360
Anal. Chem. 1984. 56,360-363
resins indicated that the complex was positively charged. Application. Determination of Platinum in Synthetic Solutions Approximating the Composition of Pt-Ir, Pt-W, and Pt-Ni Alloys, Pt-Rh Thermocouple Alloy, and Nevjanskite Mineral. As analyzed samples of platinumiridium (1&40% iridium), platinum-tungsten (4% tungsten), and platinum-nickel (40-55% nickel) alloys, platinum-rhodium (lC-20% rhodium) thermocouple alloy, and nevjanskite mineral (8-12% platinum, 6245% iridium, 13-14% osmium, 2-3% rhodium, and 1% ruthenium) were not available, synthetic mixtures containing the same compositions were prepared. The platinum content in these mixtures was determined by following the recommended procedure. The results are presented in Table 11.
ACKNOWLEDGMENT We thank May and Baker, Ltd., Dagenham, England, for supplying pure MPM as a gift sample. Registry No. MPM, 3403-42-7;platinum, 7440-06-4; copper, 7440-50-8.
(6) Howell, J. H.; Hargis, L. G. Anal. Chem. 1980, 52. 306R. (7) Howell, J. H.; Hargls, L. G. Anal. Chem. 1982, 54, 171R. (8) Radushev, A. V.; Akkermann, G. Zavod. Lab. 1977, 43, 518-546. (9) Beaupre, P. W.; Holland, W. J.; Notenboom, H. R . Mikrochlm. Acta 1979, 1 (3-4), 303-310. (10) Beaupre, P. W.; Holland, W. J. Mlkrochlm. Acta 1983, 1 , 203-206. (11) Uttarwar, R. M.; Joshi, A. P. 2.Anal. Chem. 1977, 287, 317. (12) Bhaskare, C. K.; Pawashe, R . G. Analyst (London) 1981, 106, 1005-1009. (13) Bag, S. P.; Chakrabarti, S. K. Talanta 1977, 24, 128-129. (14) Guzman, C. M.: Perez, B. D.; Pino, P. F. An. Qulm. 1974, 7 0 , 828-832. (15) Gangopadhyay, P. K.; Das, H. R.; Shome, S. C. Anal. Chim. Acta 1973, 66, 460-463. (18) Ivanov, V. M.; Gorbunova, G. N. Zh. Anal. Khlm. 1980, 35, 3363-3368. (17) Gawali, S . B.; Shinde, V. M. 2.Anal. Chem. 1978, 280, 29. (18) Sanke Gowda, H.; Padmaji, K. A. Mlcrochem. J. 1980, 2 5 , 396-402. (19) Golla, E. D.; Ayres, G. H. Talanta 1973, 2 0 , 199-220. (20) Gangopadyhyay, S . ; Gangopadhyay, P. K.; Shome, S. C. 2. Anal. Chem. 1978, 281, 143. (21) Kamil, F.; Slndhwanl, S. K.; Singh, R. P. Ann. Chim. (Rome) 1980, 7 0 , 241-256. (22) Irvlng, H.; Plerce, T. B. J. Chem. SOC. 1959, 2565-2574. (23) Job, P. C . R . Hebd. Seances Acad. Scl. 1925, 180, 928-930. (24) Yoe, J. H.; Jones, A. L. Ind. Eng. Chem. Anal. Ed. 1944, 111-115. (25) Harvey, A. E.; Mannlng, D. L. J. Am. Chem. SOC. 1950, 72, 4488-4493.
LITERATURE CITED (1) Beamish, F. E.; Van Loon, J. C. "Recent Advances in the Analytical Chemistry of the Noble Metals"; Pergamon Press: Oxford, 1972; p 375. (2) Boltz, D. F.; Mellon, M. G. Anal. Chem. 1972, 44, 300R. (3) B o k , D. F.; Mellon, M. G. Anal. Chem. 1974, 46, 227R. (4) B o k , D. F.; Mellon, M. G. Anal. Chem. 1978, 48, 216R. (5) Howell, J. H.; Hargis, L. G. Anal. Chem. 1978, 5 0 , 243R.
RECEIVED for review July 6, 1983. Accepted December 12, 1983. A.T.G. thanks the University Grants Commission, New Delhi, University of Mysore, Mysore, and D.V.S. College of Arts and Science, Shimoga, for the award of a Teacher Fellowship.
Chelation and Spectrophotometric Determination of Efrotomycin with Aluminum Ion Louis Kaplan, David W. Fink,* and Halsey C. Fink Merck Sharp & Dohme Research Laboratories, P.O. Box 2000,Rahway, New Jersey 07065 The complexation of alumlnum Ion by the ligand efrotomycln Is characterlred for analytlcal appllcatlon to spectrophotometric measurement of thls antlblotlc based upon the red shlfl of its absorptlon spectrum whlch accompanles chelatlon. thls analyte can functlon as a bldentate llgand by coordlnatlng to the metal Ion vla two oxygen donors In analogy to acetylacetone. A study and dlscusslon of several factors which affect the analytlcal reactlon and the spectral propertles of the chromophore are Included. Chelation conditions of tlme, temperature, and reagent concentratlon are Illustrated to furnish a Beer's law callbration wlth a precldon of 300 nm) remain useful for many biological and pharmaceutical applications because the aromatic and heteroaromatic extraneous interferences in these samples typically exhibit maximum absorbance in the wavelength range 240-280 nm. Because efrotomycin (I) contains two tautomeric P-dicarbonyl units, this molecule can function as a bidentate ligand by coordinating to a metal ion via two oxygen donors in
0 1984 American Chemical Society 0003-2700/84/0356-0380$01.50/0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
analogy to acetylacetone. In previous studies, the T* T phosphorescence of the acetylacetonate of A13+was reported by Crosby et al. (15);Ohnesorge and co-workers have studied the fluorescence properties of the aluminum complexes of 8-quinolinol and of its 2-methyl derivative (16-20); and White has reported several other fluorescent A13+ chelates (21), including, for example, the ligand 2-hydroxy-3-naphthoic acid which was studied by Kirkbright, West, and Woodward (22). The stability constant of the A13+chelate of the salicyclic acid prototype of this fluorescent naphthalene chelate is log K = 14.1 (23). More recently, Saari and Seitz have reported the use of immobilized morin (3,5,7,2',4/-pentahydroxyflavone) as a fluorometric reagent based upon its chelation with A13+ through oxygen donors (24). This report describes the characterization of the chelation of aluminum with efrotomycin for analytical applications. This reaction is presently used in these laboratories in support of strain improvement research. A study and discussion of several factors which affect the analytical reaction are included. These measure: (i) the effects of reagent concentration, reaction time, and temperature on the chelation, and (ii) the stability and spectral properties of the chelate. These variables are studied to describe the analytical reaction and to establish representative conditions for spectrophotometric analysis. +
EXPERIMENTAL SECTION Apparatus and Reagents. Absorption spectra were recorded on a Cary Model 15 spectrophotometer using 1.00-cm quartz cells, and reactions were run in a constant temperature water bath (Fisher Model 80). Aluminum potassium sulfate was obtained from Fisher Scientific and methyl alcohol (spectroquality) from J. T. Baker. Efrotomycin (Merck Sharp & Dohme Research Laboratories) was stored in the freezer. Anal. Calcd: C, 59.78; H, 7.57; N, 2.36. Found: C, 59.69, H, 7.78; N, 2.24. Preparation of Reagent Solution. Reagent AlK(S04)2.12H20 (1.00 g) was dissolved in 100 mL of distilled water to provide a M. A 5.00-mL aliquot of stock solution of [A13+] = 2.11 X this aqueous stock solution was diluted to 100 mL in methyl M alcohol to produce a working reagent solution of 1.06 X A13+ in the mixed solvent 5% (v/v) water in methyl alcohol. Analytical Procedure. The analytical solution was prepared to contain efrotomycin in methanolic solution over the concento 5 X M. A 10.00-mL aliquot of this tration range 4 X analytical solution was transferred to a 50-mL volumetric flask, and 4.00 mL of the A13+ reagent solution was added. The analytical mixture was then diluted to volume with methyl alcohol, mixed well, and heated at 37 " C in a water bath for 15 min. Absorbance at 380 nm was measured against methyl alcohol in the reference cell and the concentration of the drug determined from a previously prepared calibration line. RESULTS AND DISCUSSION Spectral Properties of the Ligand a n d Chelate. Figure 1presents the absorption spectrum of efrotomycin in methanolic solution. This drug exhibits a A, at 320 nm ( E = 3.33 x lo4 L/(mol cm)) with a shoulder at 285-290 nm. The structure of the chromophore of this family of antibiotics was elucidated by Maehr et al. (9); this near-UV absorption transition reflects the extended delocalization of the heteroaromatic pyridone ring augmented by conjugation with the seven carbons of the trienone system. Indeed, even the biological mode of action of this drug depends on this moiety (5, 25)-for example, Eccleston (5) has shown that spectral perturbations of this long-A absorption band accompany protein binding, showing that the pyridone-trienone chromophore is intimately involved in the protein binding of these antibiotics. This band appears at,,A 333 nm in acidic (pH < 5 ) aqueous solution and blue shifts to ,A, 325 nm a t pH >7. A plot of absorbance at 325 nm as a function of pH gives a single inflection at pK, = 6, corresponding to dissociation of the hydroxyl group on the pyridone ring. This assignment
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x,nm Figure 1. Time course of the complexation of efrotomycin with AI3+ M) in at room temperature. Spectrum A is efrotomycin (2.53X methanolic solution In the absence of AI3+. Succeeding spectra show the spectral changes following the addition of 8.48 X M AI3+. Spectra were recorded after time Intervals of 1, 5, 10,15, 20,25,30, 45, and 60 min.
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is in accord with the measured pK, of goldinomycin, 6.1 (7, 8). The direction of this spectral shift, however, is atypical among most phenols, which exhibit red shifts with formation of conjugate anionic bases. In efrotomycin, this dissociation of the pyridol proton precludes formation of the stable planar six-membered bidentate hydrogen-bonded chelate ring, which increases rigidity and coplanarity relative to the anion to enhance further the extension of conjugation and its corresponding atypical red shift of the spectrum with protonation. Upon the addition of A13+to methanolic solutions, this band red shifts (Figure 1) to A,, 328 nm and a new low-energy transition appears a t 375-385 nm of increasing absorbance with time. During the experiment described in Figure 1,the adsorbance of this low energy shoulder increased monotonically for 1 h at room temperature, a t which time the absorbance difference at 380 nm between the reaction product and the drug provided a sensitivity o f t = 8.10 x lo3L/(mol cm) efrotomycin. This reaction is characterized by isosbestic points a t 350 and 283 nm, suggesting an equilibrium between two species, and is also accompanied by the appearance of a higher energy absorption band a t A,, 275 nm. It is the former Al-Efrotomycin absorbance at 375-385 nm which is used for quantitative measurement. Rate of Formation a n d Stability of t h e Chromophore. The chelation of aluminum with efrotomycin proceeds at a rate which is directly proportional to the concentration of the analyte. This is illustrated in Figure 2, which shows the effect of reaction time on chelation a t two efrotomycin concentrations near the extremes of the range of the analytical calibration. At the lower ligand concentration (Figure 2A), absorbance increases during 15-20 min of reaction time, is nearly
362
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984 M I L Mole Ratio
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Flgure 3. Effect of reaction temperature on formation of the chelate. [Efrotomycin] = 5.06 X M, [Ai3'] = 8.48 X lo-' M; [L]/[M] = 0.60. Reaction time was 15 min. Analytical reactlon was run as
described in the analytical procedure.
constant for 20-40 min, and slowly decreases at longer times. With increased efrotomycin concentration (Figure 2B), absorbance concomitantly increases more rapidly, describing a time profile which furnishes an analytical sample suitable for measurement after 10 rnin of reaction. For purposes of the present analytical method, 15 min was seleded as the reaction time in a compromise between precision at the lower concentration range and overall analysis time. At the lower concentration in Figure 2, the range of absorbance values of the nine measurements collected for reaction times between 20 and 50 minutes was 0.03 absorbance units; hence, it is recognized that an even more rugged analytical method for this concentration could be designed by using longer reaction times. Nevertheless, the suitability of the 15-min reaction for application to the concentration range studied here is documented by the precision data below. The slow decrease in absorbance with prolonged heating could result from degradation of the drug in solution. Several possible modes of degradation of these antibiotics have been documented. These include acidic hydrolysis of the amide located between the diene and a tertiary carbon (5, IO), cleavage of the triene from the carbonyl in alkaline solution (26),and oxidative opening of the 3,4-dihydroxytetrahydrofuran ring (IO). In their study of the liquid chromatography of the analogous acetylacetonate complexes, Huber, Kraak, and Veening (27) demonstrated the slow decomposition of the aluminum chelate resulting from dissociation or hydrolysis in solution and producing three different chromatographically resolved species. However, if a similar process were extant in solutions of the present efrotomycin-aluminum chelate, these data show that the resulting mixture of species does not markedly alter the absorption spectrum or significantly affect absorbance at the analytical wavelength during the analysis. Figure 2 includes a point measured after 21 h of heating the reaction mixture a t 37 "C; even after this rigorous treatment, the decrease in absorbance from the maximum (obtained after 20-50 min of heating) is only 26% of the analytical signal. In addition, one analytical solution containing 5.06 X 10" M analyte which was held for 61/2 h after formation of the chromophore exhibited a linear decrease in absorbance at a rate of -0.006 absorbance units/h. Hence, the chromophore is sufficiently stable for measurement during the typical time frame of conventional spectrophotometric analysis; analytical solutions near the middle of the calibration range which are held for 20 min in advance of measurement will suffer a decrease of 2 / 1 in Figure 4 was 0.007 absorbance units. The data in this figure cannot be used to assign unequivocally the stoichiometry of the chelate, although the formation of either a mono or bis ligand complex is indicated by these results. That the interaction of the metal ion with the antibiotic affects the long-X absorption spectrum indicates that the A13+ion effects a change in the electronic distribution of the pyridone-trienone chromophore. The structure of this chromophore can then be represented for the 1to 1 ligandto-metal mole ratio chelate by either of the partial structure rotamers I1 or I11 or by an equilibrium between these.
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Linearity and Precision. To demonstrate the reproducibility of this analytical reaction, a calibration line was prepared from 16 points representing duplicate samples at each of eight analyte concentrations over the range 1.01 X loT5 M to 8.10 X lo4 M efrotomycin in the final analytical solution. This concentration range reflects the formation of the 1:lL/M mole ratio species by covering the ratios L/M 5 0.96:l. The linear least-squaresfit of this line is Am = (9.70X 103)[drug] + 0.022 with a correlation coefficient r = 0.999,which confirms that the reaction obeys Beer's law over this concentration range. The linear range of the Ringbom plot is 2.0 X M to 7.5X M efrotomycin. The residuals of the data points from the calibration line averaged 3.4% relative to the line. The slope of this line furnishes a sensitivity with 95% confidence limits of t = 9.44 X lo3 L/(mol cm) to 9.97 X lo3
Anal. Chem. 1984, 56,363-368
L/(mol cm). The use of duplicates a t each analyte concentration provides a convenient statistical measure of the precision of this analytical reaction, The mean difference in absorbance between the duplicates was 0.012 absorbance units. Nine replicate solutions containing 5 X lo-' M efrotomycin-near the middle of the analytical calibration line-yielded a range of 0.013 absorbance units and a reproducibility of the analytical reaction of
