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Simultaneous Determinationof Atenolol and Chlorthalidonein PharmaceuticalPreparations by Capillary-ZoneElectrophoresisKhaldun Al Azzam a , Abdalla A. Elbashir a ,Mohammed A. Elbashir a , Bahruddin Saad a &Shafida Abdul Hamid aa School of Chemical Sciences , Universiti SainsMalaysia , Penang, MalaysiaPublished online: 18 Jun 2009.

To cite this article: Khaldun Al Azzam , Abdalla A. Elbashir , Mohammed A. Elbashir ,Bahruddin Saad & Shafida Abdul Hamid (2009) Simultaneous Determination of Atenololand Chlorthalidone in Pharmaceutical Preparations by Capillary-Zone Electrophoresis,Analytical Letters, 42:10, 1458-1470, DOI: 10.1080/00032710902961065

To link to this article: http://dx.doi.org/10.1080/00032710902961065

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ELECTROPHORESIS

Simultaneous Determination of Atenolol andChlorthalidone in Pharmaceutical Preparations

by Capillary-Zone Electrophoresis

Khaldun Al Azzam, Abdalla A. Elbashir, Mohammed A. Elbashir,

Bahruddin Saad, and Shafida Abdul HamidSchool of Chemical Sciences, Universiti Sains Malaysia, Penang, Malaysia

Abstract: A capillary zone electrophoresis (CZE) method for the simultaneousdetermination of the b-blocker drugs atenolol and chlorthalidone in pharmaceu-tical formulations has been developed. The CZE separation was performed underthe following conditions: capillary temperature, 25�C; applied voltage, 25 kV;20mM H3PO4–NaOH running buffer (pH 9.0); and detection wavelength,198 nm. Phenobarbital was used as internal standard. The method was validatedand showed not only good precision and accuracy but also good robustness. Themethod has been successfully applied to the simultaneous determination of bothatenolol and chlorthalidone in pharmaceutical tablets.

Keywords: atenolol, capillary zone electrophoresis, chlorthalidone, pharmaceuti-cal preparations

Received 19 November 2008; accepted 9 April 2009.Financial support of this project by the Universiti Sains Malaysia (USM)

Research University grant scheme is acknowledged. A USM Postgraduate Fellow-ship is also acknowledged for supporting the PhD study program of K. A. Azzam.

Address correspondence to Bahruddin Saad, School of Chemical Sciences,Universiti Sains Malaysia, 11800 Penang, Malaysia. E-mail: bahrud@usm.my

Analytical Letters, 42: 1458–1470, 2009Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X onlineDOI: 10.1080/00032710902961065

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INTRODUCTION

Hypertension is a common disorder that affects nearly 972 million peopleworldwide, of which 639 million are in developing countries (Moraes et al.2008). It is a growing medical concern and one of the most serious healthproblems faced by modern society. The disease is capable of silently andprogressively affecting organs until damage is evident and irreversible,causing diminished quality of life and shortening life (Ferraro,Castellano, and Kaufman 2003).

Hypertension is defined as a repeatedly elevated blood pressureexceeding 140 over 90mmHg: systolic pressure greater than 140 with dia-stolic pressure greater than 90. Various classes of antihypertensive agentsare commonly used to reduce high blood pressure, such as b-blockers,diuretics, angiotension-converting enzymes inhibitors, angiotensin IIreceptor blockers, calcium-channel blockers, a-blockers, direct vasodila-tors, and centrally acting agents. b-Blockers are useful in managingangina and reducing mortality after myocardial infarction and in heartfailure. The b-blockers atenolol, metoprolol, and propranolol wereamong the 200 most prescribed medications in the USA in 2003 (Khurooet al. 2008).

For better treatment of hypertension, therapy combining two ormore drugs is preferred over monotherapy with a single drug. The com-bination of a b-blocker and a diuretic produces additive effects comparedwith monotherapy using either drug alone. The rationale for combiningb-blockers with diuretics is twofold: b-blockers blunt the increase in theplasma rennin level that is induced by diuretics, and diuretics decreasethe sodium and water retention caused by b-blockers (Khuroo et al.2008). Thus, a combination of atenolol (b-blocker) and chlorthalidone(diuretic) is commonly used to treat hypertension.

Liquid chromatography (LC)–mass spectroscopy (MS) methods forthe determination of b-blockers in biological specimens have beenreported (Moraes et al. 2003; Johnson and Lewis 2006). Capillary zoneelectrophoresis (CZE) methods for the determination of atenolol inhuman urine and plasma have been developed (Maguregui, Alonso,and Jimenez 1997; Arias et al. 2001). A CZE method for the determina-tion of atenolol, amiloride, hydrochlorthiazide, and bendroflumethiazidein pharmaceutical formulations and urine was reported (Maguregui,Alonso, and Jimenez 1998). Recently Balesteros, Faria, and Oliveira(2007) developed a CZE method for the determination of chlorthalidoneand losartan in capsules. Reports for determining chlorthalidone by gaschromatography (GC) (Magnar and Klas 1974; Degen and Schweizer1977) or LC for the simultaneous determination of both atenolol andchlorthalidone (Dadgar and Kelly 1988; Muirhead and Christie 1987;

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Giachetti et al. 1997; Rapado-Martinez, Garcia-Alvarez-Coque, andVillanueva-Camanas 1997; El-Gindy, Sallam, and Abdel-salam 2008;Sa’sa, Jalal, and Khalil 1988) can also be found. High-performancethin-layer chromatographic (HP-TLC) methods for the determinationof certain antihypertensive mixtures (Salem 2004) have also beenreported.

Ultraviolet (UV) spectrophotometric methods for the determinationof atenolol and metoprolol in pharmaceutical formulations (Bonazzi

Figure 1. Chemical structures of atenolol, chlorthalidone, and phenobarbital(internal standard).

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et al. 1996) and for simultaneous determination of atenolol and chlortha-lidone in pharmaceutical formulations (Wehner 2000; Vetuschi andRagno 1990) have also been reported. In some of these works, partialleast squares analysis of the UV spectral data for the determination ofatenolol and chlorthalidone in synthetic binary mixtures and pharmaceu-tical formulations (Ferraro, Castellano, and Kaufman 2003; Mohamedand Salem 2005); for the simultaneous determination of atenolol, amilor-ide hydrochloride, and chlorothalidone in pharmaceutical formulations(El-Gindy, Emara, and Mostafa 2005); and for determination ofamiloride hydrochloride, atenolol, hydrochlorothiazide, and timololmaleate in synthetic mixtures and pharmaceutical formulations (Ferraro,Castellano, and Kaufman 2004) were conducted.

However, to best of our knowledge, a CZE method for the simulta-neous determination of atenolol and chlorthalidone in coformulatedpreparations has not been reported. In this communication, a simple,sensitive CZE method that allows the simultaneous determination ofboth active ingredients is reported for the first time. The method wasfurther validated as per ICH-Q2A guidelines (ICH 1995) and was appliedto the quality control of coformulated preparations and atenolol insingle-active-ingredient preparations. Phenobarbital (Fig. 1) was usedas internal standard (IS).

EXPERIMENTAL

Chemicals and Reagents

Analytical-grade methanol was purchased from Merck (Darmstadt,Germany). Deionized water was purified by a Milli-Q system (Millipore,Bedford, USA) and was used throughout for the preparation ofsolutions. Orthophosphoric acid (85%) and sodium hydroxide werepurchased from Sigma-Aldrich (Milan, Italy). Tablets were purchasedfrom local pharmacy stores. Hypoten tablets (100mg atenolol), atenolol,chlorthalidone, and phenobarbital standards were kindly donated byHIKMA Pharmaceutical Company, Jordan.

Capillary Zone Electrophoresis Apparatus and Operating Conditions

Separations were conducted on an HP3DCE CZE system (AgilentTechnologies, Waldbronn, Germany). The unit was equipped with aphotodiode array detector. Uncoated fused-silica capillary (75 mm i.d. �65 cm; detection length 8.5 cm from the outlet end of the capillary) from

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Agilent Technologies was used. Data acquisition was performed withChemStation software. The new capillary was conditioned by flushingfor 30min with 1M NaOH, 10min with 0.1M NaOH, and 15min withwater. Between injections, it was preconditioned for 3min with 0.1MNaOH and 4min with the running buffer prior to each subsequent run.Samples and standards were injected hydrodynamically at 50mbar for5 s under the following conditions: voltage, 25 kV (positive polarity);capillary temperature, 25�C; detector wavelength, 198 nm; and therunning buffer (20mM H3PO4–NaOH solution), pH 9.0. At the end ofthe day, a final 5-min washing with water was performed. All standards,sample solutions, the running buffer, and NaOH solution were filteredthrough 0.45-mm regenerated cellulose membrane filter using Agilentsolvent filtration kit.

Preparation of Standard Solutions

Stock solutions of atenolol, chlorthalidone, and phenobarbital (500 mgmL�1 each) were prepared by adding 2mL methanol, then topped withwater to the desired concentrations. The stock solutions were used toprepare calibration standards. Working solutions for atenolol andchlorthalidone were prepared by serially diluting the stock solution withwater. All solutions were stored refrigerated in the dark when not in use.

Preparation of Sample Solutions

Ten tablets from each sample were ground into fine powder in a mortar.The powder (equivalent to about 7.5mg atenolol) were quantitativelytransferred into 50-mL volumetric flasks, dissolved with 2mL of metha-nol, and sonicated for 5min, then another 20mL water were added. Themixture was sonicated for another 5min and finally topped up to themark with water. The solution was filtered through a 0.45-mm membranefilter before being subjected to the CZE analysis.

RESULTS AND DISCUSSION

Optimization of Electrophoretic Conditions

Under high pH conditions, basic atenolol (pKa¼ 9.6) exists pre-dominantly in the negatively charged form and therefore migratestoward the anode. Moreover, because the pKa values of the analytes

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are around 9, analytes were almost completely deprotonated under therunning buffer pH used. To study the effect of pH on the peak shapeand migration time, a constant concentration of 20mM H3PO4–NaOHsolution as running buffer was investigated over the pH range 7.5–9.5.In general, the migration time decreased as pH is increased. The bestresults were achieved at pH 9.0. Therefore, a running buffer with pH9.0 was chosen for the next measurements.

The effect of buffer concentration was studied by varying both theconcentrations of H3PO4 and NaOH from 20 to 100mM at a constantpH of 9.0. The migration time increased with increasing buffer concentra-tion. When 20mM H3PO4–NaOH concentration buffer was used, goodpeak shape was obtained. Based on the migration time and the currentgenerated, 20mM H3PO4–NaOH was chosen.

The influence of voltage (15–30 kV) on the migration time wasevaluated under the optimized running buffer conditions. When theapplied voltage was increased, peak broadening and decrease in bothmigration time and resolution were observed. This is probably becauseas the applied voltage increases, the increasing Joule heat leads to areduction in migration time due to the increase in the apparent mobilitywith increasing voltage. Therefore, 25 kV was chosen as the appliedvoltage.

The influence of capillary temperature (16–30�C) was evaluatedunder the chosen running buffer conditions. When the temperature isincreased, migration time decreases.

It is imperative to control Joule heating because this parameteris directly linked to the analyte mobility, stability, and system reproduci-bility. Decreasing viscosity with temperature is responsible for the non-linearity of the dependence of velocity upon temperature, whereasincrease in the diffusion coefficient of analyte is responsible for thepoorer than expected performance at high temperatures. Therefore,25�C was chosen as the working temperature for the analysis.

Optimization of sample injection time (3–20 s) at 50 mbar wasconducted to achieve a lower detection limit without affecting the qualityof the peak shape and reproducibility, migration time, and resolution. Aninjection time of 5 s offered best results and was selected for the rest of thestudies.

Conditions for the simultaneous analysis of atenolol and chlrothali-done were 20mM H3PO4–NaOH, pH 9.0, as running buffer; injectiontime, 5 s; applied voltage, 25 kV; capillary temperature, 25�C; and thedetection wavelength, 198 nm. A typical eletrophoregram obtained underthe adopted conditions is shown in Fig. 2. The suitability of pheno-barbital as internal standard is evident because it resolved well fromthe analyte peaks. All components were eluted in less than 5min.

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Validation Procedure

Calibration Curves, Limits of Detection, and Quantitation

The working solutions containing all three standard compounds were allprepared as described previously to construct the calibration curves. Eachcalibration curve contained six different concentrations (5–250 mg mL�1)

Figure 2. Typical electropherograms obtained when operated under the adoptedconditions: (a) 25 mgmL�1 standard and (b) Tenoret tablet; 1, atenolol; 2,chlorthalidone; and 3, internal standard (phenobarbital). Conditions: 20mMH3PO4–NaOH, pH 9.0, 25 kV, 25�C, and 5-s injection time.

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and was performed in triplicate. Calibration curves with regressionequations for both atenolol and chlorthalidone were y¼ 0.03960x andy¼ 0.04792x, respectively, which were obtained by plotting the concen-tration of atenolol or chlorthalidone against the relative corrected peakarea. The limit of detection (LOD) and limit of quantitation (LOQ) foratenolol were 1.5 and 4.7 mgmL�1 respectively, and for chlorthalidonethey were 1.0 and 3.0 mgmL�1, respectively. The LOD was calculatedas the amount of the injected sample to yield a signal-to-noise ratio of3, and the LOQ was taken as the amount of the injected sample to givea signal-to-noise ratio of 10. All the standards showed good linearity(r2> 0.999) over a relatively wide concentration range (5–250 mgmL�1).As expected, the sensitivity of the proposed CE method is slightly inferiorcompared to the reported HPLC method (El-Gindy, Sallam, and Abdel-salam 2008) (LODs for atenolol and chlorothalidone were 0.30 and0.25 mgmL�1, respectively). However, the analysis time of the proposedCE is faster (5min compared to�10min in the HPLC report).

Precision

Intra- and interday variations were used to determine the precision of thedeveloped method by analyzing three concentrations (10, 25, and 100 mgmL�1) of standard solutions. The intraday variation was determined byanalyzing the nine replicates on the same day, whereas interday variationwas conducted over six consecutive days. Intra- and interday precision,expressed as the percentage of relative standard deviation (RSD), rangedfrom 1.85 to 2.85 and from 1.87 to 2.60%, respectively (Table 1), indicat-ing the good precision of the newly developed method. The precision iscomparable to the HPLC results (RSD less than 3%; El-Gindy, Sallam,and Abdel-salam 2008).

Table 1. Intra- and interday precision for the determination of atenolol- andchlorthalidone-spiked tablets

SubstanceAmount(mgmL�1)

Intraday(RSD %), n¼ 9

Interday (RSD %),n¼ 54

Atenolol 10 2.85 2.1325 1.95 1.87100 2.05 2.25

Chlorthalidone 10 2.76 2.6025 1.85 1.96100 2.20 2.31

Note. n¼ no. of introductions (three preparations for each concentration).

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Accuracy

The accuracy of the method was determined by performing recovery tests.An appropriate amount of Tenoretic tablet powder was weighed andspiked with a known amount of the standard compounds, and each sam-ple was analyzed in triplicate. Accuracy values ranged from 96.9 to 106.6%(for atenolol) and from 100.9 to 103.2% (for chlorthalidone) (Table 2). Theaccuracy values obtained indicate the potential of this method for thedetermination of both analytes in different pharmaceutical formulations.

Robustness

A robustness test was performed to investigate the reliability of resultswhen the experimental parameters were slightly changed. Varying thepH of the running buffer by�0.1, the concentration, and applied voltage

Table 2. Accuracy results for the determination of atenolol and chlorthalidone inspiked tablets

Substance Amount (mgmL�1) Accuracy (n¼ 9)

Atenolol 10 96.950 106.6100 106.4

Chlorthalidone 10 103.250 101.9100 100.9

Table 3. Determination of atenolol and chlorthalidone in tablets (Tenoret 50)under different conditions using the CZE method (n¼ 6)

CZE running conditions

Atenolol Chlorthalidone

Mean� SD RSD (%) Mean� SD RSD (%)

Adopted conditionsa 104.12� 0.38 0.37 97.35� 0.60 0.61Buffer, pH 8.9 104.98� 1.16 1.11 98.39� 0.39 0.39Buffer, pH 9.1 105.01� 0.43 0.41 99.02� 1.08 1.0919mM H3PO4–NaOHbuffer

105.64� 0.15 0.14 99.49� 1.03 1.04

21mM H3PO4–NaOHbuffer

105.30� 1.10 1.04 98.12� 1.00 1.02

24 kV 104.80� 0.29 0.28 97.96� 0.57 0.5826 kV 104.11� 0.53 0.51 97.13� 0.56 0.57

aPlease refer to text for details.

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Table

4.Assayresultsofatenololandchlorthalidonein

differentpharm

aceuticalform

ulations

Tradename

Manufacturer

Generic

name

Label

claim

(atenolol=chlorthalidone),mg

Agreem

ent(%

)

Atenolol

(%)�SD

Chlorthalidone

(%)�SD

Tenoret50

Astrazeneca

Atenololþ

Chlorthalidone

50=12.5

104.1�0.38

97.4�0.60

Tenoretic

Astrazeneca

Atenololþ

Chlorthalidone

100=25

104.9�0.38

96.2�0.74

Noten

a-alphapharm

Atenolol

50

97.6�0.55

N=D

a

Apo-atenol

Apotex

Atenolol

50

98.2�0.34

N=D

Ternolol

Hovid

Atenolol

50

97.9�0.47

N=D

Ternolol

Hovid

Atenolol

100

99.2�0.31

N=D

Norm

aten

Mim

sAtenolol

100

103.8�0.36

N=D

Hypoten

Hikma

Atenolol

100

100.0�0.82

N=D

aN=D,notdetected.

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by�1 did not have a significant effect on the results, indicating therobustness of the developed method (Table 3).

Analysis of Pharmaceutical Formulations

The developed method has been successfully applied for the simultaneousdetermination of atenolol and chlorthalidone in two coformulated tabletsand six atenolol tablets. Good agreement between the proposed methodand the manufacturer’s claimed values were found for all samples(Table 4). Figure 2b shows a typical electropherogram of the pharmaceu-tical formulations.

CONCLUSIONS

A CZE method for the simultaneous determination of atenolol andchlorthalidone was successfully developed. Under the adopted conditions,baseline separation of atenolol and chlorthalidone and the internal stan-dard were obtained in less than 5min. Good analytical performance withregards to linearity, reproducibility, accuracy, and robustness was achieved.All the validated data obtained are in compliance with the ICH-Q2A guide-lines (ICH 1995). As expected, compared to the HPLC method (El-Gindy,Sallam, and Abdel-salam 2008), the proposed method exhibited less sensi-tivity because of the shorter path length of the flow cell, but neverthelessprovided faster analysis time (<5min compared to �10min with the HPLCmethod). Greater separation efficiency and minimization of use of solventsare other inherent features of CE methods. The proposed method is there-fore recommended as quality control protocol in pharmaceutical industries.

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