HPLC and chemometric-assisted spectrophotometric methods forsimultaneous determination of atenolol, amiloride hydrochloride

and chlorthalidone

Alaa El-Gindy *, Samy Emara, Ahmed Mostafa

Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt

Received 24 June 2004; received in revised form 20 November 2004; accepted 22 November 2004

Abstract

Three methods are presented for the simultaneous determination of atenolol (AT), amiloride hydrochloride (AM) and chlorthalidone (CD).The high performance liquid chromatographic (HPLC) method depends on the separation of each drug on a reversed phase, RP 18 column.Elution was carried out with a mobile phase consisting of acetonitrile -5mM heptansulphonic acid sodium salt (20:80, v/v, pH 4.4). Quanti-tation was achieved with UV detection at 274 nm based on peak area. The other two-chemometric-assisted spectrophotometric methodsapplied were principal component regression (PCR) and partial least squares (PLS-1). These approaches were successfully applied to quantifyeach drug in the mixture using the information included in the absorption spectra of appropriate solutions in the range 240–290 nm with theintervals Dk = 0.2 nm. The three methods were successfully applied to a pharmaceutical formulation (tablets), and the results were comparedwith each other.© 2005 Elsevier SAS. All rights reserved.

Keywords: Atenolol; Amiloride hydrochloride; Chlorthalidone; HPLC; Chemometrics

1. Introduction

Chlorthalidone (CD) is a diuretic with actions and indica-tions similar to those of the thiazide diuretics [1]. It is pre-scribed with atenolol (AT), which is a cardioselectiveb-blocker and amiloride hydrochloride (AM), which is a milddiuretic. They are used in the treatment of hypertension. TheUV absorption spectra of the studied drugs show severe over-lap. Hence, their simultaneous determination is difficult whenconventional, derivative, and derivative ratio spectrophoto-metric techniques are used. No analytical method has beenreported for the simultaneous determination of AT, AM, andCD in a multicomponent mixture, while several analyticalmethods have been reported for the determination of AT orAM or CD in combination with other drugs, including spec-trophotometry [2–12], spectrofluorimetry [13–15], HPLC[11,16–20] and HPTLC [21,22].

Multivariate calibration methods, in combination with sev-eral techniques such as spectrophotometry [23–26], fluorim-

etry [27,28], polarography [29] and voltammetry [30,31], havebeen applied in analytical procedures. The application of mul-tivariate calibration to the absorbance signals produced bydrugs during their simultaneous determination in pharmaceu-tical preparations is an effective means for quality control oftheir manufacture [32]. The PLS and PCR were found to bespecially suited to multicomponent analysis, particularly formixture with highly overlapped spectra [33].

The aim of this paper is to investigate the ability of PLS-1 and PCR methods to quantify a three-component mixtureof AT, AM and CD with overlapping UV spectra, and to applythe optimized models in pharmaceutical preparations. In addi-tion, a HPLC method was developed for the assay of the com-ponents of the studied mixture. The proposed methods aresimple and accurate. They resulted in a significant reductionin analysis time and proved to be suitable for routine deter-mination of the three components of the studied mixture.

The proposed HPLC method was found to be simpler thanthe published chromatographic methods for determination ofthe studied drugs in other combined formulations. In thismethod, there is no need for internal standard while the other

* Corresponding author.E-mail address: ghhadad@hotmail.com (A. El-Gindy).

Il Farmaco 60 (2005) 269–278

http://france.elsevier.com/direct/FARMAC/

0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.doi:10.1016/j.farmac.2004.11.013

published chromatographic methods used internal standardssuch as metoprolol [16], methyl 4-hydroxybenzoate [17], felo-dipine besylate [18] and hydrochlorothiazide [22]. More-over, the proposed methods are the first publication for thesimultaneous determination of AT, AM and CD in pharma-ceutical preparations.

2. Experimental

2.1. Instrumentation

A double-beam Shimadzu (Japan) UV-visible spectropho-tometer, model UV-1601 PC equipped with 1-cm quartz cellsand connected to an IBM compatible computer.A HP600 ink-jet printer was used. The bundled software was UVPC per-sonal spectroscopy software version 3.7 (Shimadzu). Thespectral bandwidth was 2 nm and the wavelength scanningspeed was 2800 nm min–1. PLS-1 and PCR analyses werecarried out by using PLS-Toolbox software version 2.1-PC[34] for use with MATLAB 5.

The HPLC (Shimadzu, Kyoto, Japan) instrument wasequipped with a model series LC-10 ADVP pump, SCL-10 AVP system controller, DGU-12 A degasser, Rheodyne7725i injector with a 20 µl loop and a SPD-10 AVP UV-VISdetector; separation and quantitation were made on a150 × 4.6 mm (i.d.) TSK- Gel 5 µm ODS-80 TM column(Tosoh, Japan). The detector was set at 274 nm. Data acqui-sition was performed on class-VP software.

2.2. Materials and reagents

Pharmaceutical grade of AT, AM and CD was used andcertified to contain 99.8, 99.9 and 99.85%, respectively. Themethanol used was HPLC grade (BDH, Poole, UK). Heptane-sulphonic acid sodium salt, hydrochloric and acetic acids wereanalytical grade. Teklo tablet (ACAPI, Badr city, third indus-trial zone, Cairo, Egypt) was used. Each tablet was labeled tocontain 100 mg AT, 5 mg AM and 25 mg CD.

2.3. HPLC conditions

The mobile phase was prepared by mixing acetonitrile and5mM heptanesulphonic acid sodium salt in a ratio of (20:80,v/v). The pH of the mobile phase was adjusted to 4.4 usingacetic acid and the flow rate was set at 1 ml min–1. All deter-minations were performed at ambient temperature and theinjection volume was 20 µl.

2.4. Standard solutions and calibration

Standard solutions of each of AT, AM and CD were pre-pared by separately dissolving 100 mg of each drug in 100 mlmethanol then further dilutions were made in 0.1 M hydro-chloric acid (for spectrophotometric methods) or mobile phase(for HPLC method) within the concentration rang of 40–

160 µg ml–1 for AT, 2–8 µg ml–1 for AM and 10–40 µg ml–1

for CD.

2.4.1. Calibration of PLS-1 and PCR methodsA training set of 33 synthetic mixtures with different con-

centrations of each compound were prepared in 0.1 M hydro-chloric acid in the concentration ranges of 40–160 µg ml–1

for AT, 2–8 µg ml–1 for AM and 10–40 µg ml–1 for CD(Table 1). The UV absorption spectra were recorded over thewavelength range of 240–290 nm. The data points of the spec-tra were collected every 0.2 nm. The computation was madeusing PLS-Toolbox software version 2.1.

The PLS-1 and PCR models were applied to the UVabsorption spectra of these mixtures using 3 latent variablesfor AT and 4 latent variables for AM and CD by PLS-1. Fourprincipal components were used for PCR determination ofeach compound.

2.4.2. Calibration of HPLC methodTriplicate 20 µl injections were made for each concentra-

tion and chromatographed under the specified chromato-graphic conditions described previously. The peak area val-ues were plotted against corresponding concentrations. Linearrelationships were obtained (Table 2).

2.5. Sample preparation

Twenty tablets were weighed and finely powdered. Anaccurately weighed portion of the powder equivalent to about100 mg of AT, 5 mg of AM and 25 mg CD was extracted anddiluted to 100 ml with methanol. The sample solution wasfiltered. Further dilution of the filtrate was carried out with0.1 M hydrochloric acid (for spectrophotometric methods) orwith the mobile phase (for the HPLC method) to provide asolution of 100 µg ml–1 ofAT, 5 µg ml–1 ofAM and 25 µg ml–1

of CD.

2.5.1. Procedures for the determination of AT, AM and CDusing PLS-1 and PCR methods

The UV absorption spectrum of final solution was recordedover the wavelength range of 240–290 nm. The data points ofthe spectrum were collected every 0.2 nm. The PLS-1 modelwas applied using 3 latent variables for AT and 4 latent vari-ables for AM and CD. The PCR model was applied usingfour principal components. The concentration of each com-pound was calculated using each model.

2.5.2. Procedures for the determination of AT, AM and CDusing HPLC method

Triplicate 20 µl injections of the final solution were injectedand chromatographed under the specified HPLC conditionspreviously described in 2.3. The peak area values were deter-mined and the concentration of each compound was calcu-lated.

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3. Results and discussion

3.1. PCR and PLS-1 methods

Fig. 1 shows the UV absorption spectra of AT, AM andCD at their nominal concentrations in the tablet. A signifi-cant overlap in absorption bands was noticed. The simulta-neous determination of AT and CD in the tablet by conven-tional, derivative and derivative ratio spectrophotometricmethods is hindered by strong spectral overlap throughoutthe wavelength range. The PLS or PCR calibration methodswere necessary for such determination due to the presence ofinterference, while the UV spectrum of AM is not stronglyaffected by the presence of AT and CD. This is a favorablecondition and conventional zero-order or direct derivativemethods could be applied for determination ofAM in the stud-ied ternary mixture.

The quality of multicomponent analysis is dependent onthe wavelength range and spectral mode used [35]. The PLSprocedures are designated to be full spectrum computationalprocedures; however, using highly noisy, scarcely informa-tive wavelengths detract from precision. Discarding particu-larly noisy wavelengths can lessen this. This is quite sensiblein UV-Vis spectrophotometry as the pure spectra of the ana-lytes are often available and the positions of their bands arenot usually greatly affected by the presence of the excipients,so one can predict which spectral region in the sample spec-trum will contain the information relevant to the analyte [36].In this work, original and reconstructed spectra of the cali-bration matrix were compared in order to select the range ofwavelengths. The range was obtained by all regions in whichthe difference between each component of the mixture andthe others was maximum. Besides, the regions in which eachcomponent of the mixture was best reconstructed were also

Table 1Concentration data for the different mixtures used in the calibration set and internal validation for the determination of AT, AM and CD using PLS-1 and PCRmethods

Mixture composition (µg ml–1) Internal validation (% recovery)PLS-1 PCR

Mixture no AT AM CD AT AM CD AT AM CD1 60 3 15 99.5 99.7 101.0 100.0 99.8 101.02 100 5 25 100.6 101.0 99.1 100.7 101.0 99.13 140 7 35 99.3 101.6 99.6 100.1 101.5 99.64 80 5 40 100.1 101.7 100.0 100.0 101.3 100.05 160 5 25 100.1 100.4 101.5 99.4 99.5 101.66 160 3 15 99.4 99.2 99.7 98.9 100.3 100.67 120 4 25 98.8 100.0 100.6 100.6 100.1 98.48 40 2 35 100.4 99.8 98.5 100.1 98.8 100.49 100 6 10 100.2 98.9 100.4 100.9 100.0 99.210 120 6 30 101.0 100.4 99.2 99.3 100.5 99.511 140 5 25 98.9 100.6 99.5 100.0 101.5 100.512 80 3 20 99.8 100.2 100.3 99.5 99.8 101.513 160 2 35 100.2 102.0 100.4 99.9 100.6 99.914 160 5 30 99.6 99.6 101.4 99.4 100.0 100.415 160 7 10 100.2 100.6 99.9 100.2 99.1 99.716 140 8 35 99.6 99.8 100.4 98.2 99.3 100.517 120 8 25 99.0 100.1 100.4 100.7 100.6 99.318 60 8 20 100.5 99.3 99.6 99.7 100.1 100.619 140 7 40 100.9 99.4 100.5 100.4 98.8 99.520 40 4 40 99.9 100.9 99.3 100.0 98.9 100.521 100 5 40 100.6 98.5 100.6 98.9 99.9 101.522 120 7 30 99.9 98.8 99.5 100.3 99.8 100.723 100 6 35 100.2 99.3 100.5 100.9 100.9 99.724 60 3 30 101.0 100.0 101.5 100.6 99.9 98.925 40 2 15 100.2 100.0 100.7 99.7 98.0 99.026 40 5 30 99.2 100.8 99.7 98.7 100.6 101.927 60 5 35 100.2 100.2 98.9 100.1 98.8 100.428 100 2 10 100.3 100.1 99.1 100.2 98.9 100.029 80 2 15 100.2 100.1 101.9 100.2 101.6 99.230 40 3 15 99.0 98.9 100.4 99.2 101.2 101.731 120 6 30 100.4 98.7 99.9 100.2 99.2 100.232 100 7 30 100.0 101.6 99.2 99.9 99.7 99.333 80 5 35 100.6 101.0 101.2 101.0 98.7 99.7Meana 100.0 100.1 100.1 99.9 100.0 100.1S.D.a 0.59 0.90 0.83 0.66 0.92 0.87

a Mean and S.D., percentage recovery from the label claim amount.

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considered. The spectral region between wavelengths 240 and290 nm was selected for this purpose as it provided the great-est amount of information about the mixture components. Thisentailed using 251 experimental points per spectrum, as spec-tra were digitized at 0.2 nm intervals. In addition, wave-lengths less than 240 nm were rejected due to the high UVcontribution of AT and CD relative to AM and the differencebetween the synthetic mixture and pharmaceutical tablet spec-tra. Wavelengths more than 290 nm were not used because

AT and CD do not absorb in this region, so any absorbancevalues obtained at these wavelengths would have introduceda significant amount of noise in the calibration matrix, therebydecreasing the precision.

A combination of a factor design with two levels per fac-tor and a central composite design was used to statisticallymaximize the information contained in the spectra. The sim-plest design consisted of two levels per component (eight cali-bration samples). This model was too simple and the resultswere poor owing to the inadequate number of standardsemployed. The proposed central composite design does notinclude a simultaneous combination of high or low concen-trations of the components. A combination of both designswas thus considered and a training set consisting of 33 sampleswas used (Table 1).

To select the number of factors in the PLS-1 and PCR algo-rithms, a cross-validation method leaving out one sample at atime [37] was employed using the training (calibration) set of33 calibration spectra. The PLS-1 and PCR calibration on32 calibration spectra were performed and, using this calibra-tion, the concentration of the sample left out during the cali-bration process was predicted. This process was repeated33 times until each training sample had been left out once.The predicted concentrations of the components in eachsample were compared with the actual concentrations in thesetraining samples and the root mean square error of cross vali-dation (RMSECV) was calculated for each method as fol-lows:

RMSECV = �PRESS ⁄ n

Where n is the number of training samples,

PRESS = �� Ypred − Y true �2

Where Ypred and Ytrue are predicted and true concentrations inµg ml–1, respectively.

The RMSECV was used as a diagnostic test for examin-ing the errors in the predicted concentrations. It indicates boththe precision and accuracy of predictions. The RMSECV

Table 2Characteristic parameters of the calibration equations for the proposed HPLC method for simultaneous determination of AT, AM and CD

Parameters AT AM CDCalibration range (µg ml–1) 40–160 2–8 10–40Detection limit (µg ml–1) 0.003 0.004 0.002Quantitation limit (µg ml–1) 0.009 0.013 0.008Regression Eq. (Y)a:Slope (b) 17.85 × 103 190.0 × 103 22.36 × 103

Standard deviation of the slope (Sb) 24.05 3.77 × 102 27.03Relative standard deviation of the slope (%) 0.13 0.20 0.12Confidence limit of the slopeb 17.68 × 103–18.01 × 103 187.4 × 103–192.6 × 103 22.18 × 103–22.55 × 103

Intercept (a) 11.84 × 103 (–0.99 × 103) 0.77 × 103

Standard deviation of the intercept (Sa) 2.60 × 103 2.03 × 103 0.73 × 103

Confidence limit of the interceptb (–5.77 × 103)–29.46 × 103 (–14.8 × 103)–12.8 × 103 (–4.18 × 103)–5.72 × 103

Correlation coefficient (r) 0.9999 0.9999 0.9998Standard error of estimation 6.73 × 103 5.27 × 103 1.89 × 103

a Y = a + bC, where C is the concentration of compound in µg ml–1 and Y is the peak area.b 95% confidence limit.

Fig. 1. UV absorption spectra of 100 µg ml–1 of AT (_____), 5 µg ml–1 of AM(............) and 25 µg ml–1 of CD (__ - __) in 0.1 M hydrochloric acid.

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plays the same role of standard deviation in indicating thespread of the concentration errors [38].

Appropriate selection of the number of factors to be usedto construct the model is the key to achieve correct quantita-tion in PLS-1 and PCR calibrations. The most usual proce-dure for this purpose involves choosing the number of factorsthat result in the minimum RMSECV. However, this criterionis subjected to some constraints since, occasionally, theRMSECV does not reach a sharp minimum, but decreasesgradually above a given number of factors. On the other hand,it is calculated from a finite number of samples, so it is error-prone. For these reasons, the method developed by Haalandand Thomas [39] was used for selecting the optimum numberof factors, which involves selecting that model including thesmallest number of factors that result in an insignificant dif-ference between the corresponding RMSECV and the mini-mum RMSECV. Figs. 2 and 3 show the variation of theRMSECV as a function of the number of factors for the deter-mination of each compound by PLS-1 and PCR methods,respectively. As the difference between the minimum RM-SECV and other RMSECV values become smaller, the prob-ability that each additional factor is significant becomessmaller [40].

A number of factors of 3 were found to be optimum forAT, while a number of factors of 4 were found to be optimumfor AM and CD by the PLS-1 method as in Fig. 2 and a num-ber of factors of 4 were found to be optimum for AT, AM andCD by the PCR method as in Fig. 3. The selected model isthe one with the smallest number of factors such that RM-SECV for that model is not significantly greater than RM-SECV for the model with additional factor.

Plotting the actual known concentrations against the pre-dicted concentrations performed the evaluation of the predic-tive abilities of the models. The obtained results are shown inTable 3. A satisfactory correlation coefficient (r) value wasobtained for each compound (0.9999 for AT and CD and0.9997 for AM) in the training set by PLS-1 and PCR-optimized models indicating good predictive abilities of themodels. The RMSECV obtained by optimizing the calibra-tion matrix of the absorption spectra for the PLS-1 and PCRmethods is shown in Table 3 indicating good accuracy andprecision.

Plotting the concentration residuals against the predictedconcentrations carried out another diagnostic test. The residu-als appear randomly distributed around zero, indicatingadequate models as shown in Fig. 4.

The quantitative prediction abilities of PLS and PCR forspectral analyses are compared. It is difficult to generalizeabout the superiority of one method over another, becausethe relative performance of the methods is often dependenton a particular data set being analyzed [41].

3.2. HPLC method

The developed HPLC method was applied for simulta-neous determination of AT, AM and CD. The mobile phase

composition and pH were studied and optimized. A satisfac-tory separation was obtained with a mobile phase composedof acetonitrile -5mM heptanesulphonic acid sodium salt(20:80, v/v), adjusted to pH 4.4 using acetic acid. Increasingacetonitrile concentration to more than 40% lead to inad-equate separation of AT and AM. At lower acetonitrile con-centration (<10%), separation occurred but with excessive tail-ing forAM and CD peaks.Variation of pH of the mobile phaseresulted in maximum capacity factor (K′) value at pH 6.5,with loss of peak symmetry for CD. At pH 3.0–5.0, improvedresolutions for the three drugs were observed. However, atpH 4.4 optimum resolution with reasonable retention timewas observed. Quantitation based on peak area achieved withUV detection at 274 nm. The specificity of the HPLC methodis illustrated in Fig. 5 where complete separation of the three

Fig. 2. RMSECV versus latent variable for a calibration set prediction of AT,AM and CD using PLS-1 model.

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compounds was noticed. The average retentiontime ± standard deviation for AT, AM and CD were found tobe 4.2 ± 0.03, 5.1 ± 0.04 and 6.5 ± 0.02 min, respectively, forten replicates.

3.3. Analysis of pharmaceutical tablet

The proposed PLS-1, PCR and HPLC methods wereapplied to the simultaneous determination of AT, AM and CDin commercial tablets. Seven replicate determinations weremade. Satisfactory results were obtained for each compoundand were found to be in agreement with label claims (Table 4).There are no reports in the literature on the simultaneous deter-mination of the components of this mixture. Therefore, theresults of the proposed PLS-1 and PCR methods were com-pared with those of the proposed HPLC method. Statisticalcomparison between the results was performed with regardsto accuracy and precision using Student’s t-test and F-ratio at95% confidence level (Table 4). There was no significant dif-ference between the results.

3.4. Validation of the methods

3.4.1. LinearityThe linearity of the HPLC detector response for determi-

nation of AT, AM and CD was evaluated by analyzing a seriesof different concentrations of each compound. In this study,

seven concentrations were chosen, ranging from 40–160 µg ml–1 for AT, 2-8 µg ml–1 for AM and 10-40 µg ml–

1 for CD. Each concentration was repeated three times in orderto provide information on the variation in peak area betweensamples of the same concentration. The linearity of the cali-bration graphs was validated by the high value of the corre-lation coefficient and the intercept value, which was not sta-tistically (p < 0.05) different from zero. Characteristicparameters for regression equations of the HPLC methodobtained by least squares treatment of the results were givenin Table 2.

3.4.2. PrecisionFor evaluation of the precision estimates, repeatability and

intermediate precision were performed at three concentra-tion levels for each compound. The data for each concentra-tion level were evaluated by one-way ANOVA. An8 days × 2 replicates design was performed. Statistical com-parison of the results was performed using the p-value of theF-test. Three univariate analyses of variance for each concen-tration level were made. Since the p-value of the F-test isalways greater than 0.05, there is no statistically significantdifference between the mean results obtained from one levelof day to another at the 95% confidence level.

3.4.3. RangeThe calibration range was established through consider-

ation of the practical range necessary, according to each com-pound concentration present in the pharmaceutical product,to give accurate, precise and linear results. The calibrationrange of the proposed HPLC method is given in Table 2.

3.4.4. Detection and quantitation limitsAccording to ICH recommendations [42], the approach

based on the S.D. of the response and the slope was used fordetermining the detection and quantitation limits. The theo-retical values were assessed practically and given in Table 2.

3.4.5. SelectivityMethod selectivity was achieved by preparing nine syn-

thetic mixtures of the studied compounds at various concen-trations within the linearity range for HPLC (Table 5). The

Fig. 3. RMSECV versus latent variable for a calibration set prediction of AT,AM and CD using PCR model.

Table 3RMSECV and statistical parameter values for simultaneous determination of AT, AM and CD using PLS-1 and PCR methods

Item Method CompoundAT AM CD

RMSECV PLS-1 6.90 × 10–1 0.68 × 10–1 1.90 × 10–1

PCR 6.84 × 10–1 0.69 × 10–1 2.03 × 10–1

Intercept PLS-1 –4.14 × 10–2 2.71 × 10–2 11.41 × 10–2

PCR –9.22 × 10–2 1.01 × 10–2 11.56 × 10–2

Slope PLS-1 1.0006 0.9944 0.9961PCR 1.0009 0.9980 0.9961

S.E. of intercept PLS-1 3.27 × 10–1 0.24 × 10–1 0.89 × 10–1

PCR 2.71 × 10–1 0.25 × 10–1 0.95 × 10–1

S.E. of slope PLS-1 2.91 × 10–3 4.69 × 10–3 3.16 × 10–3

PCR 2.57 × 10–3 4.80 × 10–3 3.37 × 10–3

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predictive ability of the proposed PLS-1 and PCR methodswas assessed by applying the PLS-1 and PCR models to aprediction set of nine synthetic ternary mixtures. The concen-trations of AT, AM and CD were falling within the ranges ofcalibration matrix (Table 5). The synthetic mixtures were ana-lyzed according to the previous procedures described underthe proposed methods. Satisfactory results were obtained(Table 5), indicating the high selectivity of the proposed meth-ods for simultaneous determination of the studied com-pounds.

3.4.6. AccuracyThis study was performed by adding known amounts of

the studied compounds to a known concentration of the com-mercial pharmaceutical tablets (standard addition method).The resulting mixtures were analyzed and results obtainedwere compared with the expected results. The excellent recov-eries of the standard addition method (Table 6) suggested thathigh accuracy of the proposed methods.

3.4.7. RobustnessVariation of pH of the mobile phase by ± 0.1 and its organic

strength by ± 2% did not have any significant effect on chro-

Fig. 4. Concentration residuals versus predicted concentration of AT, AM and CD using PCR and PLS-1.

Fig. 5. HPLC chromatogram of 20 µl injection of tablet sample containing100 µg ml–1of AT, 5 µg ml–1 of AM and 25 µg ml–1of CD.

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matographic resolution in the HPLC method. Variation ofstrength of hydrochloric acid by ± 0.02 M did not have anysignificant effect on chemometric-assisted spectrophotomet-ric methods.

3.4.8. StabilitySolutions of the studied compounds in the mobile phase

or 0.1 M hydrochloric acid exhibited no chromatographic or

absorbance changes for 24 h when kept at room temperature,and for 4 days when stored refrigerated at 5 °C.

4. Conclusion

Two chemometric-assisted methods in spectrophotomet-ric analysis, PLS-1 and PCR were proposed for the simulta-

Table 4Determination of AT, AM and CD in commercial tablets using the PLS-1, PCR and HPLC methods

Sampleno

Concentration (µg ml–1) % RecoveryAT AM CD PLS-1 PCR HPLC

AT AM CD AT AM CD AT AM CD1 40 2 10 100.8 98.9 99.5 100.6 99.7 101.1 100.2 99.6 100.32 60 3 15 100.2 100.5 100.4 99.3 101.3 99.6 99.7 100.7 100.43 80 4 20 100.6 101.2 98.9 99.9 100.1 100.6 100.4 99.7 100.54 100 5 25 99.7 99.8 99.0 100.2 99.3 99.5 100.7 99.9 99.65 120 6 30 100.2 100.1 100.8 100.7 100.6 99.3 100.4 100.4 99.96 140 7 35 101.0 99.4 100.7 98.9 99.1 100.5 100.3 99.9 100.27 160 8 40 100.2 101.5 99.1 100.9 98.7 98.9 99.5 100.8 101.1Meana 100.4 100.2 99.8 100.1 99.8 99.9 100.2 100.1 100.3S.D.a 0.45 0.94 0.84 0.75 0.91 0.81 0.42 0.48 0.47tb 0.86 0.25 1.37 0.31 0.77 1.13Fb 1.15 3.84 3.19 3.19 3.59 2.97

a Mean and S.D., percentage recovery from the label claim amount.b Theoretical values for t and F at p = 0.05 are 2.18 and 4.28, respectively.

Table 5Determination of AT, AM and CD in synthetic mixtures using the PLS-1, PCR and HPLC methods

Mix. no. Concentration (µg ml–1) % RecoveryAT AM CD PLS-1 PCR HPLC

AT AM CD AT AM CD AT AM CD1 40 2 10 99.9 99.6 99.4 100.4 98.9 99.3 100.6 100.1 100.52 160 8 40 99.0 100.6 100.6 99.7 99.3 100.6 100.4 99.7 100.43 60 7 30 100.4 100.2 99.6 100.7 99.8 100.2 99.4 99.6 99.84 140 4 40 100.2 100.6 100.6 100.1 99.8 99.7 99.2 99.9 100.25 80 6 15 100.8 99.3 99.8 98.9 100.6 101.2 100.2 100.4 99.76 100 5 20 98.9 100.2 100.7 100.3 100.0 100.6 99.9 99.4 100.57 120 3 25 99.8 101.1 101.4 99.7 100.7 99.4 99.7 100.6 100.48 40 8 40 100.2 98.7 99.7 100.6 101.2 101.1 100.4 100.3 99.49 80 7 35 99.6 100.7 100.8 100.9 99.3 99.9 100.1 100.7 99.9Meana 99.9 100.1 100.3 100.1 99.9 100.2 100.0 100.1 100.1S.D.a 0.62 0.77 0.68 0.63 0.75 0.70 0.48 0.46 0.40

a Mean and S.D., percentage recovery from the label claim amount.

Table 6Application of standard addition technique to the analysis of AT, AM and CD using the PLS-1, PCR and HPLC methods

Sampleno

AT AM CDConcentration (µg ml–1) Concentration (µg ml–1) Concentration (µg ml–1)

Claimed Added % Found of added Claimed Added % Found of added Claimed Added % Found of addedPLS-1 PCR HPLC PLS-1 PCR HPLC PLS-1 PCR HPLC

1 20 40 99.8 100.6 100.2 1 2 99.4 100.6 99.8 5 10 99.9 101.5 99.82 20 60 100.4 99.4 99.9 1 3 101.0 98.7 100.2 5 15 99.2 99.5 100.23 20 80 100.2 98.7 99.4 1 4 100.1 99.9 99.8 5 20 100.5 100.2 100.64 20 100 101.0 100.7 100.7 1 5 99.2 99.4 100.2 5 25 100.7 100.8 100.35 20 120 99.5 99.7 99.8 1 6 101.5 100.7 100.7 5 30 99.7 99.6 99.76 20 140 100.2 100.2 100.2 1 7 99.7 101.1 100.6 5 35 100.8 100.5 99.4Meana 100.2 99.9 100.0 100.1 100.1 100.2 100.1 100.3 100.0S.D.a 0.51 0.77 0.44 0.92 0.90 0.38 0.63 0.76 0.44

a Mean and S.D., percentage recovery from the added amount.

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neous determination of AT, AM and CD in pharmaceuticaltablets. The results obtained were compared with the pro-posed HPLC method and good coincidence was observed.The three proposed methods were accurate with good repro-ducibility and sensitivity; however, better selectivity wasobtained with the HPLC method.

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