Development and
Validation of Analytical Method for Irbesartan and Atorvastatin by Simultaneous Equation Spectroscopic Method
Paras Virani1,2*, Rajanit Sojitra1,
Hasumati
Raj2, Vineet Jain2
1Research Scholar
2014, Gujarat Technological
University, Gujarat
2Quality Assurance
Department, Shree Dhanvantary
Pharmacy College,
Kim, Surat
*Corresponding Author E-mail: parasvirani@gmail.com
ABSTRACT:
A simple, accurate and precise spectroscopic method
was developed for simultaneous estimation of Irbesartan
and atorvastatin in synthetic mixture using
simultaneous equation Method. In this spectroscopic method, 226.00 nm and
246.00 nm wavelengths were selected for measurement of absorptivity.
Both the drugs show linearity in a concentration range of 05-30 μg/ml at their respective λmax.
Accuracy, precision and recovery studies were done by QC samples covering
lower, medium and high concentrations of the linearity range. The relative
standard deviation for accuracy, precision studies were found to be within the
acceptance range (<2%). The limit of determination was 0.033μg/ml and
0.125 μg/ml for Irbesartan
and atorvastatin, respectively. The limit of
quantification was 0.1008 μg/ml and 0.3792 μg/ml for Irbesartan and atorvastatin, respectively. Recovery of Irbesartan
and atorvastatin were found to be 99.75 % and 99.52%
respectively confirming the accuracy of the proposed method. The proposed
method is recommended for routine analysis since they are rapid, simple,
accurate and also sensitive and specific by no heating and no organic solvent
extraction.
KEYWORDS: Irbesartan, atorvastatin, simultaneous estimation, Simultaneous
equation method, analysis method.
INTRODUCTION:
Irbesartan, an angiotensin
II receptor antagonist [1].Is used mainly for the treatment
of hypertension. It is an orally active nonpeptidetetrazole
derivative and selectively inhibits angiotensin II receptor type 2. Angiotensin
II receptor type1 antagonists have been widely used in treatment of diseases
like hypertension, heart failure, myocardial infarction and diabetic
nephropathy. IUPAN name of Irbesartan is
2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1,3-diazaspiro[4.4]non-1-en-4-one.(2)
Figure:1
Structure of Irbesartan(3)
Irbesartan is white or almost white, crystalline powder.
Solubility is given in practically insoluble in water, sparingly soluble in
methanol, slightly soluble in methylene chloride.
Atorvastatin is used as lipid-lowering agents used in hyperlipidaemia condition. Atorvastatin
selectively and competitively inhibits the hepatic enzyme HMG-CoA reductase.(4)
As HMG-CoA reductase is
responsible for converting HMG-CoA to mevalonate in the cholesterol biosynthesis pathway, this
results in a subsequent decrease in hepatic cholesterol levels and decreases
blood cholesterol level.
Figure 2: Structure of atorvastatin(5)
Atorvastatin is
white oral most white, crystalline powder.
Solubility is given in practically insoluble
in water, soluble in methanol,
slightly soluble in methylene
chloride.
Hypertension frequently coexists with hyperlipidaemia and both are considered to be major risk
factors for developing cardiac disease ultimately resulting in adverse cardiac
events. This clustering of risk factors is potentially due to a common
mechanism. Further, patient compliance with the management of hypertension is
generally better than patient compliance with hyperlipidaemia.
It would therefore be advantageous for patients to have a single therapy which
treats both of these conditions with help of fixed dose combination of Irbesartan and atorvastatin.(6,7)
The review of literature regarding quantitative
analysis of Irbesartan and atorvastatin
revealed that no attempt was made to develop analytical methods for Irbesartan and atorvastatin. Some
spectrometric methods and chromatographic methods have been reported for the
estimation of the individual drugs. The focus of the present study was to
develop and validate a rapid, stable, specific, and economic spectroscopic
method for the estimation of Irbesartan and atorvastatinin Synthetic mixture.(8,9)
MATERIALS AND METHODOLOGY:
Atorvastatin and Irbesartan were obtained as gift samples from S Kant
pharmaceuticals and CTX life science Surat. Synthetic
Mixture contain 20mg of Atorvastatin and 160mg of Irbesartan.
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 ‘A’ volumetric glassware were used (Borosillicte)
Standard solution
of Irbesartan (IRB)
Preparation of stock solution of IRB
Accurately
weighed quantity of Irbesartan 10 mg was transferred
to 100 ml volumetric flask, dissolved and diluted up to mark with methanol to
give a stock solution having strength of 100μg/ml.
Preparation
of stock solution of ATR
Accurately
weighed quantity of Atorvastatin 10mg was transferred
to 100 ml volumetric flask, dissolved and diluted up to mark with methanol to
give a stock solution having strength of 100μg/ml.
Preparation
of standard mixture solution
From
the stock solution of IRB take 1.6ml and from stock solution of ATR take 0.2ml
and transferred in to 10ml volumetric flask and diluted up to mark with
methanol to give a solution having strength of IRB was 16 μg/ml
and ATR was 2μg/ml.
Preparation
of test solution
From
the stock solution of IRB take 1.6ml and from stock solution of ATR take 0.2ml
and transferred in to 10ml volumetric flask and diluted up to mark with
methanol to give a solution having strength of IRB was 16 μg/ml
and ATR was 2μg/ml.
Calibration
curves for Irbesartan
Pipette
out 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml of the stock solution of Irbesartan and atorvastatin
(100μg/ml) into a series of 10ml volumetric flasks and the volume was
adjusted to mark with methanol and measured absorbance at 226.00nm and 246nm. Plotte the graph of absorbance versus respective
concentration of Irbesartan and atorvastatin.
Linearity range of IRB and ATR was found with correlation co-efficient.
Development and Validation of Spectroscopic
Simultaneous Equation Method
Selection of wavelength
and method development
for determination
of irbesartan and
atorvastatin
The standard solution
of IRB and ATR were scanned
separately
between 200-400nm, and IRB showed absorbance
maxima
at 226.00nm and ATR at
246.00nm.(figure 3)
Figure3 Overlain
zero order spectra
of IRB and ATR (8:1)
ratios, respectively
VALIDATION
PARAMETERS(10)
1. Linearity and Range
The Zero order (fig.3) showed linear absorbance at 226.00 nm for IRB(05-30 µg/ml)
and
246.00nm for ATR(5-30 µg/ml)
with correlation coefficient (r2) of 0.9994 and
0.9993 for IRB and ATR, respectively.
This
method obeyed beer’s law in the concentration range
05-30µg/ml and 5-30
µg/ml for IRB and ATR, respectively. (Table 1)
Correlation coefficient (r2)
for calibration curve of
IRB and ATR was found to be 0.9994 and 0.9993, respectively
(figure
4 and 5)
The regression
line
equation for IRB and ATR are as
following,
y = 0.0983x - 0.2385 for IRB
_____________ (1)
y = 0.0642x -
0.0695 for ATR
______________ (2)
Table 1Calibration data for
IRB and ATR at 226.00 nmand246.00
nm
respectively.
*(n=6)
|
Sr. No |
Concentration (μg/ml) |
Absorbance* (226.00nm)±SD IRB |
Absorbance* (246.00nm)±SD ATR |
|
|
IRB |
ATR |
|||
|
1 |
05 |
05 |
0.3708±0.0023 |
0.2672±0.0015 |
|
2 |
10 |
10 |
0.7460±0.0020 |
0.5674±0.0017 |
|
3 |
15 |
15 |
1.2171±0.0013 |
0.8872±0.0018 |
|
4 |
20 |
20 |
1.6972±0.0015 |
1.1974±0.0012 |
|
5 |
25 |
25 |
2.2225±0.0013 |
1.5232±0.0022 |
|
6 |
30 |
30 |
2.7653±0.0025 |
1.8772±0.0016 |
Figure
4 Calibration curve for
IRB at 226.00 nm
Figure
5 Calibration curve for
ATR at 246.00 nm
2. Precision
I. Intraday
precision
The precision of the developed method was assessed by
analyzing combined standard solution containing three different
concentrations 05, 15, 30 μg/ml for IRB and 05, 15, 30 μg/ml ATR. Three replicate (n=3) each on same day.
Intraday precision data presented
in Table 2
These % RSD value
was found to be less than ±2.0 indicated that the method is precise.
II. Interday precision
The precision of the developed method was assessed by
analyzing combined standard solution
containing three different concentrations 05, 15, 30
μg/ml
for IRB and 05, 15, 30 μg/ml ATR
triplicate (n=3) per day for consecutive 3 days for inter-day precision.
Interday precision data presented
in Table 3
These % RSD
value was found to be less than ±2.0
indicated
that the method is precise.
3. Accuracy
Accuracy
of the method was determined
by recovery
study from synthetic mixture at
three
level
(80%, 100%, 120%)of standard addition.
The% recovery
values are tabulated in Table 4and
5. Percentage recovery
for IRB and ATR by this method was found in the range
of 100.07 to 100.43% and 99.21 to 100.55%, 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 excepients.
Table
2 Intraday precision data for estimation of IRB and ATR*(n=3)
|
Conc. (μg/ml) |
IRB Abs.* ± % RSD ±% RSD Abs. ±% RSD IRB |
ATR Abs.*± % RSD |
|
|
IRB |
ATR |
||
|
05 |
05 |
0.372±0.45 |
0.266±0.57 |
|
15 |
15 |
1.211±0.21 |
0.884±0.92 |
|
30 |
30 |
2.763±0.52 |
1.877±0.23 |
Table
3Interdayprecision data for estimation of IRB and ATR*(n=3)
|
Conc. (μg/ml) |
IRB Abs.* ±% RSD ±% RSD Abs. ±% RSD IRB |
ATR Abs.*±%RSD |
|
|
IRB |
ATR |
||
|
05 |
05 |
0.377±0.55 |
0.270±0.56 |
|
15 |
15 |
1.215±0.25 |
0.887±0.17 |
|
30 |
30 |
2.786±0.85 |
1.881±0.36 |
4. Limit of
detection and quantitation
The LOD
for IRB and ATR was conformed to be 0.033µg/ml and 0.125µg/ml, respectively.
The LOQ
for IRB and ATR was conformed to be 0.1008µg/ml and 0.379 µg/ml,
respectively.
The obtained LOD and LOQ results are presented in
Table 6
5. Robustness and
Ruggedness
The obtained
Ruggedness and Robustness results are presented in table 6.3.8
The % R.S.D was
found to be 0.12 – 0.84 % for IRB and 0.11 – 0.74 % for ATR.
These %RSD value
was found to be less than ± 2.0 indicated that the method is precise.
No significant
changes in the spectrums were observed, proving that the developed method is
rugged and robust.
Application of the proposed method for
analysis of IRB and ATR in combined capsule dosage form.
All the excipients were mixed in 10ml volumetric flask and sonicate for 15min. make up the volume with Distilled
Water. The solution was filtered through Whatman filter
paper No.
42. Finally the solution had concentration 1600μg/ml for IRB and 200μg/ml for ATR. from that pipette out 0.1ml in
10 ml volumetric flask and volume was
made
up to mark with methanol to obtain
final solution containing 16µg/ml of IRB and
2µg/ml of ATR. A zero order spectrum of the resulting solution was
recorded
and processed to first derivative
spectra.
Table
4Recovery data of IRB*(n=3)
|
Conc. of IRB from
formulation (µg/ml) |
Amount
of Std.IRB added (µg/ml) |
Total amount of IRB (µg/ml) |
Total
amount of IRB found
(µg/ml)* Mean± SD |
% Recovery
(n=3) |
% RSD IRB |
|
8 |
6.4 |
14.4 |
12.81±0.022 |
100.07% |
0.32% |
|
8 |
8.0 |
16.6 |
16.07±0.013 |
100.43% |
0.68% |
|
8 |
9.6 |
17.6 |
19.22±0.045 |
100.10% |
0.28% |
Table
5Recovery data ofATR*(n=3)
|
Conc. of ATR from
formulation
(µg/ml) |
Amount
of Std.ATR added (µg/ml) |
Total amount of ATR (µg/ml) |
Total
amount of ATR found
(µg/ml)* Mean± SD |
% Recovery
(n=3) |
% RSD ATR |
|
1 |
0.8 |
1.8 |
1.81±0.021 |
100.55% |
0.84% |
|
1 |
1.0 |
2.0 |
2.00±0.036 |
100.50% |
0.22% |
|
1 |
1.2 |
2.2 |
2.19±0.20 |
99.21% |
0.35% |
Table 6LOD and
LOQ
data of IRB and ATR
*(n=10)
|
Conc. (μg/ml) |
Avg.abs* ± SD (226.00nm) IRB |
% RSD |
Avg.abs*±SD (246.00nm) ATR |
% RSD |
|
|
IRB |
ATR |
||||
|
05 |
05 |
0.371 ±0.0007 |
1.93 |
0.270 ±0.0006 |
0.45 |
|
LOD (μg/ml) |
0.033 |
0.125 |
|||
|
LOQ (μg/ml) |
0.1008 |
0.3792 |
|||
Table 7 Robustness
and Ruggedness data of IRB and ATR *(n=3)
|
Condition |
Conc. (μg/ml) |
Different Instrument |
Different
Stock Solution Preparation |
||
|
UV-2450 |
UV-1800 |
Stock-1* |
Stock-2* |
||
|
Irbesartan Mean (n=3) ± % RSD |
05 |
0.376±0.32 |
0.374±0.47 |
0.376±0.12 |
0.373±0.82 |
|
15 |
1.215±0.56 |
1.216±0.22 |
1.215±0.42 |
1.216±0.56 |
|
|
30 |
2.763±0.23 |
2.765±0.84 |
2.764±0.21 |
2.763±0.32 |
|
|
Atorvastatin Mean(n=3) ± %RSD |
05 |
0.271±0.54 |
0.269±0.43 |
0.272±0.42 |
0.270±0.11 |
|
15 |
0.885±0.66 |
0.882±0.33 |
0.884±0.15 |
0.885±0.33 |
|
|
30 |
1.879±0.16 |
1.878±0.13 |
1.882±0.52 |
1.884±0.74 |
|
Stock-1 :- 10 mg dissolve in 100 ml Methanol;
Stock-2 :- 50 mg dissolve in 250 ml
Methanol
Table
8 Analysis data of commercial formulation*(n=3)
|
Sr. No |
Drug |
Formulation
(μg /ml) |
%
Assay* ± SD |
USP
limit(%) |
|
1 |
IRB |
16.0 |
99.75 ± 0.22 |
98-102% |
|
2 |
ATR |
2.0 |
99.52 ± 0.56 |
98-102% |
Summary Of Validation Parameter
Table 9 Summary of validation parameters
|
SR. NO. |
PARAMETER |
Irbesartan |
Atorvastatin |
|
1 |
Wave length Max. |
226.00
nm |
246.00
nm |
|
2 |
Linearity
(µg/ml) (n=6) |
5 to
30 µg/ml |
5 to
30 µg/ml |
|
3 |
Regression equation |
y = 0.0983x - 0.2385 |
y = 0.0642x - 0.0695 |
|
4 |
Correlation coefficient (r2) |
0.9994 |
0.9993 |
|
5 |
Accuracy(%Recovery) (n=3) |
100.26 |
100.13 |
|
6 |
Precision Intra-day (%RSD)(n=3) Inter-day (%RSD)(n=3) |
0.21-0.52 0.25-0.85 |
0.23-0.92 0.17-0.56 |
|
7 |
LOD
(µg/ml) (n=10) |
0.033 |
0.125 |
|
8 |
LOQ (µg/ml) (n=10)
|
0.1008 |
0.3792 |
|
9 |
Robustness and Ruggedness (%RSD) |
0.12-0.84 |
0.11-0.73 |
|
10 |
Assay |
99.75±0.22 |
99.52 ±0.56 |
A spectrum
of the sample solution was recorded and
the absorbance at
226.00nm and 246.00nm were
noted for estimation of IRB and
ATR, respectively. The concentrations of IRB and ATR in formulation were determined
using the corresponding calibration graph.
CONCLUSION:
A new, Simultaneous Equation method has been developed
for estimation of Irbesartan and Atorvastatin
in synthetic mixture. The method was validated by employment of ICH guidelines.
The validation data is indicative of good precision and accuracy, and prove the
reliability of the method.
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Received on 24.02.2015 Modified on 18.03.2015
Accepted on 22.03.2015 ©A&V Publications All right reserved
Res. J.
Pharm. Dosage Form. & Tech. 7(2): April-June, 2015; Page 98-102
DOI: 10.5958/0975-4377.2015.00014.2