Losartan-HCTZ-anti-beer.pdf

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Anti-beer Evaluation of Hydrochlorothiazide andLosartan by UV Derivative SpectrophotometryClaudio Vetuschi a; Almagrazia Giannandrea aa Farmaco Chimico Department, University of Bari, Bari, Italy

Online Publication Date: 05 January 2003To cite this Article: Vetuschi, Claudio and Giannandrea, Almagrazia (2003) 'Anti-beerEvaluation of Hydrochlorothiazide and Losartan by UV DerivativeSpectrophotometry', Analytical Letters, 36:5, 1051 - 1064To link to this article: DOI: 10.1081/AL-120019262URL: http://dx.doi.org/10.1081/AL-120019262

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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. 5, pp. 1051–1064, 2003

PHARMACEUTICAL ANALYSIS

Anti-beer Evaluation of Hydrochlorothiazide and

Losartan by UV Derivative Spectrophotometry

Claudio Vetuschi* and Almagrazia Giannandrea

Farmaco Chimico Department,

University of Bari, Bari, Italy

ABSTRACT

An UV derivative method for the simultaneous determination of

Hydrochlorothiazide (HCT) and Losartan (LST) in the commercial

forms was developed. The fourth derivative spectrum from the

alcoholic solution was used. HCT can be determined by a specific

signal at 330–340 nm, while LST uses the signal around 280–290 nm,

common to both products. Into the concentration range of HCT

(10.8–500)10�3 mgmL�1 and of LST (20–2400)10�3 mgmL�1, for

their mixture in the ratio 1:4, two intervals were identified, one for

lower values, for which the absorbance grows with concentration,

and one for higher values, in which the absorbance of the solution

diminishes as concentration increases. The second behaviour seems

*Correspondence: Claudio Vetuschi, Farmaco Chimico Department, University

of Bari, Via E. Orabona 4, 70125 Bari, Italy; E-mail: [email protected].

1051

DOI: 10.1081/AL-120019262 0003-2719 (Print); 1532-236X (Online)

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

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to be due to intramolecular reactions of the drugs. For both the

intervals, linear regressions were obtained by correlating the spectra

signals with the drug concentrations. The best results were obtained

by employing the regression equations from the downward section.

Key Words: Hydrochlorothiazide; Losartan; Derivative spectro-

photometry; Multiple linear regression.

INTRODUCTION

Losartan (LST), potassium salt of 2-n-butyl-4-chloro-5-hydroxy-methyl-1-[[20-(1H-tatrazol-5-yl)biphenyl-4-yl]methyl]imidazole, is theprototype of a new generation of nonpeptide angiotensin II receptorantagonists.[1–5]

Hydrochlorothiazide (HCT), 6-chloro-3,4-dihydro-2H-1,2,4-benzo-thiadiazine-7-sulfonamide-1,1-dioxide, is a diuretic in the benzothia-diazine class.

The two drugs (Fig. 1) are employed in antihypertensive pharma-ceutical formulations, alone or in association.[6–11]

The simultaneous determination of these drugs in the commercialforms is limited to a procedure based on the use of RPHPLC.[12] Apartfrom this, the literature contains only analytical procedures for HCTin the human plasma and urine, in the presence of LST but withoutany information for its quantitation.[13] The present paper proposesan UV derivative spectrophotometric method, for the simultaneousdetermination of the drugs, in pharmaceutics.

Figure 1.

1052 Vetuschi and Giannandrea

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RESULTS AND DISCUSSION

The examination of the spectra for pure HCT and LST in absorbance(Fig. 2) and in successive derivative orders indicates that the bestanalytical selectivity can be obtained in third and fourth derivatives. Par-ticularly, the fourth derivative spectrum, in alcoholic solution (Fig. 3),exhibits a characteristic peak-trough (Pc) at 330–340 nm due to HCTalone, and a peak-trough (Ps) at 280–290 nm as sum of both the products.

Investigations into the Linearity Range

Through some reference solutions of the two pure drugs and theirmixture in 1:4 ratio, in the range of: (10–500)10�3mgmL�1 for HCT, and(40–2000)10�3 mgmL�1 for LST, the relationships on Ps as function ofthe concentration of HCT, LST and their mixture were obtained andreported in Fig. 4. As it can be seen, these present a parabolic shape.

The shift away from linearity seems to be due to intramolecular reac-tions which from LST lead to the formation of two condensed dimericproducts.[14] For HCT too, some degradation products are reported.[15–17]

By examining the whole interval of concentrations, moreover, twozones could be observed. A first, at low concentrations, in which theabsorbance of the mixtures increases, and a second at high concentra-tions, in which the absorbance decreases.

Calibration

Numerous solutions of the pure drugs and their mixtures, atconcentrations within the two zones above mentioned, were preparedand the derivative spectra were recorded. All the mixtures were inthe HCT/LST ratio of 1:4� 0.5. In order to reduce or, at least, to keepreproducible the degradation reactions, all the solutions were preparedadding amounts exactly weighted of the drugs to the stirred final volumeof solvent, without any dilution. This produces a higher statistical vari-ability with a greater imprecision, but it makes the method morerigorous. Fig. 5 shows the obtained overlapped spectra.

It appears that the position of Ps signal depends on the solutionconcentration, while the �� value (�min� �max) remains constant at thevalue of 10 nm (Fig. 6).

The peak-troughs of the two selected signals were correlated with thedrug concentrations (mgmL�1) through linear regressions. Good agree-ment with the linearity was observed for the HCT concentrations

Evaluation of HCT and LST 1053

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Figure

2.

Zer

o-o

rder

ethanolicsp

ectraof:

HCT

(-s

-)10.1�

10�

3m

gm

L�1;LST

(-i

-)40.2�

10�3m

gm

L�

1;th

eir

1:4

mix

ture

(-u

-).


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Figure

3.

Fourth

der

ivative

spec

tra

of136.2�

10�3m

gm

L�

1H

CT

(A)and

542.9�

10�3m

gm

L�

1LST

(B).


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Figure

4.

Ps

as

funct

ion

of

HCT

and

LST

conce

ntrations,

and

their

1:4

mix

ture

.H

CT:

(10–500)1

0�

3m

gm

L�1;LST:(2

0–2400)1

0�3m

gm

L�

1.


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Figure

5.

Over

lappin

gofth

efo

urth

der

ivative

spec

tra

of1:4

mix

ture

ofH

CT

(4.3

–466)1

0�

3m

gm

L�

1and

LST

(10.8

–1872)1

0�

3m

gm

L�1.


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from 4� 10�3 to 60� 10�3 and for the LST from 16� 10�3 to 240� 10�3,in upward section; and from 130� 10�3 to 400� 10�3 for HCT and from500� 10�3 to 1600� 10�3 for LST, in downward section.

All the obtained regression equations, into the two linear sections,are listed in the Table 1.

– Ia and Id, which correlate Pc and HCT, and IIIa and IIId asinternal correlations between the Pc and Ps signals, wereobtained from the pure HCT spectra.

– IIa, which correlate Ps with LST, was obtained from the pureLST solutions, while for the downward section, a correspond-ing equation was not possible, because Ps and LST at highconcentrations proved to be uncorrelated (Fig. 4).

– IVa and IVd, which correlate Ps, Pc, and LST through multiplelinear regression, were finally established from themixture spectra.

An analogous relationship between the Ps position and LSTconcentration was obtained, but this is not reported here because doesnot improve the final result.

EXPERIMENTAL

Chemicals

Pure drugs, LST and HCT utilized as standards, were kindly suppliedby Merck Sharp & Dohme and Sigma-Aldrich, respectively. Ethanol(95% v/v) was spectroscopic reagent grade (C. Erba).

Figure 6. Ps position with concentration of solutions; (~) maximum; (*) right

minimum.


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Table

1.

Reg

ress

ion

equations.

HCT

(mgm

L�1)

LST

(mgm

L�

1)

Reg

ress

ion

equations

r

4–60�

10�

316–240�

10�3

IaH

CT¼

1.8

968Pc�

0.0

011

0.9

995

Upward

IIa

LST¼

1.5

082Psþ

0.0

238

0.9

899

IIIa

Pc¼

0.1

033Ps�

0.0

056

0.9

996

IVa

LST¼�

0.1

381Psþ

9.4

835Pc�

0.0

036

0.9

998

130–400�

10�3

500–1600�

10�3

IdH

CT¼

1.2

738Pcþ

0.0

716

0.9

866

Downward

IIId

Pc¼�

0.7

196Psþ

0.4

926

0.9

995

IVd

LST¼�

0.9

728Psþ

2.5

358Pcþ

0.9

974

0.9

998


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Commercial Forms

The commercial pharmaceutical forms analyzed were: LOSAZID, bySigma-Tau; HIZAAR, by Merck-Sharp Dohme; NEO LOTAN PLUS,by Neopharmed.

Instrumentation

The UV spectra were recorded over the wavelength range400–190 nm, in 10mm silica quartz cells, using a Perkin-Elmer Lambda15 Spectrophotometer.

The instrumental conditions were fixed at: scan speed 1 nm s�1,response (time constant) 1 s and spectral bandwidth 1 nm. For thefourth derivative �� was 8 nm.

Sample Solutions

Five tablets were weighed and reduced to a fine powder, and theaverage of one tablet estimated. A powder amount corresponding toone tablet, accurately weighed, was transferred into a flask containing100mL of stirred ethanol. The suspension was recorded on UV derivativeand the drugs were measured employing the Id and IVd relationships. Itwas verified that the excipients present in the commercial forms had nodetectable effect on the amplitude of the chosen signals. The obtainedresults refer to one tablet.

Analytical Procedure

HCT and LST (mgmL�1) were determined, by using the relationshipsof Table 1, as follows:

– HCT by means of Ia and Id.– LST by means of the subsequent procedures.– The HCT contribution on Ps was obtained by IIIa through the

Pc value and subtracted from Ps of the mixture, so as to obtainthe Ps value due to LST. The resulting Ps value was used toquantify LST, through IIa. An analogous procedure does notexist for the downward section.

– By regression planes IVa and IVd.


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Best analytical results were achieved by the second of theseprocedures and especially by IVd regression of downward section. Forthis reason the ‘‘anti-Beer’’ title has been used. The final results got byapplying the procedure with Id and IVd, on standard laboratory mixturesare reported on Table 2.

VALIDATION

Synthetic mixtures, with the component levels within the range ofdownward section in different ratios, were prepared in order to validate

Table 2. Analytical results on standard mixtures by employing Id and Ivd.

Nominal (mg mL�1) Found

HCT LST HCT E% LST E%

0.102 0.408 0.101 �0.98 0.416 2.01

0.118 0.502 0.118 0.00 0.495 �1.39

0.156 0.601 0.155 �0.64 0.604 0.50

0.213 0.802 0.213 0.00 0.795 �0.87

0.251 1.001 0.254 1.20 1.004 0.30

0.307 1.218 0.311 1.30 1.230 0.99

0.369 1.436 0.373 1.08 1.441 0.35

0.406 1.615 0.412 1.48 1.636 1.30

0.476 1.953 0.476 0.00 a a

aUnappreciable.

Table 3. Validation.

Nominal (mg mL�1) Found

HCT LST HCT

% recov.

LST

% recov.(mgmL�1) (mgmL�1)

0.203 1.015 0.201 99.16 1.002 98.75

0.203 0.792 0.201 98.67 0.805 101.05

0.203 1.008 0.204 100.74 0.997 98.96

0.255 0.996 0.249 97.92 1.003 100.66

0.262 0.996 0.259 98.93 0.993 99.43

0.231 0.996 0.234 101.13 1.008 101.23


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the method proposed. The procedure was applied on these solutionsand the analytical results are reported in Table 3. By means of thevalues of the table, the following results were carried out.

Accuracy: percent recoveries ð �xx� ts=ffiffiffi

np

; � ¼ 0:005Þ; were 99.4� 1.3for HCT and 100.0� 1.1 for LST.

Precision: % rsd determined for 0.203mgmL�1 of HCT was 1.1 andfor 0.996mgmL�1 of LST was 0.9.

CONCLUSION

This work offers a simple and fast method practicable in routineanalysis of title drugs in pharmaceuticals.

REFERENCES

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2. Timmermans, P.B.M.W.M.; Wong, P.C.; Chiu, A.T.; Herblin, W.F.;Benfield, P.; Carini, D.J.; Lee, R.J.; Wexler, R.R.; Saye, J.A.; Smith,R.D. Angiotensin II receptors and angiotensin II receptorantagonists. Pharmacol. Rev. 1993, 45 (2), 205–251.

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5. Stearns, R.A.; Chakravarty, P.K.; Chen, R.; Chiu, S.H.Biotransformation of losartan to its active carboxylic acid metabolitein human liver microsomes. Role of cytochrome P4502C and 3Asubfamily members. Drug Metab. Disp. 1995, 23 (2), 207–215.

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7. Ramsay, L.E.; Yeo, W.W. Double-blind comparison of losartan,lisinopril and hydrochlorothiazide in hypertensive patients with aprevious angiotensin converting enzyme inhibitor-associated cough.J. Hypertens. 1995, 13 (Suppl. 1), S73–S76.

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16. Fang, X.; Bibart, R.T.; Mayr, S.; Yin, W.; Harmon, P.A.;McCafferty, J.F.; Tyrrell, R.J.; Reed, R.A. Purification andidentification of an impurity in bulk hydrochlorothiazide.J. Pharm. Sci. 2001, 90 (11), 1800–1809.


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17. Revelle, L.K.; Musser, S.M.; Rowe, B.J.; Feldman, I.C.Identification of chlorothiazide and hydrochlorothiazide UV-Aphotolytic decomposition products. J. Pharm. Sci. 1997, 86 (5),631–634.

Received May 28, 2002Accepted December 2, 2002


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