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HPLC and Chemometric assisted

spectrophotometric methods for simultaneous
determination of atenolol, amiloride
hydrochloride and chlorthalidone
Article in Il Farmaco April 2005
DOI: 10.1016/j.farmac.2004.11.013 Source: PubMed

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Il Farmaco 60 (2005) 269278

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

HPLC and chemometric-assisted spectrophotometric methods for

simultaneous 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). Quantitation was achieved with UV detection at 274 nm based on peak area. The other two-chemometric-assisted spectrophotometric methods
applied were principal component regression (PCR) and partial least squares (PLS-1). These approaches were successfully applied to quantify
each drug in the mixture using the information included in the absorption spectra of appropriate solutions in the range 240290 nm with the
intervals Dk = 0.2 nm. The three methods were successfully applied to a pharmaceutical formulation (tablets), and the results were compared
with 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 indications similar to those of the thiazide diuretics [1]. It is prescribed with atenolol (AT), which is a cardioselective
b-blocker and amiloride hydrochloride (AM), which is a mild
diuretic. They are used in the treatment of hypertension. The
UV absorption spectra of the studied drugs show severe overlap. Hence, their simultaneous determination is difficult when
conventional, derivative, and derivative ratio spectrophotometric techniques are used. No analytical method has been
reported for the simultaneous determination of AT, AM, and
CD in a multicomponent mixture, while several analytical
methods have been reported for the determination of AT or
AM or CD in combination with other drugs, including spectrophotometry [212], spectrofluorimetry [1315], HPLC
[11,1620] and HPTLC [21,22].
Multivariate calibration methods, in combination with several techniques such as spectrophotometry [2326], fluorim* Corresponding author.
E-mail address: ghhadad@hotmail.com (A. El-Gindy).
0014-827X/$ - see front matter 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.11.013

etry [27,28], polarography [29] and voltammetry [30,31], have

been applied in analytical procedures. The application of multivariate calibration to the absorbance signals produced by
drugs during their simultaneous determination in pharmaceutical preparations is an effective means for quality control of
their manufacture [32]. The PLS and PCR were found to be
specially suited to multicomponent analysis, particularly for
mixture with highly overlapped spectra [33].
The aim of this paper is to investigate the ability of PLS1 and PCR methods to quantify a three-component mixture
of AT, AM and CD with overlapping UV spectra, and to apply
the optimized models in pharmaceutical preparations. In addition, a HPLC method was developed for the assay of the components of the studied mixture. The proposed methods are
simple and accurate. They resulted in a significant reduction
in analysis time and proved to be suitable for routine determination of the three components of the studied mixture.
The proposed HPLC method was found to be simpler than
the published chromatographic methods for determination of
the studied drugs in other combined formulations. In this
method, there is no need for internal standard while the other

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A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

published chromatographic methods used internal standards

such as metoprolol [16], methyl 4-hydroxybenzoate [17], felodipine besylate [18] and hydrochlorothiazide [22]. Moreover, the proposed methods are the first publication for the
simultaneous determination of AT, AM and CD in pharmaceutical preparations.

2. Experimental
2.1. Instrumentation
A double-beam Shimadzu (Japan) UV-visible spectrophotometer, model UV-1601 PC equipped with 1-cm quartz cells
and connected to an IBM compatible computer. A HP600 inkjet printer was used. The bundled software was UVPC personal spectroscopy software version 3.7 (Shimadzu). The
spectral bandwidth was 2 nm and the wavelength scanning
speed was 2800 nm min1. PLS-1 and PCR analyses were
carried out by using PLS-Toolbox software version 2.1-PC
[34] for use with MATLAB 5.
The HPLC (Shimadzu, Kyoto, Japan) instrument was
equipped with a model series LC-10 ADVP pump, SCL10 AVP system controller, DGU-12 A degasser, Rheodyne
7725i injector with a 20 l loop and a SPD-10 AVP UV-VIS
detector; separation and quantitation were made on a
150 4.6 mm (i.d.) TSK- Gel 5 m ODS-80 TM column
(Tosoh, Japan). The detector was set at 274 nm. Data acquisition was performed on class-VP software.
2.2. Materials and reagents
Pharmaceutical grade of AT, AM and CD was used and
certified to contain 99.8, 99.9 and 99.85%, respectively. The
methanol used was HPLC grade (BDH, Poole, UK). Heptanesulphonic acid sodium salt, hydrochloric and acetic acids were
analytical grade. Teklo tablet (ACAPI, Badr city, third industrial zone, Cairo, Egypt) was used. Each tablet was labeled to
contain 100 mg AT, 5 mg AM and 25 mg CD.
2.3. HPLC conditions
The mobile phase was prepared by mixing acetonitrile and
5mM heptanesulphonic acid sodium salt in a ratio of (20:80,
v/v). The pH of the mobile phase was adjusted to 4.4 using
acetic acid and the flow rate was set at 1 ml min1. All determinations were performed at ambient temperature and the
injection volume was 20 l.
2.4. Standard solutions and calibration
Standard solutions of each of AT, AM and CD were prepared by separately dissolving 100 mg of each drug in 100 ml
methanol then further dilutions were made in 0.1 M hydrochloric acid (for spectrophotometric methods) or mobile phase
(for HPLC method) within the concentration rang of 40

160 g ml1 for AT, 28 g ml1 for AM and 1040 g ml1

for CD.
2.4.1. Calibration of PLS-1 and PCR methods
A training set of 33 synthetic mixtures with different concentrations of each compound were prepared in 0.1 M hydrochloric acid in the concentration ranges of 40160 g ml1
for AT, 28 g ml1 for AM and 1040 g ml1 for CD
(Table 1). The UV absorption spectra were recorded over the
wavelength range of 240290 nm. The data points of the spectra were collected every 0.2 nm. The computation was made
using PLS-Toolbox software version 2.1.
The PLS-1 and PCR models were applied to the UV
absorption spectra of these mixtures using 3 latent variables
for AT and 4 latent variables for AM and CD by PLS-1. Four
principal components were used for PCR determination of
each compound.
2.4.2. Calibration of HPLC method
Triplicate 20 l injections were made for each concentration and chromatographed under the specified chromatographic conditions described previously. The peak area values were plotted against corresponding concentrations. Linear
relationships were obtained (Table 2).
2.5. Sample preparation
Twenty tablets were weighed and finely powdered. An
accurately weighed portion of the powder equivalent to about
100 mg of AT, 5 mg of AM and 25 mg CD was extracted and
diluted to 100 ml with methanol. The sample solution was
filtered. Further dilution of the filtrate was carried out with
0.1 M hydrochloric acid (for spectrophotometric methods) or
with the mobile phase (for the HPLC method) to provide a
solution of 100 g ml1 of AT, 5 g ml1 of AM and 25 g ml1
of CD.
2.5.1. Procedures for the determination of AT, AM and CD
using PLS-1 and PCR methods
The UV absorption spectrum of final solution was recorded
over the wavelength range of 240290 nm. The data points of
the spectrum were collected every 0.2 nm. The PLS-1 model
was applied using 3 latent variables for AT and 4 latent variables for AM and CD. The PCR model was applied using
four principal components. The concentration of each compound was calculated using each model.
2.5.2. Procedures for the determination of AT, AM and CD
using HPLC method
Triplicate 20 l injections of the final solution were injected
and chromatographed under the specified HPLC conditions
previously described in 2.3. The peak area values were determined and the concentration of each compound was calculated.

A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

271

Table 1
Concentration 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 PCR
methods
Mixture composition (g ml1)
Mixture no
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Meana
S.D.a
a

AT
60
100
140
80
160
160
120
40
100
120
140
80
160
160
160
140
120
60
140
40
100
120
100
60
40
40
60
100
80
40
120
100
80

AM
3
5
7
5
5
3
4
2
6
6
5
3
2
5
7
8
8
8
7
4
5
7
6
3
2
5
5
2
2
3
6
7
5

Internal validation (% recovery)

CD
15
25
35
40
25
15
25
35
10
30
25
20
35
30
10
35
25
20
40
40
40
30
35
30
15
30
35
10
15
15
30
30
35

AT
99.5
100.6
99.3
100.1
100.1
99.4
98.8
100.4
100.2
101.0
98.9
99.8
100.2
99.6
100.2
99.6
99.0
100.5
100.9
99.9
100.6
99.9
100.2
101.0
100.2
99.2
100.2
100.3
100.2
99.0
100.4
100.0
100.6
100.0
0.59

PLS-1
AM
99.7
101.0
101.6
101.7
100.4
99.2
100.0
99.8
98.9
100.4
100.6
100.2
102.0
99.6
100.6
99.8
100.1
99.3
99.4
100.9
98.5
98.8
99.3
100.0
100.0
100.8
100.2
100.1
100.1
98.9
98.7
101.6
101.0
100.1
0.90

CD
101.0
99.1
99.6
100.0
101.5
99.7
100.6
98.5
100.4
99.2
99.5
100.3
100.4
101.4
99.9
100.4
100.4
99.6
100.5
99.3
100.6
99.5
100.5
101.5
100.7
99.7
98.9
99.1
101.9
100.4
99.9
99.2
101.2
100.1
0.83

AT
100.0
100.7
100.1
100.0
99.4
98.9
100.6
100.1
100.9
99.3
100.0
99.5
99.9
99.4
100.2
98.2
100.7
99.7
100.4
100.0
98.9
100.3
100.9
100.6
99.7
98.7
100.1
100.2
100.2
99.2
100.2
99.9
101.0
99.9
0.66

PCR
AM
99.8
101.0
101.5
101.3
99.5
100.3
100.1
98.8
100.0
100.5
101.5
99.8
100.6
100.0
99.1
99.3
100.6
100.1
98.8
98.9
99.9
99.8
100.9
99.9
98.0
100.6
98.8
98.9
101.6
101.2
99.2
99.7
98.7
100.0
0.92

CD
101.0
99.1
99.6
100.0
101.6
100.6
98.4
100.4
99.2
99.5
100.5
101.5
99.9
100.4
99.7
100.5
99.3
100.6
99.5
100.5
101.5
100.7
99.7
98.9
99.0
101.9
100.4
100.0
99.2
101.7
100.2
99.3
99.7
100.1
0.87

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

3. Results and discussion

3.1. PCR and PLS-1 methods
Fig. 1 shows the UV absorption spectra of AT, AM and
CD at their nominal concentrations in the tablet. A significant overlap in absorption bands was noticed. The simultaneous determination of AT and CD in the tablet by conventional, derivative and derivative ratio spectrophotometric
methods is hindered by strong spectral overlap throughout
the wavelength range. The PLS or PCR calibration methods
were necessary for such determination due to the presence of
interference, while the UV spectrum of AM is not strongly
affected by the presence of AT and CD. This is a favorable
condition and conventional zero-order or direct derivative
methods could be applied for determination of AM in the studied ternary mixture.

The quality of multicomponent analysis is dependent on

the wavelength range and spectral mode used [35]. The PLS
procedures are designated to be full spectrum computational
procedures; however, using highly noisy, scarcely informative wavelengths detract from precision. Discarding particularly noisy wavelengths can lessen this. This is quite sensible
in UV-Vis spectrophotometry as the pure spectra of the analytes are often available and the positions of their bands are
not usually greatly affected by the presence of the excipients,
so one can predict which spectral region in the sample spectrum will contain the information relevant to the analyte [36].
In this work, original and reconstructed spectra of the calibration matrix were compared in order to select the range of
wavelengths. The range was obtained by all regions in which
the difference between each component of the mixture and
the others was maximum. Besides, the regions in which each
component of the mixture was best reconstructed were also

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A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

Table 2
Characteristic parameters of the calibration equations for the proposed HPLC method for simultaneous determination of AT, AM and CD
Parameters
Calibration range (g ml1)
Detection limit (g ml1)
Quantitation limit (g ml1)
Regression Eq. (Y)a:
Slope (b)
Standard deviation of the slope (Sb)
Relative standard deviation of the slope (%)
Confidence limit of the slopeb
Intercept (a)
Standard deviation of the intercept (Sa)
Confidence limit of the interceptb
Correlation coefficient (r)
Standard error of estimation
a
b

AT
40160
0.003
0.009

AM
28
0.004
0.013

CD
1040
0.002
0.008

17.85 103
24.05
0.13
17.68 10318.01 103
11.84 103
2.60 103
(5.77 103)29.46 103
0.9999
6.73 103

190.0 103
3.77 102
0.20
187.4 103192.6 103
(0.99 103)
2.03 103
(14.8 103)12.8 103
0.9999
5.27 103

22.36 103
27.03
0.12
22.18 10322.55 103
0.77 103
0.73 103
(4.18 103)5.72 103
0.9998
1.89 103

Y = a + bC, where C is the concentration of compound in g ml1 and Y is the peak area.
95% confidence limit.

considered. The spectral region between wavelengths 240 and

290 nm was selected for this purpose as it provided the greatest amount of information about the mixture components. This
entailed using 251 experimental points per spectrum, as spectra were digitized at 0.2 nm intervals. In addition, wavelengths less than 240 nm were rejected due to the high UV
contribution of AT and CD relative to AM and the difference
between the synthetic mixture and pharmaceutical tablet spectra. Wavelengths more than 290 nm were not used because

AT and CD do not absorb in this region, so any absorbance

values obtained at these wavelengths would have introduced
a significant amount of noise in the calibration matrix, thereby
decreasing the precision.
A combination of a factor design with two levels per factor and a central composite design was used to statistically
maximize the information contained in the spectra. The simplest design consisted of two levels per component (eight calibration samples). This model was too simple and the results
were poor owing to the inadequate number of standards
employed. The proposed central composite design does not
include a simultaneous combination of high or low concentrations of the components. A combination of both designs
was thus considered and a training set consisting of 33 samples
was used (Table 1).
To select the number of factors in the PLS-1 and PCR algorithms, a cross-validation method leaving out one sample at a
time [37] was employed using the training (calibration) set of
33 calibration spectra. The PLS-1 and PCR calibration on
32 calibration spectra were performed and, using this calibration, the concentration of the sample left out during the calibration process was predicted. This process was repeated
33 times until each training sample had been left out once.
The predicted concentrations of the components in each
sample were compared with the actual concentrations in these
training samples and the root mean square error of cross validation (RMSECV) was calculated for each method as follows:
RMSECV =

PRESS n

Where n is the number of training samples,

PRESS = Y pred Y true

Fig. 1. UV absorption spectra of 100 g ml1 of AT (_____), 5 g ml1 of AM

(............) and 25 g ml1 of CD (__ - __) in 0.1 M hydrochloric acid.

Where Ypred and Ytrue are predicted and true concentrations in

g ml1, respectively.
The RMSECV was used as a diagnostic test for examining the errors in the predicted concentrations. It indicates both
the precision and accuracy of predictions. The RMSECV

A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

plays the same role of standard deviation in indicating the

spread of the concentration errors [38].
Appropriate selection of the number of factors to be used
to construct the model is the key to achieve correct quantitation in PLS-1 and PCR calibrations. The most usual procedure for this purpose involves choosing the number of factors
that result in the minimum RMSECV. However, this criterion
is subjected to some constraints since, occasionally, the
RMSECV does not reach a sharp minimum, but decreases
gradually above a given number of factors. On the other hand,
it is calculated from a finite number of samples, so it is errorprone. For these reasons, the method developed by Haaland
and Thomas [39] was used for selecting the optimum number
of factors, which involves selecting that model including the
smallest number of factors that result in an insignificant difference between the corresponding RMSECV and the minimum RMSECV. Figs. 2 and 3 show the variation of the
RMSECV as a function of the number of factors for the determination of each compound by PLS-1 and PCR methods,
respectively. As the difference between the minimum RMSECV and other RMSECV values become smaller, the probability that each additional factor is significant becomes
smaller [40].
A number of factors of 3 were found to be optimum for
AT, while a number of factors of 4 were found to be optimum
for AM and CD by the PLS-1 method as in Fig. 2 and a number of factors of 4 were found to be optimum for AT, AM and
CD by the PCR method as in Fig. 3. The selected model is
the one with the smallest number of factors such that RMSECV for that model is not significantly greater than RMSECV for the model with additional factor.
Plotting the actual known concentrations against the predicted concentrations performed the evaluation of the predictive abilities of the models. The obtained results are shown in
Table 3. A satisfactory correlation coefficient (r) value was
obtained for each compound (0.9999 for AT and CD and
0.9997 for AM) in the training set by PLS-1 and PCRoptimized models indicating good predictive abilities of the
models. The RMSECV obtained by optimizing the calibration matrix of the absorption spectra for the PLS-1 and PCR
methods is shown in Table 3 indicating good accuracy and
precision.
Plotting the concentration residuals against the predicted
concentrations carried out another diagnostic test. The residuals appear randomly distributed around zero, indicating
adequate models as shown in Fig. 4.
The quantitative prediction abilities of PLS and PCR for
spectral analyses are compared. It is difficult to generalize
about the superiority of one method over another, because
the relative performance of the methods is often dependent
on a particular data set being analyzed [41].
3.2. HPLC method
The developed HPLC method was applied for simultaneous determination of AT, AM and CD. The mobile phase

273

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

composition and pH were studied and optimized. A satisfactory separation was obtained with a mobile phase composed
of acetonitrile -5mM heptanesulphonic acid sodium salt
(20:80, v/v), adjusted to pH 4.4 using acetic acid. Increasing
acetonitrile concentration to more than 40% lead to inadequate separation of AT and AM. At lower acetonitrile concentration (<10%), separation occurred but with excessive tailing for AM and CD peaks. Variation of pH of the mobile phase
resulted in maximum capacity factor (K) value at pH 6.5,
with loss of peak symmetry for CD. At pH 3.05.0, improved
resolutions for the three drugs were observed. However, at
pH 4.4 optimum resolution with reasonable retention time
was observed. Quantitation based on peak area achieved with
UV detection at 274 nm. The specificity of the HPLC method
is illustrated in Fig. 5 where complete separation of the three

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A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

seven concentrations were chosen, ranging from 40

160 g ml1 for AT, 2-8 g ml1 for AM and 10-40 g ml
1 for CD. Each concentration was repeated three times in order
to provide information on the variation in peak area between
samples of the same concentration. The linearity of the calibration graphs was validated by the high value of the correlation coefficient and the intercept value, which was not statistically (p < 0.05) different from zero. Characteristic
parameters for regression equations of the HPLC method
obtained by least squares treatment of the results were given
in Table 2.
Fig. 3. RMSECV versus latent variable for a calibration set prediction of AT,
AM and CD using PCR model.

compounds was noticed. The average retention

time standard deviation for AT, AM and CD were found to
be 4.2 0.03, 5.1 0.04 and 6.5 0.02 min, respectively, for
ten replicates.
3.3. Analysis of pharmaceutical tablet
The proposed PLS-1, PCR and HPLC methods were
applied to the simultaneous determination of AT, AM and CD
in commercial tablets. Seven replicate determinations were
made. Satisfactory results were obtained for each compound
and were found to be in agreement with label claims (Table 4).
There are no reports in the literature on the simultaneous determination of the components of this mixture. Therefore, the
results of the proposed PLS-1 and PCR methods were compared with those of the proposed HPLC method. Statistical
comparison between the results was performed with regards
to accuracy and precision using Students t-test and F-ratio at
95% confidence level (Table 4). There was no significant difference between the results.

3.4.2. Precision
For evaluation of the precision estimates, repeatability and
intermediate precision were performed at three concentration levels for each compound. The data for each concentration level were evaluated by one-way ANOVA. An
8 days 2 replicates design was performed. Statistical comparison of the results was performed using the p-value of the
F-test. Three univariate analyses of variance for each concentration level were made. Since the p-value of the F-test is
always greater than 0.05, there is no statistically significant
difference between the mean results obtained from one level
of day to another at the 95% confidence level.
3.4.3. Range
The calibration range was established through consideration of the practical range necessary, according to each compound concentration present in the pharmaceutical product,
to give accurate, precise and linear results. The calibration
range of the proposed HPLC method is given in Table 2.

3.4. Validation of the methods

3.4.4. Detection and quantitation limits

According to ICH recommendations [42], the approach
based on the S.D. of the response and the slope was used for
determining the detection and quantitation limits. The theoretical values were assessed practically and given in Table 2.

3.4.1. Linearity
The linearity of the HPLC detector response for determination of AT, AM and CD was evaluated by analyzing a series
of different concentrations of each compound. In this study,

3.4.5. Selectivity
Method selectivity was achieved by preparing nine synthetic mixtures of the studied compounds at various concentrations within the linearity range for HPLC (Table 5). The

Table 3
RMSECV and statistical parameter values for simultaneous determination of AT, AM and CD using PLS-1 and PCR methods
Item
RMSECV
Intercept
Slope
S.E. of intercept
S.E. of slope

Method
PLS-1
PCR
PLS-1
PCR
PLS-1
PCR
PLS-1
PCR
PLS-1
PCR

AT
6.90 101
6.84 101
4.14 102
9.22 102
1.0006
1.0009
3.27 101
2.71 101
2.91 103
2.57 103

Compound
AM
0.68 101
0.69 101
2.71 102
1.01 102
0.9944
0.9980
0.24 101
0.25 101
4.69 103
4.80 103

CD
1.90 101
2.03 101
11.41 102
11.56 102
0.9961
0.9961
0.89 101
0.95 101
3.16 103
3.37 103

A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

275

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

predictive ability of the proposed PLS-1 and PCR methods

was assessed by applying the PLS-1 and PCR models to a
prediction set of nine synthetic ternary mixtures. The concentrations of AT, AM and CD were falling within the ranges of
calibration matrix (Table 5). The synthetic mixtures were analyzed according to the previous procedures described under
the proposed methods. Satisfactory results were obtained
(Table 5), indicating the high selectivity of the proposed methods for simultaneous determination of the studied compounds.
3.4.6. Accuracy
This study was performed by adding known amounts of
the studied compounds to a known concentration of the commercial pharmaceutical tablets (standard addition method).
The resulting mixtures were analyzed and results obtained
were compared with the expected results. The excellent recoveries of the standard addition method (Table 6) suggested that
high accuracy of the proposed methods.
3.4.7. Robustness
Variation of pH of the mobile phase by 0.1 and its organic
strength by 2% did not have any significant effect on chro-

Fig. 5. HPLC chromatogram of 20 l injection of tablet sample containing

100 g ml1of AT, 5 g ml1 of AM and 25 g ml1of CD.

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A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

Table 4
Determination of AT, AM and CD in commercial tablets using the PLS-1, PCR and HPLC methods
Sample
no
1
2
3
4
5
6
7
Meana
S.D.a
tb
Fb
a
b

AT
40
60
80
100
120
140
160

Concentration (g ml1)
AM
CD
2
3
4
5
6
7
8

10
15
20
25
30
35
40

AT
100.8
100.2
100.6
99.7
100.2
101.0
100.2
100.4
0.45
0.86
1.15

PLS-1
AM
98.9
100.5
101.2
99.8
100.1
99.4
101.5
100.2
0.94
0.25
3.84

CD
99.5
100.4
98.9
99.0
100.8
100.7
99.1
99.8
0.84
1.37
3.19

AT
100.6
99.3
99.9
100.2
100.7
98.9
100.9
100.1
0.75
0.31
3.19

% Recovery
PCR
AM
CD
99.7
101.1
101.3
99.6
100.1
100.6
99.3
99.5
100.6
99.3
99.1
100.5
98.7
98.9
99.8
99.9
0.91
0.81
0.77
1.13
3.59
2.97

AT
100.2
99.7
100.4
100.7
100.4
100.3
99.5
100.2
0.42

HPLC
AM
99.6
100.7
99.7
99.9
100.4
99.9
100.8
100.1
0.48

CD
100.3
100.4
100.5
99.6
99.9
100.2
101.1
100.3
0.47

AT
100.6
100.4
99.4
99.2
100.2
99.9
99.7
100.4
100.1
100.0
0.48

HPLC
AM
100.1
99.7
99.6
99.9
100.4
99.4
100.6
100.3
100.7
100.1
0.46

CD
100.5
100.4
99.8
100.2
99.7
100.5
100.4
99.4
99.9
100.1
0.40

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

Table 5
Determination of AT, AM and CD in synthetic mixtures using the PLS-1, PCR and HPLC methods
Mix. no.

1
2
3
4
5
6
7
8
9
Meana
S.D.a
a

Concentration (g ml1)
AT
AM
CD
40
160
60
140
80
100
120
40
80

2
8
7
4
6
5
3
8
7

10
40
30
40
15
20
25
40
35

AT
99.9
99.0
100.4
100.2
100.8
98.9
99.8
100.2
99.6
99.9
0.62

PLS-1
AM
99.6
100.6
100.2
100.6
99.3
100.2
101.1
98.7
100.7
100.1
0.77

CD
99.4
100.6
99.6
100.6
99.8
100.7
101.4
99.7
100.8
100.3
0.68

AT
100.4
99.7
100.7
100.1
98.9
100.3
99.7
100.6
100.9
100.1
0.63

% Recovery
PCR
AM
CD
98.9
99.3
99.3
100.6
99.8
100.2
99.8
99.7
100.6
101.2
100.0
100.6
100.7
99.4
101.2
101.1
99.3
99.9
99.9
100.2
0.75
0.70

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

Table 6
Application of standard addition technique to the analysis of AT, AM and CD using the PLS-1, PCR and HPLC methods
Sample
no

1
2
3
4
5
6
Meana
S.D.a
a

AT
Concentration (g ml1)
Claimed Added
% Found of added
PLS-1 PCR HPLC
20
40
99.8
100.6 100.2
20
60
100.4 99.4 99.9
20
80
100.2 98.7 99.4
20
100
101.0 100.7 100.7
20
120
99.5
99.7 99.8
20
140
100.2 100.2 100.2
100.2 99.9 100.0
0.51
0.77 0.44

AM
Concentration (g ml1)
Claimed Added
% Found of added
PLS-1 PCR HPLC
1
2
99.4
100.6 99.8
1
3
101.0 98.7 100.2
1
4
100.1 99.9 99.8
1
5
99.2
99.4 100.2
1
6
101.5 100.7 100.7
1
7
99.7
101.1 100.6
100.1 100.1 100.2
0.92
0.90 0.38

CD
Concentration (g ml1)
Claimed Added
% Found of added
PLS-1 PCR HPLC
5
10
99.9
101.5 99.8
5
15
99.2
99.5 100.2
5
20
100.5 100.2 100.6
5
25
100.7 100.8 100.3
5
30
99.7
99.6 99.7
5
35
100.8 100.5 99.4
100.1 100.3 100.0
0.63
0.76 0.44

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

matographic resolution in the HPLC method. Variation of

strength of hydrochloric acid by 0.02 M did not have any
significant effect on chemometric-assisted spectrophotometric methods.

absorbance changes for 24 h when kept at room temperature,

and for 4 days when stored refrigerated at 5 C.

4. Conclusion
3.4.8. Stability
Solutions of the studied compounds in the mobile phase
or 0.1 M hydrochloric acid exhibited no chromatographic or

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

A. El-Gindy et al. / Il Farmaco 60 (2005) 269278

neous determination of AT, AM and CD in pharmaceutical

tablets. The results obtained were compared with the proposed HPLC method and good coincidence was observed.
The three proposed methods were accurate with good reproducibility and sensitivity; however, better selectivity was
obtained with the HPLC method.
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