INTRODUCTION
FINERENONE
Chemically
Finerenone is a
(4S)-4-(4-Cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide.
It is also be spelled as "Kerendia".1-7 It is available in
the Indian market as tablet dosage form in the brand names Kerendia.8
It is used to reduce the risk of kidney function decline, kidney failure,
cardiovascular death, non-fatal heart attacks, and hospitalization for heart
failure in adults with chronic kidney disease associated with type II diabetes.
It is Highly soluble in ethanol, DMSO, DMF, Water. 9
Mode
of action Finerenone is a selective antagonist of the mineralocorticoid
receptor. Activated by aldosterone and cortisol, the nonsteroidal MRA regulates
gene transcription.
Literature
survey reveals that only MS method has been developed for estimation of
Finerenone so there is no spectroscopic method available to estimate either
from bulk drug or from the pharmaceutical dosage forms. The present
investigation was proposed to estimate Finerenone in bulk and pharmaceutical
dosage form by spectroscopic method which is rather simple, sensitive,
specific, precise, accurate method. The present work was to develop and
validate as per ICH guidelines so the method was developed validated according
to ICH guidelines for accuracy, precision, reproductivity, repeatability and
robustness.10
MATERIALS
AND METHODS:
Materials
and reagents:
A
gift sample of Finerenone from Bayers Pharmaceuticals, Mumbai used
as a standard drug. Bayers (Kerendia 10mg) film coated tablet dosage form
bought from the local market. Other chemicals like Ethanol were bought from SD
fine chemicals, Mumbai, India.
APPARATUS
AND EQIUPMENTS REQUIRED:
1.
UV-Vis double beam spectrophotometer (Model; Shimadzu: 1700S, Japan), Electric
Sonicator, Volumetric flasks (10ml,50ml,100ml), Calibrated analytical pipettes,
Electronic digital balance (Techno, Mumbai)
Preparation
of concentration range (Beer’s limit):
Determination
of concentration range which obeys the Lambert and Beer’s law is necessary for
accuracy and reproducibility in Spectrophotometric analysis for quantitative
determination of any drug. For this; Finerenone stock solution (100µg/ml) was
prepared using pure drug in Ethanol. Further dilutions were made using 0.2ml, 0.4ml,
0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml and 1.8ml of above solution was
transferred to a series of 10ml volumetric flasks this gave a series of
concentrations ranging from 2 to 18 µg/ml of Finerenone. The final volume was
made up to 10ml mark using Ethanol, sonicated for 5 minutes. The resultant
solutions were measured using a double beam uv-vis-spectrophotometer at wave
length of 239.8nm against a reagent blank. A calibration curve was plotted with
concentration against absorbance. From the graph it was clear that Beer’s law
was obeyed in concentration range of 2-14µg/ml and deviation was observed above
these concentrations.
Preparation
of standard calibration curve:
100
mg of pure Finerenone drug was dissolved in little quantity of Ethanol in a 100
ml volumetric flask, the volume was made up to the mark using Ethanol. The
solution was sonicated for 10 minutes. This gave a Finerenone solution with
concentration of 1mg/ml (1000µg/ml). 10ml of this solution was further diluted
100ml in volumetric flask using Ethanol to obtain a concentration of 100µg/ml.
Further dilutions were made using 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and
1.8ml solution was transferred to a series of 10ml volumetric flasks (to Obtain
a series of concentrations ranging from 2-14 µg/ml of Finerenone). The final
volume was made up to 10ml mark using Ethanol, sonicated for 5 minutes, the
absorbances were measured at of 239.8nm against a reagent blank. A calibration
curve was plotted with concentration against absorbance.
Estimation
of Finerenone in tablet dosage forms:
Twenty
tablets were weighed accurately and powdered. The tablet powder equivalent to
100 mg of Finerenone was transferred into a 100mL volumetric flask and
dissolved in little quantity of Ethanol. Then the solution was sonicated for 30
minutes and filtered using whatman filter paper No#41. The filtrate so obtained
was diluted with Ethanol to produce 100ml. Further dilutions were made with
Ethanol to get required concentrations within Beer’s - Lambert limits. The
resultant solutions were measured at wave length of 239.8nm against a reagent
blank. The concentration of drug was calculated with the help of standard
calibration curve.
ANALYTICAL
METHOD VALIDATION:
Validation
of an analytical procedure is the process by which it is established, by
laboratory studies, that the performance characteristics of the procedure meet
the requirements for its intended use. All analytical methods that are intended
to be used for analyzing any clinical samples will need to be validated.
Validation of analytical methods is an essential but time consuming activity
for most analytical development laboratories. It is therefore important to wave
length. Understand the requirements of method validation in more detail and the
options that are available to allow for optimal utilization of analytical
resources in a development laboratory.
RESULTS
AND DISCUSSION:
Results
of determination of beer's limit:
Figure:1
showing Beer’s limit for Finerenone pure drug:
(Figure:1
showing Beer's limit for Finerenone at 239.8nm)
Table:1
showing Beer’s range for Finerenone:
|
Sl. No.
|
Concentration in µg/ml
|
**Absorbance at λmax 239.8nm
|
|
1.
|
0.0
|
0.000
|
|
2.
|
2
|
0.180
|
|
3.
|
4
|
0.350
|
|
4.
|
6
|
0.535
|
|
5.
|
8
|
0.740
|
|
6.
|
10
|
0.920
|
|
7.
|
12
|
1.101
|
|
8.
|
14
|
1.300
|
|
9.
|
16
|
1.420
|
|
10.
|
18
|
1.860
|
(**
Average of three determinations)
Standard
calibration curve of finerenone :
Table:2
Showing absorbance of Finerenone at various concentrations:
|
Sl.No.
|
Concentration in µg/ml
|
Absorbance at λmax 239.8nm
|
|
1.
|
0.0
|
0.000
|
|
2.
|
2
|
0.180
|
|
3.
|
4
|
0.350
|
|
4.
|
6
|
0.535
|
|
5.
|
8
|
0.740
|
|
6.
|
10
|
0.920
|
|
7.
|
12
|
1.101
|
|
8.
|
14
|
1.300
|
|
|
Results
of determination of absorption maxima:
|
|
Figure
2 showing standard calibration curve for Finerenone
|
Figure
3 showing absorption maxima of Finerenone at 239.8nm
|
Estimation
of finerenone in tablet dosage form:
Table
3 showing absorption of drug from tablet dosage form:
|
Volume
of stock Solution Used
|
Amount
of drug (label claim) (µg/ml)
|
Absorbance
at 239.8nm
|
Amount
of drug found (µg/ml)
|
Percentage
purity found ±S.D** (%w/w)
|
|
0.2ml
|
2
|
0.1088
|
2.014
|
100.70±0.11
|
|
0.6ml
|
6
|
0.312
|
6.038
|
100.63±1.41
|
|
1.0ml
|
10
|
0.509
|
9.980
|
99.80±0.61
|
|
1.4ml
|
14
|
0.693
|
13.979
|
99.85±0.46
|
(**
Average of three determinations)
DETERMINATION
OF ACCURACY:
Table:4
Accuracy results for Finerenone :
|
Brands
|
Initial
amount (µg/ml)
|
Amount
of pure drug added (µg/ml)
|
Amount
recovered (µg/ml)
|
%
Recovery ±S.D**
|
|
|
10
|
8(80%)
|
8.026
|
100.32±0.144
|
|
Kerendia
|
10
|
10(100%)
|
9.896
|
98.96±0.226
|
|
|
10
|
12(120%)
|
12.025
|
100.20±0.416
|
(**Average
of six determinations, n=6)
DETERMINATION
OF PRECISION:
Table:5
Precision results for Finerenone:
|
Sl.
|
Conc.
in(µg/ml)
|
Inter-day
absorbance
Mean±S.D**
|
%
C.V
|
Intra-day
absorbance
Mean±S.D**
|
%
C.V
|
|
1.
|
8
|
0.412±0.004
|
0.37
|
0.411±0.016
|
0.64
|
|
2.
|
10
|
0.511±0.025
|
0.36
|
0.511±0.020
|
0.25
|
|
3.
|
12
|
0.604±0.018
|
0.62
|
0.607±0.026
|
0.26
|
(**Average
of three determinations, n=3)
Determination
Of Ruggedness Parameters:
Table:6
Showing Ruggedness parameters:
|
Parameters
|
Laboratory
|
Name
of the instrument
|
Manufacturer
of the
chemicals
used
|
|
Lab. 1
with analyst
I
|
M.M.U.
College of Pharmacy, Ramanagara
|
Shimadzu-(model:
1700S,Japan) double beam UV-Vis spectrophotometer
|
S.D
Fine Chemicals, Mumbai.
|
|
Lab. 2
with analyst
II
|
Dr. H.L.T.
College of Pharmacy, Kengal, Channapatna
|
Systronic
UV-Vis Double beam spectrophotometer
|
Loba Chemicals,
Mumbai
|
Table:7
Showing Ruggedness results for Finerenone:
|
Sl.No
|
Brand
|
Label
claim(mg)
|
Lab.
1* with analyst I
|
Lab.
2* with analyst II
|
|
Amount
found(mg)
|
%Recovery±S.D**
|
Amount
found(mg)
|
%Recovery±S.D**
|
|
1.
|
Kerendia
|
10
|
10.07
|
100.7±0.747
|
9.95
|
99.5±0.347
|
(Lab 1*
MMU College of pharmacy, Lab 2* Dr.HLT College of pharmacy,.**Average of six
determinations, n=6)
DETERMINATION
OF ROBUSTNESS:
Table
8 Showing Robustness results for Finerenone:
|
Type
|
Sl.
No.
|
Conc.
In
(µg/ml)
|
Change
in temperature
|
Change
in PH
|
|
+50C
|
-50C
|
2drops
of 0.1N
NaOH
|
2drops
of 0.1N
HCl
|
|
Absorbance
at 239.8nm Mean±S.D**
|
|
Pure
|
1
|
8
|
0.406±0.027
|
0.407±0.017
|
0.405±0.012
|
0.407±0.0121
|
|
Drug
|
2
|
10
|
0.516±0.023
|
0.514±0.018
|
0.515±0.011
|
0.516±0.028
|
|
|
3
|
12
|
0.610±0.023
|
0.611±0.027
|
0.610±0.050
|
0.609±0.049
|
(**Average
of three determinations, n=3)
Table:9
showing calibration data for Finerenone at 239.8nm:
|
 Parameters
|
Calibration
data at 239.8nm
|
|
λmax
|
239.8nm
|
|
Beer’s
law limit (µg/ ml)
|
2
-14µg/ml
|
|
Molar
Absorptivity
|
1.1156Lmol-1cm-1
|
|
Regression
Equation(Y=a+bc)
|
Y=
0.050X+0.011
|
|
Slope
(b)
|
0.01421
to 0.01467
|
|
Intercept(a)
|
-
0.004515 to 0.0016752
|
|
Correlation
Coefficient (R2)
|
0.997
|
|
Limit
of detection (LOD)
|
1.220µg/ml
|
|
Limit
of quantitation (LOQ)
|
5.220µg/ml
|
Determination
of Beer’s limit:
The
Beer’s limit felled in the range of 2-14µg/ml under given experimental
conditions.
Determination
of absorption maxima:
100
µg/ml stock solution of Finerenone was prepared and absorbances were measured
from 200nm to 340nm. The optimum wave length was found to be 239.8nm
Assay:
Marketed
tablets contained Finerenone were used for the assay. After extraction, proper
dilution, measurement, the concentration was determined using standard
calibration curve. The amount of drug found in the range of 99.80 – 100.70%w/w
Method
validation:
The
proposed method was validated in accordance to ICH guidelines.
a)
Accuracy: Percentage
of recoveries of Finerenone in tablets was found in the range of 99.94 –
100.30% w/w
b)
precision:
The percent coefficient of variations (% C.V) was between 0.34-0.60 for
intra-day and 0.23-0.62 for inter-day absorbances.
c)
Repeatability: Repeatability was determined by analyzing the sample
at the given concentration wavelength for at least six times and it was found
that the variability in the results was not more than 0.5%.
d)
Reproducibility: The standard solution of Finerenone by analyst-I and
analyst-II separately. The values obtained were evaluated using F-test and
t-test to verify their reproducibility. Calculated value for t-test was found
to be less than the tabulated (standard) value it can calculated that no
significant difference was observed in the result of analysis.
e)
Ruggedness: Ruggedness of the developed method was determined by
changing the analytical toolssuch as laboratory, instruments, analyst and
chemicals. The result (in terms of %RSD) of six determinations indicated that
there were no significant variations in the data.
f)
Robustness: Robustness of the method was established by slightly
changing the temperature and PH of the reaction mixture. The data so obtained
showed no significant variation in the absorption pattern.
g)
Limit of detection and limit of quantitation: Were determined
from the standard deviation of y – intercepts of six calibration curves and
average slope of six calibration curves. LOD and LOQ of Finerenone was found to
be 1.220µg/ml and 5.220µg/ml respectively.
CONCLUSION:
A
new spectrophotometric method was developed to estimate Finerenone in pure and
tablet dosage forms. Ethanolic solutions of Finerenone was estimated by using
UV-spectrophotometer (Shimadzu 1700S, Japan) with matched 1cm quartz cell. It
showed maximum absorption at the range of 2-14µg/ml at 239.8nm with coefficient
of correlation (R2) of 0.997. The method so developed was validated according
to ICH guide lines for accuracy, precision (inter and intra-day precisions),
repeatability, reproducibility, ruggedness, robustness etc.
The
proposed method was found to be simple, accurate, sensitive, precise,
reproducible and rapid.
This
method can be successfully employed for routine quantitative analysis of Finerenone
in bulk and tablet dosage form.
ACKNOWLEDGMENT:
The
authors are thankful to head of the department of chemistry.
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