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Variable-angle scanning fluorescence spectrometry for the simultaneous determination of three diuretic drugs
ANALYTICA CHIMICA
ACTA ELSEVIER
Analytica Chimica Acta 306 (1995) 313-321
Variable-angle scanning fluorescence spectrometry for the simultaneous determination of three diuretic drugs Francisco Garcia SBnchez a, Albert0 Fernindez Carmen Cruces Blanc0 b**
Gutierrez b,
a Department ofAnalytical Chemistry, University of Mblaga, Milaga, Spain ’ Department of Analytical Chemistry Faculty of Sciences, University of Granada, C / Fuentenueva s/n, Received 2 August 1994; revised 2 November
1994; accepted 8 December
18071 Granada, Spain
1994
Abstract
The applicability of variable-angle synchronous scanning (VASS) fluorescence spectroscopy has been demonstrated for the resolution of mixtures of three diuretics (furosemide, triamterene, piretamide) with closely overlapping fluorescence profiles. This technique permits linear or non-linear paths to be scanned at preselected angles through the excitation-emission matrix by scanning both monochromators at different speeds, in order to obtain the best variable-angle scanning spectra (highest signal values and interference-free bands). Such an approach has permitted the simultaneous determination of the three compounds at the pg to ng ml - ’ level, with a relative standard deviation I 5% (n = 10). When applied to commercial pharmaceutical dosage forms, recoveries of the original drug between 98-101% have been obtained. Keywords: Fluorimetry;
Diuretics;
Pharmaceutical
analysis
1. Introduction When substances with overlapping spectra have to be determined simultaneously by spectrofluorimetry, it could be quite difficult to choose a pair of excitation and emission wavelengths that would permit the observation of one of them without the interference by the other. For such a reason, it is evident that good methods of reducing spectral band widths or, otherwise, improving spectral selectivity, will be of great benefit in spectrofluorimetric analysis. Several methods have been proposed to resolve such problems (overlapping) without manipulation of
* Correspondingauthor. 0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDi 0003-2670(94)00689-X
the samples or using time-consuming and highly expensive separation techniques. Synchronous fluorescence alone [1,2] or in combinations with derivative techniques [3,4] have provided a convenient means for implementing sensitivity [5-131 and selectivity 114-231 in both inorganic and organic chemistry. One drawback to conventional synchronous scanning is that it is limited to producing 45” sections cut through the excitation-emission matrix (EEM), whose locus is defined by the constant wavelength interval (Ah) employed. In recent years, advances in analytical instrumental and the general use of computers have promoted the application of mathematical algorithms for treating the large amount of data
314
F. Garcia Srinchez et al. /Analytica
that modem instrumentation can provide, increasing the selectivity of analytical methods. A novel approach of conventional synchronous scanning, proposed by Kubic et al. [24], and which offers considerable flexibility, is variable-angle synchronous scanning (VASS), where the wavelength separation between the two monochromators is varied. There are three different instrumental configurations for performing VASS. First, the speed of the monochromators can be manipulated by two different motors scanned at different rates [25]. A second approach consists of acquiring the EEM and storing the data on the interfaced microcomputer, and the desired angle (linear or nonlinear) is determined by using the appropriate software [26]. Recently, Garcia Sanchez and co-workers [27,28] have modified a commercial digital instrument to generate the VASS directly from the spectrofluorimeter output. With this approach, applied in the present work, a few minutes will be sufficient to obtain the variable-angle spectra. As it is observed, VASS does not require highly sophisticated equipment but the applications found in the literature are fairly uncommon [29-311, due to the fact that the VASS mode is not yet widely accessible. In the present work, the increased selectivity afforded by the VASS technique, has been well demonstrated in studies on the determination of three diuretic drugs (furosemide, triamterene, piretamidel, which permits to avoid previous separation techniques, which have normally been used for this compounds in real samples [32].
2. Experimental 2.1. Apparatus Two spectrofluorimeters have been used. The three-dimensional spectra and their corresponding contour maps are all recorded on a Perkin-Elmer MPF-66 spectrofluorimeter, equipped with a 150-W xenon arc lamp and a R-928 photomultiplier. The spectrofluorimeter was connected to a Perkin-Elmer Model 7300 Professional computer provided with PETLS application software (C 646-0280). The scanning speed and the response time were set at 240 nm
Chimica Acta 306 (1995) 313-321
min-’ and 0.5 s, respectively. Slit-widths were set at 5 and 3 nm, for the excitation and emission monochromators, respectively. The variable-angle fluorescence spectra were recorded on a Perkin-Elmer LS-5 spectrofluorimeter which includes a xenon discharge lamp (9.9 W) pulsed at line frequency and f/3 Monk-Guillieon monochromators. The spectrofluorimeter was operated in the computer-controlled mode via the RS232C serial interface by either of the following systems. In one, an IBM-PC/XT microcomputer equipped with a 3.5-in. floppy disk, provided with the Fluoropack package [27] which controls the spectrofluorimeter and the acquisition of variable-angle fluorescence data. In the other, a Perkin-Elmer Model 3600 Data Station microcomputer was used. With the software package used, a variable-angle scan at the desired angle can be obtained by selecting the appropriate value and sign for each of the different increments. With this method, it is possible to obtain a number of sequential segments, each at different angles. Each pair of adjacent segments can be linked together, thereby defining any desired trajectory throughout the EEM. Slit-widths for both monochromators were set at 5 nm, with a scanning speed of 240 nm min- ’ . In both spectrofluorimeters, the measurements were made in standard 1 X l-cm path-length quartz cells, thermostatically controlled at 25 + 0.5” C with a water-bath circulator (Frigiterm S-382). 2.2. Reagents All the experiments were performed with analytical-reagent grade chemicals and pure solvents. Doubly distilled, demineralized water was used throughout. Triamterene (TAT) was obtained from Sigma (T4143). Stock solutions were prepared in a 50-ml calibrated flask by dissolving 5 mg of the compound in absolute methanol. Furosemide (FUR) was also purchased from Sigma (F-4381) and its stock solutions were prepared in a 25-ml calibrated flask by dissolving 10 mg in absolute ethanol. Piretamide (PIR) was synthetized and purified in our laboratories. A stock solution was prepared in a 25-ml calibrated flask by dissolving 5 mg of the compound in absolute ethanol. These stock solutions were pre-
315
F. Garcia Srinchez et al. / Analytica Chimica Acta 306 (1995) 313-321
pared weekly and were used to prepare standard solutions daily by suitable dilutions with absolute ethanol.
3. Results and discussion
3.1. Selection of experimental
and instrumental pa-
rameters
2.3. General procedure Place aliquots of the working solutions of each of the three diuretics, containing between 100 and 400 pg of FUR, 0.5 to 1.5 ng of TAT and 10 and 100 pg of PIR into 10-1111volumetric flasks and dilute to final volume with absolute ethanol. Record the three-dimensional fluorescence spectra and their corresponding contour maps at a scan speed of 240 nm mine ’ with 90 scans and 2 nm increments, using an excitation slit of 5 nm and an emission slit of 3 nm. Measure the fluorescence intensity in the VASS maps at the following wavelength excitation and emission pairs: 359-429,385-417 and 290-459 nm, for FUR, TAT and PIR, respectively, and plot against concentration of each diuretic. 2.4. Procedure
for pharmaceutical
compounds
anal-
ysis
Furosemide was determined in tablets: Seguril (Hoechst Ibtrica, Barcelona) with a nominal content of 40 mg of furosemide. Triamterene was also determined in tablets: Triniagar (Lab. Farmasimes, Barcelona) containing triamterene (50 mg) and mebuticide (50 mg>, respectively. Piretamide was determined in capsules: Perbilen (Hoechst IbCrica) containing piretamide (6 mg). For FUR and TAT analysis, twenty tablets were weighed and powdered and an amount of the powder containing ca. the equivalent of one tablet was shaken with absolute ethanol and methanol, respectively, and diluted to 100 ml with the same solvent. The extract was filtered through Whatman No. 1 filter paper, the first 20 ml of filtrate were discarded and a suitable aliquot of the filtrate was diluted to 25 ml with absolute ethanol. The contents of piretamide capsules were directly dissolved in ethanol and the standard solutions of the three compounds were conveniently diluted with absolute ethanol to obtain working solutions in the concentration range of their calibration graphs. Aliquots of these solutions were treated as indicated under General procedure.
Furosemide and piretamide are readily soluble in ethanol, while triamterene could only be dissolved in methanol. For such a reason, native fluorescence of the three compounds was studied in aqueous, methanolic or ethanolic solutions. Fluorescence emission was strongly dependent on solvent composition, the best results being obtained using ethanol as solvent. Different proportions of water-ethanol mixtures were studied. It was observed that the fluorescence intensity of the three compounds increased as the ethanol concentration increased. So, a 100% ethanol solution was the most satisfactory, giving the highest fluorescence intensity, and was chosen for subsequent experiments. A study of the stability of the solutions at 25” C showed that the compounds were stable for, at least, 3 h. In order to avoid possible fluorescence signal modifications, a thermostatically controlled waterbath at this temperature is recommended for the rest of the experimental work. The object of this work was to demonstrate the applicability of the VASS technique to the resolution
:,o
A\ 350
‘\--__ 430
390
470
” 510
x nm
Fig. 1. Fluorescence excitation (A, B, C) and emission (A’, B’, C’) spectra of piretamide (A, A’) ( A,, = 348 nm, A,, = 437 nm), furosemide (B, B’ ) (A,, = 365 nm, A,, = 410 nm) and triamterene (C, C’) (A,, = 372 nm, Aem = 436 nm). Concentrations of each compound: 1.2 pg ml-‘; 2.0 Kg ml-’ and 20 ng ml-‘, respectively. RF1 = Relative fluorescence intensity.
316
F. Garcia Srinchez et al. /Analytic0
Chimico Acta 306 (1995) 313-321
, . 1
F. Garcia Srinchez et al. /Analytica
of a mixture of compounds with closely overlapping fluorescence spectra. The excitation and emission spectra for FUR, TAT and PIR are shown in Fig. 1. As can be observed, because of spectral overlapping, the analysis of mixtures of the three compounds would not be feasible by conventional spectrofluorimetry at their wavelength maxima.
These conventional excitation and emission spectra are not complete descriptions of the spectral distribution of these compounds, because the excitation spectra are obtained at only a single emission wavelength and the emission spectra at a single excitation wavelength. A complete description requires a three dimensional spectrum [32], the total (-)
Chirrioe
(+)
L hin)
/
A,
(b)
Em.
(nm)
= 260 nfn
(min) = 350 nm
A.. km) L
330
317
Chimica Acta 306 (1995) 313-321
= 390 nm
(max) = 487 “In
550 416
Fig. 3. Three-dimensional VASS spectrum (a) and its corresponding contour map (b) of an isoemissive mixture of the three diuretics with VASS route selected. Concentrations of each compound: piretamide (0.6 Kg ml-’ 1, furosemide (1.4 pg ml-‘) and triamterene (120 ng ml-’ ).
318
F. Garcia S&chez et al./Analytica
luminescence spectrum (TLS). Sequential scans of the emission spectra (90 scans) were carried out between 330 and 550 nm at different excitation wavelengths ranging from 220 to 428 nm. The corresponding TLS of each of the three compounds are shown in Fig. 2a. All the necessary data are collected in the computer-interfaced spectrometer, and the corresponding contour maps may also be obtained (Fig. 2b). Previously, in order to select the appropriate route to minimize the spectral interference with the minimum loss of sensitivity, a detailed inspection of each of the contour maps, is necessary. As is observed in Fig. 2b, the zones of maximum fluorescence intensity appear at the main peaks of each compound centred on their respective wavelength excitationemission pairs which are (280 nm, 407 nm) and (336 nm, 414 nm) for PIR, (276 nm, 379 nm) and (336 nm, 401 nm) for FUR and (368 nm, 432 nm) and (278 nm, 371 nm) for TAT, respectively. To determine the optimum VASS route, a careful examination of a contour map corresponding to a standard solution of the three diuretics in near isoemissive concentrations, has been carried out (Fig. 3b). The variable-angle scanning route shown traverses those parts of the contour map with the least overlap. Although some sensitivity is lost because no peak maxima are traversed, nearly interference-free signals of the three components may be obtained because the route avoids the regions of maximum interference between the three compounds. The corresponding three-dimensional variable-angle spectrum of the same three-component mixture of the diuretics, to illustrate the potential utility of this technique for mixture analysis, is shown in Fig. 3a. The software package used [27] displays the required data in 5 min, in comparison with periods of several hours required to obtained the three-dimensional data matrices, which indicates the ready applicability of the present method to the quantitative analysis of different compounds in routine analysis. 3.2. Analytical characteristics In order to test the mutual independence of the analytical signals of FUR, TAT and PIR , the following calibration graphs were obtained for standards containing 1.0-10.0 pg ml-’ of piretamide in the
Chimica Acta 306 (1995) 313-321
2rm 4x9
R.F.I.
I
FUR
487
Lhm)
TAT
R.F.I.
480.8
c)
PKR
R.F.I.
350
40s
4
Fig. 4. Two -dimensional projections of several miXhIreS of piretamide, furosemide and triamterene: (a) piretamide (l-10 pg ml-‘), furosemide (20.0 pg ml-‘), triamterene (100 ng ml-‘); (b) furosemide (lo-40 @g ml-‘), piretamide (4.0 pg ml-‘), triamterene (100 ng ml-‘); (c) triamterene (SO-150 ng ml-‘), piretamide (4.0 pg ml-‘), furosemide (20.0 pg ml-‘).
F. Garcia Srinchez et al./Analytica Table 1 Analytical
parameters
of the proposed
Diuretic Furosemide
Piretamide
Triamterene
319
methods Calibration
Other diuretics present
Diuretic determined
a Arbitrary h n = 10.
Chimica Acta 306 (1995) 313-321
Corm. ( pg ml
_
_
TAT
0.1
PIR _
4.0
TAT
0.1
FUR _ PIR
20.0 _ 4.0
FUR
20.0
’1
Intercept
Slope a
a
Corr. coeff. b
2.3
+ 50.9
0.988
1.4
+61.3
0.981
28.7
- 19.6
0.993
27.1
+5.4
0.996
1.8
-7.1
0.999
1.2
+ 12.9
0.988
units.
absence and presence of 100 ng ml-’ and 20.0 pg ml-’ of triamterene and furosemide, respectively. Similarly, graphs were prepared for standards containing 10.0-40.0 pg ml-’ of furosemide in the absence and presence of 4.0 pg ml-’ and 100 ng as well as a ml-’ of piretamide and triamterene, calibration graph for triamterene (50-150 ng ml-‘) in the absence and presence of 20.0 pg ml-’ and 4.0 pg ml-’ of furosemide and piretamide, respectively. The utility of the selected VASS route was demonstrated by obtaining the two-dimensional projections of these calibration graphs (see Fig. 4a, b and cl. As it can be deduced from these VASS spectra, large errors would be obtained when measuring conventionally the different mixtures indicated in the calibration graphs; by avoiding the wavelength pairs of maximum fluorescence intensity of each
Table 2 Results obtained
for synthetic
Mixture composition
compound and selecting for their measurement those corresponding to less fluorescence intensity and better resolution (see General procedure), the interferences are greatly decreased. Some of the features of the proposed methods are summarized in Table 1. The relative standard deviation (n = 10) for determination of 20.0 pg ml-’ of FUR; 100 ng ml-’ of TAT and 4.0 pg ml-’ of PIR were 5.2, 3.4 and 1.4%, respectively. The VASS spectrofluorimetric method has been applied to the analysis of several synthetic mixtures of the three compounds in different ratios. Table 2 summarizes the results calculated from the calibration graphs. Despite a detailed selection of the variable-angle route through the EEM, the strong overlap between the spectra of the three selected compounds causes some error in the simultaneous determination of the three compounds. However, as can be deduced from
mixtures of the three compounds
( Fg ml-’ )
Concentration
found a ( pg ml-
’ 1a
Recovery (%I
FUR
PIR
TAT
FUR
PIR
TAT
FUR
PIR
TAT
20.0 15.0 10.0 20.0 15.0 7.0
4.0 6.0 2.0 2.0 4.0 2.0
0.1 0.1 0.06 0.1 0.06 0.15
19.1 16.8 9.1 18.6 15.9 6.6
4.2 6.0 1.8 2.3 4.1 1.9
0.11 0.12 0.06 0.10 0.07 0.16
95.5 112.0 91.0 93.0 106.0 94.3
105.0 100.0 90.0 115.0 102.5 95.0
110.0 112.0 100.0 110.0 116.6 106.7
a Mean of three determinations
320
F. Garcia Scinchez et al./Analytica
Table 3 Results for the assays of pharmaceutical Pharmaceutical preparation a
preparations
Nominal content (mg)
by the VASS method
Amount added (mg)
Amount found b (mg)
Recovery (%)
FUR
TAT
FUR
FUR
39.8
-
0.2 _ 0.2 _ -
42.9
0.9 5.9 6.1 _ 0.9
FUR
PIR
TAT
Seguril
40.0
-
_
_
Perbilen
40.0 _ _
6.0 6.0 _ _
_ 50.0 50.0
0.8 _
Triniagar -
Chimica Acta 306 (1995) 313-321
_ 40.0 40.0
PIR
0.8
41.8 42.0
PIR
TAT 0.22 0.21 50.3 50.8
PIR
99.5
-
107.4 _ 104.5 _ 105.0
112.5 98.3 101.7 112.5
TAT _ 110.0 105.0 100.6 101.6
a Commercially available dosage forms. b Mean of seven determinations.
Table 2, satisfactory results were obtained for the three compounds in all the cases tested, which would not be possible by any conventional fluorescence method. 3.3. Applications In order to check the usefulness of the proposed method, and because there are no pharmaceutical dosage forms commercially available containing the three diuretics simultaneously, the proposed methods have been applied to their determination in three different pharmaceutical preparations which contain only one of the three diuretics. These preparations were conveniently spiked with different amounts of the other compounds, so individual and mixtures analyses were carried out. Recoveries achieved by means of the proposed VASS method are summarized in Table 3. The results indicate that the amount of the parent drugs in each of the three dosage forms were in reasonable agreement with the declared label strength, and that the recovery percentages achieved in all cases vary between 97-110%.
References [l] [2] [3] [4] [5]
J.B.F. Lloyd and I.W. Evett, Anal. Chem., 50 (1977) 1710. T. Vo-Dinh, Anal. Chem., 50 (1978) 50 396. G.L. Green and T.C. O’Haver, Anal. Chem., 46 (1974) 2191. P. John and I. Soutar, Anal. Chem., 48 (1976) 520. J.C. Andre, M. Bouchy and M.L. Viriot, Anal. Chim. Acta, 105 (1979) 297. [6] A.F. Fell, Trends Anal. Chem., 2 (1983) 63.
[7] C. Cruces Blanc0 and F. Garcia Sanchez, Anal. Chem., 56 (1984) 2035. [8] C. Cruces Blanc0 and F. Garcia Sanchez, Anal. Chim. Acta, 166 (1984) 277. [9] F. Garcia Sanchez, A. Navas and M. Santiago, Anal. Chim. Acta, 167 (1985) 217. [lo] F. Garcia Sanchez and C. Cruces Blanco, Anal. Chem., 58 (1986) 73. [ll] F. Garcia Sanchez, M. Hernandez Lopez and J.C. MQrquez Gomez, Talanta, 33 (1986) 785. [12] C. Cruces Blanco and F. Garcia Sanchez, J. Assoc. Off. Anal. Chem., 69 (1986) 105. [13] F. Garcia Sanchez and C. Cruces Blanco, Anal. Chim. Acta, 222 (1989) 177. [14] J.N. Miller, T.A. Ahmad and A.F. Fell, J. Proc. Anal. Div. Chem. Sot., 19 (1982) 37. [15] S. Rubio, A. Gomez-Hens and M. Valclrcel, Anal. Chem., 57 (1985) 1101. [16] F. Garcia Sanchez, J.C. Mdrquez Gdmez and M. Hernlndez Lopez, Analyst, 112 (1987) 649. [17] F. Garcia Sanchez, M. Hemindez Lopez and J.C. Marquez G6mez, Spectrochim. Acta, 43A (1987) 101. [18] F. Garcia Sanchez and C. Cruces Blanco, Anal. Chem., 60 (1988) 323. [19] F. Garcia Sanchez and C. Cruces Blanco, Talanta 37 (1990) 573. [20] F. Salinas, A. Mufioz de la Peiia, I. Duran-Meras and M.S. Durbn, Analyst, 115 (1990) 1007. [21] F. CapitLn, G. Sanchez-Palencia, A. Navalbn, L.F. CapitlnVallvey and J.L. Vilchez, Anal. Chim. Acta, 259 (1992) 345. [22] F. Capitan, E. Alonso, R. Avidad, L.F. Capitin-Vallvey and J.L. Vilchez, Anal. Chem., 65 (1993) 1336. [23] J.A. Murillo Pulgarin and A. Alai& Molina, Talanta, 41 (1994) 21. [24] T.A. Kubic, T. Kanabrocki and J. Dwyer, Communication presented at the 32nd Annual Congress of the American Academy of Forensic Sciences, 1980. [25] J.N. Miller, Analyst, 109 (1984) 191. [26] B.J. Clark, A.F. Fell, I.E. Aitchison, D.M.G. Pattie, M.H. Williams and J.N. Miller, Spectrochim. Acta, 38B (1983) 61.
F. Garcia Skhez
et al. /Analytica
[27] M.T. Oms, V. Cerda, F. Garcia Sanchez and A.L. Ramos Rubio, Talanta, 35 (1988) 671. [28] F. Garcia Sanchez, A.L. Ramos Rubio, V. Cerda and M.T. Oms, Talanta, 35 (1988) 335. [29] B.J. Clark, A.F. Fell, K.T. Milne, D.G. Pattie and H. Williams, Anal. Chim. Acta, 170 (1985) 35. [30] F. Garcia Sanchez, A.L. Ramos Rubio, V. Cerdl and M.T. Oms, Anal. Chim. Acta, 228 (1990) 293.
Chimica Acta 306 (1995) 313-321 [31] M.T. Oms, R. Fortaleza, V. Cerda, S. Maspoch Coello Blanco, Anal. Chim. Acta, 233 (1990) 159.
321 and M.
[32] G.D. Christian, J.B. Callis and E.R. Davidson, in E.L. Wehry (Ed.), Modern Fluorescence Spectroscopy, Vol. 4, Plenum Press, New York, 1981, p. 111.
ACTA ELSEVIER
Analytica Chimica Acta 306 (1995) 313-321
Variable-angle scanning fluorescence spectrometry for the simultaneous determination of three diuretic drugs Francisco Garcia SBnchez a, Albert0 Fernindez Carmen Cruces Blanc0 b**
Gutierrez b,
a Department ofAnalytical Chemistry, University of Mblaga, Milaga, Spain ’ Department of Analytical Chemistry Faculty of Sciences, University of Granada, C / Fuentenueva s/n, Received 2 August 1994; revised 2 November
1994; accepted 8 December
18071 Granada, Spain
1994
Abstract
The applicability of variable-angle synchronous scanning (VASS) fluorescence spectroscopy has been demonstrated for the resolution of mixtures of three diuretics (furosemide, triamterene, piretamide) with closely overlapping fluorescence profiles. This technique permits linear or non-linear paths to be scanned at preselected angles through the excitation-emission matrix by scanning both monochromators at different speeds, in order to obtain the best variable-angle scanning spectra (highest signal values and interference-free bands). Such an approach has permitted the simultaneous determination of the three compounds at the pg to ng ml - ’ level, with a relative standard deviation I 5% (n = 10). When applied to commercial pharmaceutical dosage forms, recoveries of the original drug between 98-101% have been obtained. Keywords: Fluorimetry;
Diuretics;
Pharmaceutical
analysis
1. Introduction When substances with overlapping spectra have to be determined simultaneously by spectrofluorimetry, it could be quite difficult to choose a pair of excitation and emission wavelengths that would permit the observation of one of them without the interference by the other. For such a reason, it is evident that good methods of reducing spectral band widths or, otherwise, improving spectral selectivity, will be of great benefit in spectrofluorimetric analysis. Several methods have been proposed to resolve such problems (overlapping) without manipulation of
* Correspondingauthor. 0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDi 0003-2670(94)00689-X
the samples or using time-consuming and highly expensive separation techniques. Synchronous fluorescence alone [1,2] or in combinations with derivative techniques [3,4] have provided a convenient means for implementing sensitivity [5-131 and selectivity 114-231 in both inorganic and organic chemistry. One drawback to conventional synchronous scanning is that it is limited to producing 45” sections cut through the excitation-emission matrix (EEM), whose locus is defined by the constant wavelength interval (Ah) employed. In recent years, advances in analytical instrumental and the general use of computers have promoted the application of mathematical algorithms for treating the large amount of data
314
F. Garcia Srinchez et al. /Analytica
that modem instrumentation can provide, increasing the selectivity of analytical methods. A novel approach of conventional synchronous scanning, proposed by Kubic et al. [24], and which offers considerable flexibility, is variable-angle synchronous scanning (VASS), where the wavelength separation between the two monochromators is varied. There are three different instrumental configurations for performing VASS. First, the speed of the monochromators can be manipulated by two different motors scanned at different rates [25]. A second approach consists of acquiring the EEM and storing the data on the interfaced microcomputer, and the desired angle (linear or nonlinear) is determined by using the appropriate software [26]. Recently, Garcia Sanchez and co-workers [27,28] have modified a commercial digital instrument to generate the VASS directly from the spectrofluorimeter output. With this approach, applied in the present work, a few minutes will be sufficient to obtain the variable-angle spectra. As it is observed, VASS does not require highly sophisticated equipment but the applications found in the literature are fairly uncommon [29-311, due to the fact that the VASS mode is not yet widely accessible. In the present work, the increased selectivity afforded by the VASS technique, has been well demonstrated in studies on the determination of three diuretic drugs (furosemide, triamterene, piretamidel, which permits to avoid previous separation techniques, which have normally been used for this compounds in real samples [32].
2. Experimental 2.1. Apparatus Two spectrofluorimeters have been used. The three-dimensional spectra and their corresponding contour maps are all recorded on a Perkin-Elmer MPF-66 spectrofluorimeter, equipped with a 150-W xenon arc lamp and a R-928 photomultiplier. The spectrofluorimeter was connected to a Perkin-Elmer Model 7300 Professional computer provided with PETLS application software (C 646-0280). The scanning speed and the response time were set at 240 nm
Chimica Acta 306 (1995) 313-321
min-’ and 0.5 s, respectively. Slit-widths were set at 5 and 3 nm, for the excitation and emission monochromators, respectively. The variable-angle fluorescence spectra were recorded on a Perkin-Elmer LS-5 spectrofluorimeter which includes a xenon discharge lamp (9.9 W) pulsed at line frequency and f/3 Monk-Guillieon monochromators. The spectrofluorimeter was operated in the computer-controlled mode via the RS232C serial interface by either of the following systems. In one, an IBM-PC/XT microcomputer equipped with a 3.5-in. floppy disk, provided with the Fluoropack package [27] which controls the spectrofluorimeter and the acquisition of variable-angle fluorescence data. In the other, a Perkin-Elmer Model 3600 Data Station microcomputer was used. With the software package used, a variable-angle scan at the desired angle can be obtained by selecting the appropriate value and sign for each of the different increments. With this method, it is possible to obtain a number of sequential segments, each at different angles. Each pair of adjacent segments can be linked together, thereby defining any desired trajectory throughout the EEM. Slit-widths for both monochromators were set at 5 nm, with a scanning speed of 240 nm min- ’ . In both spectrofluorimeters, the measurements were made in standard 1 X l-cm path-length quartz cells, thermostatically controlled at 25 + 0.5” C with a water-bath circulator (Frigiterm S-382). 2.2. Reagents All the experiments were performed with analytical-reagent grade chemicals and pure solvents. Doubly distilled, demineralized water was used throughout. Triamterene (TAT) was obtained from Sigma (T4143). Stock solutions were prepared in a 50-ml calibrated flask by dissolving 5 mg of the compound in absolute methanol. Furosemide (FUR) was also purchased from Sigma (F-4381) and its stock solutions were prepared in a 25-ml calibrated flask by dissolving 10 mg in absolute ethanol. Piretamide (PIR) was synthetized and purified in our laboratories. A stock solution was prepared in a 25-ml calibrated flask by dissolving 5 mg of the compound in absolute ethanol. These stock solutions were pre-
315
F. Garcia Srinchez et al. / Analytica Chimica Acta 306 (1995) 313-321
pared weekly and were used to prepare standard solutions daily by suitable dilutions with absolute ethanol.
3. Results and discussion
3.1. Selection of experimental
and instrumental pa-
rameters
2.3. General procedure Place aliquots of the working solutions of each of the three diuretics, containing between 100 and 400 pg of FUR, 0.5 to 1.5 ng of TAT and 10 and 100 pg of PIR into 10-1111volumetric flasks and dilute to final volume with absolute ethanol. Record the three-dimensional fluorescence spectra and their corresponding contour maps at a scan speed of 240 nm mine ’ with 90 scans and 2 nm increments, using an excitation slit of 5 nm and an emission slit of 3 nm. Measure the fluorescence intensity in the VASS maps at the following wavelength excitation and emission pairs: 359-429,385-417 and 290-459 nm, for FUR, TAT and PIR, respectively, and plot against concentration of each diuretic. 2.4. Procedure
for pharmaceutical
compounds
anal-
ysis
Furosemide was determined in tablets: Seguril (Hoechst Ibtrica, Barcelona) with a nominal content of 40 mg of furosemide. Triamterene was also determined in tablets: Triniagar (Lab. Farmasimes, Barcelona) containing triamterene (50 mg) and mebuticide (50 mg>, respectively. Piretamide was determined in capsules: Perbilen (Hoechst IbCrica) containing piretamide (6 mg). For FUR and TAT analysis, twenty tablets were weighed and powdered and an amount of the powder containing ca. the equivalent of one tablet was shaken with absolute ethanol and methanol, respectively, and diluted to 100 ml with the same solvent. The extract was filtered through Whatman No. 1 filter paper, the first 20 ml of filtrate were discarded and a suitable aliquot of the filtrate was diluted to 25 ml with absolute ethanol. The contents of piretamide capsules were directly dissolved in ethanol and the standard solutions of the three compounds were conveniently diluted with absolute ethanol to obtain working solutions in the concentration range of their calibration graphs. Aliquots of these solutions were treated as indicated under General procedure.
Furosemide and piretamide are readily soluble in ethanol, while triamterene could only be dissolved in methanol. For such a reason, native fluorescence of the three compounds was studied in aqueous, methanolic or ethanolic solutions. Fluorescence emission was strongly dependent on solvent composition, the best results being obtained using ethanol as solvent. Different proportions of water-ethanol mixtures were studied. It was observed that the fluorescence intensity of the three compounds increased as the ethanol concentration increased. So, a 100% ethanol solution was the most satisfactory, giving the highest fluorescence intensity, and was chosen for subsequent experiments. A study of the stability of the solutions at 25” C showed that the compounds were stable for, at least, 3 h. In order to avoid possible fluorescence signal modifications, a thermostatically controlled waterbath at this temperature is recommended for the rest of the experimental work. The object of this work was to demonstrate the applicability of the VASS technique to the resolution
:,o
A\ 350
‘\--__ 430
390
470
” 510
x nm
Fig. 1. Fluorescence excitation (A, B, C) and emission (A’, B’, C’) spectra of piretamide (A, A’) ( A,, = 348 nm, A,, = 437 nm), furosemide (B, B’ ) (A,, = 365 nm, A,, = 410 nm) and triamterene (C, C’) (A,, = 372 nm, Aem = 436 nm). Concentrations of each compound: 1.2 pg ml-‘; 2.0 Kg ml-’ and 20 ng ml-‘, respectively. RF1 = Relative fluorescence intensity.
316
F. Garcia Srinchez et al. /Analytic0
Chimico Acta 306 (1995) 313-321
, . 1
F. Garcia Srinchez et al. /Analytica
of a mixture of compounds with closely overlapping fluorescence spectra. The excitation and emission spectra for FUR, TAT and PIR are shown in Fig. 1. As can be observed, because of spectral overlapping, the analysis of mixtures of the three compounds would not be feasible by conventional spectrofluorimetry at their wavelength maxima.
These conventional excitation and emission spectra are not complete descriptions of the spectral distribution of these compounds, because the excitation spectra are obtained at only a single emission wavelength and the emission spectra at a single excitation wavelength. A complete description requires a three dimensional spectrum [32], the total (-)
Chirrioe
(+)
L hin)
/
A,
(b)
Em.
(nm)
= 260 nfn
(min) = 350 nm
A.. km) L
330
317
Chimica Acta 306 (1995) 313-321
= 390 nm
(max) = 487 “In
550 416
Fig. 3. Three-dimensional VASS spectrum (a) and its corresponding contour map (b) of an isoemissive mixture of the three diuretics with VASS route selected. Concentrations of each compound: piretamide (0.6 Kg ml-’ 1, furosemide (1.4 pg ml-‘) and triamterene (120 ng ml-’ ).
318
F. Garcia S&chez et al./Analytica
luminescence spectrum (TLS). Sequential scans of the emission spectra (90 scans) were carried out between 330 and 550 nm at different excitation wavelengths ranging from 220 to 428 nm. The corresponding TLS of each of the three compounds are shown in Fig. 2a. All the necessary data are collected in the computer-interfaced spectrometer, and the corresponding contour maps may also be obtained (Fig. 2b). Previously, in order to select the appropriate route to minimize the spectral interference with the minimum loss of sensitivity, a detailed inspection of each of the contour maps, is necessary. As is observed in Fig. 2b, the zones of maximum fluorescence intensity appear at the main peaks of each compound centred on their respective wavelength excitationemission pairs which are (280 nm, 407 nm) and (336 nm, 414 nm) for PIR, (276 nm, 379 nm) and (336 nm, 401 nm) for FUR and (368 nm, 432 nm) and (278 nm, 371 nm) for TAT, respectively. To determine the optimum VASS route, a careful examination of a contour map corresponding to a standard solution of the three diuretics in near isoemissive concentrations, has been carried out (Fig. 3b). The variable-angle scanning route shown traverses those parts of the contour map with the least overlap. Although some sensitivity is lost because no peak maxima are traversed, nearly interference-free signals of the three components may be obtained because the route avoids the regions of maximum interference between the three compounds. The corresponding three-dimensional variable-angle spectrum of the same three-component mixture of the diuretics, to illustrate the potential utility of this technique for mixture analysis, is shown in Fig. 3a. The software package used [27] displays the required data in 5 min, in comparison with periods of several hours required to obtained the three-dimensional data matrices, which indicates the ready applicability of the present method to the quantitative analysis of different compounds in routine analysis. 3.2. Analytical characteristics In order to test the mutual independence of the analytical signals of FUR, TAT and PIR , the following calibration graphs were obtained for standards containing 1.0-10.0 pg ml-’ of piretamide in the
Chimica Acta 306 (1995) 313-321
2rm 4x9
R.F.I.
I
FUR
487
Lhm)
TAT
R.F.I.
480.8
c)
PKR
R.F.I.
350
40s
4
Fig. 4. Two -dimensional projections of several miXhIreS of piretamide, furosemide and triamterene: (a) piretamide (l-10 pg ml-‘), furosemide (20.0 pg ml-‘), triamterene (100 ng ml-‘); (b) furosemide (lo-40 @g ml-‘), piretamide (4.0 pg ml-‘), triamterene (100 ng ml-‘); (c) triamterene (SO-150 ng ml-‘), piretamide (4.0 pg ml-‘), furosemide (20.0 pg ml-‘).
F. Garcia Srinchez et al./Analytica Table 1 Analytical
parameters
of the proposed
Diuretic Furosemide
Piretamide
Triamterene
319
methods Calibration
Other diuretics present
Diuretic determined
a Arbitrary h n = 10.
Chimica Acta 306 (1995) 313-321
Corm. ( pg ml
_
_
TAT
0.1
PIR _
4.0
TAT
0.1
FUR _ PIR
20.0 _ 4.0
FUR
20.0
’1
Intercept
Slope a
a
Corr. coeff. b
2.3
+ 50.9
0.988
1.4
+61.3
0.981
28.7
- 19.6
0.993
27.1
+5.4
0.996
1.8
-7.1
0.999
1.2
+ 12.9
0.988
units.
absence and presence of 100 ng ml-’ and 20.0 pg ml-’ of triamterene and furosemide, respectively. Similarly, graphs were prepared for standards containing 10.0-40.0 pg ml-’ of furosemide in the absence and presence of 4.0 pg ml-’ and 100 ng as well as a ml-’ of piretamide and triamterene, calibration graph for triamterene (50-150 ng ml-‘) in the absence and presence of 20.0 pg ml-’ and 4.0 pg ml-’ of furosemide and piretamide, respectively. The utility of the selected VASS route was demonstrated by obtaining the two-dimensional projections of these calibration graphs (see Fig. 4a, b and cl. As it can be deduced from these VASS spectra, large errors would be obtained when measuring conventionally the different mixtures indicated in the calibration graphs; by avoiding the wavelength pairs of maximum fluorescence intensity of each
Table 2 Results obtained
for synthetic
Mixture composition
compound and selecting for their measurement those corresponding to less fluorescence intensity and better resolution (see General procedure), the interferences are greatly decreased. Some of the features of the proposed methods are summarized in Table 1. The relative standard deviation (n = 10) for determination of 20.0 pg ml-’ of FUR; 100 ng ml-’ of TAT and 4.0 pg ml-’ of PIR were 5.2, 3.4 and 1.4%, respectively. The VASS spectrofluorimetric method has been applied to the analysis of several synthetic mixtures of the three compounds in different ratios. Table 2 summarizes the results calculated from the calibration graphs. Despite a detailed selection of the variable-angle route through the EEM, the strong overlap between the spectra of the three selected compounds causes some error in the simultaneous determination of the three compounds. However, as can be deduced from
mixtures of the three compounds
( Fg ml-’ )
Concentration
found a ( pg ml-
’ 1a
Recovery (%I
FUR
PIR
TAT
FUR
PIR
TAT
FUR
PIR
TAT
20.0 15.0 10.0 20.0 15.0 7.0
4.0 6.0 2.0 2.0 4.0 2.0
0.1 0.1 0.06 0.1 0.06 0.15
19.1 16.8 9.1 18.6 15.9 6.6
4.2 6.0 1.8 2.3 4.1 1.9
0.11 0.12 0.06 0.10 0.07 0.16
95.5 112.0 91.0 93.0 106.0 94.3
105.0 100.0 90.0 115.0 102.5 95.0
110.0 112.0 100.0 110.0 116.6 106.7
a Mean of three determinations
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F. Garcia Scinchez et al./Analytica
Table 3 Results for the assays of pharmaceutical Pharmaceutical preparation a
preparations
Nominal content (mg)
by the VASS method
Amount added (mg)
Amount found b (mg)
Recovery (%)
FUR
TAT
FUR
FUR
39.8
-
0.2 _ 0.2 _ -
42.9
0.9 5.9 6.1 _ 0.9
FUR
PIR
TAT
Seguril
40.0
-
_
_
Perbilen
40.0 _ _
6.0 6.0 _ _
_ 50.0 50.0
0.8 _
Triniagar -
Chimica Acta 306 (1995) 313-321
_ 40.0 40.0
PIR
0.8
41.8 42.0
PIR
TAT 0.22 0.21 50.3 50.8
PIR
99.5
-
107.4 _ 104.5 _ 105.0
112.5 98.3 101.7 112.5
TAT _ 110.0 105.0 100.6 101.6
a Commercially available dosage forms. b Mean of seven determinations.
Table 2, satisfactory results were obtained for the three compounds in all the cases tested, which would not be possible by any conventional fluorescence method. 3.3. Applications In order to check the usefulness of the proposed method, and because there are no pharmaceutical dosage forms commercially available containing the three diuretics simultaneously, the proposed methods have been applied to their determination in three different pharmaceutical preparations which contain only one of the three diuretics. These preparations were conveniently spiked with different amounts of the other compounds, so individual and mixtures analyses were carried out. Recoveries achieved by means of the proposed VASS method are summarized in Table 3. The results indicate that the amount of the parent drugs in each of the three dosage forms were in reasonable agreement with the declared label strength, and that the recovery percentages achieved in all cases vary between 97-110%.
References [l] [2] [3] [4] [5]
J.B.F. Lloyd and I.W. Evett, Anal. Chem., 50 (1977) 1710. T. Vo-Dinh, Anal. Chem., 50 (1978) 50 396. G.L. Green and T.C. O’Haver, Anal. Chem., 46 (1974) 2191. P. John and I. Soutar, Anal. Chem., 48 (1976) 520. J.C. Andre, M. Bouchy and M.L. Viriot, Anal. Chim. Acta, 105 (1979) 297. [6] A.F. Fell, Trends Anal. Chem., 2 (1983) 63.
[7] C. Cruces Blanc0 and F. Garcia Sanchez, Anal. Chem., 56 (1984) 2035. [8] C. Cruces Blanc0 and F. Garcia Sanchez, Anal. Chim. Acta, 166 (1984) 277. [9] F. Garcia Sanchez, A. Navas and M. Santiago, Anal. Chim. Acta, 167 (1985) 217. [lo] F. Garcia Sanchez and C. Cruces Blanco, Anal. Chem., 58 (1986) 73. [ll] F. Garcia Sanchez, M. Hernandez Lopez and J.C. MQrquez Gomez, Talanta, 33 (1986) 785. [12] C. Cruces Blanco and F. Garcia Sanchez, J. Assoc. Off. Anal. Chem., 69 (1986) 105. [13] F. Garcia Sanchez and C. Cruces Blanco, Anal. Chim. Acta, 222 (1989) 177. [14] J.N. Miller, T.A. Ahmad and A.F. Fell, J. Proc. Anal. Div. Chem. Sot., 19 (1982) 37. [15] S. Rubio, A. Gomez-Hens and M. Valclrcel, Anal. Chem., 57 (1985) 1101. [16] F. Garcia Sanchez, J.C. Mdrquez Gdmez and M. Hernlndez Lopez, Analyst, 112 (1987) 649. [17] F. Garcia Sanchez, M. Hemindez Lopez and J.C. Marquez G6mez, Spectrochim. Acta, 43A (1987) 101. [18] F. Garcia Sanchez and C. Cruces Blanco, Anal. Chem., 60 (1988) 323. [19] F. Garcia Sanchez and C. Cruces Blanco, Talanta 37 (1990) 573. [20] F. Salinas, A. Mufioz de la Peiia, I. Duran-Meras and M.S. Durbn, Analyst, 115 (1990) 1007. [21] F. CapitLn, G. Sanchez-Palencia, A. Navalbn, L.F. CapitlnVallvey and J.L. Vilchez, Anal. Chim. Acta, 259 (1992) 345. [22] F. Capitan, E. Alonso, R. Avidad, L.F. Capitin-Vallvey and J.L. Vilchez, Anal. Chem., 65 (1993) 1336. [23] J.A. Murillo Pulgarin and A. Alai& Molina, Talanta, 41 (1994) 21. [24] T.A. Kubic, T. Kanabrocki and J. Dwyer, Communication presented at the 32nd Annual Congress of the American Academy of Forensic Sciences, 1980. [25] J.N. Miller, Analyst, 109 (1984) 191. [26] B.J. Clark, A.F. Fell, I.E. Aitchison, D.M.G. Pattie, M.H. Williams and J.N. Miller, Spectrochim. Acta, 38B (1983) 61.
F. Garcia Skhez
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[27] M.T. Oms, V. Cerda, F. Garcia Sanchez and A.L. Ramos Rubio, Talanta, 35 (1988) 671. [28] F. Garcia Sanchez, A.L. Ramos Rubio, V. Cerda and M.T. Oms, Talanta, 35 (1988) 335. [29] B.J. Clark, A.F. Fell, K.T. Milne, D.G. Pattie and H. Williams, Anal. Chim. Acta, 170 (1985) 35. [30] F. Garcia Sanchez, A.L. Ramos Rubio, V. Cerdl and M.T. Oms, Anal. Chim. Acta, 228 (1990) 293.
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321 and M.
[32] G.D. Christian, J.B. Callis and E.R. Davidson, in E.L. Wehry (Ed.), Modern Fluorescence Spectroscopy, Vol. 4, Plenum Press, New York, 1981, p. 111.