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Simultaneous Determination
of Valsartan and
Hydrochlorothiazide in
Tablets by High-Performance
Liquid Chromatography
Giuseppe Carlucci , Valeria Di Carlo & Pietro
Mazzeo
a
Dipartimento di Chimica, Ingegneria Chimica
e Materiali, Università di L'Aquila, Via Vetoio,
67010 Coppito (L'Aquila), Italy
Published online: 27 Feb 2008.

To cite this article: Giuseppe Carlucci , Valeria Di Carlo & Pietro Mazzeo (2000):
Simultaneous Determination of Valsartan and Hydrochlorothiazide in Tablets by
High-Performance Liquid Chromatography, Analytical Letters, 33:12, 2491-2500

To link to this article: http://dx.doi.org/10.1080/00032710008543204

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 ANALYTICAL LETTERS, 33(12), 2491-2500 (2000)

SIMULTANEOUS DETERMINATION O F VALSARTAN AND

HYDROCHLOROTHIAZIDE IN TABLETS BY HIGH-PERFORMANCE
LIQUID C H R O M A T O G R A P H Y
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Key Words: HPLC, Valsartan, ^Hydrochlorothiazide, Simultaneous

Determination,.

Giuseppe Carlucci, Valeria Di Carlo and Pietro Mazzeo

Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università di

L'Aquila, Via Vetoio, 67010 Coppito (L'Aquila), Italy.

ABSTRACT

A method for the simultaneous determination of valsartan and

hydrochlorothiazide in tablets is described. The procedure, based on the use of
reversed-phase high-performance liquid chromatography, is linear in the
1 1
concentration range 5.0-10.0 jig ml" for valsartan and 0.5-2.0 ug ml" for
hydrochlorothiazide, is simple and rapid and allows accurate and precise results.
1 1
The limit of detection was 1.0 ug ml" for valsartan and 0.05 ug ml" for
hydrochlorothiazide.

2491

Copyright © 2 0 0 0 by Marcel Dekker, Inc. www.dekker.com

 2492 CARLUCCI, DI CARLO, AND MAZZEO

H3C
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H 2 NO 2 S' so2

Fig. 1: Chemical structure of valsartan (A), hydrochlorothiazide (B) and

furprofen (C).

INTRODUCTION

Valsartan, or N-(l-oxopentyl)-N-[[2'-(lH-tetrazol-5-yl)[l,l'-biphenyl]-4-
yl]methyl]-L-valine (Fig. 1A), is a prototype of the new generation of effective
and orally active non peptide angiotensin II ATi receptor antagonists, which
 VALSARTAN AND HYDROCHLOROTHIAZIDE 2493

have been developed in sequence to the angiotensin converting enzyme

inhibitors as a further therapeutic action on the renin-angiotensin-aldosterone
system, one of the most important regulators of blood pressure1'2.
Hydrochlorothiazide, or 6-chloro-3,4-dihydro-2H-l,2,4-benzothiadiazine-7-
sulfonamide 1,1-dioxide (Fig. IB), is a diuretic of the class of benzothiadiazines
widely used in antihypertensive pharmaceutical formulations, alone or in
combination with other drugs, which decreases active sodium reabsorption and
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reduces peripheral vascular resistance.

The two drugs are successfully used in association in the treatment of
hypertension3'5.
The determination of valsartan has been carried out in plasma by high-
performance liquid chromatography6 and in urine by gas chromatography-mass
spectrometry7.
Several analytical procedures have been described for the individual
determination of hydrochlorothiazide, most frequently by using electrochemical
or spectrophotometric methods8, and jointly with other pharmaceutical
substances, including spectrophotometric9"" and HPLC10'16"18 procedures.
So far no method available for the simultaneous determination of these two
drugs in pharmaceutical forms or in biological fluids has been described.
This paper describes a method for the simultaneous determination of
hydrochlorothiazide and valsartan in tablets. The procedure, based on the use of
reversed-phase high-performance liquid chromatography, is simple and rapid
and provides accurate and precise results.

MATERIALS AND METHODS

Materials
Valsartan and furprofen were kindly supplied by the Department of Internal
Medicine of the University of L'Aquila; hydrochlorothiazide was purchased
from Sigma-Aldrich (Milan, Italy). Acetonitrile (HPLC grade) and all other
 T
2494 CARLUCCI, DI CARLO, AND MAZZEO

analytical-grade reagents were obtained from Farmitalia-Carlo Erba (Milan,

Italy). Water (HPLC grade) was obtained by distillation in glass and passage
through a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Chromatographic System and Conditions

HPLC analysis was carried out using a Waters (Waters, Milford, MA, USA)
system composed of the following: a Model 510 pump, a Model 996 diode array
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detector equipped with a Millennium 2.10 data elaborator. A Model 7125 sample
injector (Rheodyne, Cotati, CA, USA) equipped with a 20 \d loop was used.
The analysis was performed on an analytical 250 x 4.6 mm I.D. reversed-
phase Hypersil ODS (5 nm particle size) column (Alltech Associates, Deerfield,
IL, USA), protected by a 20 x 4.6 mm I.D. disposable (40 \an particle size)
Pelliguard precolumn (Supelco, Bellefonte, PA.USA). Separations were
performed at room temperature (range 20-25° C).
The mobile phase consisted of a mixture of acetonitrile and acetate buffer (pH
4.0; 0.1 M)(40:60, v/v). Phosphate buffer prior to use was filtered through an HA
0.45 pm filter, while acetonitrile through a FA 0.5 jun filter (Millipore, Bedford,
MA, USA). The mobile phase was prepared daily, sonicated before use and
delivered at a flow rate of 1.0 ml min'1.
The spectra were registered in the range 200-400 run; column eluate for
calibration curves was monitored to 220 nm.

Standard Solutions and Calibration Curves

Stock solutions containing valsartan (1.0 mg ml'1) and hydrochlorothiazide
(1.0 mg ml"1) were prepared by dissolving a weighed amount of substance in the
mobile phase. Standard solutions were prepared by dilution of the above stock
solutions with mobile phase and by varying the valsartan concentration in the
range 5.0-10.0 ng ml"1 (maintaining the hydrochlorothiazide concentration at a
constant level of 1.5 |ig ml"1) and the hydrochlorothiazide concentration in the
 VALSARTAN AND HYDROCHLOROTHIAZIDE 2495

range 0.5-2.0 jig ml"1 (maintaining the valsartan concentration at a constant level
of 9.6 jig ml"1).
The stock solution of internal standard (furprofen; Fig. 1C)(1.O mg ml"1) was
prepared with mobile phase. An aliquot of the internal standard solution, after
appropriate dilution, was added to each standard solution so as to give a final
concentration of 10 ug ml'1.
The standard solutions could be stored at 4°C for over one month with no
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evidence of decomposition.
The calibration curves for HPLC analysis were obtained by plotting the
peak-area ratio of each drug to internal standard versus its concentration.The
equations, obtained through regressional analysis of data for the above standard
solutions (each datum average of a minimum number of five determinations)
were: for valsartan y = 0.25 lx - 0.099 (r = 0.9998) and for hydrochlorothiazide y
= 0.468x - 0.002 (r = 0.9995), where y is the peak-area ratio in the arbitrary
units of the detector used and x is the drug concentration (ug ml"1).
The RSD values of the slope were 1.5% (valsartan) and 2.4%
(hydrochlorothiazide); those of the intercept were very high (until 20%) because
of their low numerical values.

Analysis of Tablets
Five tablets were crushed and combined. An amount of material was
accurately weighed, added with acetonitrile and centrifuged at 3000 rpm for 15
min. The clear supernatant was completely transferred into a 100 ml calibrated
flask and diluted to volume with acetonitrile.
The solution obtained was diluted with the mobile phase so as to obtain a
concentration of the two drugs in the range of linearity previously determined.
An aliquot of the internal standard solution was added to the sample solution
prior to the dilution so as to give a final concentration of internal standard of
10.0 ng ml"1. HPLC analysis was carried out on aliquots of 20 jxl by using the
corresponding calibration curves.
 2496 CARLUCCI, DI CARLO, AND MAZZEO

B
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0 * 8 12
time(min)
Fig. 2: HPLC profile of a standard solution containing hydrochlorothiazide (A;
1.5 ng ml'1), furprofen (B; 10 ng ml*1) and valsartan (C; 5 \ig ml 1 ). Vertical
axis: UV detector response at 220 run.

RESULTS AND DISCUSSION

Fig. 2 illustrates a typical HPLC chromatogram obtained from a standard

solution; Fig. 3 the relative 3D elution profile. The retention times were 3.6, 6.4
and 11.2 min for hydrochlorothiazide, furprofen (internal standard) and
valsartan, respectively. The corresponding capacity factors were 2, 4.3 and 8.3
 VALSARTAN AND HYDROCHLOROTHIAZIDE 2497
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0.00 2.00 4.00 S.OO S.OO 10.00 12.00 14

Fig. 3: 3D elution profile of the standard solution of Fig. 2.

(column void marker utilized: sodium nitrate). No interfering substance was

observed in the samples chromatograms.
Table 1 shows the results obtained in the analysis of a commercial
antihypertensive formulation.
No potential interference may derive from their composition. A comparison
with a reference determination method has not been possible because so far no
other procedure for the simultaneous quantitation of these drugs has been
reported.
The degree of reproducibility of the results obtained through small deliberate
variations in method parameters and by changing instruments and operators has
been very satisfactory.
 2498 CARLUCCI, DI CARLO, AND MAZZEO

TABLE 1
Results Obtained in the Analysis of Pharmaceutical Tablets1

Commercial sample I nominal I found II nominal II found

(mg) (mg) (mg) (mg)

1 80.0 79.8 12.5 12.4

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I: valsartan
II: hydrochlorothiazide
" Analyses in triplicate (RSD: 0.2% for I and 0.3% for II)

The proposed method shows a good linearity in the concentration ranges

examined. The RSD for valsartan is comprised between 0.1 and 0.6 %, for
hydrochlorothiazide between 0.2 and 0.4 %. The limit of detection was 1.0 ng
ml'1 for valsartan and 0.05 ng ml"1 for hydrochlorothiazide.
The accuracy of the method was determined by investigating with the
described procedure mixtures of accurately weighed amounts of the two drugs.
The relative error is variable in the range 0.1-0.4 % for valsartan and 0.1-2.0 %
for hydrochlorothiazide.

CONCLUSIONS

This method described for the simultaneous determination of valsartan and

hydrochlorothiazide in antihypertensive pharmaceutical forms is very simple and
rapid, provides accurate and precise results, and, through an appropriate
biological samples extraction procedure, should be of value for the concomitant
quantitation of the two drugs in human plasma and for the study of their
pharmacokinetics.
 VALSARTAN AND HYDROCHLOROTHIAZIDE 2499

ACKNOWLEDGEMENTS
This research was supported by a grant from the Ministero dell'Universita e
della Ricerca Scientifica e Tecnologica.

REFERENCES

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(1994).

Received: February 29, 2000

Accepted: May 17, 2000