1. INTRODUCTION:
Cilnidipine
is a dual blocker of L-type voltage-gated Ca2+ channels in
vascular smooth muscle and N-type Ca2+ channels in sympathetic nerve terminals
that supply blood vessels. It inhibits the Ca2+ influx in both in
vessel and in the nerve. So causes the vasodilation and inhibits the release of
norepinephrine, which causes the vasodilation and decreases the heart rate and
also decreases cardiac contraction in heart. So, used in treatment of
hypertension. It is chemically 3-(2-methoxyethyl) 5-[3-phenylprop-2-enyl]
2,6-dimethyl-4-(3-nitrophenyl)-1,4 dihydropyridine- 3,5-dicarboxylate
Cilnidipine is Yellow Crystalline Solid having molecular weight 492.52g/mol.(1-3)
Fig. 1 Chemical structure of cilnidipine
Valsartan
is an ARB that selectively inhibits the binding of angiotensin II to AT1, which
is found in many tissues such as vascular smooth muscle and the adrenal glands.
This effectively inhibits the AT1-mediated vasoconstrictive and
aldosterone-secreting effects of angiotensin II and results in a decrease in
vascular resistance and blood pressure.Inhibition of aldosterone secretion may
inhibit sodium and water reabsorption in the kidneys while decreasing potassium
excretion. It is chemically
3-methyl-2-[pentanoyl-[[4-[2-(2h-tetrazol-5-yl)phenyl]phenyl] methyl] amino]-butanoic
acid.
Valsartan
is a White Crystalline Powder having molecular weight435.52g/mol.(3,4)
![3-Methyl-2-[pentanoyl-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]amino]-butanoic acid Structure](4_AJPA_5_1_2015_files/image006.gif)
Fig. 2 Chemical Structure of Valsartan
The
review of literature regarding quantitative analysis of Cilnidipine and
Valsartan revealed that no Simultaneous
Equation
method attempt was made to develop analytical methods forCilnidipine and
Valsartan. Some spectrometric methods and chromatographic methods have been
reported for the estimation of the individual and combination of drugs.(5-14)
The focus of the present study was to develop and validate a rapid, stable,
specific, and economic Spectroscopic method for the estimation of Cilnidipine
and Valsartan in Synthetic Mixture.
1.1. Theory:
We can find out concentration of both the drug from combination mixture
using the simultaneous equation method. In this method using the absorbance of
both the drug and mixture at their wavelength and put this value in following
equation and we can find out the concentration of drugs present in combination.
Cx= (A2 × Ay1) – (A1 × Ay2)
--------------------------------- ------------------------ (1)
(Ay1 × Ax2)
– (Ay2 × Ax1)
Cy = (A1 × Ax2) – (A2 × Ax1)
--------------------------------- ------------------------ (2)
(Ax2 × Ay1) – (Ax1 × Ay2)
Where,
Cx= Concentration of drug X
Cy = Concentration of drug
A1 = Absorbance of mixture at wavelength 1
A2 = Absorbance of mixture at wavelength 2
Ax1 = Absorptivity of drug A at wavelength 1
Ax2 = Absorptivity of drug A at wavelength 2
Ay1 = Absorptivity of drug B at wavelength 1
Ay2 =Absorptivity of drug B at wavelength 2
2. MATERIALS AND METHODOLOGY:
2.1. Apparatus
A double beam
UV/ Visible spectrophotometer
(Shimadzu
model 2450, Japan) with spectral
width of 2nm, 1 cm quartz cells
was used to measure absorbance
of all the solutions. Spectra
were automatically
obtained by UV-Probe system software.
2.2. Reference samples
CIL and VAL reference standard
are kindly supply by Nikshan Pharmaceuticals, Ankleswar and Cipla
Pharmaceuticals, Ankleswar as a gift sample respectively.
2.3. Materials and
reagents
Methanol AR grade (RANKEM)
2.4. Preparation
of Standard Solution and Synthetic Mixture
2.4.1 Preparation of stock solution
of Cilnidipine:
An accurately weighed quantity
equivalent to 10mg of Cilnidipine was transferred to 100 ml volumetric flask made up to the mark with the
methanol. Sonicate for 15 mins. Filter Stock solution (100μg/ml).
2.4.2 Preparation of standard stock
solution of Valsartan:
An accurately weighed quantity of VAL (10 mg) was transferred to a
separate 100 ml volumetric flask and dissolved and diluted to the mark with
methanol to obtain standard solution having concentration of VAL(100μg/ml).
2.4.3.Preparation of Standard
Mixture Solution (CIL+ VAL):
0.2
ml of working standard stock solution of CIL (100μg/ml) and 1.6ml of
standard Stock solution of VAL (100μg/ml) were pipetted out into 10ml
volumetric flask and volume was adjusted to the mark with methanol to get
2μg/ml of CIL and 16μg/ml of VAL.
2.4.4 Preparation of Test Solution
The
preparation of synthetic mixture was as per patent:
Ø Cilnidipine:10 mg
Ø Valsartan: 80 mg
Ø Croscamellose Sodium: 10 mg
Ø Hydroxypropyl Cellulose: 10 mg
Ø Hydrated Silicone Dioxide: 10 mg
Ø Macrogol (PEG) 6000: 30 mg
All
the excipients were mixed in 100 ml volumetric flask containing 25 ml of
Methanol and sonicated for 15min. make up the volume with Methanol. The
solution was filtered through Whatman filter
paper No.
42.CIL100μg/ml andVAL800μg/ml). Finally the solution had concentration
100μg/ml for CIL and 800μg/ml for CIT from that pipette out 2ml in
100ml volumetric flask and make up to the mark with Methanol.
2.5 Procedure:
2.5.1 Selection of Wavelength
for Estimation of Cilnidipine and Valsartan:
The
Standard Stock Solutions of Cilnidipine and Valsartan were scanned in the range
of 200 to 400nm against methanol as a blank. Maximum absorbance was obtained at
240.00nm and 250.00 nm for Cilnidipine and Valsartan, respectively.
2.5.2 Calibration curves for
Cilnidipine:
This series consisted of five
concentrations of standard CIL solution ranging from 2 to 10μg/ml. The
solutions were prepared by pipetting out Standard CIL stock solution
(100μg/ml). Then pipetting out (0.2ml, 0.4ml, 0.6ml, 0.8ml, and 1.0ml) was
transferred into a series of 10 ml volumetric flask and volume was adjusted up
to mark with methanol. A zero order spectrum of the resulting solution was
recorded, measured the absorbance at 240.00 nm against a reagent blank solution
(methanol). Calibration curve was prepared by plotting absorbance versus
respective concentration of CIL.
2.5.3 Calibration curve
for VAL
This series consisted of five
concentrations of standard VAL solution ranging from 16 to 80μg/ml. The
solutions were prepared by pipetting out Standard VAL stock solution (1.6ml,
3.2ml, 4.8ml, 6.4ml, and 8.0ml) was transferred into a series of 10 ml
volumetric flask and volume was adjusted up to mark with methanol. A zero order
spectrum of the resulting solution was recorded, measured the absorbance at
250.00 nm against a reagent blank solution (methanol). Calibration curve was
prepared by plotting absorbance versus respective concentration of VAL.
Figure 3
Overlain zero
Order
spectra
of CIL and VAL (1:8) Ratios, Respectively
3.
RESULTS AND DISCUSSION:
Validation Parameters
3.1 Linearity:
Five
point calibration curves were obtained in the concentration range of 2-10μg/ml
for Cilnidipine and 16-80μg/ml for Valsartan. The response of drug was
found to be linear in investigation range and the regression equations was
found to be y = 0.103x + 0.005 for CIL (n=6) and y = 0.028x-0.064for VAL (n=6),
with the correlation coefficient 0.9999 and 0.9993(n=6) respectively, is
listed in Table 1.
Table.1 Calibration
data for CIL and VAL at 240.00nm and 250.00nm, respectively. *(n=6)
|
Cilnidipine
|
Absorbance
± S.D
(n=6)
|
Valsartan
|
Absorbance
±S.D. (n=6)
|
|
2
|
0.202
± 0.001
|
16
|
0.421
±0.001
|
|
4
|
0.408
±0.003
|
32
|
0.842
±0.001
|
|
6
|
0.611
±0.003
|
48
|
1.034
±0.002
|
|
8
|
0.824
±0.002
|
64
|
1.792
±0.001
|
|
10
|
1.031
±0.006
|
80
|
2.262
±0.001
|
Fig. 4 Calibration curve for CIL at 240.00nm
Fig. 5 Calibration
curve for VAL at 250.00nm
3.2
Precision:
The
precision of the method was evaluated in terms of inter-day and intra-day by
carrying out independent assays of three concentrations chosen from range of
the standard curves (2, 4, and 6μg/ml of CIL and 16, 32, 48μg/ml of
VAL) and the %RSD of assay (inter-day and intra-day) was calculated. The
results of study are shown in Table 2 and 3.
Table 2.Intraday Precision data for
estimation of CIL and VAL *(n=3)
|
Conc. (μg/ml)
|
CIL
Abs.*
±%RSD
±% RSD Abs.
±% RSDTZN
|
VAL
Abs.*±%RSD
|
|
CIL
|
VAL
|
|
2
|
16
|
0.613±0.24
|
0.554±0.27
|
|
4
|
32
|
1.059±0.09
|
1.012±0.15
|
|
6
|
48
|
1.791±0.06
|
1.781±0.08
|
Table 3.Interday Precision data for
estimation of CIL and VAL *(n=3)
|
Conc. (μg/ml)
|
CIL
Abs.*
±%RSD
±% RSD Abs.
±% RSDTZN
|
VAL
Abs.*±%RSD
|
|
CIL
|
VAL
|
|
2
|
16
|
0.612±0.33
|
0.557±0.37
|
|
4
|
32
|
1.063±0.14
|
1.013±0.19
|
|
6
|
48
|
1.794±0.14
|
1.784±0.11
|
3.3
Accuracy:
The
accuracy of the method was determined by spiking of CIL and VAL to
prequantified sample solutions of CIL (2μg/ml) and VAL (16 μg/ml) in
triplicate at three concentration level of 80, 100, 120% of the specified
limit. The percentage recoveries of CIL and VAL were calculated and the result
is nearer to 100% shown in Table 4 and 5.
3.4
Limit of Detection and Limit of Quantification
The
limit of detection (LOD) and limit of quantitation (LOQ) of the method were
evaluated by standard deviation of response and slope method. LOQ and LOD were
calculated by the equation LOD = 3.3 × N/B and LOQ = 10
× N/B, where “N” is standard deviation of the absorbance, and “B”
is the slope of the corresponding calibration curve. The limit of detection
(LOD) were found to be 0.0736 μg/ml for CIL and 0.2667μg/ml for VAL
and respectively and limit of quantitation (LOQ) were found to be 0.2232 μg/ml
for CIL and 0.8082μg/ml for VAL presented in Table 6.
3.5 Robustness and
Ruggedness
Robustness
was done by different instrument and difference in wavelength. The result was
decided by %RSD which is in the limit which is mentioned in Table no 7.
4. Application of
the proposed method for
analysis of CIL and VAL in synthetic
mixture:
A
zero order spectrum of the sample solution containing
2µg/ml of CIL and
16µg/ml of VAL
was recorded and
the absorbance at 240.00nm
and 250.00nm were noted for estimation of CIL and VAL,
respectively. The concentration
of CIL and VAL in mixture was determined
using the corresponding calibration
graph. The results from the analysis of
synthetic mixture containing Cilnidipine (10mg)and Valsartan (80mg)
in combination were
presented
in Table in 8. The percent assay
shows that there was no interference
from excipients and
the proposed method can successfully
applied to analysis
of commercial formulation containing CIL and
VAL. The % assay values were tabulated
in Table 8.
Table
8 Analysis
data
of Synthetic Mixture*(n=3)
|
Drugs
|
% Assay ± SD
|
% RSD(n=3)
|
|
Cilnidipine
|
99.00 ± 0.0015
|
0.23
|
|
Valsartan
|
99. 68 ± 0.001
|
0.16
|
Table.9 Summary of Validation Parameters
|
SR. NO.
|
Parameter
|
Cilnidipine
|
Valsartan
|
|
1
|
Wave
length Max.
|
240.00nm
|
250.00nm
|
|
2
|
Linearity (µg/ml) (n=6)
|
2 to
10 µg/ml
|
16 to
80 µg/ml
|
|
3
|
Regression
equation
|
y = 0.103x - 0.005
|
y = 0.028x - 0.064
|
|
4
|
Correlation
coefficient (r2)
|
0.9999
|
0.9993
|
|
5
|
Accuracy(%Recovery)
(n=3)
|
101.63%
|
101.86%
|
|
6
|
Precision
Intra-day
(%RSD)(n=3)
Inter-day
(%RSD)(n=3)
|
0.06-0.24
0.14-0.33
|
0.08-0.27
0.11-0.37
|
|
7
|
LOD (µg/ml)
(n=10)
|
0.054
|
0.136
|
|
8
|
LOQ
(µg/ml) (n=10)
|
0.165
|
0.414
|
|
9
|
Robustness
and Ruggedness (%RSD)
|
0.08-0.25
|
0.09-0.81
|
|
10
|
Assay
|
99.00%
|
99.68%
|
5. CONCLUSION:
A new, Simultaneous Equation method has
been developed for estimation of Cilnidipine and Valsartan. The method was
validated by employment of ICH(15)guidelines. The validation data is
indicative of good precision and accuracy, and prove the reliability of the
method. The method involves the generation of absorbance spectra followed by
measurement of the absorbance. The proposed method does not require any
sophisticated mathematical treatment for the absorption data, and it exhibits
several advantages over other Spectrophotometric methods for resolution of
binary mixtures. Therefore, the presented methodology is adequate for the
routine quality control analysis of these fixed-dose combinations.
6. CONFLICT OF INTEREST:
The
authors confirm that this article content has no conflict of interest.
7. 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.
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