Anal. Chem. 1988, 60, 2493-2496
(8) Yang, S. S.; Gilpin, R. K. J . Chromatogr. 1987, 394, 295. (9) Sindorf, D. W.; Maciel, G. E. J . Am. Chem. SOC. 1983, 105, 3767. (10) Avnir, D.; Busse, R.; Ottolenghi, M.; Wellner, E.; Zachariasse, K. J . fhys. Chem. 1985, 8 9 , 3521. (11) Lochmuiler, C. H.; Hunnicutt, M. L. J . fhys. Chem. 1988, 9 0 , 4318. (12) Carr, J. W.; Harris, J. M. Anal. Chem. 1988, 5 8 , 626. (13) Lochmuller, C. H.; Colborn, A. S.; Hunnicutt, M. L.; Harris, J. M. J . Am. Chem. SOC. 1984, 106, 4077. (14) Bogar, R. G.; Thomas, J. C.; Caiiis, J. B. Anal. Chem. 1984, 5 6 , 1080. (15) StAhlberg, J.; Almgren, M. Anal. Chem. 1985, 5 7 , 817. (16) Birks, J. B. fhotophysics of Aromatic Molecules ; Wiley-Interscience: London, 1970; Chapter 7. (17) Birks, J. B.; Dyson, D. J.; Munro, 1. H. R o c . R . SOC.,London, A 1983, 275, 575. (18) Bauer, R. K.; Borenstein, R.; de Mayo, P.; Okada, K.; Rafalska, M.; Ware, W. R.; Wu, K. C. J . Am. Chem. SOC. 1982, 104, 4635. (19) Bauer, R. K.; de Mayo, P.; Okada, K.; Ware, W. R.; Wu, K. C. J . fhys. Chem. 1983, 8 7 . 460.
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(20) Moelwyn-Hughes, E. A. The Chemical Statics and Kinetics of Solutions ; Academic: London, 197 1. (21) Ha, L. N.; Ungvaral, J.; Kovats, E. Anal. Chem. 1982, 5 4 , 2410. (22) Morel. D.; Serpinet, J.; Letoffe, J. M.; Claudy, P. Chromatographia 1988, 22, 103. (23) Van Miltenburg, J. C.; Hammers, W. E. J . Chromatogr. 1983, 268, 147. (24) Selinger, B. K.; Watkins, A. R. J . Photochem. 1982, 2 0 , 319. (25) Alwattar, A. H.; Lumb, M. D.;Birks, J. B. In Organic Molecular fhotophysics; Birks, J. B., Ed.; Wiley-Interscience: London, 1973; Vol. I,p 402. (26) Farin, D.;Volpert, A.; Avnir, D. J . Am. Chem. SOC.1985, 107, 3368. (27) Avnir, D. J . Am. Chem. Soc. 1987, 109, 2931.
RECEIVED for review November 6,1987. Accepted August 1, 1988. This work was supported by the Swedish National Science Research Council.
Simultaneous Determination of Salicylic and Salicylur c Acids n Urine by First-Derivative Synchronous Fluorescence Spectroscopy Arsenio Muiioz de la Peiia,* Francisco Salinas, and Isabel Durfin Mer& Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain A sensitive, rapid, and specific assay has been developed for the simultaneous determination of salicylic (SA) and saiicylurk (SU) acids in urine, based on its natural fluorescence. The range of application is between 0.02 and 0.25 pg mL-l for salicylic and between 0.02 and 0.22 pg mL-’ for saiicyiuric acid. The urlne Is extracted into elhyl ether In acid medium and reextracted with a glycine-NaOH buffer of pH 1I , prior to Instrumental determination. Overlapping of conventional fluorescence spectra is resolved by using first-derivative synchronous fluorometry, thus making the use of separation techniques unnecessary. Overall recovery of both compounds was about 103% for salicylic and 105% for salicyluric.
Salicylates are used as prescription items for a variety of medical conditions. Their therapeutic action and toxicity are still the subject of considerable research, and the use of high doses of these compounds in the treatment of rheumatoid arthritis makes necessary its analytical control in biological fluids. The main metabolite of salicylic acid (SA) is salicyluric acid (SU), because the major route of SA elimination is via conjugation with a glycine to form SU (1, 2). The variety of published assay methods (3-6) present problems as to the directness, specificity, sensitivity, and ease of operation required for the analysis of a large number of samples. In fluorometric methods, high sensitivity and selectivity are generally expected. However, problems of selectivity can occur in multicomponent analysis because of the overlap of the broad-band spectra observed. Specificity is a particular problem in the determination of fluorescent drugs. Generally, these compounds are determined by using a prior separation step, which is rather time-consuming for routine analysis and in some cases requires special and expensive instrumentation. The fluorescent spectra of salicylic and salicyluric acids overlap considerably so that the conventional fluorometric method does not permit simultaneous determination of these compounds. This problem has been partially resolved by synchronous fluorescence spectroscopy (7-ll), in which the excitation and 0003-2700/88/0360-2493$0 1.50/0
emission monochromators are simultaneously scanned, separated by a constant wavelength interval, Ax. The spectral distribution is a function of the difference between the excitation and emission wavelengths. The maximum fluorescence intensity of a particular component occurs when AA corresponds to the difference between the wavelengths of the excitation and emission maxima. The resulting spectra represent the intensity profile of a 45’ section cut through the excitation-emission matrix (EEM). The synchronous spectra are generally characterized by a narrowing of the spectral bands. Although direct synchronous spectra are often sufficiently resolved for analytical purposes, the first or second derivative of these spectra would be helpful to closely differentiate bands. This technique was introduced by John and Soutar (12),and its analytical applications have recently been reviewed (13, 14). As the effectiveness of derivative spectroscopy is a function of the bandwidth of the zero-order spectrum, derivative synchronous fluorometry provides a satisfactory method of resolving mixtures of components with spectra that strongly overlap. The combination of synchronous and derivative techniques results in an increased selectivity and in an increment of the SNR values obtained by differentiation of the conventional spectrum. This is so because the amplitude of the derivative signal is inversely proportional to the bandwidth of the original spectrum, and as stated above, a characteristic of synchronous fluorometry is the narrowing of the spectral bandwidth in relation to conventional fluorometry. In the method described here, salicylic and salicyluric acids are determined directly and simultaneously by its natural fluorescence. However, as the conventional and synchronous spectra of both compounds completely overlap, the determination is done by first-derivative synchronous fluorometry and provides a clear example of the high resolving power of this technique. EXPERIMENTAL SECTION Apparatus. All the spectrometric measurements were con-
ducted with a Perkin-Elmer spectrofluorometer (Model MPF-43). The spectrometer used a 150-W xenon arc lamp as the excitation 0 1988 American Chemical Society
2494 * ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
light source and a R-508 photomultiplier (Hamamatsu Co.) as the detector. For synchronous excitation measurements, both excitation and emission monochromators were locked together and scanned simultaneously. Standard 1 X 1 cm quartz cells were used for fluorescence measurements. The spectrofluorometer was interfaced to a Commodore 64 niicrocomputer for spectral acquisition and subsequent calculation of the excitation-emission matrices and of the derivative spectra ( 1 5 ) . A homemade &bit analog to digital (A/D) converter was constructed. The A/D converter is based on the successive approximation technique. The sampling rate of the A/D converter was synchronized with the starting of the spectrofluorometer and with the scan speed of the monochromators. Smoothed and derivative spectra were calculated by the Savitzky and Golay method (16,17) and contour plots in the excitation-emission plane were produced, linking points of equal fluorescence intensity. Reagents. All experiments were performed with analyticalreagent grade chemicals and pure solvents. Doubly distilled and demineralized water was used throughout. Salicylic acid (2-hydroxybenzoic acid) was obtained from E. Merck, Darmstadt, and salicyluric acid (o-hydroxyhippuricacid) from Aldrich-Chemie,Steinheim, West Germany, and these were used as analytical standards. A stock solution of each acid is prepared in 50-mL volumetric flasks by dissolving 50 mg in water. These stock solutions (containing 1 mg/mL) are used to prepare standard solutions by suitable dilutions. Procedure. Place a known volume of diluted urine (0.5 mL), containing 0.50-6.25 c(g of salicylic acid and 0.50-5.5 pg of salicyluric acid, into a separatory funnel. Add 1 mL of HCl1 M and enough water to make a total volume of 3 mL. Extract with 10 mL of ethyl ether by shaking for 10 min. Reextract the organic phase with 1 mL of glycine-NaOH buffer of pH 11 and 2 mL of water. The aqueous phase is transferred to a 25-mL volumetric flask, and the volume is increased to the mark with doubly distilled water. Record synchronous fluorescence spectra by scanning both monochromators simultaneously at a 90-nm constant difference. The excitation monochromator is scanned from 200 to 440 and the emission monochromator from 290 to 530 nm. The scan speed and the response time of the spectrofluorometerare 240 nm min-' and 1.5 s, respectively. Archive the spectra on a disk file, and calculate the first-derivative spectra by the Savitzky and Golay method with a width in nanometers of 15. First-derivativemeasurements are made as the vertical distance from the first-derivative synchronous spectrum at A,,/A, = 3?6/416 nm to the base line for salicylic acid and from the first-derivative synchronous spectrum at Aex/Aem = 302/392 nm for the salicyluric acid.
RESULTS AND DISCUSSION The determination of salicylic acid and salicyluric acid in biological fluids is possible by various methods. Among these, fluorometry would be expected to be suitable, although the method in its conventional implementation suffers from the fact that the peaks of interest of both compounds overlap. The excitation and emission spectra of salicylic and salicyluric acids are shown in Figure 1. Salicylic acid shows an excitation maximum at 297 nm, and salicyluric acid shows an excitation maximum a t 323 nm. The emission spectra show maxima a t 397 and 402 nm, respectively. Absorption spectra of salicylic acid show a maximum a t 295 nm and that of salicyluric acid a maximum a t 324 nm, which explains the longer excitation wavelength of salicyluric acid. I n f l u e n c e of Experimental Variables. Studies on the influence of the pH of solutions of salicylic and salicyluric acids in water show that the singly ionized form of salicylic acid (18) is present over the pH range of 5-13 and that the fluorescent singly ionized form of salicyluric acid is present for pH values greater than 10. At p H 11, the two species coexist in the medium with the greatest fluorescence intensity, and both analytes are stable a t that p H a t least for 1 h. We have proved that the fluorescence signals obtained for salicylic and salicyluric acids at pH 11 are the same by ad-
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Figure 1. Fluorescence excitation and emission spectra of aqueous solutions of (a) saticylic acid, 1 pg mL-', A, = 297 nm, k,, = 397 = 402 nm. nm, and (b) salicyluric acid, 1 pg mL-', A, = 323 nm, A,
justing the pH with NaOH or with a glycine-NaOH buffer. In accordance with this, a 0.1 M glycine-NaOH buffer of pH 11 was elected as the optimum to ensure a sufficient buffer capacity. This buffer has been selected because it has been proposed for the determination of salicylic and salicyluric acids by other authors (19). It is evident that pH control could be used as an alternative to our method to distinguish between the signals from the two acids, but it implies that two aliquots of the sample be prepared for one analysis. We think that our method is simpler because only one scan of the sample is required. The dependence of the fluorescence intensity of both compounds on the temperature is critical. I t is therefore recommended that a thermostat with a measurement temperature of 20 "C, i.e., about room temperature, be used. Election of Optimum Ak. The extent of the overlap of these compounds can usefully be examined by interfacing a microcomputer to the spectrofluorometer to obtain the total fluorometric information available in the excitation emission matrix. With suitable computer programs, three-dimensional spectra can be obtained and presented as the isometric projection, where the emission spectra a t stepped increments of the excitation wavelength are recorded and plotted, and also the three-dimensional spectra can be effectively transformed into a plot in the two dimensions of excitation and emission wavelengths by joining points of equal intensity (Figure 2). The synchronous scan described a 45' cut in the excitationemission matrix, and the main difficulty encountered in the application of this technique is that the best AA value must be known beforehand for optimum results, and in some multicomponent systems, several different AA values might be necessary to achieve complete identification. The contour representation is of greatest interest in indicating the most suitable trajectory to follow in the excitation-emission matrix,
ANAL.YTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
2495
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Flgure 2. Contour plot of the excitation-emission matrix of (a)salicylic acid, (b) salicyluric acid, and (c) a mixture of both compounds in glycine-NaOH buffer (pH 11). Data were obtained by using the PerkinElmer MPF-43 interfaced to a Commodore 64 microcomputer. The contours join points wlth fluorescence intensities l o % , 20 %, etc., of the maximum. The synchronousfluorescence path (-) slices the data matrix at 45' (AA = 90 nm). in order to obtain conventional synchronous fluorescence spectra for the complete resolution of overlapping component peaks. On the other hand, the three-dimensional plot, by itself, will often be more complicated than necessary for specific analytical applications. We have adapted the two methods for our analytical application. We used the synchronous scan method, but by first generating a three-dimensional plot to completely characterize the sample spectrum and determining the most useful values of AA for subsequently more rapid and quantitative analysis (20). Figure 2 shows the two-dimensional fluorescence contour plots of salicylic acid, salicyluric acid, and of a mixture of the two compounds under optimum reaction conditions. The
320/400
400/480
A exc/Aem (nm) Figure 3. Synchronous fluorescence spectra of (a) salicylic acid, (b) salicyluric acid, and (c)a mixture of both compounds (AA = 90 nm).
parallel diagonal lines superimposed on the spectrum represent the scan paths through the excitation-emission matrix that would be obtained with synchronous scans at the wavelength interval shown. The optimum AA to simultaneously determine salicylic and salicyluric acids is immediately evident. A AA of 90 nm seems the optimum to pass around the maximum of both compounds without considerable loss of sensitivity. Figure 3 shows synchronous spectra of salicylic acid, salicyluric acid, and a mixture of both compounds, maintaining a constant interval between the emission and excitation wavelengths AX = A,, - A,, = 90 nm. Because of the large overlap of the spectra, the determination of salicylic and salicyluric acids by synchronous fluorometry is still not feasible (subject to considerable difficulties). This overlap has been resolved by using first-derivative synchronous fluorometry. Figure 4 shows the first-derivative synchronous fluorescent spectra of salicylic acid, salicyluric acid, and a mixture of both compounds. From an examination of Figure 4, it appears that because of the closeness of the two overlapping spectra of both compounds, they are not sufficiently well-resolved to generate two distinct peaks in the first-derivative synchronous spectra of the mixture of both complexes. The technique used to choose suitable wavelengths to take the measurements proportional to salicylic and salicyluric acid concentrations for the preparation of calibration graphs has been the "zero-crossing measures". This technique involves the measurement of the absolute value of the total derivative spectrum a t an abcissa value (wavelength) corresponding to the zero-crossing of the spectrum of the interfering component: measurements of the value of the derivative spectrum of one of the two components would be a function only of the concentration of the other component. In fact, the height h, &,/Aem = 326/416 nm) is proportional to the salicylic acid concentration, and h2 (Aex/Aem = 3021392 nm) is proportional to the salicyluric acid concentration (Figure 4). Instrumental Parameters. The most appropriate parameters of the procedure to register synchronous derivative spectra were selected. A scan speed of 240 nm min-' and a response time of the spectrofluorometer of 1.5 s were selected after verifying that these parameters do not affect practically the derivative signal obtained, because the differentiation is
2496
ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
Table I. Resolution of Salicylic-Salicyluric Acid Mixtures
salicyluric, pg
40
salicylic/salicyluric ratio
30
1:3 1:2 1:1 3.3:l 2: 1
20
a Average
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taken
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0.055 0.082 0.120 0.208 0.163
0.180 0.160 0.120 0.060 0.080
0.191 0.161 0.125 0.058 0.085
0.060 0.080 0.120 0.200 0.160
mL-'
of three determinations.
Table 11. Recovery Test of Salicylic and Salicyluric Acids Added to Urine
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attained numerically and not electronically. For the calculation of the derivative spectra by the Savitzky and Golay method, a width of 15 nm was selected as give the best signal to noise ratio. Analytical Parameters. The determination of binary mixtures of salicylic acid and salicyluric acid is carried out in only one scan. The method involved the construction of independent calibration curves for each component. The /, , = 326/416 varies signal for the first derivative a t hX linearly with salicylic acid concentration in the range 0.02-0.25 pg mL-', and the variation of the signal at X/, = 3021392 with the salicyluric acid concentration is linear in the range 0.02-0.22 pg mL-l. The relative standard deviation (P = 0.05; n = 11)for 0.16 pg mL-* of salicylic acid is 3.3% and for 0.14 pg mL-' of salicyluric acid is 1.9%. The first-derivative synchronous fluorescence method has been applied to the analysis of several synthetic mixtures of salicylic and salicyluric acids in different ratios. Table I summarizes the results calculated from the calibration curves. Urine Samples. The first-derivative synchronous fluorometric method has been applied to the analysis of several synthetic mixtures of salicylic and salicyluric acids in urine from normal adults. Detection limits of the method are well below therapeutic levels (100-500 pg mL-l), allowing a dilution of the urine. The interference of other normal components is avoided by the extraction step ( 4 , 19, 21). Reextraction could be avoided by evaporating the ethyl ether to dryness at 50 O C and dissolving the residue in the basic buffer ( 2 2 ) , but we have found more reproducible results by reextraction into the buffer.
Table I1 summarizes the results obtained by adding different amounts (between 7.5 and 20 pg) of salicylic and salicyluric acids to the urine and diluting ten times prior to the application of the proposed procedure. Discussion. The results obtained in the determination of salicylic and salicyluric acids prove that derivative synchronous fluorescencespectroscopy can be useful in dispensing with the chromatographic step required in many quantitative analyses currently utilizing high-performance liquid chromatography to improve selectivity. These chromatographic procedures are rather time-consuming for routine assays and require a special and expensive apparatus. Therefore, derivative synchronous fluorescence spectroscopy is applicable wherever simplicity, speed, and cost-effectiveness are sought.
LITERATURE CITED (1) Nelson, E.; Hanano, M.; Levy, G. J. fharmacol. E x . Ther. 1962, 153, 159-164. (2) Curnrnings, A. J.; Martin, B. K.; Renton. R. Br. J. fharmacol. Chemother. 1966, 26, 461-467. (3) Schacter, D.; Manis. J. G. J. Clin. Invest. 1958, 3 7 , 800-807. (4) Chirigos, M. A.; Udenfriend, S.J. Lab. Clin. Med. 1959, 54, 769-772. (5) Umberger, C. J.; Fiorese, F. F. Clin. Chem. (Wlnston-Salem, N.C.) 1963, 9. 91-97. (6) Rowland. M.; Riegelrnan, S.J. fharm. Sci. 1967, 56. 717-720. (7) Vo-Dinn, T.; Garnmage. R. B.; Hawthorne, A. R.; Thorgate. J. H. Environ. Sci. Techno/. 1976, 72, 1297-1302. (8) Lloyd. J. B. F.: Evett. 1. W. Anal. Chem. 1977, 4 9 , 1711-1715. (9) Lloyd, J. B. F. Analyst (London) 1975, 100, 82-95. (10) Vo-Dinh, T. Anal. Chem. 1978, 5 0 , 396-401. (11) Vo-Dinh. T.; Garnmage. R. B. Anal. Chem. 1978, 5 0 , 2054-2058. (12) John. P.; Soutar, I . Anal. Chem. 1976, 48, 520-524. (13) Rubiq, S . ; G6mez-Hens, A.: ValcBrcel. M. Talanta 1986, 3 3 , 633-640. (14) Garcia SBnchez, F.; Cruces. C.; Ramos Rubio. A. L. J. Mol. Struct. 1986, 143, 473-476. (15) Mufioz de la Peira, A.; Murillo. J. A,; Vega, J. M.; Baringo. F. Comput. ch8m. 1986, 12, 213-217. (16) Savitzky, A,; Golay. M. J. E. Anal. Chem. 1964, 3 6 , 1627-1639. (17) Steinier. J.; Termonia, Y.; Deltour. J. Anal. Chem. 1972. 4 4 , 1906-1909. (18) Thornmes, G. A.; Leininger, E. Anal. Chem. 1958, 3 0 , 1361-1363. (19) Putney, J. W.. Jr.; Borzelleca, J. F. Arch. Int. Pharmamdyn. Ther. 1970, 188. 119-126. (20) Weiner, E. R. Anal. Chem. 1978, 5 0 , 1583-1585. (21) Truin, E. B.. Jr.; Morgan, A. M.; Little, J. M. J. A m . Pharm. Assoc.. Sci. Ed. 1955, 4 4 . 142-148. (22) Bekersky, I.; Boxenbaurn. H. G.; Witson. M. H.: Puglisi, R. P.; Stanley, A. K. Anal. Lett. 1979, 51. 539-550.
RECEIVED for review May 3, 1988. Accepted August 5 , 1988.
