for Ranolazine and Dronedarone was
performed from 15-90µg/ml and 3-18µg/ml
respectively.
2.8.6 System precision:
Precision is the measure of closeness of the data values
to each other for a number of measurements under the
same analytical conditions. Standard solution of
Ranolazine (60µg/ml) and Dronedarone (12µg/ml)
were prepared as per test method and injected for 3
times. Results are shown in Table-4.
Table-4: System precision
AREAS(Ranolazine)
619758
620658
621384
618932
621554
620524
620468
992
0.16
AREAS(Dronedarone)
636653
646380
630940
651186
654454
638932
643091
9075.9
1.4
Table-5: Method precision
% Assay
100.60
99.35
99.39
100.99
100.95
100.29
99.73
100.10
101.01
100.61
99.33
100.02
2.8.7 Method precision:
Three samples were prepared and analyzed as per the
test method on same day and three different days and
calculated the % RSD for assay of five preparations.
Results were shown in Table- 5.
2.8.8 Robustness:
Robustness studies were carried out by variations in
flow rate, mobile phase compositions and temperature.
It was observed that the small changes in these
operational parameters did not lead to changes of
retention time of the peak interest. The degree of
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Mean
S.D
% R.S.D
100.26
0.73
0.73
100.13
0.602
0.60
reproducibility of the results proven that the method is
robust.
2.8.9 System suitability test:
The system suitability was determined by making six
replicate injections from freshly prepared standard
solutions. The observed RSD values were well within
usually accepted limits (≤2%). Theoretical plates,
tailing factor, resolution between Ranolazine and
Dronedarone were determined. The results are all
within acceptable limits summarized in Table-6.
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Drug
Ranolazine
Dronedarone
Table-6: Characteristics of HPLC method
Parameters defined
Linearity range (µg/ml)
Slope
Intercept
Regression coefficient(r2)
LOD ( µg/ml)
LOQ (µg/ml)
Tailing factor
Plate count
Linearity range (µg/ml)
Slope
Intercept
Regression coefficient(r2)
LOD (µg/ml)
LOQ (µg/ml)
Tailing factor
Plate count
Obtained value
15-90µg/ml
10541
516.4
0.999
0.19
0.56
1.31
3397
3-18 µg/ml
54254
927
0.999
0.0150
0.0455
1.49
4346
Figure-4: Standard chromatogram of Ranolazine and Dronedarone
3. RESULTS AND DISCUSSION
The nature of sample, its molecular weight and
solubility decides the proper selection of stationary
phase. The drugs Ranolazine and Dronedarone were
preferably analyzed by reverse phase chromatography
and accordingly C18 column was selected. The elution of
the compounds from column was influenced by polar
mobile phase. The ratio of ammonium acetate buffer to
Acetonitrile was optimized to 50:50 to give well
resolved and good symmetrical peaks with short run
time. The retention time of Ranolazine and
Dronedarone were found to be 2.152 and 6.213 min
respectively. The calibration curve was linear over the
concentration range of 15-90µg/ml (Ranolazine) and 318µg/ml (Dronedarone).
The linearity of the method was statistically confirmed.
RSD values for accuracy and precision studies obtained
were less than 2% which revealed that developed
method was accurate and precise. The system
suitability parameters were given in table-5. The
analytical recovery at five different concentrations of
Ranolazine and Dronedarone was determined and the
recovery results were in the range of 50-150µg/ml.
Therefore proposed validated method was successfully
applied to determine Ranolazine and Dronedarone in
tablet dosage form.
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4. CONCLUSION
The developed method is accurate, simple, rapid and
selective for the simultaneous estimation of Ranolazine
and Dronedarone in pharmaceutical dosage form. The
excipients of the commercial sample analyzed did not
interfere in the analysis, which proved the specificity of
the method for these drugs. The sample preparation is
simple, the analysis time is short and the elution is by
isocratic method. Hence the proposed method can be
conveniently adopted for the routine quality control
analysis in the combined formulation.
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1.
2.
3.
4.
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Ranolazine Using Reverse Phase High Performance Liquid
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Ramanaiah G.Ramachandran D, Srinivas, Jayapal G,
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Validation of Stability Indicating RP-LC Method for
Estimation of Ranolazine in Bulk and Its Pharmaceutical
Formulations." AJAC. 3: 378-384.
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© 2015; AIZEON Publishers; All Rights Reserved
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