simultaneous determination of atenolol, propranolol, dipyridamole and amiloride by means of...

Simultaneous determination of atenolol, propranolol, dipyridamoleand amiloride by means of non-linear variable-angle synchronous

¯uorescence spectrometry

JoseÂ A. Murillo PulgarõÂn*, Aurelia AlanÄoÂn Molina, Pablo FernaÂndez LoÂpez

Department of Analytical Chemistry and Foods Technology, University Castilla La Mancha, 13071 Ciudad Real, Spain

Received 3 February 1998; received in revised form 8 April 1998; accepted 14 April 1998

Abstract

First derivative non-linear variable-angle synchronous ¯uorescence spectrometry has been developed to improve the

selectivity of ¯uorescence measurements without loss of sensitivity. It allows the rapid simultaneous determination of different

substances in a mixture from a single spectrum based on a single scan. This method was applied for the simultaneous

determination of atenolol (ATE), propranolol (PRO), amiloride (AMI) and dipyridamole (DIP) at concentrations between 10±

400, 6±200, 5.6±280 and 5±100 ng mlÿ1, respectively, by means of absolute values of ®rst derivative of non-linear variable-

angle synchronous scan at �ex/�em�228.8/300, 287.2/340, 366.4/412.8 and 288/487.2 nm for ATE, PRO, AMI and DIP,

respectively. In order to obtain maximum sensitivity and an adequate selectivity, factors affecting ¯uorescence intensity were

studied. As a result, the analyses were performed in an ethanol±water (70%(v/v)) medium at a pH 7.5, adjusted by using

trishydroxymethyl amino methane (0.08 M) as a buffer solution. Analytical parameters of the proposed method were

calculated according to the error propagation theory. The sensitivity, repeatability, reproducibility and limit of detection

achieved with the proposed method are adequate for the determination of these doping substances. # 1998 Elsevier Science

B.V. All rights reserved.

Keywords: Atenolol; Propranolol; Amiloride; Dipyridamole; Fluorimetry

1. Introduction

Atenolol (ATE), propranolol (PRO), amiloride

(AMI) and dipyridamole (DIP) are widely used in

the treatment of several diseases. It is well known that

the fraudulent consumption of this type of substance is

quite common these days, therefore they have been

included in the list of forbidden substances [1] by the

International Olympic Committee. ATE, PRO and

AMI [2] are antihypertensive substances, whereas

DIP [2] is an antithrombotic agent. ATE and PRO

are beta-adrenoceptors that are abused in sports with

little physical activity. These compounds, act on the

heart reducing the cardiac frequency, the contraction

force and the coronary ¯ow. AMI is a weak diuretic

which retains potassium, since it promotes sodium

excretion and potassium reabsorption. Further, low

doses of AMI lead to a high-volume urine excretion

producing a diminution of arterial tension since a great

quantity of electrolytes is eliminated. DIP can act as a

Analytica Chimica Acta 370 (1998) 9±18

*Corresponding author. Fax: 0034 9 26295318.

0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.

P I I S 0 0 0 3 - 2 6 7 0 ( 9 8 ) 0 0 2 6 4 - 5

vasodilator which stimulates a rise in the blood ¯ow

through the coronary circulation. This vasodilator

agent is classi®ed in doping terms as stimulant.

Fluorescence spectrometry is widely used in quan-

titative analysis because of its great sensitivity and

selectivity as well as its relative low cost. This tech-

nique has not, however, been widely applied to the

simultaneous direct determination of several ¯uores-

cent components in mixtures, mainly because the

¯uorescence spectra of individual substances contain

broad bands which often overlap.

Several methods have been proposed to resolve

such problems without manipulation of the samples

or using time-consuming and highly expensive separa-

tion techniques. Among them, synchronous [3] and

derivative [4] ¯uorescence spectrometry are the most

popular. Synchronous spectrometry consists essen-

tially of the simultaneous scanning of both mono-

chromators, while maintaining a constant wavelength

interval (��) between them. The simpli®cation of the

spectral pro®le together with the reduction of band

width are its main characteristics [5,6]. The synchro-

nous spectrum which is a 458 section through the

contour map is of limited use.

To achieve greater selectivity, variable-angle ¯uor-

escence spectrometer was developed [7]. The vari-

able-angle ¯uorescence spectra are obtained by

scanning the monochromators (excitation and emis-

sion) at different speeds, thus the wavelength differ-

ence between them is not constant. Because the

recorded planes are not restricted to 458, any path

can be traced, depending on the sample under study.

With variable-angle ¯uorimetry, several compounds

can be determined simultaneously from a single spec-

trum.

In the non-linear variable-angle synchronous ¯uor-

escence (NLVASF) method the trajectory of the scan is

varied continuosly through the excitation and emis-

sion matrix. This technique offers several possibilities:

1. in a complex system, the maximum and minimum

emission intensities can be explored by traversing

the peaks and valleys;

2. a curved trajectory can be followed through the

emission±excitation matrix, allowing light scatter-

ing peaks to be avoided;

3. overlapping systems that cannot be resolved by

linear scanning can be resolved by this technique.

Nowadays, this technique, in its pure concept, has

not been applied ever due to the mechanical dif®culty

that entrails the synchronization of the movement

of the excitation and emission monochromators.

Therefore, several methods described in bibliography

intend to take advantage of the characteristics

described above, realizing a synchronous scan which

is a combination of several linear variable syn-

chronous scans with different angles [8±11]. The

resulting spectra represent the intensity pro®le of a

cut through the excitation±emission matrix, the tra-

jectory of which is a continuous function. It is not

possible to take the derivative because of the different

values of the limits in the point where the trajectory

angle is varied. Therefore an appropriate empirical

equation involving the effect of each compound or

a further treatment of the data by a multilinear

regression program [10], for resolving complex

mixtures may be needed. This scan is obtained by

means of the Ftotal program [12], which permits

the theoretical NLVASF scan to be obtained from

the data stored in the contour map (the curved trajec-

tory can be varied through the emission±excitation

matrix, to describe any desired path). This step helps

to optimize the route that will produce the best

NLVASF scanning spectra (highest signal value, smal-

lest band width at half-maximum intensity and inter-

ference-free bands).

The derivative ¯uorescence technique, as described

by Green and O'Haver [13], can also be used to

enhance minor spectral features.

The combination of synchronous scanning spectro-

¯uorimetry with derivative techniques is advanta-

geous in terms of sensitivity, when compared with

results obtained by differentiation of the conventional

spectrum, because the amplitude of the derivative

signal is inversely proportional to the band width of

the original spectrum. Moreover, the selectivity of

spectro¯uorimetric analysis is greatly improved by

using both techniques in conjunction, as suggested

by John and Soutar [4] and as recently reported in

[3,14±21].

In this work, a method for analyzing a mixture of

¯uorescent doping substances with broad and highly

overlapped excitation and emission spectra is

described. The application of the ®rst derivative

NLVASF to multicomponent analysis provides a clear

example of the high resolving power of this technique

10 J.A. Murillo PulgarõÂn et al. / Analytica Chimica Acta 370 (1998) 9±18

without resorting to expensive or time-consuming

procedures.

2. Experimental

2.1. Apparatus

All ¯uorimetric measurements were performed on

an Aminco Bowman Series 2 connected to the `̀ AB2''

Software which runs on the OS2 operating system.

The instrument is equipped with a continuous 150 W

Xenon lamp. Quartz glass cuvettes with a pathlength

of 1.0 � 1.0 cm have been used.

2.2. Software

The AB2 program allows the instrument control,

operation and acquisition of excitation, emission and

total ¯uorescence spectra.

The Ftotal program [12] provides the spectral char-

acterization of analytical interest of the ¯uorescence

of any luminescent compound by generating informa-

tion through the isometric representation of the three-

dimensional spectrum as a level curve. Besides, it

processes the spectral data to obtain any type of

bidimensional spectrum. For the NLVASF spectrum

Ftotal obtains the intensity values by applying Lagran-

ge's interpolation method to the excitation wave-

lengths calculated by means of the particular

function that relates emission wavelengths at 0.8 nm

intervals with the excitation wavelengths since they

are not equidistant.

The statistical analysis is totally covered by means

of a program that has a menu which includes proce-

dures such as least median of squares regression

(detection of outlier and leverage points), least squares

regression with and without replicates, weighted least

squares regression, tests of regression and correlation,

detection and determination limits (IUPAC [27,28],

Error Propagation Theory [29,30], and Clayton et al.

[31]), ellipse graph for the 95% con®dence region for

the true slope and intercept on the y-axis estimated

from the regression method, dispersion and con®dence

bands for the calibration graph and ANOVA test for

linearity and for comparison of several regression

lines.

2.3. Reagents

All experiments were performed with analytical

reagent grade chemicals, pure solvent and Milli-Q

water. The stock solution of ATE (Aldrich, 125 mg

dissolved in 500 ml water), PRO (Aldrich, 125 mg

dissolved in 500 ml water), AMI (Sigma, 125 mg

dissolved in 500 ml water) and DIP (Aldrich,

125 mg dissolved in 500 ml ethanol) were diluted

(DIP with ethanol) to prepare standard solutions by

suitable dilutions. The stock solutions were stored and

protected from light. Under these conditions, the

solutions were stable for at least three months.

Working samples of ATE, PRO, AMI and DIP were

stable for at least 2 h at room temperature.

Pharmaceutical preparations of Normopresil

(Serma S.A., Barcelona, Spain), Atenolol Leo (Byk

Elmu S.A., Madrid, Spain), Neatenolol diu (Fides-

Rottapharma, S.A., Valencia, Spain), Sumial 10,

Ameride (Du Pont Pharma, S.A., Madrid, Spain),

Kalten (Zeneca Farma, S.A., Pontevedra, Spain) and

Persantin (Boehringer Ingelheim, S.A., Barcelona,

Spain) with different nominal contents, were ran-

domly purchased from local pharmacies.

2.4. Procedure

2.4.1. Sample preparation

For the preparation of the calibration graph, place

an aliquot of ATE, PRO, AMI and DIP equivalent to

0.25±10, 0.15±5, 0.14±7 and 0.125±2.5 mg, respec-

tively, in a 25 ml volumetric ¯ask, add 2 ml of buffer

solution (pH 7.5), 17.5 ml of analytical grade ethanol,

and dilute with water to a ®nal volume of 25 ml and

shake.

Record for each sample 61 emission spectra of

384 nm width in steps of 0.8 nm, varying the excita-

tion wavelength in 6.4 nm steps. Obtain the total

luminescence spectra by means of the Ftotal program

[12]. Apply the function that passes through the

excitation and emission maxima selected for ATE,

PRO, AMI and DIP to obtain the NLVASF spectrum.

Calculate the ®rst derivative, according to the Savitzky

and Golay algorithm [22,23]. The absolute values of

the ®rst derivative of these synchronous spectra were

measured at �ex/�em�228.8/300, 287.2/340, 366.4/

412.8 and 288/487.2 nm for determination of ATE,

PRO, AMI and DIP, respectively.

J.A. Murillo PulgarõÂn et al. / Analytica Chimica Acta 370 (1998) 9±18 11

For the analysis of the formulations: Normopresil,

Atenolol Leo, Neatenolol diu, Sumial 10, Ameride,

Kalten and Persantin introduce three tablets of each

formulation in three different 1 l volumetric ¯asks.

Add water suf®ciently to dissolve them. Shake and

maintain them in an ultrasonic bath for 15 min. Dilute

with water to a ®nal volume of 1 l. Persantin, which

contains DIP, must be dissolved and diluted with

ethanol.

In both cases, the assay was completed as described

for calibration graph. The percentage recovery of

every compound is computed from regression equa-

tions for pure drugs.

3. Results and discussion

3.1. Spectral characteristics

The best characterization of the ¯uorescence of the

compounds is achieved by obtaining the three-dimen-

sional spectrum. This spectrum can be obtained and

presented as the isometric projection, where the exci-

tation spectra at stepped increments of emission wave-

length are recorded and plotted, as shown in Fig. 1(a)±

(d), where Rayleigh scattering has been removed. A

reversed projection (where emission spectra are

plotted at decreased excitation wavelength) of the

data can sometimes indicate emission peaks hidden

by the foreground. Alternatively, the three-dimen-

sional spectra can be effectively transformed to a plot

in two dimensions of the excitation and emission

wavelength by linking points of equal intensity to

form contours. In general, this contour presentation

is more useful than the isometric projection for indi-

cating the presence of hidden emission peaks and

providing the selection of the best trajectory to obtain

optimum results in the application of the synchronous

scan technique.

Fig. 2 shows the total ¯uorescence spectra of ATE

((A), solid line), PRO ((B), broken line), AMI ((C),

solid line) and DIP ((D), broken line). As can be

Fig. 1. Isometric plot of the excitation±emission matrix (backward projection) of (a) ATE, (b) PRO, (c) AMI and (d) DIP.


observed, owing to spectral overlapping, the analysis

of mixtures of the four compounds would not be

feasible by conventional spectro¯uorimetry at their

wavelength maxima.

3.2. Influence of experimental variables

Chemical variables were studied and optimized to

obtain the best measurement conditions, maximum

¯uorescence sensitivity and an adequate selectivity.

ATE, PRO and AMI are soluble in water, but DIP is

not, and consequently, it was necessary to use an

organic solvent. DIP is highly soluble in ethanol,

therefore it was necessary to study the variation in

the ¯uorescence of ATE, PRO, AMI and DIP with

changes of the ethanol percentage. In this way the

effect of ethanol content in the medium was investi-

gated by preparing samples varying the ethanol per-

centage between 10% and 90%(v/v). In all cases, the

¯uorescence intensity increases with the percentage of

ethanol. A 70% of ethanol was selected as adequate.

The in¯uence of pH on the ¯uorescence intensity

was studied by adding different amounts of HCl and

NaOH. Hardly any variation is observed in the ¯uor-

escence intensity of ATE, PRO and AMI, while the

¯uorescence intensity of DIP is constant up to a pH

value of 4.0. It strongly increases for pH values

between 4.0 and 6.5 and the maximum ¯uorescence

intensity is reached for apparent pH values higher than

6.5. An apparent pH value of 7.7 is adequate for the

determination because the maximum intensity is

achieved. The pH selected was adjusted by adding

trishydroxymethyl amino methane buffer solution.

This buffer was soluble in the ethanol medium and

besides, the ¯uorescence intensity of ATE, PRO, AMI

and DIP was not affected by this and its concentration.

A 0.08 M concentration of the buffer was therefore

selected to get a suf®cient buffering capacity.

Another factor that affects the ¯uorescence inten-

sity is the temperature. The ¯uorescence intensity

showed a decrease when the temperature increases

from 58C to 658C. The temperature coef®cient is

obtained by performing the least squares regression

of the relative signal increment versus the tempera-

ture. The slope is the temperature coef®cient. The

results were 0.6%, 0.5%, 1.2%, and 0.2% 8Cÿ1 for

ATE, PRO, AMI and DIP, respectively. ATE, PRO and

DIP show a clear linear behavior with the temperature,

while AMI does not. Therefore, the use of a thermostat

is recommended and a measurement temperature of

208C was chosen.

The in¯uence of analytes concentration on the

¯uorescence intensity was studied under these condi-

tions. The best range for the relation ¯uorescence

intensity versus concentration was up to 400, 200,

280 and 100 ng mlÿ1 for ATE, PRO, AMI and DIP,

respectively.

3.3. Selection of optimum route

The major dif®culty encountered in the application

of the synchronous scan technique is that the best route

must be known beforehand to get optimum results.

Consequently, a detailed inspection of each of the

contour maps is necessary. The optimum route is

immediately obvious even to relatively inexperienced

operators. Moreover, the advances in analytical instru-

ments and the use of computers permits to obtain

analytical results (contour maps and its treatment to

obtain the ®rst derivative of NLVASF scan) in less than

10 min.

To determine the optimum NLVASF scan route, a

careful examination of a contour map corresponding

to a standard solution of the four compounds at

concentration levels which produce equal intensity

of ¯uorescence, has been carried out (Figs. 1 and

Fig. 2. Contour plot of ATE ((A), solid line), PRO ((B), broken

line), AMI ((C), solid line) and DIP ((D), broken line). The route is

a cubic equation that passes through the points �ex1�228.8, �em1�300 nm (ATE), �ex2�228.8, �em2�340 nm (PRO), �ex3�366.4,

�em3�412.8 nm (AMI) and �ex4�288, �em4�487.2 nm (DIP). This

route corresponds to the following function: �ex�1984.89ÿ18.4371��em�5.99749�10ÿ2��em2ÿ6.01003�10ÿ5��em3.


2). The route is optimized to minimize the spectral

interference caused by every compound in the mixture

with no loss of sensitivity.

From the contour plots it is evident that a route

which is obtained by means of the cubic equation that

passes through the points �ex1�228.8, �em1�300 nm

(ATE), �ex2�228.8, �em2�340 nm (PRO), �ex3�366.4, �em3�412.8 nm (AMI) and �ex4�288, �em4�487.2 nm (DIP), is the optimum to pass through the

excitation and emission maximum of the four com-

pounds. This route corresponds to the following func-

tion: �ex � 1984:89ÿ 18:4371� �em � 5:99749�10ÿ2 � �2

em ÿ 6:01003� 10ÿ5 � �3em. This function

is represented in Fig. 2.

3.4. Simultaneous determination of ATE, PRO, AMI

and DIP

Fig. 3 shows NLVASF spectra for ATE, PRO, AMI

and DIP. The determination by measuring the max-

imum intensity produces errors in the quanti®cation of

PRO by the presence of ATE and in DIP by the

presence of AMI. Owing to this overlap of the spectra,

the determination of these doping substances only by

NLVASF was not feasible. This overlap has been

resolved by taking the ®rst derivative of the spectrum.

The ®rst derivative of NLVASF spectra for ATE,

PRO, AMI and DIP are shown in Fig. 4. Although the

®rst derivative does not separate the bands, satisfac-

tory results could be obtained by applying the zero-

crossing technique. We selected 238.6/307.2 (A),

301.4/349.6 (B), 354.6/393.6 (C) and 312.4/

477.6 nm/nm (D) as optimal �ex/�em for the determi-

nation of ATE, PRO, AMI and DIP, respectively.

The number of points through which the derivative

is obtained was optimized, and it was concluded that

derived spectra with a suitable signal-to-noise ration

are obtained with 25 points.

3.5. Statistical analysis results

In order to test the mutual independence of the

analytical signal of ATE, PRO, AMI and DIP, i.e., to

show that zero-crossing wavelengths selected are

independent of the compounds present, three calibra-

tion graphs were constructed from ®rst derivative

signals for each one in the absence and in the presence

of different concentration of the rest of the com-

pounds. The concentration interval of the calibration

graphs and the concentration of the rest of the com-

pounds are given in the second and third column of

Tables 1 and 2. The concentration levels of the cali-

bration graphs were selected according to maximum

Fig. 3. NLVASF spectra of (A) ATE, (B) PRO, (C) AMI and (D)

DIP. (ATE)�400 ng mlÿ1; (PRO)�200 ng mlÿ1; (AMI)�280 ng mlÿ1; (DIP)�100 ng mlÿ1.

Fig. 4. First derivative of NLVASF spectra of (A) ATE, (B) PRO,

(C) AMI and (D) DIP. (ATE)�400 ng mlÿ1; (PRO)�200 ng mlÿ1;

(AMI)�280 ng mlÿ1; (DIP)�100 ng mlÿ1.


sensitivity for the four compounds in the instrumental

conditions. First, the voltage of the photomultiplier

was optimized at the wavelength selected for the

determination for each compounds at same concen-

tration levels (100 ng mlÿ1). The minimum adjusted

voltage of the photomultiplier was selected, which

was in the case of DIP, then, the upper limit of the

calibration graph concentration for the other sub-

stances (ATE, PRO and AMI) was optimized accord-

ing to this voltage.

In order to test the presence of outlier the least

median of squares regression (LMS) [24] was applied.

As LMS is a robust regression method, it is able to

detect outlier points. These outliers cause errors in the

true line when experimental data are ®tted according

to the least squares regression. No outliers were

detected. Table 1 shows the outstanding results of

the statistical analysis.

The proposed method was evaluated by a statistical

analysis of experimental data by ®tting the least-

squares line according to y�a�bx [25,26]. Table 2

shows the outstanding results of the statistical analysis

of data.

To obtain the most representative calibration graph,

a classic overall least squares regression was devel-

oped, including all the data pairs. In order to test the

validity of the overall regression line an ANOVA test

comparing the three lines proposed for every analyte

was performed [26]. The F1 value compares the total

deviations from the overall linear region with the

deviation within each set from the set line testing

the suitability of the single overall regression line. If

the experimental value F1 is less than the theoretical

value F1, the departure of the individual sets from the

overall regression line is not signi®cant. The F2 value

compares the differences among the regression coef-

®cients, i.e., the slopes with the deviation within each

set from the set line testing the differences between the

regression coef®cients. If the experimental value F2 is

less than the theoretical value F2, then there are no

signi®cant differences between the individual slopes

and the overall regression slope may be taken as the

representative one. In all cases the validity of the

overall regression lines is proven by the values of

experimental F1 and F2 values which are less than the

theoretical ones at 95% con®dence level.

The residual values about the regression lines pro-

posed show a uniform variance (the errors of measure-

ments are independent with the concentration of the

drugs), therefore the regression lines meet the require-

ment of homocedasticity [25,26].

The signi®cance of the intercept on the y-axis was

established by testing the 95% joint con®dence inter-

vals for the parameters of the linear regression model

Table 1

Application of least median of squares regression (y�a�bx) to the data of the calibration sets obtained by means of first derivative of non-

linear variable-angle synchronous fluorescence

Compound

determined

Concentration

interval (ng mlÿ1)

Compound

present (ng mlÿ1)

Intercept (a) Slope (b) Determination

coefficient (r2)

SD of

estimation

ATE 10±400 Absence of the others 8.805�10ÿ4 6.041�10ÿ4 0.9999 4.1�10ÿ4

PRO 100, AMI 140, DIP 80 4.622�10ÿ3 5.628�10ÿ4 0.9999 1.9�10ÿ4

PRO 18, AMI 16.8, DIP 20 ÿ3.108�10ÿ4 5.808�10ÿ4 0.9999 1.8�10ÿ4

PRO 6±200 Absence of the others ÿ4.410�10ÿ4 1.450�10ÿ3 0.9999 9.5�10ÿ4

ATE 300, AMI 140, DIP 80 1.510�10ÿ3 1.418�10ÿ3 0.9999 6.5�10ÿ4

ATE 40, AMI 16.8, DIP 20 ÿ4.660�10ÿ4 1.440�10ÿ3 0.9999 7.5�10ÿ4

AMI 5±280 Absence of the others ÿ1.409�10ÿ4 1.108�10ÿ3 0.9999 2.9�10ÿ4

ATE 300, PRO 100, DIP 80 ÿ7.756�10ÿ6 1.074�10ÿ3 0.9999 3.6�10ÿ4

ATE 40, PRO 18, DIP 20 7.458�10ÿ4 1.028�10ÿ3 0.9999 1.5�10ÿ4

DIP 5±100 Absence of the others ÿ2.713�10ÿ4 3.952�10ÿ3 1.0000 1.3�10ÿ4

ATE 300, PRO 100, AMI 140 ÿ1.293�10ÿ3 4.061�10ÿ3 1.0000 5.1�10ÿ4

ATE 40, PRO 18, AMI 16.8 1.487�10ÿ4 4.107�10ÿ3 1.0000 3.0�10ÿ4

ATE (atenolol), PRO (propranolol), AMI (amiloride), DIP (dipyridamole).


[26]. If the intercept on the y-axis `̀ a'' is plotted

against the slope `̀ b'' for repeated random samples

the points will fall elliptically around the true center

(�, �), and conversely, any con®dence interval for the

true combination of � and � will take the form of an

elliptical region around the best estimates (a,b) as

center [19]. If the null intercept on the y-axis falls

within the joint con®dence region, the intercept is not

signi®cantly different from zero. Therefore a propor-

tional relation between analytical signal and concen-

tration can be assumed. All intercepts found are not

signi®cantly different from zero.

By applying the IUPAC [27,28] de®nition, based

only on three times the standard deviation of the blank

a detection limit of 5.2, 1.9, 2.1 and 0.1 ng mlÿ1 was

obtained for ATE, PRO, AMI and DIP, respectively.

The propagation of errors will give a detection limit

consistent with the reliability of the blank measure-

ments (IUPAC) and besides the signal measurements

of the standards [29,30]. In this case a detection limit

of 5.9, 2.0, 2.8 and 1.0 ng mlÿ1, respectively, was

obtained for each. Moreover, Clayton [31] considers

the probability of positive false and negative false, the

detection limit being 6.7, 2.3, 3.2 and 1.2 ng mlÿ1 for

ATE, PRO, AMI and DIP, respectively. Because of the

limit of detection according to Clayton includes all the

possible errors described above, it is suitable to

assume this type of detection limit.

Table 2

Application of least squares regression (y�a�bx) to the data of the calibration sets obtained by means of first derivative of non-linear variable-

angle synchronous fluorescence (comparison of several regression lines by means of ANOVA test (F1 and F2))

Compound

determined

Concentration

interval

(ng mlÿ1)

Compound

present

(ng mlÿ1)

Intercept (a) Slope (b) Determination

coefficient (r2)

SD of

estimation

F1 F2

ATE 10±400 Absence of the others 2.849�10ÿ3 5.796�10ÿ4 0.9985 3.4�10ÿ3

PRO 100, AMI 140,

DIP 80

2.381�10ÿ3 5.729�10ÿ4 0.9994 2.1�10ÿ3 0.78 0.77

PRO 18, AMI 16.8,

DIP 20

4.585�10ÿ4 5.852�10ÿ4 0.9995 2.0�10ÿ3

Overall 1.896�10ÿ3 5.792�10ÿ4 0.9991 2.5�10ÿ3

PRO 6±200 Absence of the others 3.073�10ÿ4 1.446�10ÿ3 0.9997 1.9�10ÿ3

ATE 300, AMI 140,

DIP 80

2.105�10ÿ3 1.422�10ÿ3 0.9998 1.5�10ÿ3 2.18 1.92

ATE 40, AMI 16.8,

DIP 20

2.854�10ÿ4 1.416�10ÿ3 0.9994 2.5�10ÿ3

Overall 8.993�10ÿ4 1.428�10ÿ3 0.9995 2.2�10ÿ3

AMI 5±280 Absence of the others 1.569�10ÿ3 1.059�10ÿ3 0.9992 3.0�10ÿ3

ATE 300, PRO 100,

DIP 80

5.021�10ÿ5 1.074�10ÿ3 0.9999 6.4�10ÿ4 1.71 2.72

ATE 40, PRO 18,

DIP 20

1.311�10ÿ3 1.046�10ÿ3 0.9996 2.1�10ÿ3

Overall 9.766�10ÿ4 1.060�10ÿ3 0.9995 2.3�10ÿ3

DIP 5±100 Absence of the others ÿ9.366�10ÿ4 4.023�10ÿ3 0.9996 2.8�10-3

ATE 300, PRO 100,

AMI 140

ÿ2.029�10ÿ3 4.104�10ÿ3 0.9999 1.3�10ÿ3 2.85 2.47

ATE 40, PRO 18,

AMI 16.8

1.383�10ÿ3 4.040�10ÿ3 0.9996 2.8�10ÿ3

Overall ÿ5.276�10ÿ4 4.056�10ÿ3 0.9996 2.9�10ÿ3

ATE (atenolol), PRO (propranolol), AMI (amiloride), DIP (dipyridamole). Theoretical F1 (4, 15, 0.05)�3.06. Theoretical F2 (2, 15,

0.05)�3.68.


Tab

le3

Res

ult

from

the

assa

ys

of

ph

arm

aceu

tica

lp

rep

arat

ions

by

firs

tder

ivat

ive

of

non-l

inea

rvar

iable

-angle

synch

ronous

fluore

scen

cete

chniq

ue

Ph

arm

aceu

tica

l

pre

par

atio

n

No

min

alco

nte

nt

(mg

)A

mount

added

(mg)

Am

ount

found

(mg)

Rec

over

y(%

)

AT

EP

RO

AM

ID

IPA

TE

PR

OA

MI

DIP

AT

EP

RO

AM

ID

IPA

TE

PR

OA

MI

DIP

Kal

ten

50

.0Ð

2.5

ÐÐ

13.3

Ð8.3

48.4

14.2

2.6

58.2

96.8

106.9

106.0

99.3

Nea

ten

olo

ld

iu1

00

.0Ð

ÐÐ

Ð50.0

43.8

31.2

99.3

53

45.4

31.4

99.3

106.1

103.6

100.6

Ate

no

lol

leo

10

1.3

ÐÐ

ÐÐ

50.0

43.8

31.2

105.5

53.6

45.8

32.5

104.1

107.2

104.5

104.3

No

rmo

pre

sil

10

0.0

ÐÐ

ÐÐ

50.0

43.8

31.2

110.0

54.0

45.8

31.6

110.0

108.1

104.5

101.4

Su

mia

l1

0Ð

10

.0Ð

Ð25.0

Ð11.7

8.3

26.9

10.9

12.1

8.6

107.5

109.1

103.4

103.2

Per

san

tin

ÐÐ

Ð1

01.5

312.5

166.7

145.8

Ð332.2

166.7

132.8

96.1

106.3

100.0

91.1

94.7

AT

E(a

ten

olo

l),

PR

O(p

rop

ran

olo

l),

AM

I(a

mil

ori

de)

DIP

(dip

yri

dam

ole

).


In order to study the precision of the method, a

series of ten solutions was prepared containing 80, 24,

22.4 and 40 ng mlÿ1 of ATE, PRO, AMI and DIP,

respectively, and they were measured 15 times in a day

(repeatability study) and once a day for 10 days

(reproducibility study). The standard deviation, and

coef®cients of variation of repeatability at each of

these levels were 2.0, 0.35, 0.19, and 0.32 ng mlÿ1 and

2.5%, 1.5%, 0.85% and 0.80%, respectively. The

standard errors and coef®cients of variation of repro-

ducibility at each of these levels were 9.2, 0.99, 0.19,

and 1.43 ng mlÿ1 and 11.5%, 4.1%, 0.85% and 3.6%,

respectively (95% con®dence level).

In order to check the usefulness of the proposed

method, and because there are no pharmaceutical

dosage forms commercially available containing

ATE, PRO, AMI and DIP simultaneously, the pro-

posed method has been applied to their determination

in six different pharmaceutical preparations which

contains at least one of the four drugs. These prepara-

tions were conveniently spiked with different amounts

of the other compounds and the mixture analysis was

carried out. The assay results expressed as a percen-

tage of the nominal contents (R%) resulting from the

average of the determination of three different tablets

are summarized in Table 3. The recoveries (from

96.8% up to 110.0%) agree well enough with the

nominal content and the precision is quite satisfactory.

Acknowledgements

The authors gratefully acknowledge ®nancial sup-

port from the `̀ DireccioÂn General de InvestigacioÂn

CientõÂ®ca y TeÂcnica'' (Project no. PB 94-0743).

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simultaneous determination of atenolol, propranolol, dipyridamole and amiloride by means of...

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