simultaneous determination of irbesartan and hydrochlorothiazide in tablets by derivative...

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Simultaneous Determination of Irbesartan andHydrochlorothiazide in Tablets by DerivativeSpectrophotometryJ. Joseph-Charles a , S. Brault a , C. Boyer a , M.-H. Langlois a , L. Cabrero a & J.-P. Dubost aa Université Victor Segalen Bordeaux 2, UFR des Sciences Pharmaceutiques , Laboratoire deChimie Analytique , Bordeaux Cedex, FrancePublished online: 02 Feb 2007.

To cite this article: J. Joseph-Charles , S. Brault , C. Boyer , M.-H. Langlois , L. Cabrero & J.-P. Dubost (2003) SimultaneousDetermination of Irbesartan and Hydrochlorothiazide in Tablets by Derivative Spectrophotometry, Analytical Letters, 36:11,2485-2495, DOI: 10.1081/AL-120024337

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©2003 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

ANALYTICAL LETTERS

Vol. 36, No. 11, pp. 2485–2495, 2003

PHARMACEUTICAL ANALYSIS

Simultaneous Determination of Irbesartan

and Hydrochlorothiazide in Tablets by

Derivative Spectrophotometry

J. Joseph-Charles, S. Brault, C. Boyer,

M.-H. Langlois, L. Cabrero, and J.-P. Dubost*

Universite Victor Segalen Bordeaux 2, UFR des

Sciences Pharmaceutiques, Laboratoire de Chimie

Analytique, Bordeaux Cedex, France

ABSTRACT

A second-derivative spectrophotometric method for the simultaneous

determination of irbesartan (IRB) and hydrochlorothiazide (HCT) in

tablets is described. Measurements were made at the zero-crossing

wavelengths at 230.1 nm for IRB and 232.7 nm for HCT. The calibra-

tion graphs were linear in the range of 14.4–33.6mgL�1 for IRB and

1.2–2.8mgL�1 for HCT. The limits of quantitation were 5.0 and

1.1mgL�1. The proposed method was successfully applied to the

*Correspondence: J.-P. Dubost, Universite Victor Segalen Bordeaux 2, UFR des

Sciences Pharmaceutiques, Laboratoire de Chimie Analytique, Case 110, 146,

Rue Leo Saignat, F-33076 Bordeaux Cedex, France; Fax: þ335 5757 4684;E-mail: [email protected].

2485

DOI: 10.1081/AL-120024337 0003-2719 (Print); 1532-236X (Online)

Copyright & 2003 by Marcel Dekker, Inc. www.dekker.com

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simultaneous determination of IRB and HCT in commercial tablets

with a high percentage of recovery, good accuracy, and precision.

Key Words: Irbesartan; Hydrochlorothiazide; Derivative spectro-

photometry; Simultaneous determination; Tablets.

1. INTRODUCTION

Irbesartan, 2-butyl-3-[[20-(1H-tetrazol-5-yl)[1,10-biphenyl]-4-yl]methyl]-1,3-diazaspiro[4,4]non-1-en-4-one (IRB) is a member of a new chemicalclass of effective and orally active nonpeptide angiotensin II receptorantagonists. It is a potent, long-lasting selective antagonist of theangiotensin II type 1 receptor that is indicated for the treatment of essentialhypertension.[1–3] Also known as SR 47436 and BMS-186295, IRB wasdiscovered by Sanofi Recherche and clinically developed by SanofiPharmaceuticals and Bristol-Myers Squibb.

Hydrochlorothiazide, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadia-zine-7-sulphonamide-1,1-dioxide (HCT) is one of the oldest thiazidediuretics used worldwide in combination with other antihypertensives.IRB is more effective when given in combination with HCT,[4,5] andthe two substances are marketed as combination drugs by Sanofi andBristol-Myers Squibb.

As an essential part of the drug control, a rapid and simple assayis required to monitor IRB and HCT in pharmaceuticals. Two high per-formance liquid chromatographic (HPLC) methods have been developedfor determination of IRB in human plasma.[6,7] IRB has been separatedfrom other angiotensin II receptor antagonists by capillary electrophoresis(EC),[8,9] micellar electrokinetic capillary chromatography (MEKC).[10]

Several analytical procedures have been described for the quantitativedetermination of HCT individually or in combination with other drugs,includingHPLC,[11–15] capillary electrophoresis,[16] polarography,[17] flow-injection analysis,[18] and spectrophotometry.[19–22] There are two reportedmethods for the simultaneous determination of IRB and HCT. A HPLCmethod has been described for determination of both compounds inhuman plasma samples.[23] Spectrophotometric methods have beenproposed for the assay of the two drugs in combined tablets by applyingfirst-derivative spectrophotometry for IRB and direct spectrophotometryfor HCT.[24] HCT is co-formulated with IRB in a medicinallyrecommendedc ratio of 1:12. Analysis of such mixture is challengingbecause the amount of the major component (IRB) is too high with respectto the amount of the minor component (HCT) even if HCT is strongly

2486 Joseph-Charles et al.

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absorbing light in the UV region. The values ofA1%1cm at 224 and 270 nm are

1180 and 640 for HCT, 570 and 140 for IRB.This article introduces a direct method using only derivative

spectrophotometry for the simultaneous determination of IRB andHCT in pharmaceutical forms. The aim of this work is to develop asimple, rapid, sensitive, and accurate method that could be applied inquality control laboratories for the simultaneous determination of IRBand HCT in pharmaceuticals without prior separation.

2. EXPERIMENTAL

2.1. Apparatus and Conditions

The spectrophotometric measurements were performed on aSecomam S1000 PC UV–VIS spectrophotometer using 1.0 cm quartzcell against solvent blank under the following operating conditions:scan speed 100 nmmin�1, scan range 210–300 nm, fixed slit width 2 nm,and derivation interval (��) 9 nm. The recorder scale expansion wasoptimized to facilitate readings.

2.2. Chemicals and Reagents

All chemicals and solvents were used without any further purification.Analytical grade methanol (Merck, Darmstadt, Germany) and hydro-chloric acid (Prolabo, Fontenay s/bois, France) were used. Water wasdoubly distilled. IRB and HCT and CoAprovel tablets were kindlysupplied by Sanofi Pharma (Ambares, France). Each CoAprovel tabletwas labelled to contain 150.0mg of IRB and 12.5mg of HCT in additionto excipients consisting of microcrystalline cellulose, croscarmellosesodium, lactose monohydrate, magnesium stearate, colloidal hydratedsilica, pregelatinized maize starch, and red and yellow ferric oxides.

2.3. Standard Solutions and Calibration

Amixture ofmethanol-0.01MHCl (70:30, v/v) was used as the solventfor the proposed method. Stock solutions of IRB (400.0mgL�1) and HCT(80.0mgL�1) were prepared in the solvent. The working standardsolutions were prepared by dilution of the above solutions with the samesolvent to reach concentration ranges of 14.4–33.6mgL�1 for IRB,

Determination of Irbesartan and Hydrochlorothiazide 2487

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1.2–2.8mgL�1 for HCT. Similarly, two series of each mixture were alsoprepared from the stock solutions. The first series was obtained by varyingIRB concentration (14.4–33.6mgL�1) and maintaining HCT con-centration at a constant level of 2.0mgL�1. The second series containeda constant concentration of IRB (24.0mgL�1) and a varying con-centration of HCT (1.2–2.8mgL�1).

The standard solutions were measured during a period of two daysand found to be stable. The second-order derivative spectra of thesesolutions were scanned over the wavelength range of 210–300 nm againstmethanol-0.01M hydrochloric acid (70:30, v/v) as a blank. Calibrationcurves were constructed by assaying standard solutions of IRB alone andHCT alone, and second order derivative absorbance values (2D) at 230.1and 232.7 nm were plotted against the corresponding concentrations.Through these calibration curves 2D values at 230.1 nm (zero-crossingof HCT) were measured for the determination of IRB in the presenceof HCT and 2D values at 232.7 nm (zero-crossing of IRB) were used forthe determination of HCT in the presence of IRB.

2.4. Analysis of Tablets

Ten tablets were separately weighed and finely powdered. About90mL of methanol-0.01M hydrochloric acid (70:30, v/v) was addedto an accurately weighed amount of the powder equivalent to approxi-mately 30mg of IRB and 2.5mg of HCT in a 100mL calibrated flask.The mixture was shaken by magnetic stirrer for 15min and finally dilutedto volume with the same solvent. A portion of the resulting suspensionwas centrifuged at 3500 revmin�1 for 5min. A 1.0mL of the clearsupernatant was transferred into a 10mL volumetric flask and dilutedto volume with the solvent.

3. RESULTS AND DISCUSSION

3.1. Spectrophotometric Measurements

A thorough investigation was carried out in order to choose theoptimum solvent medium for the spectrophotometric determination ofIRB and HCT. Zero-order absorption spectra to fourth-order derivativespectra of both drugs in methanol, ethanol, and methanol-0.01M hydro-chloric acid (70:30, v/v) at appropriate concentrations were tested.


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The second-derivative spectra of drug solutions in methanol-0.01Mhydrochloric acid (70:30, v/v) were the best for their simultaneous deter-mination in tablet formulations.

Figure 1 shows the zero-order absorption spectra of IRB, HCT, anda mixture of HCT and IRB in a 1:12 HCT:IRB ratio. Because of thelarge overlapping with the absorbance spectra of the two compounds,conventional UV spectrophotometry is not suitable for analyzingsimultaneously IRB and HCT in mixtures. The high content of IRBand the small content of HCT in commercial tablets (the HCT:IRB ratio is 1:12) result in a large contribution of the spectrum ofIRB to the maxima in the spectrum of HCT. Interference could notbe overcome by the use of first-derivative spectrophotometry. In thesecond-derivative spectra (Fig. 2) it was considerably reduced. Thesecond-derivative spectra of IRB and HCT showed sharp bands withlarge amplitudes that seemed more selective for identification and deter-mination of the two compounds. Owing to the overlapping spectra, thezero-crossing method was considered to be the most suitable approachfor resolving mixtures of these compounds and it was used in this workwith satisfactory results. The measurements selected for the preparationof analytical calibration graphs were those which exhibited the bestlinear response, a zero or near zero intercept on the ordinate and aresponse unaffected by the concentration of any other component.Zero-crossing wavelengths at 230.1 and 232.7 nm were selected res-pectively for the simultaneous determination of IRB and HCT sincereproducible readings were obtained at these wavelengths.

3.2. Selection of Optimum Instrumental Conditions

The main instrumental parameters that affect the shape of thederivative spectra and the signal-to-noise ratio are the wavelengthscanning speed, the wavelength increment over which the derivative isobtained (��) and the smoothing. All of these parameters need to beoptimized to give good selectivity and better sensitivity in the determi-nation. Various values were tested for the wavelength scanning speedand a scan speed of 100 nmmin�1 was chosen as the best. Increasing ��improves the noise level, which leads to less pronounced fluctuations inthe derivative spectrum. However, spectral resolution is very poor if thevalue of �� is too high. The value of �� should be optimized by takinginto account the noise level, the spectrum resolution, and the sampleconcentration. Several �� values were tested and ��¼ 9 nm wasselected as the optimum for a satisfactory signal-to-noise ratio.


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220

230

240

250

260

270

280

290

Wav

elen

gth

(n

m)

Absorbance

HC

T 2

mg/

l

IRB

24

mg/

l

HC

T 2

mg/

l

+IR

B 2

4 m

g/l

0

0,51

1,52

2,5

Figure

1.

Zeroorder

derivativespectraofIR

B24mg/L,HCT2mg/L,and(H

CT2mg/L

þIR

B24mg/L).


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-0,0

08

-0,0

06

-0,0

04

-0,0

020

0,00

2

0,00

4

0,00

6 220

230

240

250

260

270

280

290

Wav

elen

gth

(n

m)

Derivative values

IRB

24

mg/

l

HC

T 2

mg/

l

HC

T 2

mg/

l+ IR

B 2

4 m

g/l

Figure

2.

Secondorder

derivativespectraofIR

B24mg/L,HCT2mg/L,and(H

CT2mg/L

þIR

B24mg/L).


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3.3. Statistical Analysis of Spectrophotometric Data

Under the experimental conditions described, the linearity of thecalibration graphs for IRB and HCT in the pure solutions was validatedby the high values of the correlation coefficient for regression equations.The value of the intercepts on the ordinate was found not statisticallydifferent to zero. The second-derivative spectra of the two series of samplescontaining different concentrations (14.4–33.6mgL�1) of IRB, and aconstant concentration (2.0mgL�1) of HCT or different concentrations(1.2–2.8mgL�1) of HCT, and a constant concentration (24.0mgL�1) ofIRB were recorded at the wavelengths mentioned in order to investigatethe effect of HCT and IRB on the simultaneous determination of the twocompounds. Linear relationships with negligible intercepts were alsoobtained, confirming the mutual independence of the two componentsderivative signals. Moreover the similarity observed between regressionequations of pure IRB or HCT solutions and their mixture solutionssuggested no interferences in the estimation of one drug in the presenceof the other. The standard deviations of the slope (Sa), the intercept (Sb),and the correlation coefficient (Sr) were calculated (Table 1). The detectionand quantification limits achieved as defined by International conferenceof harmonization (ICH)[25] were 1.6 and 5.0mgL�1 for IRB while thecorresponding values for HCT were 0.4 and 1.1mgL�1. The within-runprecision of the method was tested by analyzing three replicates ofsynthetic mixtures containing 24.0mgL�1 of IRB and 2.0mgL�1 of HCT.The relative standard deviations (RSD) were 0.53 and 1.03%, respectively.The between-run precision of same concentrations of IRB and HCT wasevaluated over three days by performing three measurements on each day.The RSD were 1.37 and 2.52% for IRB and HCT, respectively. Theaccuracy and selectivity of the method were verified by means of recoverystudies for IRB andHCT in their synthetic mixtures with different portionsof the two drugs. Three successive determinations of each solution werecarried out. Satisfactory results were obtained for the recovery of bothdrugs. Mean recovery (� %RSD) was found to be 99.5� 1.95 and96.9� 1.96 for the determination of IRB and HCT respectively.

3.4. Applications

The proposed method was applied to the determination of com-mercially available tablets containing a mixture of HCT and IRB in aratio 1:12. The effect of possible interferences from the excipients wasstudied by assaying CoAprovel tablets labelled to contain 150mg of IRB


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Table

1.

Calibration

equations

for

the

determination

ofIR

Band

HCT

instandard

solutions

by

second-derivative

spectrophotometry.

Compound

Linearity

range(m

gL�1)

Regressionequationsa

rbSc a

Sd b

IRB

14.4–33.6

2D230.1=0.00lC

IRBþ0.001

0.999

2.08�10�5

0.0005

IRB(H

CT2.0mgL�1)

14.4–33.6

2D230.1=0.00lC

IRBþ0.002

0.998

3.99�10�5

0.0009

HCT

1.2–2.8

2D232.7=0.012CHCT�0.001

0.996

0.0006

0.0013

HCT(IRB24.0mgL�1)

1.2–2.8

2D232.7=0.011CHCT

0.999

0.0002

0.0005

aDerivativevalue(2D)atthecorrespondingwavelength

vs.concentration(C

)ofthecompoundmeasured(m

gL�1);n¼15.

bCorrelationcoeffi

cient.

cSaStandard

deviationoftheslope.

dSbStandard

deviationoftheintercept.


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and 12.5mg of HCT per tablet. The mean percentage recoveries were101.7� 0.45 with a %RSD of 1.84 for IRB, and 97.0� 0.03 with a%RSD of 1.55 for HCT. The indicated values were the mean of differentanalyses of the same commercial batch. These results indicated that therewas no interference from the sample matrix. The proposed methodproved to be suitable for the content uniformity test.

4. CONCLUSION

The method developed in this study is easily carried out for simulta-neous determination of IRB and HCT in tablets. It does not need anyspecial equipment, is simple and rapid for sample preparation and assayprocedure, and provides accurate and precise results. It can be used forroutine quality controls.

ACKNOWLEDGMENT

The authors would like to thank Sanofi Pharma for providingstandard drug samples and commercial tablets.

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simultaneous determination of irbesartan and hydrochlorothiazide in tablets by derivative...

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