Absorbance
Correction Method for Simultaneous Estimation of Amlodipine
Besylate and Simvastatin in
Synthetic Mixture
Vandana M. Patel*, Hasumati A. Raj, Vineet C. Jain
Department of Quality Assurance,
Shree Dhanvantary College of Pharmacy, Kim, Surat, Gujarat, India.
*Corresponding Author E-mail: vandanapatel@gmail.com
ABSTRACT:
A simple, accurate and precise
spectroscopic method was developed for simultaneous estimation of Amlodipine besylate and Simvastatin in synthetic mixture using Absorbance
correction method. At 360.80 nm (λmax of Amlodipine besylate) Simvastatin has zero absorbance so Amlodipine
besylate is directly estimate at 360.80 nm. At 237.60
nm (λmax of Simvastatin)
both drugs have some absorbance so Simvastatin is
estimate at 237.60 nm using absorbance correction method. The method was found
to be linear (r2>0.999) in the range of 5-10 μg/ml
for Amlodipine besylate at
360.80 nm. The linear correlation was obtained (r2>0.999) in the range of
5-10 μg/ml for Simvastatin
at 237.60 nm. The limit of determination was 0.17 μg/ml
and 0.10μg/ml for Amlodipine besylate
and Simvastatin, respectively. The limit of
quantification was 0. 54μg/ml and 0. 32μg/ml
for Amlodipine besylate and Simvastatin, respectively. The accuracy of these method were evaluated by recovery studies and good
recovery result were obtained greater than 99%. The method was successfully
applied for simultaneous determination of Amlodipine besylate and Simvastatin in
binary mixture.
KEY WORDS:
Amlodipine besylate, Simvastatin, Absorption correction Method.
INTRODUCTION:
Hypertension (HTN) or high blood
pressure, is a very common disorder. Blood pressure
is summarised by
two measurements, systolic and diastolic, which depend on whether the
heart muscle is contracting (systole) or relaxed between beats (diastole). Both
the drug used for cholesterol induced
hypertension disease.[1]
Amlodipine besylate and Simvastatin combination was approved on 28 February, 2008[8]
Figure 1:
Chemical Structure of Amlodipine besylate
Amlodipine besylate is long-acting dihydropyridine-type (DHP) calcium channel blocker used to
lower blood pressure and to treat
anginal chest pain.
IUPAC name of Amlodipine besylate
is 3-ethyl 5-methyl(4RS)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl
-1,4-dihydropyridine -3,5-dicarboxylate benzene sulphonate.
Amlodipine besylate acts
primarily on vascular smooth muscle cells by stabilizing voltage-gated L-type
calcium channels in their inactive conformation. By inhibiting the influx of
calcium in smooth muscle cells, amlodipine prevents
calcium-dependent myocyte contraction and
vasoconstriction.
Simvastatin[5-7]
Figure 2: Chemical structure of Simvastatin
Simvastatin is HMG-CoA reductase
inhibitor (3-hydroxy-3-methylglutaryl-coA Reductase
Inhibitor). Simvastatin is used along with a
proper diet to help lower "bad" cholesterol and fats (such as LDL, triglycerides)
and raise "good" cholesterol (HDL) in the blood.
Simvastatin used In Hyperlipidaemia
Inhibitor of HMG –CoA Reductase
Enzyme, HMG-CoA reductase catalyzes the
conversion HMG to mevalonate, which is the rate
determining step in the biosynthesis of cholesterol, thus inhibition leads to a
reduction in the concentration of cholesterol in the liver.
Amlodipine serves as an anti-hypertensive medicine and also increases
the lipid-lowering activity of simvastatin through
synergistic activity with the lipid-lowering agent. Simvastatin
serves as a lipid-lowering agent and also has an activity of decreasing blood
pressure through a synergistic effect with amlodipine.[8]
MATERIALS
AND METHODOLOGY:[9]
·
Amlodipine besylate and Simvastatin were
obtained as gift samples from Prudence Pharmachem, Ankleshwar and
Praveen Laboratories ,Surat. Synthetic Mixture
contains 10mg of Amlodipine besylate
and 10mg of Simvastatin.
·
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)
MATERIALS
AND REAGENTS:
Preparation of stock
solution of AML:
Accurately
weighed quantity of Amlodipine besylate
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 SIM:
Accurately
weighed quantity of Simvastatin 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 (AML + SIM):
1ml of
standard stock solution of AML (100μg/ml) and 1ml of standard stock
solution of SIM (100μg/ml) were pipetted out
into two 10ml volumetric flasks and volume was adjusted to the mark with
methanol to get 10μg/ml of AML and 10μg/ml of SIM.
Preparation
of test solution:
The preparation of
synthetic mixture was as per patent:
·
Amlodipine besylate : 10 mg
·
Simvastatin : 10
mg
·
Sodium Starch glycolate:
20 mg
·
Starch : 20 mg
·
Talc : 40 mg
·
All the excipients
were mixed in 100ml volumetric flask. make up the
volume with Methanol up to 25 ml. and sonicated for
15min .The solution was filtered through Whatman filter paper. and make up the volume up to 100 ml with methanol. Finally
the solution had concentration 100μg/ml for Amlodipine
besylate and 100μg/ml for Simvastatin.
RESULT AND DISCUSSION:
Selection of wavelength and Method Development
for
Determination of
Amlodipine Besyate and Simvastatin
The standard solution of AML and SIM were scanned separately
between 200-400nm, and zero-order spectra were not showed overlapping peaks.
|
AML at
360.80nm SIM at
237.60 nm Linearity
in methanol 5-10 nm |
From spectra at 360.80 nm (λmax
of Amlodipine besylate) Simvastatin has zero absorbance so Amlodipine besylate is
directly estimate at 360.80 nm.
At 237.60 nm (λmax of Simvastatin) both drugs have some absorbance so Simvastatin is estimate at 237.60 nm using absorbance
correction method.
VALIDATION OF PROPOSED METHOD[10]
Parameters to be
considered for the validation of methods are:
1) LINEARITY
AND
RANGE:
Procedure:
The linearity response was determined by analyzing 6 independent levels of calibration curve in the range of
5-10 μg/ml and 5-10 μg/ml for AML and SIM respectively (n=6)
Calibration curve
for Amlodipine besylate:
This series
consisted of five concentrations of standard Amlodipine
besylate solution ranging from 5 to
25 μg/ml. The solutions were prepared by pipetting out Standard Amlodipine
besylate stock solution (0.5ml, 1ml, 1.5ml, 2.0ml,
2.5ml) was
transferred into a series of 10 ml volumetric flask and volume was adjusted up
to mark with methanol. A zero order derivative spectrum of the resulting
solution was recorded, measured the absorbance at 360.80 nm against a reagent
blank solution (methanol). Calibration curve was prepared by plotting
absorbance versus respective concentration of Amlodipine
besylate.
Calibration curve for Simvastatin:
This series consisted of five concentrations of standard
Simvastatin solution ranging from 5 to 25 μg/ml. The solutions were
prepared by pipetting out Standard Simvastatin stock solution (0.5ml, 1ml, 1.5ml, 2.0ml, 2.5ml ) was transferred into a series of 10 ml volumetric
flask and volume was adjusted up to mark with methanol. A zero order derivative
spectrum of the resulting solution was recorded, measured the absorbance at
237.60 nm against a reagent blank solution (methanol). Calibration curve was
prepared by plotting absorbance versus respective concentration of Simvastatin.
2) PRECISION:
i.
Intraday precision:
Procedure:
·
The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations
5, 15, 25 μg/ml
for AML and 5, 15, 25 μg/ml for
SIM. Three replicate
(n=3) each on same day.
·
For zero order spectra absorbance was
measured at 360.80 nm for AML and 237.60 nm for SIM.
·
The % RSD value of the results
corresponding to the absorbance wasexpressed for intra-day
precision.
Table.1 Calibration data for mixture of AML, and SIM
at 360.80nm, 237.80nm, respectively *(n=6)
|
AML (µg/ml) |
ABSORBANCE* Avg ± SD |
AML (µg/ml) |
ABSORBANCE* Avg ± SD |
SIM (µg/ml) |
ABSORBANCE* Avg ± SD |
|
5 |
0.094± 0.00098 |
5 |
0.283±0.00083 |
5 |
0.356 ± 0.00089 |
|
10 |
0.175 ± 0.00154 |
10 |
0.553±0.0016 |
10 |
0.721 ± 0.0026 |
|
15 |
0.246 ± 0.001095 |
15 |
0.785±0.0021 |
15 |
1.116 ± 0.0037 |
|
20 |
0.325 ± 0.001169 |
20 |
1.061±0.0010 |
20 |
1.512 ± 0.0013 |
|
25 |
0.411 ± 0.000816 |
25 |
1.284±0.0010 |
25 |
1.851 ± 0.0015 |
|
Amlodipine
besylate at 360.80 nm |
Amlodipine
besylate at 237.60 nm |
Simvastatin
at 237.60 nm |
Table.2 Intraday precision data for
estimation of AML and SIM* (n=3)
|
Precision |
Con |
AML |
Con |
SIM |
|
Abs. ±% RSD |
5 µg/ml |
0.093 ± 0.22 |
5 µg/ml |
0.619± 0.16 |
|
15 µg/ml |
0.275± 0.41 |
15 µg/ml |
1.847± 0.11 |
|
|
25 µg/ml |
0.449± 0.12 |
25 µg/ml |
2.813± 0.12 |
ii. Interday
Precision:
Procedure:
·
The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations
5,15,25 μg/ml
for AML and 5,15,25 μg/ml
for
SIM triplicate (n=3) per day for consecutive 3 days for inter-day
precision.
·
For zero order
spectra absorbance was
measured at
360.80 nm for AML and 237.60 nm for SIM. The % RSD value
of the results corresponding to the
absorbance was expressed
for
inter-day precision.
Table.3 Interday
precision data for estimation of AML and SIM* (n=3)
|
Precision |
Con |
AML |
Con |
SIM |
|
Abs.±% RSD |
5 µg/ml |
0.094± 0.32 |
5 µg/ml |
0.623± 0.27 |
|
15 µg/ml |
0.278 ± 0.54 |
15 µg/ml |
1.850± 0.13 |
|
|
25 µg/ml |
0.451± 0.22 |
25 µg/ml |
2.816± 0.16 |
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 and Percentage recovery for AMLO and
SIM by this method was found in the range of 100.37 to 102.12%and 98-101.81% 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.
4) LOD and LOQ:
The Limit of detection and quantitation of the developed method was assessed by
analyzing 10 replicates of standard solutions containing concentrations 5 μg/ml for AML and SIM.
The LOD and LOQ were calculated as LOD =
3.3*σ/S, and LOQ = 10*σ/S, where σ is the standard deviation
of the lowest standard concentration and S is the slope of the standard curve.%
RSD was calculated
Application
of
the proposed method for
analysis of EDA and ARG in synthetic mixture (ASSAY):
·
A zero order derivative spectrum of the
resulting solution was recorded and absorbances at
360.80nm and 237.60nm were noted for estimation of AML and SIM, respectively.
·
The concentration of AML and SIM in
mixture was determined using the corresponding calibration graph. The results from the analysis of synthetic mixture containing Amlodipine besylate (10mg) and Simvastatin (10mg)
in combination are presented. The
percent assay shows that there is no interference from excipients and the proposed method can successfully applied to analysis of commercial
formulation containing AML and SIM. The % assay values are tabulated.
6) Robustness:
Robustness and Ruggedness of
the method was determined by subjecting the method to slight change in the
method condition, individually, the:
Ø Change in Wavelength (± 0.2 nm)
·
AML at 360.60 nm
and 361 nm and SIM at 237.40 nm and 237.80 nm
·
Change in
instrument (UV-Vis Spectrophotometer model 1800 and 2450),
Three replicates were made for the
concentration (5,15,25μg/ml of AML and 5,15,25 μg/ml of SIM) with different stock solution
preparation.
% RSD was calculated.
Table.4 Recovery data of AML*and SIM*(n=3)
|
Initial conc. (µg/ml) |
Level of recovery |
Quantity of Std. Added (µg/ml) |
Total Amount (µg/ml) |
Result of recovery study |
||||||
|
Total Quantity Found* (µg/ml)± %RSD |
% recovery ± %RSD |
|||||||||
|
AML |
SIM |
AML |
SIM |
AML |
SIM |
AML |
SIM |
AML |
SIM |
|
|
10 |
10 |
0% |
- |
- |
10 |
10 |
10.01±0.15 |
10.12±0.11 |
100.41±0.51 |
100.55±0.49 |
|
10 |
10 |
80 % |
8 |
8 |
18 |
18 |
18.02±11 |
17.86±14 |
100.62±0.45 |
99.50±0.47 |
|
10 |
10 |
100 % |
10 |
10 |
20 |
20 |
20.14±15 |
20.09±21 |
100.80±0.16 |
100.65±0.29 |
|
10 |
10 |
120 % |
12 |
12 |
22 |
22 |
22.05±22 |
22.08±18 |
100.46±0.41 |
100.49±0.44 |
|
Mean of 3 Determination |
100.62% |
100.21% |
||||||||
Table.5
LOD and LOQ data of AML and SIM*(n=10)
|
Drugs |
LOD (µg/ml) |
LOQ (µg/ml) |
|
Amlodipine besylate |
0.17 |
0.54 |
|
Simvastatin |
0.10 |
0.32 |
Table. 6 Analysis data
of commercial
formulation *(n=3)
|
Drugs |
% Assay ± SD |
% RSD(n=3) |
|
Amlodipine besylate |
100.45 ± 0.0058 |
0.58 |
|
Simvastatin |
100.85 ± 0.0061 |
0.60 |
Table 7:
Robustness and Ruggedness data of AML and SIM*(n=3)
|
Condition |
Conc. (µg/ml) |
Different Instrument |
Wavelength (±0.2 nm) |
||
|
UV-2450 |
UV-1800 |
360.60 nm |
361 nm |
||
|
AML Mean (n=3) ± % RSD |
5 |
0.094±0.10 |
0.096±0.13 |
0.094±0.17 |
0.098±0.21 |
|
15 |
0.276±0.17 |
0.274±0.15 |
0.275±0.19 |
0.278±0.16 |
|
|
25 |
0.448±0.18 |
0.445±0.27 |
0.449±0.10 |
0.452±0.18 |
|
|
SIM Mean(n=3) ± %RSD |
|
UV-2450 |
UV-1800 |
237.40 nm |
237.80 nm |
|
5 |
0.620±0.33 |
0.624±0.27 |
0.625±0.19 |
0.627±0.29 |
|
|
15 |
1.848±0.22 |
1.845±0.23 |
1.846±0.21 |
1.850±0.20 |
|
|
25 |
2.812±0.20 |
2.814±0.32 |
2.817±0.17 |
2.820±0.25 |
|
SUMMARY
OF VALIDATION PARAMETER
|
PARAMETERS |
Absorbance correction method |
|
|
Amlodipine besylate |
Simvastatin |
|
|
Concentration
range(µg/ml) |
5-25 |
5-25 |
|
Regression
equation |
y = 0.0157+0.015 |
y = 0.0756x - 0.0235 |
|
Correlation Coefficient(r2) |
0.9991 |
0.9993 |
|
Accuracy(%Recovery) (n=3) |
100.62% |
100.21% |
|
Intra-day Precision (%RSD) (n=3) |
0.12-0.41 |
0.11-0.16 |
|
Inter-day precision (%RSD) (n=3) |
0.22-0.54 |
0.16-0.27 |
|
LOD(µg/ml) |
0.17 |
0.10 |
|
LOQ(µg/ml) |
0.54 |
0.32 |
|
Ruggedness and Robustness |
0.10-0.27 |
0.19-0.33 |
|
% Assay |
100.45% |
100.85% |
CONCLUSION:
All the parameters
are validated as per ICH guidelines for the method validation and found to
be suitable for routine quantitative analysis in
pharmaceutical dosage forms. The
result of linearity, accuracy, precision proved to be
within limits with lower limits of
detection and quantification. Ruggedness and Robustness of method was confirmed as no significant were observed
on analysis by subjecting the method
to slight change in the method condition. Assay results obtained
by
proposed method are in
fair agreement.
ACKNOWLEDGEMENT:
We are
sincerely thankful to Shree Dhanvantary Pharmacy
College, Kim, Surat, for providing us Infrastructure facilities and moral support
to carry out this research work. We are also thankful to SDPARC for giving us
their special time and guidance for this research work. We also thank our
colleagues for their helping hand.
REFERENCES:
1. Hypertension-KD
Tripathi. Medical Pharmacology; 6th
Edn; Jaypee Brothers
Medical Publishers (p) Ltd, New Delhi, 2008, pp 539.
2. Drug profile available from http://en.wikepedia.org/wiki/
Amlodipine besylate
3 Drug Profile available from: www.chemicalland
21.com/Amlodipine besylate
4 Mechanism action available from: http://www.drugs
bank.com/Amlodipine besylate
5 Drug profile available from:
http://www.drugsbank.com/Simvastatin
6 Introduction available
on :www.medicine net.com
7 Moshe FB, Itzhak
EL, Naomi M, Vered R and Teva
pharma. Simvastatin formulations and methods of
making same. WIPO WO2005044258 A1, 2004.
8 Sung W, Sung S, Youg G, Ja-seongkod and Jin W. Combined pharmaceutical formulation
with controlled-release comprising dihydropyridine
calcium channel blockers and HMG-Coa reductase inhibitors. WIPO WO 2008023958 A1,2007.
9 Beckett
AH, Stenlake JB, Davidson AG. Ultraviolet-Visible
absorption spectrophotometry, Practical
Pharmaceutical Chemistry, 4th Edn; New Delhi, CBS
Publishers, 2002, pp 275-300.
10 International Conference on Harmonization,
Harmonized Tripartite Guideline, Validation of Analytical Procedures Text and
Methodology, ICH Q2(R1), 2005.
Received on 24.03.2015 Accepted on 11.04.2015
© Asian Pharma
Press All Right Reserved
Asian J. Pharm.
Res. 5(2): April-June 2015;
Page 78-82
DOI: 10.5958/2231-5691.2015.00011.8