Q-Absorbance Ratio
Spectrophotometric method for simultaneous determination of Atenolol
and Ivabradine HCl in
synthetic mixture
Pooja A. Patil1*, Dr. Hasumati
A. Raj1, Dr. Gautam B. Sonara2
1Department of Quality
Assurance, Shree Dhanvantary Pharmacy College, Kim, Surat,
Gujarat.
2Department of Pharmacognosy,
Shree Dhanvantary Pharmacy College, Kim, Surat, Gujarat.
*Corresponding
Author E-mail: patilpooja1611@gmail.com
ABSTRACT:
It describes Simultaneous
estimation of simple, accurate, precise, robust and economical Q-Absorbance
ratio spectrophotometric method for Atenolol and Ivabradine HCl in synthetic
mixture.
Objective: To Delivered
information related to Ivabradine HCl
and Atenolol combination’s analytical method. Both
drugs in combination have not any analytical method done by other or nor
published anywhere. So we developed and validated it which is novel method for
drugs.
Method: Absorbance ratio
method for the ratio of absorbance at two selected wavelengths, one which is an
iso-absorbtive point and other being the λ-max
of one of the two component. Atenolol
and Ivabradine HCl show an iso-absorbtive point at 286.40nm in methanol. The second wavelength
used is 276nm which is λ-max of Atenolol in
methanol. So it is economic in nature. The linearity was obtained in the
concentration range of 20-100µg/ml for Atenolol and
2-10µg/ml for Ivabradine HCl.
The concentration of the drugs was determined by using ratio of absorbance at iso-absorbtive point and at the λ-max of Atenolol.
Result: This method is linear
for both drugs; in range 2-10µg/ml for Ivabradine HCl and 20-100µg/ml for Atenolol
found at λmax of Atenolol
276nm (R2= 0.9990) and at Iso-absorptive
point 286.40nm (R2= 0.9998). % recovery for Ivabradine
HCl found 100.47% and Atenolol
100.32%. And all validation parameter (Robustness,
Ruggedness, Interday, Intraday) show %RSD >2%. And Limit of detection
for Ivabradine HCl and Atenolol at λ1(maximum
wavelength) and λ2(Iso-absorptive
point) was found 0.309 and 0.181 respectively. % Assay for Ivabradine
HCl and Atenolol found to
be 100.58% and 100.13% respectively.
Conclusion: The method was
successfully applied to pharmaceutical synthetic mixture which is considered in
approved patent which show no interference. The result of analysis has been
validated statistically and by recovery studies. So this
method accurate, precise, robust, rugged and economic in nature.
KEYWORDS: Atenolol, Ivabradine HCl, Absorbance ratio
method, Iso-absorbtivepoint.
1. INTRODUCTION:
Atenolol
chemically (RS)
- 4 - (2-hydroxy -3-isopropyl
amino propoxy) phenyl Acetamide is a β-adrenoreceptor
blocker primarily used for hypertension, angina pectoris and myocardial infraction. It mainly acts by inhibition of renin release and angiotensin-II and aldosterone
production. (1)
Figure 1: Chemical structure of Ivabradine
HCl
Figure 2: Chemical structure of Atenolol
Ivabradine
HCl (S)-3- {3- [(3,4-dimethoxy bicycle [4.2.0]
octa-1,3,5-triene-7-ylmethyl) Methyl amino] propyl}-7,
8-dimethoxy-2, 3, 4, 5-tetrahydro-1H-3-benzazepine-2-one is If channel blocker
which present at sinus node responsible for generating the early phase of
spontaneous diastolic depolarization in pacemaker cells, thereby reducing the
frequency of action potential initiation and increasing the time required to
reach the voltage threshold for action potential initiation(2). Hence improving blood pressure and pain in angina pectoris.
Both drugs show beneficial effect of combined form without any adverse effect. (3)-(6)
In
this present study Methanol AR grade used as solvent for Q-Absorbance ratio
method for both drug using UV spectrophotometry. The present method used cheap solvent. It
shows accurate and precise method, it is economic and reproducible.
2.
MATERIALS
AND METHODS:
2.1 Instrument
Spectroscopic
Analysis was carried out on a UV/Visible
2450 and UV/Visible 1800 (Shimadzu)
double beam UV-Visible spectrophotometer with software of UV Probe version 2.34.
A Semi-Micro Analytical Balance (Sartorius
CD 2250, Germany) was used for
weighing purpose.
2.2
Material
Analytically
pure Ivabradine HCl raw
material was received as gift sample from Torrent Pharmaceutical, Ahmedabad and
Atenolol raw material as gift sample from Meridian
Enterprise Pvt. Ltd., Navsari.
Methanol
AR Grade (from Finar Pvt. Ltd., Ahmedabad), Distilled
Water (from SDPC research laboratory, Surat), NaOH AR Grade (from RANKEM Pvt. Ltd., Ahmedabad), HCl AR Grade (from ASTRON Pvt. Ltd., Ahmedabad) were used
for development purpose.
2.3 Methods
Preparation
of standard solution
A
10 mg of standard Atenolol and Ivabradine
HCl were weighed and transferred to 100ml separate
volumetric flask and dissolved in methanol. The flask was shaken and volumes
were made up to mark with methanol to give solution containing 100µg/ml each of
Atenolol and Ivabradine HCl.
Methodology
Absorbance
ratio method uses ratio of absorbance at two selected wavelengths, one which is
an iso-absorbtive point and other being the
λ-max of one of two components. From overlay spectra of 2 drugs, it is
evident that Atenolol and Ivabradine
HCl show an iso-absorbtive
point at 286.40nm. The second wavelength used is 276nm of λ-max of Atenolol. Working standard solution having concentration
20, 40, 60, 80, 100 µg/ml for Atenolol and 2, 4, 6,
8, 10 µg/ml for Ivabradine HCl
(taken ratio 10:1 Atenolol: Ivabradine
HCl according to US patent (3)) were
prepared in methanol and the absorbance at 286.40nm iso-absorbtive
point and 276nm λ-max of Atenolol were measured
and absorptivity were calculated using calibration
curve.
The
concentration of 2 drugs of mixture in 10:1 ratio at 286.40nm and 276nm can be
calculated using following equation(8):
Where,
A1 and A2 are absorbance of mixture at 276nm and 286.40 nm;
ax1 and ay1 are absorptivity of Atenolol
and Ivabradine HCl at 276nm
and ax2 and ay2 at 286.40 nm respectively.
QM
= A2/ A1, QX = ax2/ ax1 and QY = ay2/ ay1.
2.4 Validation of proposed method
The
proposed method was validated according to the International Conference on
Harmonization (ICH) guideline(4).
Linearity
The
calibration curve was plotted over a concentration range 20-100 µg/ml for Atenolol and 2-10 µg/ml for Ivabradine
HCl. Appropriate aliquots from the standard stock
solution of Atenolol and Ivabradine
HCl were used to prepare 2 different sets of
dilution: Series A and B as follow, series A consisted of different
concentration of Atenolol (20 -100 µg/ml). Aliquots
from stock solution of Atenolol in methanol (100
µg/ml) was pipette out in to a series of 10 ml volumetric flask and diluted
with methanol to get final concentration 20, 40, 60, 80, 100 µg/ml (2, 4, 6, 8,
10 ml). Series B consisted of different concentration of Ivabradine
HCl (2-10µg/ml). Aliquots from stock solution of Ivabradine HCl in methanol (100
µg/ml) was pipette out in to a series of 10 ml volumetric flask and diluted
with methanol to get final concentration 2, 4, 6, 8, 10 µg/ml (0.2, 0.4, 0.6,
0.8, 1.0 ml).
Method
precision
The
precision of instrument was checked by repeated scanning and measurement of
absorbance of solution (n=6) 3 concentration (lower, middle, highest) and 3
replicates for Atenolol and Ivabradine
mixture without changing the parameter of proposed spectrophotometry
method.
The
intraday and inter day precision of proposed method was determined by analyzing
the corresponding response 3 times on same day on 3 different days of 3
different concentrations (2+20 µg/ml, 6+60 µg/ml, 10+100 µg/ml). The result was
reported in terms of relative standard deviation (RSD%).
Accuracy
The
accuracy of method was determined by calculating the recoveries of Atenolol and Ivabradine HCl by standard spiking method. Known amount of standard
solution for Atenolol and Ivabradine
HCl were spiked at 80%, 100% and 120% level to
pre-quantified sample solution of Atenolol and Ivabradine HCl (40 µg/ml for Atenolol and 4 µg/ml
for Ivabradine HCl). The
amount of Atenolol and Ivabradine
HCl were estimated by applying obtained values to the
respective regression line equation.
This
recovery study done in synthetic mixture which was made according to US patent(7)
and amount taken equivalent to 10 mg of Ivabradine
HCl other side Atenolol
directly weighed 100 mg in mixture. Amount taken and study data given in table
5 and 6.
Limit of detection and limit
of quantification
The limit of detection (LOD)
and the limit of quantitation (LOQ) of the drug were
derived by calculating the signal-to-noise ratio (S/N) i.e. 3.3 for LOD and 10
for LOQ) using the following equation designated by International conference on
Harmonization guidelines.
LOD = 3.3 × σ/S, LOQ = 10
× σ/S
Where, σ = standard
deviation response and
S = slope of the calibration
curve.
Robustness
and Ruggedness
The
sample solution was prepared and then analyzed with change in typical
analytical condition like stability of analytical solution.
Analysis of drugs in sample
The absorbance of the sample
solution i.e. A1 and A2 were recorded at 276.00 nm (λ-max of Atenolol) and 286.40 nm (iso-absorbtive
point) respectively. Ratio of absorbance were calculated, i.e. A2/A1. Relatives concentration of two drugs in the sample was
calculated using below equations. The analysis procedure was repeated three
times with synthetic mixture.
For synthetic
mixture, 400 mg taken in which it
equivalent to 10 mg IVA and 100 mg ATN. Take it in 100 ml Volumetric flask dissolved in 25 ml
of Methanol and sonicate for 15 min. Make up to 100 ml with Methanol and shake vigorously,
Filter it. From it take 0.4 ml dilute up to 10 ml of Methanol. TO
make 4 µg/ml of Ivabradine HCl
and 40 µg/ml of Atenolol.
3.
RESULT AND DISCUSSION
In absorbance ratio method
(Q-analysis), the primary requirement for developing a method for analysis is
that the entire spectra should follow the Beer’s law at all the wavelength
which was fulfilled in case of both these drugs were 276.00 nm (λmax of Atenolol) and 286.40
nm (iso-absorbtive point) at which the calibration
curve were prepared for both drugs. The overlain UV absorption was found to be
simple, accurate, precise, robust, rugged and economic. Hence the method can be
employed for the routine analysis of these two drugs in combined form.
Figure 3: Overlay spectra of Atenolol
and Ivabradine HCl in
methanol
Figure 4: Calibration curve of Atenolol(276nm) and Ivabradine HCl (286.40nm)
Figure 5: Calibration curve of Atenolol
at 286.40 nm
Table
1: Regression data of Atenolol(276nm)
and Ivabradine HCl (at
286.40nm)
|
At 276.00 nm Conc.(µg/ml)
|
Absorbance ±SD
|
%RSD
|
At Isobestic point 286.40
nm Conc. (µg/ml)
|
Absorbance ±SD
|
%RSD
|
|
2 : 20
|
0.075±0.004
|
0.933
|
2 : 20
|
0.036±0.002
|
0.586
|
|
4 : 40
|
0.171±0.015
|
0.876
|
4 : 40
|
0.068±0.005
|
0.798
|
|
6 : 60
|
0.281±0.016
|
0.710
|
6 : 60
|
0.101±0.008
|
0.826
|
|
8 : 80
|
0.398±0.017
|
0.880
|
8 : 80
|
0.135±0.014
|
0.835
|
|
10 : 100
|
0.506±0.003
|
0.511
|
10 : 100
|
0.169±0.011
|
0.677
|
Table
2: Regression data for Atenolol at 286.40 nm
|
At Isobestic point
286.40 nm Conc.(µg/ml)
|
Absorbance ±SD
|
%RSD
|
|
2 : 20
|
0.036±0.002
|
0.586
|
|
4 : 40
|
0.068±0.005
|
0.798
|
|
6 : 60
|
0.101±0.007
|
0.826
|
|
8 : 80
|
0.135±0.013
|
0.835
|
|
10 : 100
|
0.169±0.015
|
0.677
|
Table
3: Data of precision study for Atenolol and Ivabradine HCl
|
Wavelength
|
Conc.(µg/ml)
Ivabradine HCl: Atenolol
|
Precision
|
|
Intraday
|
Interday
|
|
Mean(n=3)
|
% RSD
|
Mean(n=3)
|
% RSD
|
|
286.5 nm
|
2 : 20 μg/ml
|
0.126
|
0.457
|
0.127
|
0.453
|
|
6 : 60 μg/ml
|
0.358
|
0.161
|
0.356
|
0.486
|
|
10 : 100 μg/ml
|
0.598
|
0.194
|
0.595
|
0.349
|
|
276 nm
|
2 : 20 μg/ml
|
0.067
|
0.149
|
0.067
|
0.853
|
|
6 : 60 μg/ml
|
0.195
|
0.518
|
0.196
|
0.590
|
|
10 : 100 μg/ml
|
0.327
|
0.466
|
0.321
|
0.351
|
Table
4: Synthetic mixture for Atenolol & Ivabradine HCl in 1:10 ratio as per US Patent (3)
|
INGREDIENT
|
QUANTITIY
|
USE
|
|
Ivabradine HCl
|
100 mg
|
Antianginal
|
|
Atenolol
|
1000 mg
|
Antihypertensive
|
|
Starch
|
120 mg
|
Binder
|
|
Magnesium Stearate
|
40 mg
|
Lubricant
|
|
MCC
|
1000 mg
|
Filler
|
|
Lactose
|
1732 mg
|
Diluent
|
|
Hydrophobic colloidal
silica
|
8 mg
|
Non adherent Agent
|
|
Total
|
4000 mg (4 gm)
|
Table 5: Amount weighed for
recovery study in synthetic mixture
|
Level of
recovery
|
Amt. of
Synthetic mixture taken(mg)
|
Amt. of Drug in synthetic mixture(mg)
|
Spiked Amt. of API in mixture(mg)
|
Total Amt. (mg)
|
|
IVA
|
ATN
|
IVA
|
ATN
|
IVA
|
ATN
|
|
0%
|
400
|
10
|
100
|
-
|
-
|
10
|
100
|
|
80 %
|
400
|
10
|
100
|
8
|
80
|
18
|
180
|
|
100 %
|
400
|
10
|
100
|
10
|
100
|
20
|
200
|
|
120 %
|
400
|
10
|
100
|
12
|
120
|
22
|
220
|
Table 6: Data for recovery
study of Atenolol and Ivabradine
HCl
|
Level of
recovery
|
Total Conc.
(µg/ml)
|
Result of
recovery study
|
|
Total Quantity
Found (µg/ml)
|
% Recovery
|
|
IVA
|
ATN
|
IVA
|
%RSD
|
ATN
|
%RSD
|
IVA
|
%RSD
|
ATN
|
%RSD
|
|
0%
|
4
|
40
|
4.02
|
0.517
|
40.05
|
0.062
|
100.58
|
0.517
|
100.13
|
0.065
|
|
80 %
|
7.2
|
72
|
7.21
|
0.080
|
72.23
|
0.211
|
100.51
|
0.178
|
100.72
|
0.474
|
|
100 %
|
8
|
80
|
8.01
|
0.190
|
80.10
|
0.049
|
100.33
|
0.380
|
100.25
|
0.099
|
|
120 %
|
8.8
|
88
|
8.83
|
0.173
|
88.10
|
0.011
|
100.48
|
0.319
|
100.20
|
0.019
|
|
Mean of 3 Determination
|
100.47
|
0.348
|
100.32
|
0.164
|
Table 7: Data for LOD and LOQ
of Atenolol and Ivabradine HCl (n= 10)
|
Parameter
|
IVA
|
%RSD
|
ATN
|
%RSD
|
|
LOD (µg/ml)
|
0.181
|
0.697
|
0.309
|
0.216
|
|
LOQ (µg/ml)
|
0.550
|
0.938
|
Table 8: Data of robustness
at λ-max and iso-absorbtive point
|
Condition
|
Conc.
(µg/ml)
IVA: ATN
|
Different Instrument
|
Different Analyst
|
|
UV-2450
|
UV-1800
|
A
|
B
|
|
Mean (n=3)
|
% RSD
|
Mean (n=3)
|
% RSD
|
Mean (n=3)
|
% RSD
|
Mean (n=3)
|
% RSD
|
|
Isobestic point 286.40 nm
|
4 : 40
|
0.131
|
0.431
|
0.136
|
0.846
|
0.131
|
0.834
|
0.126
|
0.836
|
|
276.00 nm
|
4 : 40
|
0.256
|
0.676
|
0.257
|
0.896
|
0.256
|
0.686
|
0.257
|
0.896
|
Table 9: Data of ruggedness
at λ-max and iso-absorbtive point
|
Condition
|
Conc.
(µg/ml)
IVA: ATN
|
Different Solvent
|
Change in
Wavelength ± 0.5 nm
|
|
2 % water in Methanol
|
5 %water in Methanol
|
285.90 nm
|
289.90 nm
|
|
Mean (n=3)
|
% RSD
|
Mean (n=3)
|
% RSD
|
Mean (n=3)
|
%RSD
|
Mean (n=3)
|
% RSD
|
|
Isobestic point
286.40 nm
|
4 : 40
|
0.213
|
0.540
|
0.104
|
0.553
|
0.131
|
0.439
|
0.084
|
0.680
|
|
|
|
|
|
|
275.50 nm
|
276.50 nm
|
|
276.00 nm
|
4 : 40
|
0.236
|
0.645
|
0.181
|
0.956
|
0.246
|
0.473
|
0.255
|
0.977
|
Table 10: Result of
formulation analysis of both drugs
|
DRUGS
|
Result of Formulation Analysis (n=3)
|
|
Conc. of Drug
|
%Assay
|
|
IVA
|
4 µg/ml
|
101.13
|
|
ATN
|
40 µg/ml
|
100.62
|
Table 11: Validation parameters
results of both drugs at 276 nm (λ-max of Atenolol)
and at 286.40 nm (iso-absorbtive point)
|
SR. NO.
|
PARAMETER
|
AT λ2= ISOBESTIC POINT
(286.40 nm)
|
ATλ1 = ATENOLOL(276.00
nm)
|
|
1
|
Wavelength Max.
|
286.41 nm
|
276.00 nm
|
|
2
|
Linearity (µg/ml) (n=6)
|
2-10 µg/ml
|
20-100 µg/ml
|
|
3
|
Regression equation
|
y = 0.0299x + 0.0091
|
y = 0.0058x + 0.0088
|
|
4
|
Correlation coefficient (R2)
|
0.9993
|
0.9990
|
|
5
|
Accuracy(%Recovery) (n=3)
|
100.47 ± 0.348
|
100.32 ± 0.164
|
|
6
|
Precision
Intra-day (%RSD) (n=3)
Inter-day (%RSD) (n=3)
|
0.161 – 0.457
0.349 – 0.486
|
0.149 – 0.518
0.351 – 0.853
|
|
7
|
LOD (µg/ml) (n=10)
|
0.181
|
0.309
|
|
8
|
LOQ (µg/ml) (n=10)
|
0.550
|
0.938
|
|
9
|
Robustness
Different Instrument
(%RSD) (n=3)
Different Analyst (%RSD)
(n=3)
|
0.431 – 0.846
0.676 – 0.896
|
0.834 – 0.836
0.686 – 0.896
|
|
10
|
Ruggedness
Different Solvent (%RSD)
(n=3)
Change in Wavelength(%RSD) (n=3)
|
0.645 - 0.956
0.439 - 0.680
|
0.540 – 0.553
0.007 – 0.473
|
|
11
|
Assay
|
100.58%
|
100.13%
|
4.
CONCLUSION:
The proposed
spectrophotometric method was to be simple, sensitive, accurate, precise,
robust, rugged and economic for determination of Atenolol
and Ivabradine HCl in
synthetic mixture. The method utilizes easily available and cheap solvent for
analysis from synthetic mixture. This both drug show more beneficial effect in anginal patient compare to monotherapy
an its analytical method for combination is not
available at any other, so this method is novel for combine form. The common excipient and other additives are usually present in the
synthetic mixture do not interfere in analysis of Atenolol
and Ivabradine HCl in
method, hence it can be conveniently adopted for routine quality control
analysis of drug in combined pharmaceutical formulation.
5.
ACKNOWLEDGMENT:
Authors are very much thankful Shree Dhanvantary Pharmacy College, Kim, Surat for giving permission to carry out my research
work. The corresponding author very thankful to guide Dr. Hasumati Raj (QA Department), Co-guide Dr. Gautam Sonara and Principle Dr. Noolvi for guidance, help and encouragement at every step
during the progress of work.
6.
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