INTRODUCTION:
An Arrhythmia is an abnormality of rate,
rhythm or site of the cardiac impulse or an abnormality in the impulse
conduction. Disturbance of impulse generation may be due to altered normal and
abnormal automaticity or after-depolarization. In disturbances of impulse
conduction, an impulse may recirculate in the heart
and cause repeated activation (re-entry).or there
should be conduction block.
·Atrial
Fibrillation:
Atrial fibres are activated asynchronously at a rate of 350-550/min (due to
electrophysiological in homogeneity of atrial
fibres), associated with grossly irregular and often fast (100-160/min)
ventricular response.
·Amiodarone
and Ranolazine combination was approved on 3 Sep, 2009.,
·Patented
by Gilead Sciences Inc, Europe Patent No: EP 2337559 A2 (2) Combination
of both these drugs may help to treating the atrial
fibrillation. Both the drugs were approved by the US Government, and have
frequently been used for the treatment of Atrial
fibrillation in US. (Clinical trials)(3,4)
A. Amiodarone
Hydrochloride (5-9)
Figure
1: Chemical Structure of Amiodarone Hydrochloride
Chemical name :-
2-butylbenzofuran-3-yl-4-(2-diethylaminoethoxy)-3,5-diiodophenylketone
hydrochloride(5)
Amiodarone Hydrochloride appears as white or almost
white crystalline powder. The drug is slightly soluble in Distilled Water. Freely soluble in methanol.
Amiodarone Hydrochloride melts at 158-165 °C(6)
The pKa value of Amiodarone
Hydrochloride is 6.56(7)
The molecular formula of Amiodarone
Hydrochloride is C25H29I2NO3.HCL..
Amiodarone is categorized as a class III Antiarrhythmic agent and prolongs phase 3 of the
cardiac action potential, the repolarization phase
where there is normally decreased calcium permeability and increased Potassium
permeability.(8-9)
B. Ranolazine
Figure
2: Chemical structure of Ranolazine
Chemical name:-
N-(2,6-dimethylphenyl)-2{4-[2-hydroxy-3-(2-methoxyphenoxy)propyl
piprazine-1-yl}acetamide(10).
Ranolazine is a white to off-white crystalline powder that is
freely soluble in Methanol and slightly soluble in water.
Ranolazine melts at 122-124°C(11)
The pKa value of Amiodarone
Hydrochloride is 13.6(12)
The molecular formula of Ranolazine
is C24H33N3O4
Ranolazine a piperazine derivative is a new
anti-ischemic drug for the treatment of angina and atrial
fibrillation. The effect of ranolazine to block late INa has the potential to disrupt this cycle by
reducing intracellular calcium accumulation and the accompanying decrease in
ventricular wall tension.(13-14)
MATERIAL AND METHODS:
APPARATUS
AND INSTRUMENT:
A
double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with
spectral width of 2 nm, 1 cm quartz cells was used to measure absorbance of all
the solutions.
Spectra
were automatically obtained by UV-Probe system software.
An
analytical balance (Sartorius CD2250, Gottingen, Germany) was used for weighing
the samples.
Sonicator(D120/2H, TRANS-O-SONIC)
Class
‘B’ volumetric glassware were used (Borosillicte)
All
instruments and glass wares were calibrated.
REAGENTS
AND MATERIAL
Amiodarone HCl
(Gift sample from CTX Life Sciences, Sachin)
Ranolazine (Gift sample from CTX Life
Sciences, Sachin)
Methanol
AR Grade (FINAR), Distilled Water, NaOH AR Grade
(RANCHEM), HCl (ASTRON) was used for development
purpose.
SECOND
DERIVATIVE CONDITIONS
Mode :
Spectrum
Scan speed : Fast
Wavelength range : 200-400 nm
Derivative order : Second
Scaling factor: 50
PREPARATION
OF STANDARD SOLUTIONS
Standard
solution of Edaravone (AMI)
Preparation
of stock solution of AMI:
Accurately
weighed quantity of
AMI 10 mg was transferred to 100 ml volumetric flask, dissolved,
and diluted up to mark with methanol to give a stock solution having strength
100 µg/ml.
Standard
solution of Argatroban (RAN)
Preparation
of stock
solution of RAN:
Accurately
weighed quantity of
RAN 100 mg was transferred
into 100 ml volumetric flask, dissolved and diluted up to mark with methanol to
give a stock solution having strength 1000 µg/ml.
Preparation
of Standard Mixture Solution (AMI + RAN):
1ml
of standard stock solution of AMI (100 μg/ml) and 1ml of standard stock solution of RAN (1000 μg/ml)
were pipetted out into two 10 ml volumetric flasks and
volume was adjusted to the mark with methanol to get 10
μg/ml of AMI and 100 μg/ml of RAN.
Preparation
of test solution:
The
preparation of synthetic mixture was as per patent:
Amiodarone HCl : 25 mg
Ranolazine : 250 mg
Hydroxy Propyl
Methyl cellulose :30 mg
Micro
Crystalline Cellulose : 38 mg
Magnesium
Stearate :
7 mg
All
the excipients were mixed in 100ml volumetric flask
dissolved in 25 ml of distilled water and sonicated
for 15 min. make up the volume with methanol up to 100 ml. The solution was
filtered through Whatman filter paper No. 42.
Finally
the solution had concentration 100μg/ml
for AMI and 1000μg/ml for RAN.
VALIDATION
OF PROPOSED METHOD
Parameters
to be considered for the validation of methods are:
1)
LINEARITY AND RANGE
Procedure:
The
linearity response was determined by analyzing 5 independent levels of
calibration curve in the range of 1-20 μg/ml and 10-200 μg/ml for AMI and RAN
respectively (n=6).
Calibration
curves for AMI:
This series consisted of five concentrations of
standard AMI solution ranging from 1-20μg/ml. The solutions were prepared
by pipette out Standard AMI stock solution (0.1ml,
0.5ml, 1.0ml, 1.5ml and 2.0ml) was transferred into a series of 10 ml
volumetric flask and volume was adjusted up to mark with Methanol. A Second
order derivative spectrum, measured the absorbance at 263.20nm against a
reagent blank solution (Methanol).
Calibration
curve for RAN :
This series consisted of five concentrations of
standard RAN solution ranging from 10-200μg/ml. The solutions were
prepared by pipette out Standard RAN stock solution (0.1ml,
ml, 0.5ml, 1.0ml, 1.5ml and 2.0ml) was transferred into a series of 10 ml
volumetric flask and volume was adjusted up to mark with Methanol. A Second order derivative spectrum measured
the absorbance at 249.20 nm against a reagent blank solution (Methanol).
2)
PRECISION
I.
Intraday precision
Procedure
The
precision of the developed method was assessed by
analyzing combined standard solution containing three different concentrations
1, 10, 20 μg/ml for AMI and 10, 100, 200 μg/ml RAN.
Three replicate (n=3) each on same day
Second-derivative
absorbance (D1) was measured at 249.20 nm for RAN and 263.20 nm for
AMI.
The
% RSD value of the results corresponding to the absorbance was expressed for
intra-day precision.
II.
Interday Precision
Procedure
The
precision of the developed method was assessed by analyzing combined standard
solution containing three different
concentrations 1, 10, 20 μg/ml for AMI and 10, 100, 200 μg/ml for RAN
triplicate (n=3) per day for consecutive 3 days for inter-day precision.
Second-derivative
absorbance (D1) was measured at 249.20 nm for RAN and 263.20 nm for
AMI.
The
% RSD value of the results corresponding to the absorbance was expressed for
inter-day precision.
III. Repeatability:
The
precision of the developed method was assessed by analyzing combined standard
solution containing three different
concentrations 1, 10, 20 μg/ml for AMI and 10, 100, 200 μg/ml for RAN
triplicate (n=3) per day for consecutive 3 days for Repeatability precision.
Second-derivative
absorbance (D1) was measured at 249.20 nm for RAN and 263.20 nm for
AMI.
The
% RSD value of the results corresponding to the absorbance was expressed for
inter-day precision.
(3)
ACCURACY
From
the Synthesis Mixture weigh accurately equivalent about 10mg of AMI. Take Four
100ml Volumetric Flask and in each flask add synthetic mixture equivalent to 10mg
of AMI. Flask 1 form as a Placebo and remaining flask 2, 3, 4 spike with 80,
100 and 120 % of Solid API. Repeat the same procedure for RAN as per Table.
Take content in 100 ml volumetric flask dissolved in 25 ml Methanol and Sonicate for 15min. make up the volume with Methanol up to
100 ml. The solution was filtered through Whatman filter paper No. 42.
Finally
the solution had concentration 100μg/ml for AMI and
1000μg/ml for RAN. From that pipette out 0.5 ml in 10 ml volumetric flask
and volume was made up to mark with Methanol to make final concentration AMI (5
µg/ml) and RAN (50 µg/ml).
Data
from nine determinations over three concentration levels covering the specified
range was determined and % recovery was calculated.
Procedure
Table 1: Mixture for Accuracy Study
|
Concentration of Formulation (mg) |
Concentration of API Spiking (mg) |
Total concentration (mg) |
|||
|
AMI |
RAN |
AMI |
RAN |
AMI |
RAN |
|
10 |
100 |
- |
- |
10 |
100 |
|
10 |
100 |
8 |
8 |
18 |
180 |
|
10 |
100 |
10 |
100 |
20 |
200 |
|
10 |
100 |
12 |
120 |
22 |
220 |
From
that withdraw 0.5 ml of Solution and diluted with Methanol up to 10ml
volumetric flask and scanned between 200nm to 400nm against Methanol as a
blank. The amount of AMI and RAN was calculated at each level and % recoveries
were computed.
4)
LOD (LIMIT OF DETECTION) and LOQ (LIMIT OF QUANTIFICATION)
The
Limit of detection and Limit of Quantification of the developed method was
assessed by analyzing ten replicates of standard solutions containing
concentrations 5 μg/ml
for AMI and 50 μg/ml for RAN.
The
LOD and LOQ may be calculated as
Where,
SD = ten replicates of absorbance
Slope
= the mean slope of the 6 calibration curves
5)
ROBUSTNESS AND RUGGEDNESS
Robustness
and Ruggedness of the method was determined by subjecting the method to slight
change in the method condition, individually, the:
Change
in instrument (UV-Vis Spectrophotometer model 1800 and 2450),
Change
in Analyst,
Change
in Ratio,
Change
in solvent like 2% Water in MeOH and 5% water in MeOH
Three
replicates were made for the concentration (5 μg/ml of AMI and 50 μg/ml
of RAN) with different stock solution preparation and the recording of absorbances were done on both the UV-Vis spectrophotometer.
% RSD was calculated.
ANALYSIS
OF AMI AND RAN IN SYNTHETIC MIXTURE
Composition
of synthetic mixture
Prepared the
Synthetic Mixture as per Patent.
Amiodarone Hydrochloride-142.8
Ranolaizne-1428.57
HPMC-171.42
MCC-217.14
Magnesium Stearate-40 (total=2000 mg)
Take Powder equivalent to 10 mg of Synthetic
Mixture in 100 ml Volumetric Flask.
Dissolve in 25 ml of Methanol. Sonicate for 15
min. Dilute up to the 100 ml with Solvent. Shake Vigoursly.
Filter the solution and further dilute.
Finally
the solution had concentration 100 μg/ml for AMI and 1000 μg/ml for RAN.
From
that pipette out 1 ml in 10 ml volumetric flask and volume was made up to mark
with methanol to obtain final solution containing 5 µg/ml of AMI and 50 µg/ml
of RAN. A zero order derivative spectrum of the resulting solution was recorded
and processed to first derivative spectra. A Second order derivative spectrum
of the sample solution was recorded and the absorbance at 249.20 nm and 263.20 nm were
noted for estimation of AMI and RAN, respectively. The concentrations of AMI
and RAN in formulation were determined using the corresponding calibration
graph.
RESULT AND DISCUSSION:
Selection
of wavelength and method development for determination of Amiodarone
HCl and Ranolazine(15)
The
standard solution of AMI and RAN were scanned separately between 200-400nm, and
zero-order spectra were not showed overlapping peaks.
Figure 3: Overlain zero order spectra of AMI and
RAN, respectively
Thus
obtained spectra were then processed to obtain second-derivative spectra.
Second-order
derivative spectrum for AMI showed zero crossing points at 229.20 nm and 249.20
nm but at 229.20 nm RAN absorbance is not properly estimated. So 249.20 nm is
selected which give r2=0.9996.
Second
order derivative s pectrum for RAN showed zero crossing
point at 263.20 nm.
Figure
4: Overlain Second order spectra of AMI and RAN
Figure
5: Overlain second order spectra of AMI and RAN in 1:10 ratios, respectively
with the combination solution (1:10)
The
overlain second order spectra (fig.3) of AMI and RAN reveal that AMI showed
zero crossing at 249.20 nm, while RAN showed zero crossing at 263.20 nm. At
zero crossing point (ZCP) of AMI (249.20 nm), RAN showed an absorbance, whereas
at ZCP of RAN (263.20 nm), AMI absorbance.
VALIDATION
PARAMETERS (16)
1.
Linearity and Range
The
second-derivative spectra (fig.3) showed linear absorbance at 263.20nm (ZCP of
RAN) for AMI (1-20 µg/ml) and 249.20 nm (ZCP of AMI) for RAN (10-200 µg/ml)
with correlation coefficient (r2) of 0.9996 and 0.9996 for AMI and
RAN, respectively.
This
method obeyed beer’s law in the concentration range 1-10 µg/ml and 10-200 µg/ml
for AMI and RAN, respectively. (Table 2)
Correlation
coefficient (r2) form calibration curve of AMI and RAN was found to
be 0.9996 and 0.9996, respectively.
The
regression line equation for AMI and RAN are as following,
y =
0.006x +0.014 for AMI _____________ (1)
y =
0.002x +0.003 for RAN ______________ (2)
Figure 6: Overlain linear second order spectra of
AMI (Blue) and RAN (Pink) in 1:10 ratios
From
the combination solution of AMI and RAN the dilution were made in ratio of 1:10
and absorbance were recorded (Table 2) and correlation coefficient (r2)
of 0.9996 and 0.9996 (figure 7) for AMI
and RAN, respectively.
Table
2: Calibration data for AMI and RAN at
280.47nm
and 351.0nm, respectively. *(n=6)
|
Sr. No |
Concentration (μg/ml) |
Absorbance*
(263.20 nm) ± SD AMI |
Absorbance*
(249.20 nm)± SD RAN |
|
|
AMI |
RAN |
|||
|
1 |
1 |
10 |
0.021 ± 0.0001 |
0.020 ± 0.0001 |
|
2 |
5 |
50 |
0.044 ± 0.0003 |
0.109 ± 0.001 |
|
3 |
10 |
100 |
0.078 ± 0.0006 |
0.218 ± 0.0014 |
|
4 |
15 |
150 |
0.109 ± 0.0009 |
0.317 ± 0.0016 |
|
5 |
20 |
200 |
0.138 ± 0.0010 |
0.416 ± 0.0014 |
Figure 7: Calibration curve for AMI at 263.20 nm
and RAN at 249.20 nm
2.
Precision
I.
Intraday precision
The
data for intraday precision for combined standard solution of AMI and RAN is
presented in Table 3.
The
% R.S.D was found to be 0.60- 0.83% for AMI and 0.36 – 0.89% for RAN.
These
%RSD value was found to be less than 1.0 indicated that the method is precise.
Table 3: Intraday precision data for estimation of
EDA and ARG *(n=3)
|
Conc. (μg/ml) |
Abs. At 263.20 nm (Mean) |
% RSD |
Abs. At 249.20 nm (Mean) |
% RSD |
|
AMI: RAN |
||||
|
1:10 |
0.016 |
0.72 |
0.017 |
0.89 |
|
10:100 |
0.077 |
0.83 |
0.216 |
0.80 |
|
20:200 |
0.133 |
0.60 |
0.422 |
0.36 |
II.
Interday precision
The
data for Interday precision for combined standard solution of AMI and RAN is
presented in Table 4.
The
% R.S.D was found to be 0.71-0.92% for AMI and 0.36-0.96% for ARG.
These
%RSD value was found to be less than ± 2.0 indicated that the method is
precise.
Table 4: Interday precision data for estimation of
AMI and RAN *(n=3)
|
Conc. (μg/ml) |
Abs. At 263.20 nm (Mean) |
% RSD |
Abs. At 249.20 nm (Mean) |
% RSD |
|
AMI: RAN |
||||
|
1:10 |
0.0161 |
0.71 |
0.017 |
0.89 |
|
10:100 |
0.078 |
0.92 |
0.216 |
0.96 |
|
20:200 |
0.133 |
0.75 |
0.421 |
0.36 |
III. Repeatability:
The
data for Interday precision for combined standard solution of AMI and RAN is
presented in Table 5.
Table 5 - Repeatability precision data for
estimation of AMI and RAN (n=3)
|
Conc. (μg/ml) |
Abs. At 263.20 nm (Mean) |
% RSD |
Abs. At 249.20 nm (Mean) |
% RSD |
|
AMI: RAN |
||||
|
1:10 |
0.016 |
0.95 |
0.017 |
0.34 |
|
10:100 |
0.075 |
0.70 |
0.217 |
0.70 |
|
20:200 |
0.133 |
0.87 |
0.422 |
0.47 |
3.
Accuracy
Accuracy
of the method was determined by recovery study from synthetic mixture at three
levels (80%, 100%, and 120%) of standard addition.
The
% recovery values are tabulated in Table 6 and 7.
Percentage
recovery for AMI and RAN by this method was found in the range of 99.33 to
100.45% and 100.43-100.96%, respectively,
The
value of %RSD within the limit indicated that the method is accurate and
percentage recovery shows that there is no interference from the excipients.