Publication Ref No.

: IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-5/JULY/013 ISSN 0974 – 9446

SIMULTANEOUS ESTIMATION OF NEBIVOLOL HYDROCHLORIDE AND AMLODIPINE

BESYLATE IN COMBINED TABLET DOSAGE FORM BY Q-ANALYSIS METHOD

Chandan Kumar Giri1*,

M.S. Kondawar and D.D. Chougule1

1
Appasaheb Birnale College of Pharmacy,
Sangli, Maharashtra, India
Chandan Kumar Email: chandanqa@gmail.com

ABSTRACT

A simple, accurate, precise, rapid and economical spectrophotometric method for simultaneous
estimation of Nebivolol hydrochloride and Amlodipine besylate in combined tablet dosage form has
been developed utilizing Q-analysis method or Absorbance ratio method. In this method, absorbance is
measured at two wavelengths, one being the iso-absorptive point 268nm (λ1) and the other being λmax
of one of the sample components 282nm (λ2). Both the drugs obey Beer’s law in the concentration
ranges employed for this method. Result of the method was validated statistically as well as by
recovery studies. Proposed method for simultaneous estimation of NH and AB in combined sample
solutions was found to be simple, accurate and reproducible. Once the equations are determined,
analysis required only the measurement of the absorbances of the sample solution at the two
wavelengths selected, followed by a few simple calculations. It is a novel method that can be employed
for routine analysis in quality control or R & D laboratory.

Key Words: Nebivolol hydrochloride, Amlodipine besylate, Spectrophotometric analysis, Q-Analysis

method.

INTRODUCTION adrenergic blocker. Nebivolol selectively blocks

Nebivolol hydrochloride (NH), chemically α, α’-
[Iminobis (methylene)] bis [6-fluoro-3, 4-dihydro-
2H-1-benzopyran-2-methanol] is a beta-
the β1- adrenoceptor. Nebivolol reduces heart
rate, rate of myocardial contractility and
systemic blood pressure while increasing
diastolic pause. β-blockers are useful
1

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prophylactic agents in stable and unstable types
of angina. Nebivolol is preferable in patients
with bronchi spasm, diabetes, peripheral
vascular disease or Raynaud’s phenomenon.
The drug is not official in any pharmacopoeia. A
survey of literature revealed that there are a
number of methods for the individual
determination of Nebivolol hydrochloride such
as a HPLC- fluorescence method, a UV-
colorimetric method, and HPTLC method.
Amlodipine besylate is chemically R,S-2-[(2-
aminoethoxy) methyl]-4-(2-chloroethyl)-3-ethoxy
carbonyl-5-methoxy carbonyl-6-methyl-1,4-
dihydropyridine benzene sulphonate used in the
treatment of hypertension and congestive heart
failure. Literature survey reveals that there are a
number of methods for the individual
determination of Amlodipine like RP-HPLC,
difference spectrophotometry, colorimetry,
HPTLC, GC, LC Mass spectrometry and few
spectrophotometric methods. There is no such
method available for the estimation of Nebivolol
hydrochloride and Amlodipine besylate in
combined tablet dosage forms.
MATERIALS AND METHOD:
Instrument: UV/Visible spectrophotometer
(JASCO) V-550, Digital balance (SHIMADZU)
AUX-220
Reagents: Methanol of Analytical grade
Standard drug: Nebivolol hydrochloride and
Amlodipine besylate from Alkem Laboratory,
Mumbai.
Tablet dosage form: AMLOPRES-NB Tablets
from CIPLA, Each uncoated tablet contains
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Amlodipine besylate equivalent to Amlodipine 5
mg and Nebivolol hydrochloride equivalent to
Nebivolol 5 mg.
Preparation of standard stock solution:
Standard stock solutions containing Nebivolol
hydrochloride (NH) and Amlodipine besylate
(AB) were prepared individually by dissolving 10
mg of NH and 10mg of AB separately in 50ml of
methanol. It was then sonicated for 10 minutes
and the final volume of both the solutions were
made up to 100 ml with methanol to get stock
solutions containing 100µg/mL each of NH and
AB in two different 100 ml volumetric flasks. NH
exhibited λmax at 282 nm and AB showed λmax
at 238 and 360 nm. Both of these drugs show
isobestic points at 268 nm. Wavelength 282 nm
with reference to 268 nm (isobestic point) was
selected as the working wavelength, as at this
wavelength there is minimum interference of the
other drug.
Optimization of concentrations of the
standards:
Concentrations of 10.0 µg/mL of Nebivolol
hydrochloride and 10.0 µg/mL of Amlodipine
besylate were used as standard 1 and standard
2. The Q values and absorptivities for both the
drugs were calculated as follows:
Fig. 1: Overlain spectrum of Nebivolol
hydrochloride and Amlodipine besylate
2
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Regression and optical characteristics of Analysis of tablet formulation:

Nebivolol hydrochloride and Amlodipine
besylate (Refer Table No: 1) Twenty tablets of Nebivolol hydrochloride and
Amlodioine besylate combination were weighed
and their average weight was determined.
Absoptivity of AB at 282nm Afterwards, tablets were crushed to powder
QAB= = 0.833 sample. From the triturate, tablet powder
Absorptivity of AB at 268nm equivalent to 10.0 mg of NH and 10 mg of AB
Absoptivity of NH at 282nm was weighed and transferred to 100 ml
QNH= =1.4545 volumetric flask and dissolved in 50 ml
Absoptivity of AB at 282nm methanol. The content was kept in
Absorbance of Mixture at 282nm ultrasonicator for 20 min. Finally, the volume
QM= was made up to the mark with methanol. The
Absorbance of mixture at 268nm solution was filtered through Whatman filter
paper No.41.
At 268nm the standard equations are as
follows: This tablet solution was further diluted to obtain
Amlodipine besylate - y = 0.012x + 0.002 10 µg/ml for NH and 10 µg/ml for AB. The
Nebivolol hydrochloride- y = 0.011x + 0.001 mixed sample solutions were analyzed to obtain
Amount of each drug was determined using spectra. Absorbance values at 268 nm
following formulas: (isobestic point) and 282 nm were noted. The
QM – QNH A concentrations of Nebivolol hydrochloride and
CAB = x ------------------ 1 Amlodipine besylate were calculated from the
QAB – QNH aAB equation (1) and (2). The results of the analysis
QM– QAB A of tablet formulation are reported in Table No.3
CNH = x ------------------ 2 and data for statistical validation are given in
QNH – QAB aNB Table No.4.

Where, Recovery Studies:

CAB = Concentration of AB in gm/100 mL.
CNH = Concentration of NH in gm/100 mL.
QM = Ratio of absorbance of mixture at λmax
and λiso
QAB = Ratio of absorptivity of AB at λiso and
λmax
QNH = Ratio of absorptivity of NH at λiso and
λmax
A = Absorbance of mixture at isoabsorptive
point.
aAB = E (1%, 1cm) of AB at λiso
aNB = E (1%, 1cm) of NH at λiso
Recovery studies were carried out by applying
the method to pre-analyzed drug sample in
tablet dosage form to which known amount of
NH and AB corresponding to 80%, 100% and
120% of label claim was added (standard
addition method). The mixed sample solutions
were analyzed as per tablet formulation. At
each level, three determinations were per-
formed. Results of recovery studies and
statistical evaluation are shown in Table No. 5
and 6, respectively

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Amlodipine besylate and Nebivlol hydrochloride.

RESULTS: The authors also thankful to Cipla
pharmaceutical for providing gift sample of
Standardization of the method by analysis of Tablet dosage form
powder mixture of known composition (Refer
Table No: 2), Analysis of Tablet Formulation REFERENCES:
(Refer Table No: 3), Statistical validation of
Tablet (Refer Table No: 4), Recovery
1. ICH Q2A; Guidelines on validation of
Studies(Refer Table No: 5), Statistical
analytical procedure; Definitions and
Validation for the Recovery of Tablet
terminology, Federal Register, 1995, 60,
Formulation(Refer Table No: 6)
11260.
2. ICH Q2B; Guidelines on validation of
DISCUSSION :
analytical procedure; Methodology, Federal
Register, 1996, 60, 27464.
The proposed method for simultaneous 3. PS Tarte*, SP Wate, PB Khedikar, BT
estimation of AB and NH dosage forms were Satpure and G Pawnikar Simultaneous
found to be simple, accurate, economical and
Estimation of Nebivolol and
rapid. The method was validated as per the ICH
Hydrochlorothiazide in Combined Tablet
and USP guidelines. The values of SD or RSD
Dosage Form by Multicomponent Mode of
are within the prescribed limit of 2 %, showing
Analysis www.ajrconline.org ISSN 0974-
high precision of method and recovery was
4169; AJRC
close to 100% for both the drugs. During the
4. Singh, M. Bakshi, “Understanding Analytical
linearity stud y it was observed that absorbance
Method Validation.” Pharma. Times, August,
values of AB and NB in the marketed
1999, pg. 15-20.
formulation were linear in the range of 80% to
5. ICH Guidance on Analytical Method
120% of test concentration with R2 close to
Validation, International Convention on
one. Quality for the Pharmaceutical Industry,
Toronto, Canada, September, 2002
CONCLUSION:
6. Rao A. L. *, Rajeswari K.R. And Sankar
G.G. (2009) Spectrophotometric Method for
Proposed method for simultaneous estimation the Determination of Nebivolol Hydrochloride
of NH and AB in combined sample solutions
in Bulk and Pharmaceutical
was found to be simple, accurate and
FormulationsISSN: 0973-4945; CODEN
reproducible. Once the equations are
ECJHAO E-Journal of Chemistry
determined, analysis required only the
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measurement of the absorbances of the sample
448.
solution at the two wavelengths selected,
7. Dhandapani B. *, Thirumoorthy N. And
followed by a few simple calculations. It is a
Prakash D.J. (2009) Development and
novel method that can be employed for routine
Validation for the Simultaneous
analysis in quality control or R & D laboratory.
Quantification of Nebivolol Hydrochloride
and Hydrochlorothiazide by UV
ACKNOWLEDGEMENTS:
Spectroscopy, RP-HPLC and HPTLC in
Tablets , ISSN: 0973-4945; CODEN
The authors are thankful to the Alkem lab, ECJHAO E-Journal of Chemistry
Mumbai for providing gift sample of standard

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http://www.e-journals.net 2010, 7(2), 341-
348
8. Joshi S. J. , Karbhari P. A. and Bhoir
S. I.( 2009) RP-LC Simultaneous
Determination of Nebivolol Hydrochloride
and Amlodipine Besilate in Bi-Layer Tablets,
Journal of Chromatographia, ISSN0009-
5893, Volume 70, 557-561
9. V. Juyal, Chaudhary M., Kumar
P., Gnanarajan G. , P. K. Yadav (2008)
Method development and its validation for
simultaneous estimation of Atorvastatin and
Amlodipine in combination in tablet dosage
form by UV spectroscopy, using multi-
component mode of analysis Journal of
Pharmacy
Research.01/01/2008; WWW.DOAJ.ORG,IS
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10. Birajdar A. S. *, Meyyanathan S. N., and
Bhojraj Suresh (2009) Simultaneous
Estimation of Nebivolol Hydrochloride and
Valsartan and Nebivolol Hydrochloride and
Hydrochlorothiazide in Pharmaceutical
Formulations by UV Spectrophotometric
Methods, Indian J.Pharm. Educ. Res. 44(2),
Apr-Jun, 2010
11. SHAH K. V. *, Thumber B. L., Desai T. R.
(2009) Simultaneous Estimation of Nebivolol
Hydrochloride and Hydrochlorothiazide in
Bulk and Tablet Dosage Form by Q-Analysis
Method International Journal of
Pharmaceutical Science & Research (2009)
1(2) 80-85
12. Patil P. R. *, Rakesh S. U., Dhabale P.N.,
and Burade K.B. ,development and
statistical validation of uv spectrophotometric
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ISSN 0974 – 9446
method for estimation of amlodipine besylate
in tablet dosage form, ISSN 0975 – 2633
Arch Pharm Sci & Res Vol 1 No 2 158 - 161
October 2009
13. Garg G., Saraf S. and Saraf S. (2008)
Development and validation of simultaneous
estimation of enalapril maleate and
amlodipine besylate in combined dosage
forms. Trends Applied Sci. Res., 3: 278-284.
14. Kakde R.B. *, Kotak V.H., Barsagade A.G.,
NK Chaudhary and DL Kale (2008)
Spectrophotometric Method for
Simultaneous Estimation of Amlodipine
Besylate and Bisoprolol Fumarate in
Pharmaceutical Preparations, ISSN 0974-
3618 www.rjptonline.org, 513
15. Kamble A. Y.; Mahadik M. V.; Khatal L. D.;
Dhaneshwar S. R. (2010) Validated HPLC
and HPTLC Method for Simultaneous
Quantitation of Amlodipine Besylate and
Olmesartan Medoxomil in Bulk Drug and
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Issue 2 January 2010 , pages 251 - 258
16. Rahman Nafisur and Azmi Syed Najmul
Hejaz (2001) Spectrophotometric method for
the determination of amlodipine besylate
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Tables and Figures:

Table 1: Regression and optical characteristics of Nebivolol hydrochloride and Amlodipine besylate.

Parameters AB NH

λmax in methanol 238 282

Beer’s law limit (µg/mL) 2-40 2-80

Correlation coefficient (r) 0.9996 0.9995

Molar absorptivity (lit/mole/cm) 21378.65 53997.32

Slope 0.0376 0.0165

Intercept -0.0047 -0.0158

Table 2: Standardization of the method by analysis of powder mixture of known composition:

Concentration present Concentration found

% Estimated
(µg/ml) (µg/ml)

AB NH AB NH AB NH

10 10 10.12 9.94 101.2 99.4

10 10 10.08 9.97 100.8 99.7

10 10 10.02 9.98 100.2 99.8

10 10 10.04 9.99 100.4 99.9

10 10 10.10 10.03 101.0 100.3

10 10 10.07 10.06 100.7 100.6

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Table 3: Analysis of Tablet Formulation

Label claim Amount found*

Percentage found
(mg/ tablet) (mg/ tablet)

AB NH AB NH AB NH

5 5 5.05 4.97 101.0 99.4

5 5 5.07 4.98 101.4 99.6

5 5 5.00 5.03 100.0 100.6

5 5 5.05 4.99 101.0 99.8

5 5 5.01 5.00 100.2 100.0

5 5 5.06 5.02 101.2 100.4

Table 4: Statistical validation of Tablet

Standard Coefficient
Drug mean Standard error
deviation of variation

Amlodipine 100.80 0.516398 0.512 0.21

Nebivolol 99.96 0.422953 0.423 0.172

Table 5: Recovery Studies

Standard Coefficient
Drug mean Standard error
deviation of variation

Amlodipine 100.80 0.516398 0.512 0.21

Nebivolol 99.96 0.422953 0.423 0.172

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Table 6: Statistical Validation for the Recovery of Tablet Formulation

Level of mean of % Standard Coefficient of

Standard error
%recovery recovery deviation variation

DRUG AB NH AB NH AB NH AB NH

80 100.88 99.07 0.273 0.455 0.271 0.460 0.111 0.186

100 100.26 100.2 0.612 0.294 0.611 0.293 0.25 0.12

120 100.6 100.08 0.224 0.199 0.223 0.198 0.0916 0.0812

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