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Direct determination of triamterene in urine by matrix isopotential synchronous fluorescence spectrometry
ANALYTICA CHIMICA ACTA ELSEVIER
Analytica Chimica Acta 326 (I 996) 117-I 26
Direct determination of triamterene in urine by matrix isopotential synchronous fluorescence spectrometry Amelia AlaiXh Molina,
Josh A. Murillo Pulgarin”, Department
Pablo Fernhdez
L6pez
of Analytical Chemistry and Foods Technology, University Custilla La Mancha, 13071 Ciudad Real,
Spin
Received 14 November 1995; revised 18 January 1996; accepted 21 January 1996
Abstract A new method for the determination of triamterene for concentrations between 10 and 1000 ng ml-’ by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This new method is useful for the determination of compounds in samples with unknown background fluorescence, such as triamterene in urine, without the need of tedious pre-separation. The determination was performed in an aqueous medium adjusted at a pH value of 6.2, provided by adding sodium citrate-citric acid buffer solution. Since triamterene is widely used as a doping substance in sport, the method was successfully applied to the determination of triamterene in urine. Better sensitivity and repeatability are achieved for these matrices with the proposed method than with the fluorimetric ones described in the literature. An extensive statistical analysis has been developed to all calibration graphs. This treatment includes robust regression such as least median of squares which also detects outliers and leverage points. The weighted least squares regression has been applied to find the more exact straight line which fits the experimental data. The error propagation has been used for the calculation of the detection limit, determination limit and the repeatability of the method. Keywords: Triamterene; Urine; Fluorimetry
1. Introduction Triamterene a yellow 9, gives
(2,4,7-triamino-6-phenylpteridine)
crystalline
powder.
Its solutions
is
up to a pH
a blue fluorescence.
Triamterene
is a natriuretic
agent
which
is much
used in the treatment of several diseases. It can also be applied as doping substance. In sports this diuretic is abused mainly for two reasons: to obtain a rapid diminution of corporal weight, important in sports
* Corresponding author. Fax: +34 9 262953 18 SOOO3-2670/96/$15.008~:)1996 Elsevier Science B.V. All rights reserved PII SOOO3-2670(96)00065-7
which are divided in different weight categories, and to reduce the concentration of medical drugs in urine by dilution by means of a rapid production of an elevated quantity of urine, thus trying to diminish the possibility to detect other doping substances. No medical reason can justify in any sport a rapid decrease of weight, whereas on the other hand this abuse causes grave dangers for health because of possible serious secondary effects. Triamterene is rapidly but incompletely absorbed after oral administration. Once the drug is in the body, about 30-70% of an oral dose is excreted in the urine [l]. Variable amounts are excreted in the bile.
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First measurements of triamterene have been made directly in pharmaceutical preparations by perchloric acid tritations [2,3]. Spectrophotometric absorption measurements at 360 nm [2] and at 375 nm [3] have been used. An estimation of triamterene and deoxycholic acid by planimetric measurement of thin layer chromatograms is also described [4]. Guerello [5] developed a spectrophotometric method for the determination of triamterene with furosemide using 50% of ethanol at 275 and 370nm. Rao [6] determined this drug and its dosage formed by a fluorimetric method using 85% of formic acid to increase sensitivity and reproducibility of the estimation. Sastry [7] analysed a mixture of triamterene and amiloride hydrochloride in pure and pharmaceutical preparations by fluorimetry. Also a polarographic method [8] has been described for triamterene and its analogues in biological fluids. Usually, the specific determination of this natriuretic agent in biological fluids involves liquid chromatography (LC) [9-121. Normally the estimation of triamterene at therapeutic levels by LC includes various preceding procedures, such as extractions and concentrations in the organic solvents [9-121 with all the disadvantages that this may cause, since all of these procedures are based on equilibrium reactions. Some authors have attempted the direct injection of the sample onto the LC column [13-l 51. However, due to the irreversible adsorption of some components of biological fluids to the column, direct injection decreases the chromatographic resolution, and greatly reduces the operative lifetime of the column. Instead of the LC methods described above [9-151, we propose a fast, simple, easy, sensitive and straightforward fluorimetric method to estimate triamterene in biological fluids like urine samples, reaching the same sensitivity as direct determination, without the need for prior separation and preconcentration or derivatization procedures. Recently in this department a new fluorimetric synchronous technique called matrix isopotential synchronous fluorescence spectrometry (MISF) has been developed [16]. This technique is particularly useful for removing fluorescence matrix background effects and enables the determination of individual compounds in complex samples. It is essential that the matrix has an almost invariable composition. It is
Chimicu Acta 326 (1996) I1 7-126
possible to maintain a constant background signal, even though its fluorescence intensity may vary, if a cut is made in the total fluorescence spectrum following one of the trajectories which join points of equal intensity (isopotential trajectory) from an initial point to final excitation and emission wavelengths. This trajectory is obtained by means of a program developed in BASIC [17]. It is always possible to find the matrix trajectory which passes through the maximum fluorescence excitation and emission wavelengths of the component to be determined and so the same sensitivity is achieved as in direct determination in the absence of background fluorescence. This technique can be improved by applying derivative methods. Human urine is composed of numerous organic substances, some of which are fluorescent [ 181, which provide a high background fluorescence and interfere with the direct determination of triamterene. Recent works in our laboratory dealt with the application of this new fluorimetric technique to the determination of different drugs in biological fluids with excellent results [ 16,19-221.
2. Experimental 2.1. Apparatus All tluorimetric measurements were performed on a Aminco Bowman Series 2 equipped with a continuous 150 W Xenon lamp, connected to software which runs on the OS2 operating system. Quarzglas cuvettes with pathlength of 1.Ox 1 .Ocm have been used. Thermostatic equipment and a Crison Model 2001 pH-meter with a glass-saturated calomel combination electrode and a Selecta centrifuge (Model Mixtaxel) were also used. 2.2. Software A program was developed which enabled us to obtain the values of X,, and X,, for any constant value of fluorescence intensity from a three-dimensional spectrum. As the values obtained for a particular curve were not equidistant, the Lagrange
J.A. Murillo
Pulgan’n
et al./Analytica
interpolation method was applied to all points, which were placed in order, using emission wavelengths at 0.6nm intervals. Once the trajectory had been defined, the spectrum was obtained by means of the Ftotal program 1171, so that the spectra obtained from it displayed the same format as those performed directly from the Aminco Bowman Series 2 spectrofluorimeter. The Ftotal program not only generates information on a fluorescent compound through the isometric representation of the three-dimensional spectrum in the form of a level curve, but it also processes the spectral data to obtain any type of spectrum of the socalled new fluorimetric techniques. The statistical analysis is covered by means of a program developed by us, which has a menu that includes all procedures mentioned in this paper.
Chimica
Acta 326 (1996)
117-126
119
(pH 6.2) and 5 ml of solution of urine free from triamterene, and dilute with water to a final volume of 25 ml. Record 61 emission spectra of 288 nm width in steps of 0.6nm, varying the excitation wavelength in 4.8 nm steps. Obtain the total luminescence spectra, select the adequate trajectory, which passes through the excitation and emission maxima of triamterene, and obtain matrix isopotential synchronous spectra by means of Ftotal 1171. Calculate the first derivative, according to the Savitzky and Golay algorithm [23,24]. Finally, determine the triamterene content by measuring the derivative signal at emission wavelength of 406.8 nm, and using the appropriate calibration graph.
3. Results and discussion 2.3. Reagents 3.1. Factors affecting fluorescence All experiments were performed with analytical reagent grade chemicals, pure solvents and Mini-Q water. The standard triamterene was obtained from Sigma. A stock solution of triamterene was prepared in a 500 ml volumetric flask by dissolving 25 mg in water. This stock solution was used to prepare standard solutions by suitable dilutions. A 1.25 M buffer solution of pH 6.2 was prepared by mixing adequate amounts of citric acid with sodium hydroxide. The stock solution of triamterene was stored, protected from the light and maintained below 5°C. Under these conditions, the solution of triamterene was stable for two months. The working samples of triamterene were stable for at least 2 h at room temperature. Urine samples were obtained from fasting and healthy people in the morning. 2.4. Procxdure 2.4. I. Sample preparation Centrifuge urine for 15 min at 3800rpm and transfer the clear supematant solution into a flask, store and maintain below 5°C. For the preparation of calibration graph, place an aliquot of triamterene equivalent to 0.25-25 pg in a 25 ml volumetric flask, add 10 ml of buffer solution
intend,
Chemical variables were studied to obtain the best measurement conditions and maximum fluorescence sensitivity. Triamterene is highly soluble in water, so it was not necessary to use an organic solvent like ethanol. Nevertheless it was suitable to study the variation of triamterene and urine fluorescence with changes in the dielectric constant of the media. In this way the effect of ethanol content in the medium was investigated by preparing samples of triamterene and urine, and by varying the ethanol percentage between 0% and 98% V/V for the triamterene solutions and between 0% and 80% for the urine solutions. The fluorescence intensity due to triamterene does not change when the ethanol percentage in the medium increases (Fig. 1b) while fluorescence intensity due to urine increases (Fig. la). Because of these reasons it is not necessary to use ethanol. The influence of pH on the fluorescence intensity was also studied by adding different amounts of HCl and NaOH to a urine solution and a triamterene solution. As can be seen in Fig. 2b, the fluorescence intensity of triamterene is nearly constant with pH values between 2 and 10. The fluorescence intensity due to urine decreases slowly from pH 4 to 1 1 (Fig. 2a). A pH 6.2 was selected as adequate: this is approximately the pH value of urine of healthy
J.A. Murillo Pulgan’n et al./Analytica
a)
-c+-cc
Urine
b) -
1
,
I
Chimica Acta 326 (1996) I 17-126
2
Triarnterene
1
,
40
20
%
1
I
,‘I’1 60
1
80
100
a)
w
1
00 0
2
4
6
8
IO
I2
.,
1.J
PH Fig. 2. Influence of the pH on fluorescence intensity of (a) urine and (b) triamterene. Excitation at 365nm. Emission at 436.8nm. Concentration of triamterene lOOOngmll’, urine (15). Urine data scaled to 10 with regard to maximum fluorescence intensity.
3.2. Determination people. It was proportioned by adding sodium citrate-citric acid buffer solution. The fluorescence intensity of triamterene and urine was not affected by the buffer and its concentration. A 0.5 M concentration of buffer was therefore selected to get an adequate buffering capacity. Another factor that affects the fluorescence intensity is temperature. In this way, in both cases, fluorescence intensity showed a decrease when the temperature increases from 4 to 75°C. The temperature coefficients are about 0.46%“C’ for triamterene and about 0.61%“C~’ for urine. This effect can be explained by higher internal conversion as temperature increases, facilitating non-radiative deactivation of the excited singlet state 1251 Therefore, the use of a thermostat is recommended and a measurement temperature of 20°C is chosen. The influence of triamterene concentration on the fluorescence intensity was studied under these conditions. The best range for the relation fluorescence intensity vs. concentration was found for concentrations between 10 and triamterene 1OOOngmll’.
Triamterene
b) -
Ethanol
Fig. 1. Influence of the ethanol content on fluorescence intensity of (a) urine and (b) triamterene. Excitation at 365 nm. Emission at 436.8 nm. Concentration of triamterene lOOOngmll’, urine (1:5). Urine data scaled to 10 with regard to maximum fluorescence intensity.
Urine
of triamterene
in urine
All fluorimetric three-dimensional spectra were performed by varying emission wavelength from 307.2 to 595.2nm and excitation wavelength from 200 to 488nm. Both excitation and emission bandpass were 8 nm. Scan rate was 50 nm s-‘. Under these conditions, the time taken to determine contour spectra is 438 s. MISF was applied to the analysis of triamterene in urine, a very fluorescent matrix, under the optimum chemical conditions established above. The fluorescence maxima characteristic of triamterene are located in the UV region. As can be observed in Fig. 3, in this region, urine shows one broad peak which prevents determination of this compound without prior separation. Previous experiments have shown that the qualitative composition of the fluorescent metabolites of urine from healthy people of both sexes and different diets, aged between 25 and 35 yr, are practically constant, a necessary condition for the application of this new technique. The different samples of urine display the same type of fluorescence, with hardly any variation in the form of the spectrum and in the
J.A. Murillo Pulgarin et al./Analytica
121
Chimica Acta 326 (1996) 117-126
440 x E 344
x n In
248
355 2
4512 x
3JJ
mw
Fig. 3. Total fluorescence spectra of IOOOng ml (solid line) and urine I:5 (broken line).
‘
.,.
_
._,
‘
X EM@)
’ of triamterene
location of the fluorescence maxima, although it is possible to observe some variations in their intensity. The spectrum corresponding to the arithmetical mean of ten total fluorescence spectra of the different urine samples was obtained by means of the Ftotal program [ 171. In order to determine the triamterene we calculated the isopotential trajectory in the averaged urine spectrum, which passes through the excitation and emission maxima of the triamterene, as shown in Fig. 4. Urine sample solutions containing triamterene gave signals smaller than those obtained with aqueous standard solutions, owing to some type of binding with other components of the urine. Total luminescence spectra of triamterene were obtained in different urine samples in order to construct calibration graphs and to carry out recovery experiments. Fig. 5 shows the effect of background fluorescence intensity (urine) on triamterene MISF and derivatives spectra. It can be seen that the spectra are identical in form although their intensities are different by constant terms. These correspond to the values of the fluorescence intensity due to the three urines in the isopotential trajectory applied. It is easy to observe that derivation totally removes the background effect. The first derivative of MISF spectra was applied to all samples. The number of points through which the derivative is obtained was optimized, with the conclusion that derivative spectra with a suitable signal-noise ratio were obtained with 2.5 points.
Fig. 4. Total fluorescence spectrum of triamterene (broken and the selected isopotential trajectory of urine (solid line).
line)
In the same way we obtained the total luminescence spectra of triamterene in aqueous solution at the same concentrations, to which we applied the above mentioned trajectory (Fig. 4) in order to obtain their MISF spectra. We also obtained their first derivative, as with the urine samples. As can be readily seen in Fig. 6a, which shows the MISF spectra of triamterene obtained in urine, it is not posible to determine this drug by measuring the maximun fluorescence intensity (which is located at X,,=436.8nm) with regard to the final extremes (X+.,=392.4 nm and X,,=454.2nm) of the selected trajectory, since the fluorecence intensity does not reach a constant value. The first derivative technique is therefore applied. The calibration graph was constructed by measuring the first derivative at X,,=406.8 nm where maximum sensitivity is achieved. Fig. 6b shows the spectra derived from the calibration of triamterene in urine. In order to test the independence of the analytical signal of triamterene, i.e., to show that the signal measured is independent of the urine, three calibration graphs from the first derivative signals were constructed with different urine samples. The proposed method was evaluated by a statistical analysis of experimental data by fitting the least squares line according to y = a + bx, after discarding outliers with the help of the least median of squares regression (LMS) [26], since LMS is a robust regression method. Table 1 shows the outstanding results of the statistical analysis. To verify if the intercepts on the ),-axis were negligible, significances were studied by applying the
122
J.A. Murillo Pulgarin et al./Analytica
Chimica Acta 326 (1996) 117-126
a)
0.0
, 392
,
I
,
400
I
,
408
I
416
,
I
424
,
I
432
Emission Wavelength
I I40
’
I
448
-I 456
,
,
392
,
(
400
(mn)
,
,
408
,
,
416
Emission
,
s/ ;
,
424
,
,
432
,
,
,
440
Wavelength
,
448
456
(nm)
b)
lcmngd’
I
I2- 7CQngmL' 3- 4fXingmL 4. 2OOngmL'
0.10
0.05 1
A = 406.6 nm
0.00
398
40s
412
419
426
433
440
.o.os , 398
Emission Wavelength
,
, 40s
\s
’, , , , , 412
419
(
426
,
,
433
,
440
(mn)
Fig. 5. (a) Set of MISF spectra of 4OOng ml-’ of triamterene different urine samples. (b) Their first derivative spectra.
in
Student t-test at 95% confidence level and suitable degrees of freedom [27]. If the intercepts on the yaxis for the lines calculated by the least squares technique are negligible, it is necessary to perform the least squares regression again according to the function y = b’x and, therefore the new value of the slopes of calibration graphs (b’) may be calculated. As can be seen from Table 1, the intercepts on the yaxis were negligible in the range of concentration, since the experimental t is smaller than the theoretical
Emission
Wavelength
(nm)
Fig. 6. (a) Set of MISF spectra of triamterene in urine. Triamterene concentrations: (1)lOOO; (2)700; (3)400; (4)200; (5)lOO; (6)50; (7)25 and (8)lOngml~‘. (b) The corresponding first derivative spectra.
t and therefore the new slopes were calculated. This shows that by applying the first derivative of MISF spectra, the signal due to urine is removed. To obtain the most representative calibration graph, an overall least squares was developed, in which, the regression is obtained by including all the data pairs and performing the classic least squares regression. Due to the heterocedasticity of residuals
-
I
0.9966 0.9992 0.9996 0.9976 0.9996 0.9998 0.9960 0.9978 0.9988 0.9993 -4.478~10~’
7.759x10
5
2.058x lo- A 7.362x 10m4 -4.661~10~’
regression line according to y = regression line according to y= squares regression line according squares regression line according squares regression line according squares regression line according
IT III I 11 III
1 - Least squares 2 Least squares 3 - Overall least 4 - Overall least 5 Weight least 6 - Weight least
3 4 5 6
2
Urine Urine Urine Urine Urine Urine -
1
Intercepton y-axis
data of the determination
Correlation coefficient
statistical
Sample
Fitting used
Table 1 Outstanding
4 4 4 (4.932~10
(2.153xlO~‘4m -4.036~10
-6.013x 4,
IO-‘)
matrix interval
isopotential
fluorescence
0.244
1.168
1.785 1.424 1.318
Experimental t value
synchronous
(1.142x10 4- 1.015~10~~) (1.071x10 4-1.014x10~4) (1.170x10-4-1.131x104) (l.158xlO-4-l.O6OxlO 4, (l.o74xlo.4 l.033xlo~~4) (1.158x10 4--l.l3OxlO 4, (l.l21xlO-4-l.O61xlO4) (l.l24xlO-4-1.O8OxlO4) (l.159x10-4-l.o14xlo4) (1.148~10-~ l.014x10~4)
Confidence for slope
of lirst derivative
(4.879x IO-j- -7.632x 10. 4, (2.001 x 10m3p -5.289~ IO 4, 4) (3.991 X IO 4 -1.331x10
Confidence interval for intercept
in urine by means
a + bx. Theoretical t value: 2.447. b’x. Theoretical t value: 2.365. to y = ti + bx. Theoretical I value: 2.074. to y = b’x. Theoretical f value: 2.069. t value: 2.447. to y=a + ky. Theoretical to y=b’x. Theoretical r value: 2.365.
1.079x10 1.043xlO~4 l.151x10~4 1.109x10~4 1.053x10 1.144x10 1.091x10 1.102x10~4 1.124~10-~ l.122xlo~4
‘l
of triamterene Slope
2.3x10 3 1.0x10 3 7.1 x 1om4 2.7x IO-’ l.lxlO~’ 7.6x10 4 2.4x10 ’ 2.4x 10m3 4.3x IOwr 4.0x 10-4
Standard deviaton of estimation
E
124
J.A. Murillo Pulgarin et al./Analytica
Chimica Acta 326 (1996) I 17-126
; R 6.fJE-02 I
S.D.
v
1.60E+1 2.50E*l !G.BBE*i 1 .em+z 2 .fmE+Z 4.88E+2 7 .eaB+Z 1.8BE*3
1
I I 3.81-82
e
OS
M1
3.1824E-4 3.298BE-4 5.0107E-4 2.0472E-4 1.70791-3 1.85!i0E-3 1.6843E-3 Z.-E-3
i b.BE+flZ
3.BE42
CONCWIRbTlON
b)
8.1915E-4 2.7ell.E-3 5.77493-3 l.l335E-2 2.4469E-2 4.!i795E-2 7.787lE-2 1.0848E-1 9.BEM2
1.2E*B3
9.4eE-84
A T R"4.48E-04 C E P I -6.88E-85 no
SLOPE Fig, 7. (a)Calibration graph of triamterene. The first derivative data are represented by the mean values and its standard deviation. The solid line is the weighted least square regression according to y = a + bx and the broken line is the confidence band. (b) The ellipse is 95% confidence region for the true slope and intercept on y-axis estimated from the weighted least squares regression.
obtained, we propose a fit of the experimental data to weight least squares line (WLS) [28,29] according to y = a + bx, shown in Fig. 7a together with experimental data. As the heterocedasticity is a non-uniform variance response, first derivative values are weighted in accordance with their mean standard deviation. The non-uniform variance is caused by random errors, the so-called noise sources, like noise due to fluctuations in the light source (proportional to the signal), or the shot noise arising from photomultiplier detectors (proportional to the square root of the signal). Other random errors come from errors of pipetting, volume, etc.
The 95% confidence region for true slope and intercept [27] estimated is represented in Fig. 7b. The null intercept on the y-axis falls within the joint confidence region, near to the ellipse center. The confidence interval for the slope (shown in Fig. 7b with solid line) corresponding is 1.160x 10P41.083x 10P4. The intercept significance is tested by applying the Student t-test. The result was not significant different from zero. It is therefore possible to calculate the weight least squares line according to y = b’x. The standard deviation of estimation values are smaller by applying WLS method instead of an overall least squares regres-
J.A. Murillo Pulgarin et al./Analytica
sion, therefore the precision achieved is high as can be observed through confidence intervals for slope and intercept. When the first derivative of matrix isopotential synchronous spectrofluorimetry is used, a detection limit of 5.43 ngml ’ and determination limit of 18.47 ng ml ’ were obtained by applying the IUPAC [30] definition, in which only the standard deviation of the blank is considered. The propagation of errors approach will give values of detection and determination limits consistent with the reliability of the blank measurements and the signal measurements of the standards 1301. In this case a detection limit of 6.02 ngml ’ and a determination limit of 20.06 ng ml ’ was obtained. In order to study the repeatability of the method, a series of ten solutions was prepared containing 200ngml ’ of triamterene in urine. By applying the IUPAC definition, the standard deviation of replicates mean was 1.8 ngmll’ and the relative error 2.13%, while based on error propagation, the standard deviation obtained was 2.8 ngmll’ and the relative error 3.30% (95% confidence level).
Chimica Acra 326 (1906) 117-126
An exhaustive statistical analysis has been developed to all calibration graphs, including the LMS robust regression and least squares both classical and weighted. Due to the the heterocedasticiy of residuals of the overall least squares regression, the weighted version is recomended, increasing major accuracy in the regression, i.e., the confidence interval for slope and intercept are smaller, the standard deviation of measurements being the main error source. The intercept on y-axis significance was investigated through the ellipse and the Student t-test at 95% confidence level and was found not significant.
Acknowledgements The authors gratefully acknowledge financial support from the “Direction General de Investigacion Cientifica y Tecnica” (Project N” PB 94-0743).
References Clarke‘s Isolation and Identification of Drugs, The Pharmaceutical Press, London, 1986, pp. 1037-l 03X. 121 Her Majerty’s Stationery Office, London. The British Pharmacopoeia, 1973, 48 I. 131 Rockville, MD, The United States Pharmacopoeia, XIX, The United States Pharmacopeial Convention, 20852 ( 1975) S 17. [41 W. Bruehl, Jr and 13. Schmid, Arzneim. Forxh.. 20 (1970) 485. 151 Lilo 0. Gerello and Jose Dobrezky, Rev. Farm., I I I (1969) 13. [61 Ramana G. Rao, Kanjilal Geetha and Rama K. Mohan, Indian J. Pharm. Sci., 41 (1979) 1.56. A.S.R.P. Tiptrneni and T. [71 C.S.P. Sastry. M. Suryanarayana, Satyanarayana, Indian Drugs, 26 (1989) 6.5 I. 181 E. Gonzilez, R. Montes and J.J. Lasema, Anal. Chim. Acta. 2X2 (1993) 687. [91 S. Sved, J.A.A. SertiC and L.J. McCilveray. J. Chromatogr.. 162 (1979) 474. [101 R.B. Brodie, L.F. Chasseaud, T. Taylor and L.M. Walmsley. J. Chromatogr.. 164 (1979) 527. G.J. Yakatan and J.E. Cruz, J Pharm. Sci.. 70 ( I981 ) 949. IllI 1121 E.T. Lin, Clin. Liq. Chromatogr., I (1984) 123. [I31 F. Soergel, E.T. Lin, J. Hasegawa and L.Z. Benet, J. Pharm. SCI., 73 (1984) 831. [I41 K.J. Swart and H. Botha, J. Chromatogr., 413 (1987) 315. R. Herriez-Hemindez and A. Sevillano[I51 P. Campins-Falc6. CabeLa, Chromatographia, 38 (I 994) 29. [I61 J.A. Murillo and A. 4laii6n, A. Anal. Chim. Acta. 296 (1994) x7. 1171 J.A. Murillo and A. Alar?&, Computer Chem., I7 (1993) 34.
III
4. Conclusions A new method for direct fluorimetric determination of triamterene in urine by matrix isopotential synchronous fluorescence, without the need of prior separation has been described. The determination of this natriuretic drug in urine can be performed at 406.8nm of emission wavelength in the first derivative matrix isopotential synchronous scan. As can be readily observed through detection limits, the major source of error is in the blank (90%), therefore the IUPAC definition with a standard deviation proportional factor of three can be accepted. The standard deviation obtained for a concentration of 200ng ml-’ confirms that, to calculate the confidence interval of a measurement, error propagation may be used. In this case, the contidence interval obtained from weight least squares regression for ten replicates of 200ng ml ’ is (205.6.. ,196.l.. -186.5) ngmll’. Excellent repeatability and sensitivity, better than those of other spectrofluorimetric methods, were obtained without pre-separation procedures.
125
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[18] M.J.P. Leiner, M.R. Hubmann and O.S. Woltbeis, Anal. Chim. Acta, 198 (1987) 13. [19] J.A. Murillo and A. Alafion, Analyst, 119 (1994) 1915. [20] J. J Berzas, J. A Murillo and M.A. Gomez, Analyst, 120 (1995) 171. [21] J.A. Murillo, A. Alai&t and P. Fer&ndez, Talanta, (1995) in press. [22] J.A. Murillo and A. Alarion, Anal. Chim. Acta, (1995) in press. 1231 A. Savitzky and M.J.E. Golay, Anal. Chem., 36 (1964) 1627. [24] J. Steinier, Y. Termonia and J. Deltour, Anal. Chem., 44 (1972) 1906.
Chimica Acta 326 (1996) 117-126 [25] [26] [27] [28] [29]
[30]
W.R. Seitz, in P.J. Elvin, E.J. Meehan and I.M. Kolthoff (Eds.), Treatise Anal. Chem., Wiley, 1981, pp 194196. P.D. Lark, B.R. Craven and R.C.L. Bosworth, The Handling of Chemical Data, Pergamon, Exeter, 1968, Chap. 4. P.J. Rousseeuw and A.M. Leroy, Robust Regression and Outlier Detection, Wiley, New York, 1987. J.N. Miller, Analyst, 116 (1991) 3. D.L. Massart, B.G.M. Vandeginste, S.N. Deming and L. Kaufman, in B.G.M. Vandeginste and L. Kaufman, Chemometrics: a textbook, Oxford, 1988. L. Gary and J.D. Winefordner, Anal Chem., 55 (1983) 712.
Analytica Chimica Acta 326 (I 996) 117-I 26
Direct determination of triamterene in urine by matrix isopotential synchronous fluorescence spectrometry Amelia AlaiXh Molina,
Josh A. Murillo Pulgarin”, Department
Pablo Fernhdez
L6pez
of Analytical Chemistry and Foods Technology, University Custilla La Mancha, 13071 Ciudad Real,
Spin
Received 14 November 1995; revised 18 January 1996; accepted 21 January 1996
Abstract A new method for the determination of triamterene for concentrations between 10 and 1000 ng ml-’ by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This new method is useful for the determination of compounds in samples with unknown background fluorescence, such as triamterene in urine, without the need of tedious pre-separation. The determination was performed in an aqueous medium adjusted at a pH value of 6.2, provided by adding sodium citrate-citric acid buffer solution. Since triamterene is widely used as a doping substance in sport, the method was successfully applied to the determination of triamterene in urine. Better sensitivity and repeatability are achieved for these matrices with the proposed method than with the fluorimetric ones described in the literature. An extensive statistical analysis has been developed to all calibration graphs. This treatment includes robust regression such as least median of squares which also detects outliers and leverage points. The weighted least squares regression has been applied to find the more exact straight line which fits the experimental data. The error propagation has been used for the calculation of the detection limit, determination limit and the repeatability of the method. Keywords: Triamterene; Urine; Fluorimetry
1. Introduction Triamterene a yellow 9, gives
(2,4,7-triamino-6-phenylpteridine)
crystalline
powder.
Its solutions
is
up to a pH
a blue fluorescence.
Triamterene
is a natriuretic
agent
which
is much
used in the treatment of several diseases. It can also be applied as doping substance. In sports this diuretic is abused mainly for two reasons: to obtain a rapid diminution of corporal weight, important in sports
* Corresponding author. Fax: +34 9 262953 18 SOOO3-2670/96/$15.008~:)1996 Elsevier Science B.V. All rights reserved PII SOOO3-2670(96)00065-7
which are divided in different weight categories, and to reduce the concentration of medical drugs in urine by dilution by means of a rapid production of an elevated quantity of urine, thus trying to diminish the possibility to detect other doping substances. No medical reason can justify in any sport a rapid decrease of weight, whereas on the other hand this abuse causes grave dangers for health because of possible serious secondary effects. Triamterene is rapidly but incompletely absorbed after oral administration. Once the drug is in the body, about 30-70% of an oral dose is excreted in the urine [l]. Variable amounts are excreted in the bile.
118
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First measurements of triamterene have been made directly in pharmaceutical preparations by perchloric acid tritations [2,3]. Spectrophotometric absorption measurements at 360 nm [2] and at 375 nm [3] have been used. An estimation of triamterene and deoxycholic acid by planimetric measurement of thin layer chromatograms is also described [4]. Guerello [5] developed a spectrophotometric method for the determination of triamterene with furosemide using 50% of ethanol at 275 and 370nm. Rao [6] determined this drug and its dosage formed by a fluorimetric method using 85% of formic acid to increase sensitivity and reproducibility of the estimation. Sastry [7] analysed a mixture of triamterene and amiloride hydrochloride in pure and pharmaceutical preparations by fluorimetry. Also a polarographic method [8] has been described for triamterene and its analogues in biological fluids. Usually, the specific determination of this natriuretic agent in biological fluids involves liquid chromatography (LC) [9-121. Normally the estimation of triamterene at therapeutic levels by LC includes various preceding procedures, such as extractions and concentrations in the organic solvents [9-121 with all the disadvantages that this may cause, since all of these procedures are based on equilibrium reactions. Some authors have attempted the direct injection of the sample onto the LC column [13-l 51. However, due to the irreversible adsorption of some components of biological fluids to the column, direct injection decreases the chromatographic resolution, and greatly reduces the operative lifetime of the column. Instead of the LC methods described above [9-151, we propose a fast, simple, easy, sensitive and straightforward fluorimetric method to estimate triamterene in biological fluids like urine samples, reaching the same sensitivity as direct determination, without the need for prior separation and preconcentration or derivatization procedures. Recently in this department a new fluorimetric synchronous technique called matrix isopotential synchronous fluorescence spectrometry (MISF) has been developed [16]. This technique is particularly useful for removing fluorescence matrix background effects and enables the determination of individual compounds in complex samples. It is essential that the matrix has an almost invariable composition. It is
Chimicu Acta 326 (1996) I1 7-126
possible to maintain a constant background signal, even though its fluorescence intensity may vary, if a cut is made in the total fluorescence spectrum following one of the trajectories which join points of equal intensity (isopotential trajectory) from an initial point to final excitation and emission wavelengths. This trajectory is obtained by means of a program developed in BASIC [17]. It is always possible to find the matrix trajectory which passes through the maximum fluorescence excitation and emission wavelengths of the component to be determined and so the same sensitivity is achieved as in direct determination in the absence of background fluorescence. This technique can be improved by applying derivative methods. Human urine is composed of numerous organic substances, some of which are fluorescent [ 181, which provide a high background fluorescence and interfere with the direct determination of triamterene. Recent works in our laboratory dealt with the application of this new fluorimetric technique to the determination of different drugs in biological fluids with excellent results [ 16,19-221.
2. Experimental 2.1. Apparatus All tluorimetric measurements were performed on a Aminco Bowman Series 2 equipped with a continuous 150 W Xenon lamp, connected to software which runs on the OS2 operating system. Quarzglas cuvettes with pathlength of 1.Ox 1 .Ocm have been used. Thermostatic equipment and a Crison Model 2001 pH-meter with a glass-saturated calomel combination electrode and a Selecta centrifuge (Model Mixtaxel) were also used. 2.2. Software A program was developed which enabled us to obtain the values of X,, and X,, for any constant value of fluorescence intensity from a three-dimensional spectrum. As the values obtained for a particular curve were not equidistant, the Lagrange
J.A. Murillo
Pulgan’n
et al./Analytica
interpolation method was applied to all points, which were placed in order, using emission wavelengths at 0.6nm intervals. Once the trajectory had been defined, the spectrum was obtained by means of the Ftotal program 1171, so that the spectra obtained from it displayed the same format as those performed directly from the Aminco Bowman Series 2 spectrofluorimeter. The Ftotal program not only generates information on a fluorescent compound through the isometric representation of the three-dimensional spectrum in the form of a level curve, but it also processes the spectral data to obtain any type of spectrum of the socalled new fluorimetric techniques. The statistical analysis is covered by means of a program developed by us, which has a menu that includes all procedures mentioned in this paper.
Chimica
Acta 326 (1996)
117-126
119
(pH 6.2) and 5 ml of solution of urine free from triamterene, and dilute with water to a final volume of 25 ml. Record 61 emission spectra of 288 nm width in steps of 0.6nm, varying the excitation wavelength in 4.8 nm steps. Obtain the total luminescence spectra, select the adequate trajectory, which passes through the excitation and emission maxima of triamterene, and obtain matrix isopotential synchronous spectra by means of Ftotal 1171. Calculate the first derivative, according to the Savitzky and Golay algorithm [23,24]. Finally, determine the triamterene content by measuring the derivative signal at emission wavelength of 406.8 nm, and using the appropriate calibration graph.
3. Results and discussion 2.3. Reagents 3.1. Factors affecting fluorescence All experiments were performed with analytical reagent grade chemicals, pure solvents and Mini-Q water. The standard triamterene was obtained from Sigma. A stock solution of triamterene was prepared in a 500 ml volumetric flask by dissolving 25 mg in water. This stock solution was used to prepare standard solutions by suitable dilutions. A 1.25 M buffer solution of pH 6.2 was prepared by mixing adequate amounts of citric acid with sodium hydroxide. The stock solution of triamterene was stored, protected from the light and maintained below 5°C. Under these conditions, the solution of triamterene was stable for two months. The working samples of triamterene were stable for at least 2 h at room temperature. Urine samples were obtained from fasting and healthy people in the morning. 2.4. Procxdure 2.4. I. Sample preparation Centrifuge urine for 15 min at 3800rpm and transfer the clear supematant solution into a flask, store and maintain below 5°C. For the preparation of calibration graph, place an aliquot of triamterene equivalent to 0.25-25 pg in a 25 ml volumetric flask, add 10 ml of buffer solution
intend,
Chemical variables were studied to obtain the best measurement conditions and maximum fluorescence sensitivity. Triamterene is highly soluble in water, so it was not necessary to use an organic solvent like ethanol. Nevertheless it was suitable to study the variation of triamterene and urine fluorescence with changes in the dielectric constant of the media. In this way the effect of ethanol content in the medium was investigated by preparing samples of triamterene and urine, and by varying the ethanol percentage between 0% and 98% V/V for the triamterene solutions and between 0% and 80% for the urine solutions. The fluorescence intensity due to triamterene does not change when the ethanol percentage in the medium increases (Fig. 1b) while fluorescence intensity due to urine increases (Fig. la). Because of these reasons it is not necessary to use ethanol. The influence of pH on the fluorescence intensity was also studied by adding different amounts of HCl and NaOH to a urine solution and a triamterene solution. As can be seen in Fig. 2b, the fluorescence intensity of triamterene is nearly constant with pH values between 2 and 10. The fluorescence intensity due to urine decreases slowly from pH 4 to 1 1 (Fig. 2a). A pH 6.2 was selected as adequate: this is approximately the pH value of urine of healthy
J.A. Murillo Pulgan’n et al./Analytica
a)
-c+-cc
Urine
b) -
1
,
I
Chimica Acta 326 (1996) I 17-126
2
Triarnterene
1
,
40
20
%
1
I
,‘I’1 60
1
80
100
a)
w
1
00 0
2
4
6
8
IO
I2
.,
1.J
PH Fig. 2. Influence of the pH on fluorescence intensity of (a) urine and (b) triamterene. Excitation at 365nm. Emission at 436.8nm. Concentration of triamterene lOOOngmll’, urine (15). Urine data scaled to 10 with regard to maximum fluorescence intensity.
3.2. Determination people. It was proportioned by adding sodium citrate-citric acid buffer solution. The fluorescence intensity of triamterene and urine was not affected by the buffer and its concentration. A 0.5 M concentration of buffer was therefore selected to get an adequate buffering capacity. Another factor that affects the fluorescence intensity is temperature. In this way, in both cases, fluorescence intensity showed a decrease when the temperature increases from 4 to 75°C. The temperature coefficients are about 0.46%“C’ for triamterene and about 0.61%“C~’ for urine. This effect can be explained by higher internal conversion as temperature increases, facilitating non-radiative deactivation of the excited singlet state 1251 Therefore, the use of a thermostat is recommended and a measurement temperature of 20°C is chosen. The influence of triamterene concentration on the fluorescence intensity was studied under these conditions. The best range for the relation fluorescence intensity vs. concentration was found for concentrations between 10 and triamterene 1OOOngmll’.
Triamterene
b) -
Ethanol
Fig. 1. Influence of the ethanol content on fluorescence intensity of (a) urine and (b) triamterene. Excitation at 365 nm. Emission at 436.8 nm. Concentration of triamterene lOOOngmll’, urine (1:5). Urine data scaled to 10 with regard to maximum fluorescence intensity.
Urine
of triamterene
in urine
All fluorimetric three-dimensional spectra were performed by varying emission wavelength from 307.2 to 595.2nm and excitation wavelength from 200 to 488nm. Both excitation and emission bandpass were 8 nm. Scan rate was 50 nm s-‘. Under these conditions, the time taken to determine contour spectra is 438 s. MISF was applied to the analysis of triamterene in urine, a very fluorescent matrix, under the optimum chemical conditions established above. The fluorescence maxima characteristic of triamterene are located in the UV region. As can be observed in Fig. 3, in this region, urine shows one broad peak which prevents determination of this compound without prior separation. Previous experiments have shown that the qualitative composition of the fluorescent metabolites of urine from healthy people of both sexes and different diets, aged between 25 and 35 yr, are practically constant, a necessary condition for the application of this new technique. The different samples of urine display the same type of fluorescence, with hardly any variation in the form of the spectrum and in the
J.A. Murillo Pulgarin et al./Analytica
121
Chimica Acta 326 (1996) 117-126
440 x E 344
x n In
248
355 2
4512 x
3JJ
mw
Fig. 3. Total fluorescence spectra of IOOOng ml (solid line) and urine I:5 (broken line).
‘
.,.
_
._,
‘
X EM@)
’ of triamterene
location of the fluorescence maxima, although it is possible to observe some variations in their intensity. The spectrum corresponding to the arithmetical mean of ten total fluorescence spectra of the different urine samples was obtained by means of the Ftotal program [ 171. In order to determine the triamterene we calculated the isopotential trajectory in the averaged urine spectrum, which passes through the excitation and emission maxima of the triamterene, as shown in Fig. 4. Urine sample solutions containing triamterene gave signals smaller than those obtained with aqueous standard solutions, owing to some type of binding with other components of the urine. Total luminescence spectra of triamterene were obtained in different urine samples in order to construct calibration graphs and to carry out recovery experiments. Fig. 5 shows the effect of background fluorescence intensity (urine) on triamterene MISF and derivatives spectra. It can be seen that the spectra are identical in form although their intensities are different by constant terms. These correspond to the values of the fluorescence intensity due to the three urines in the isopotential trajectory applied. It is easy to observe that derivation totally removes the background effect. The first derivative of MISF spectra was applied to all samples. The number of points through which the derivative is obtained was optimized, with the conclusion that derivative spectra with a suitable signal-noise ratio were obtained with 2.5 points.
Fig. 4. Total fluorescence spectrum of triamterene (broken and the selected isopotential trajectory of urine (solid line).
line)
In the same way we obtained the total luminescence spectra of triamterene in aqueous solution at the same concentrations, to which we applied the above mentioned trajectory (Fig. 4) in order to obtain their MISF spectra. We also obtained their first derivative, as with the urine samples. As can be readily seen in Fig. 6a, which shows the MISF spectra of triamterene obtained in urine, it is not posible to determine this drug by measuring the maximun fluorescence intensity (which is located at X,,=436.8nm) with regard to the final extremes (X+.,=392.4 nm and X,,=454.2nm) of the selected trajectory, since the fluorecence intensity does not reach a constant value. The first derivative technique is therefore applied. The calibration graph was constructed by measuring the first derivative at X,,=406.8 nm where maximum sensitivity is achieved. Fig. 6b shows the spectra derived from the calibration of triamterene in urine. In order to test the independence of the analytical signal of triamterene, i.e., to show that the signal measured is independent of the urine, three calibration graphs from the first derivative signals were constructed with different urine samples. The proposed method was evaluated by a statistical analysis of experimental data by fitting the least squares line according to y = a + bx, after discarding outliers with the help of the least median of squares regression (LMS) [26], since LMS is a robust regression method. Table 1 shows the outstanding results of the statistical analysis. To verify if the intercepts on the ),-axis were negligible, significances were studied by applying the
122
J.A. Murillo Pulgarin et al./Analytica
Chimica Acta 326 (1996) 117-126
a)
0.0
, 392
,
I
,
400
I
,
408
I
416
,
I
424
,
I
432
Emission Wavelength
I I40
’
I
448
-I 456
,
,
392
,
(
400
(mn)
,
,
408
,
,
416
Emission
,
s/ ;
,
424
,
,
432
,
,
,
440
Wavelength
,
448
456
(nm)
b)
lcmngd’
I
I2- 7CQngmL' 3- 4fXingmL 4. 2OOngmL'
0.10
0.05 1
A = 406.6 nm
0.00
398
40s
412
419
426
433
440
.o.os , 398
Emission Wavelength
,
, 40s
\s
’, , , , , 412
419
(
426
,
,
433
,
440
(mn)
Fig. 5. (a) Set of MISF spectra of 4OOng ml-’ of triamterene different urine samples. (b) Their first derivative spectra.
in
Student t-test at 95% confidence level and suitable degrees of freedom [27]. If the intercepts on the yaxis for the lines calculated by the least squares technique are negligible, it is necessary to perform the least squares regression again according to the function y = b’x and, therefore the new value of the slopes of calibration graphs (b’) may be calculated. As can be seen from Table 1, the intercepts on the yaxis were negligible in the range of concentration, since the experimental t is smaller than the theoretical
Emission
Wavelength
(nm)
Fig. 6. (a) Set of MISF spectra of triamterene in urine. Triamterene concentrations: (1)lOOO; (2)700; (3)400; (4)200; (5)lOO; (6)50; (7)25 and (8)lOngml~‘. (b) The corresponding first derivative spectra.
t and therefore the new slopes were calculated. This shows that by applying the first derivative of MISF spectra, the signal due to urine is removed. To obtain the most representative calibration graph, an overall least squares was developed, in which, the regression is obtained by including all the data pairs and performing the classic least squares regression. Due to the heterocedasticity of residuals
-
I
0.9966 0.9992 0.9996 0.9976 0.9996 0.9998 0.9960 0.9978 0.9988 0.9993 -4.478~10~’
7.759x10
5
2.058x lo- A 7.362x 10m4 -4.661~10~’
regression line according to y = regression line according to y= squares regression line according squares regression line according squares regression line according squares regression line according
IT III I 11 III
1 - Least squares 2 Least squares 3 - Overall least 4 - Overall least 5 Weight least 6 - Weight least
3 4 5 6
2
Urine Urine Urine Urine Urine Urine -
1
Intercepton y-axis
data of the determination
Correlation coefficient
statistical
Sample
Fitting used
Table 1 Outstanding
4 4 4 (4.932~10
(2.153xlO~‘4m -4.036~10
-6.013x 4,
IO-‘)
matrix interval
isopotential
fluorescence
0.244
1.168
1.785 1.424 1.318
Experimental t value
synchronous
(1.142x10 4- 1.015~10~~) (1.071x10 4-1.014x10~4) (1.170x10-4-1.131x104) (l.158xlO-4-l.O6OxlO 4, (l.o74xlo.4 l.033xlo~~4) (1.158x10 4--l.l3OxlO 4, (l.l21xlO-4-l.O61xlO4) (l.l24xlO-4-1.O8OxlO4) (l.159x10-4-l.o14xlo4) (1.148~10-~ l.014x10~4)
Confidence for slope
of lirst derivative
(4.879x IO-j- -7.632x 10. 4, (2.001 x 10m3p -5.289~ IO 4, 4) (3.991 X IO 4 -1.331x10
Confidence interval for intercept
in urine by means
a + bx. Theoretical t value: 2.447. b’x. Theoretical t value: 2.365. to y = ti + bx. Theoretical I value: 2.074. to y = b’x. Theoretical f value: 2.069. t value: 2.447. to y=a + ky. Theoretical to y=b’x. Theoretical r value: 2.365.
1.079x10 1.043xlO~4 l.151x10~4 1.109x10~4 1.053x10 1.144x10 1.091x10 1.102x10~4 1.124~10-~ l.122xlo~4
‘l
of triamterene Slope
2.3x10 3 1.0x10 3 7.1 x 1om4 2.7x IO-’ l.lxlO~’ 7.6x10 4 2.4x10 ’ 2.4x 10m3 4.3x IOwr 4.0x 10-4
Standard deviaton of estimation
E
124
J.A. Murillo Pulgarin et al./Analytica
Chimica Acta 326 (1996) I 17-126
; R 6.fJE-02 I
S.D.
v
1.60E+1 2.50E*l !G.BBE*i 1 .em+z 2 .fmE+Z 4.88E+2 7 .eaB+Z 1.8BE*3
1
I I 3.81-82
e
OS
M1
3.1824E-4 3.298BE-4 5.0107E-4 2.0472E-4 1.70791-3 1.85!i0E-3 1.6843E-3 Z.-E-3
i b.BE+flZ
3.BE42
CONCWIRbTlON
b)
8.1915E-4 2.7ell.E-3 5.77493-3 l.l335E-2 2.4469E-2 4.!i795E-2 7.787lE-2 1.0848E-1 9.BEM2
1.2E*B3
9.4eE-84
A T R"4.48E-04 C E P I -6.88E-85 no
SLOPE Fig, 7. (a)Calibration graph of triamterene. The first derivative data are represented by the mean values and its standard deviation. The solid line is the weighted least square regression according to y = a + bx and the broken line is the confidence band. (b) The ellipse is 95% confidence region for the true slope and intercept on y-axis estimated from the weighted least squares regression.
obtained, we propose a fit of the experimental data to weight least squares line (WLS) [28,29] according to y = a + bx, shown in Fig. 7a together with experimental data. As the heterocedasticity is a non-uniform variance response, first derivative values are weighted in accordance with their mean standard deviation. The non-uniform variance is caused by random errors, the so-called noise sources, like noise due to fluctuations in the light source (proportional to the signal), or the shot noise arising from photomultiplier detectors (proportional to the square root of the signal). Other random errors come from errors of pipetting, volume, etc.
The 95% confidence region for true slope and intercept [27] estimated is represented in Fig. 7b. The null intercept on the y-axis falls within the joint confidence region, near to the ellipse center. The confidence interval for the slope (shown in Fig. 7b with solid line) corresponding is 1.160x 10P41.083x 10P4. The intercept significance is tested by applying the Student t-test. The result was not significant different from zero. It is therefore possible to calculate the weight least squares line according to y = b’x. The standard deviation of estimation values are smaller by applying WLS method instead of an overall least squares regres-
J.A. Murillo Pulgarin et al./Analytica
sion, therefore the precision achieved is high as can be observed through confidence intervals for slope and intercept. When the first derivative of matrix isopotential synchronous spectrofluorimetry is used, a detection limit of 5.43 ngml ’ and determination limit of 18.47 ng ml ’ were obtained by applying the IUPAC [30] definition, in which only the standard deviation of the blank is considered. The propagation of errors approach will give values of detection and determination limits consistent with the reliability of the blank measurements and the signal measurements of the standards 1301. In this case a detection limit of 6.02 ngml ’ and a determination limit of 20.06 ng ml ’ was obtained. In order to study the repeatability of the method, a series of ten solutions was prepared containing 200ngml ’ of triamterene in urine. By applying the IUPAC definition, the standard deviation of replicates mean was 1.8 ngmll’ and the relative error 2.13%, while based on error propagation, the standard deviation obtained was 2.8 ngmll’ and the relative error 3.30% (95% confidence level).
Chimica Acra 326 (1906) 117-126
An exhaustive statistical analysis has been developed to all calibration graphs, including the LMS robust regression and least squares both classical and weighted. Due to the the heterocedasticiy of residuals of the overall least squares regression, the weighted version is recomended, increasing major accuracy in the regression, i.e., the confidence interval for slope and intercept are smaller, the standard deviation of measurements being the main error source. The intercept on y-axis significance was investigated through the ellipse and the Student t-test at 95% confidence level and was found not significant.
Acknowledgements The authors gratefully acknowledge financial support from the “Direction General de Investigacion Cientifica y Tecnica” (Project N” PB 94-0743).
References Clarke‘s Isolation and Identification of Drugs, The Pharmaceutical Press, London, 1986, pp. 1037-l 03X. 121 Her Majerty’s Stationery Office, London. The British Pharmacopoeia, 1973, 48 I. 131 Rockville, MD, The United States Pharmacopoeia, XIX, The United States Pharmacopeial Convention, 20852 ( 1975) S 17. [41 W. Bruehl, Jr and 13. Schmid, Arzneim. Forxh.. 20 (1970) 485. 151 Lilo 0. Gerello and Jose Dobrezky, Rev. Farm., I I I (1969) 13. [61 Ramana G. Rao, Kanjilal Geetha and Rama K. Mohan, Indian J. Pharm. Sci., 41 (1979) 1.56. A.S.R.P. Tiptrneni and T. [71 C.S.P. Sastry. M. Suryanarayana, Satyanarayana, Indian Drugs, 26 (1989) 6.5 I. 181 E. Gonzilez, R. Montes and J.J. Lasema, Anal. Chim. Acta. 2X2 (1993) 687. [91 S. Sved, J.A.A. SertiC and L.J. McCilveray. J. Chromatogr.. 162 (1979) 474. [101 R.B. Brodie, L.F. Chasseaud, T. Taylor and L.M. Walmsley. J. Chromatogr.. 164 (1979) 527. G.J. Yakatan and J.E. Cruz, J Pharm. Sci.. 70 ( I981 ) 949. IllI 1121 E.T. Lin, Clin. Liq. Chromatogr., I (1984) 123. [I31 F. Soergel, E.T. Lin, J. Hasegawa and L.Z. Benet, J. Pharm. SCI., 73 (1984) 831. [I41 K.J. Swart and H. Botha, J. Chromatogr., 413 (1987) 315. R. Herriez-Hemindez and A. Sevillano[I51 P. Campins-Falc6. CabeLa, Chromatographia, 38 (I 994) 29. [I61 J.A. Murillo and A. 4laii6n, A. Anal. Chim. Acta. 296 (1994) x7. 1171 J.A. Murillo and A. Alar?&, Computer Chem., I7 (1993) 34.
III
4. Conclusions A new method for direct fluorimetric determination of triamterene in urine by matrix isopotential synchronous fluorescence, without the need of prior separation has been described. The determination of this natriuretic drug in urine can be performed at 406.8nm of emission wavelength in the first derivative matrix isopotential synchronous scan. As can be readily observed through detection limits, the major source of error is in the blank (90%), therefore the IUPAC definition with a standard deviation proportional factor of three can be accepted. The standard deviation obtained for a concentration of 200ng ml-’ confirms that, to calculate the confidence interval of a measurement, error propagation may be used. In this case, the contidence interval obtained from weight least squares regression for ten replicates of 200ng ml ’ is (205.6.. ,196.l.. -186.5) ngmll’. Excellent repeatability and sensitivity, better than those of other spectrofluorimetric methods, were obtained without pre-separation procedures.
125
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[18] M.J.P. Leiner, M.R. Hubmann and O.S. Woltbeis, Anal. Chim. Acta, 198 (1987) 13. [19] J.A. Murillo and A. Alafion, Analyst, 119 (1994) 1915. [20] J. J Berzas, J. A Murillo and M.A. Gomez, Analyst, 120 (1995) 171. [21] J.A. Murillo, A. Alai&t and P. Fer&ndez, Talanta, (1995) in press. [22] J.A. Murillo and A. Alarion, Anal. Chim. Acta, (1995) in press. 1231 A. Savitzky and M.J.E. Golay, Anal. Chem., 36 (1964) 1627. [24] J. Steinier, Y. Termonia and J. Deltour, Anal. Chem., 44 (1972) 1906.
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[30]
W.R. Seitz, in P.J. Elvin, E.J. Meehan and I.M. Kolthoff (Eds.), Treatise Anal. Chem., Wiley, 1981, pp 194196. P.D. Lark, B.R. Craven and R.C.L. Bosworth, The Handling of Chemical Data, Pergamon, Exeter, 1968, Chap. 4. P.J. Rousseeuw and A.M. Leroy, Robust Regression and Outlier Detection, Wiley, New York, 1987. J.N. Miller, Analyst, 116 (1991) 3. D.L. Massart, B.G.M. Vandeginste, S.N. Deming and L. Kaufman, in B.G.M. Vandeginste and L. Kaufman, Chemometrics: a textbook, Oxford, 1988. L. Gary and J.D. Winefordner, Anal Chem., 55 (1983) 712.