Rapid Fluorimetric Determination of Phenothiazines Employing in situ Photochemical Oxidation V. I?.White' and Christopher S. Frings Medical Laboratory Associates, A Damon laboratory, 1025 South 78th Street, Birmingham, Ala. 35205
J. E. Villafranca and J. M. Fitzgerald* Department of Chemistry, University of Houston, Houston, Texas 77004
Exposure of five phenothiazine drugs (perphenazine, chlorpromazine, fluphenazine, trifluoperazine, and thioridazine) to an intense ultraviolet source results in simultaneous photooxidation of the drugs and fluorescence excitation of the products. This photochemical-fluorimetric method is unique because no addltlon of diluting reagents is needed; lowered detection limits result in convenient determinations between 10 and 1000 ppb of perphenazine, chlorpromazine, fluphenazine, and trifluoperazine. Correlation coefficients for linear regression of either digitally integrated intensities or analog fluorescence peak intensities vs. initial drug concentrations are all greater than 0.9990. Thioridazine yields nonlinear callbratlon curves, but a satisfactory (correl. coeff. of 0.9996) linear approximation can be used between 10 and 200 ppb. Optimum values of pH, emission wavelength, and photolysis time are reported for each drug.
Phenothiazine derivatives are among t h e most widely used drugs for treatment of psychiatric patients ( I ) . T h e dosage required for each patient depends on variables including t h e particular phenothiazine chosen and severity of t h e disease involved. Analytical methods for phenothiazines have been reviewed (2);many methods d o not possess sufficient limits of detection t o measure therapeutic concentrations in blood a n d urine (3-7). Methods based on t h e fluorescence of both the unoxidized or oxidized compounds have shown the most promise (8-14). A recently reported phosphorimetric method has detection limits of about 30 p p b for 14 different phenothiazines, but requires liquid nitrogen a n d instrumentation with ms response times ( 1 5 ) . Previously, oxidization of phenothiazines t o their more fluorescent products has been carried out with chemical reagents (8-13), which necessarily dilute t h e sample, and thus increases the limit of detection. Chemical preoxidation is also time consuming (ca. 20 min). Photochemical decomposition of the sample may be regarded as a hindrance in fluorimetric analysis, but recently, both digital and analog readout methods have been applied so as to overcome photochemical complications ( 16-18). Linear calibration curves, measuring either native fluorescence (16) or fluorescent photoproducts (17, 1 8 ) can be obtained over several orders of magnitude at p p b concentrations. In t h e case of phenothiazines, in situ photochemical oxidation and simultaneous fluorimetry appeared promising for a rapid, sensitive, a n d convenient quantitation.
EXPERIMENTAL Apparatus. The apparatus used has been previously described in detail (16).The photolysis-fluorescence source was a Hanovia Model
Present address, National Health Laboratories Inc., 3730 Dacoma, Houston, Texas 77092. 1314
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
901Bll200 W Xe-Hg arc, filtered by a Corning 7-54 broad-band uv filter. All the drugs are known to absorb within the wavelengths transmitted by this filter (13). Better precision was obtained when the contents of the 25-ml cell were agitated, so stirring was used for all samples (16).The integrator time interval control (16)was modified to provide integration periods of 0.5,1, and 2 min. The PMT was operated at 750 V (highest voltage consistent with low noise) and the PMT turret slits were set at maximum (5 mm). Reagents. The drugs studied were all the highest purity available from the following sources: chlorpromazine and trifluoperazine, Smith, Kline & French; thioridazine, Sandoz; fluphenazine, National Formulary; perphenazine, Schering. The structures of these drugs are available (13,15),as are the structures of the oxidation products (19). Stock solutions (5 mg/250 ml) were prepared in distilled water, with the exception of perphenazine, which was dissolved in 12.5 ml of ethanol and then diluted to volume with distilled water. The photolysis-fluorescence behavior of perphenazine samples prepared from this stock solution were identical for subsequent dilutions with either 5% (v/v) ethanol or distilled water. Therefore, all drug samples were prepared by dilution with water and adjusted to pH 1 (HCl), pH 10 (as. "3) or run at natural pH (5.5). Procedures. The conditions which yielded the largest fluorescence signal above background ( 1 6 ) were established as follows: Samples of ca. 1 ppm were prepared at each of the three pH's, and irradiated. The fluorescence emission was monitored at previously published wavelengths (13) until an intensity plateau was reached (3-5 min). Then the wavelength of the emission monochromator was varied as previously described (16) to locate that combination of pH and wavelength which gave the maximum difference between signal and background. The integration time was then selected by recording the emission from a fresh sample at the selected pH and wavelength settings. At their respective best settings, the emission from chlorpromazine and perphenazine reached a maximum after 45 s of irradiation, and then decayed slowly; a 1-min integration time was selected to obtain the digital signal. The emissions from fluphenazine, trifluoperazine and thioridazine reached a stable value after 2 min of irradiation; for convenience, a 2-min integration interval was selected. Monochromator slits were set at either 2.25 or 2.75 mm in order to satisfy two criteria: detection limit of 10 ppb, and integrated background blank smaller than 25 V. The conditions selected for establishing calibration curves are summarized in Table I. Data for analytical working curves were collected with both digital and analog readouts (16-18). The analog signal magnitude was measured by taking the difference, in chart divisions, between a blank solution and the maximum fluorescence signal recorded from a given sample. Digital data were obtained as described previously (16).Electronic blank subtract was not used so that both digital and analog data could be obtained simultaneously with the same sample. Both the analog peak intensity and the digital integral of fluorescence ingrowth were then subjected to linear least squares treatment vs. the initial phenothiazine concentration taken. A summary of statistical parameters for the calculated calibration lines is given in Table I.
RESULTS AND DISCUSSION As can be seen from data summarized in Table I, t h e photochemical-fluorimetric method described here is capable of determination of five of the most commonly prescribed phenothiazines down t o 10 ppb. In situ photooxidation is particularly advantageous because the fluorophor is formed without sample dilution. The limits-of-detection obtained here are less
Table I. Analytical Conditions a n d Statistical Summary of Results for Digital a n d Analog Measurement of Phenothiazines Summary of results Conditionsn Concn Concn InterResol.f, Run No., Slope,e A, nm (slit, Time, range, cepte Mode sensitivity mm) b min Compound PPb PPb PH Perphenazine
5.5
385 (2.75)
2.0 +1.og
I-D I-A 11-D 11-A
Corr. Coeff.e
45-900 45-900 20-900 20-900
22.643 4.004 20.979 3.452
25.432 59.045 22.203 18.852
6.2 13.7 6.0 9.3
0.9998 0.9990 0.9998 0.9996
10
469 (2.75)
1.0
111-D 111-A IV-D IV-A
10-1000 10-1000 10-1000 10-1000
64.018 14.015 65.594 14.378
1.781 -66.613 2.603 -76.862
4.1 2.5 5.2 3.9
0.9999 0.9999 0.9999 0.9999
Chlorpromazine
10
469 (2.75)
1.0
I-D I-A 11-D 11-A
10-1000 20-1000 10-1000 10-1000
60.012 12.864 54.773 11.672
3.437 227.81 3.177 110.97
10.7 17.2 7.9 13.3
0.9994 0.9988 0.9998 0.9993
Fluphenazine
5.5
402 (2.25)
2.0
I-D I-A 11-D 11-A 111-D 111-A IV-D IV-A
10-800 10-800 10-800 10-800 18-630 18-630 10-800 10-800
76.999 7.450 67.364 6.992 70.471 7.312 74.378 7.537
35.383 49.984 29.236 3.704 25.394 2.241 27.670 72.156
7.2 4.3 4.9 2.5 3.4 2.6 6.1 6.7
0.9997 0.9999 0.9998 0.9999 0.9998 0.9999 0.9998 0.9997
Trifluoperazine
5.5
408 (2.25)
2.0
I-D I-A 11-D 11-A 111-D 111-A IV-D IV-A
10-770 10-770 10-800 10-800 10-800 10-800 10-220 10-220
98.629
20.645 7.367 20.078 16.321 21.441 7.562 19.890 32.385
4.9 11.3 8.2 9.4 7.o 6.9 2.8 2.6
0.9998 0.9991 0.9996 0.9994 0.9996 0.9997 0.9993 0.9993
90.881 9.389 87.214 9.116 87.432 8.828
0.9972 85.998 13.801 7.4 0.9989 52.304 22.724 6.8 0.9996 12.141 82.377 1.9 0.9941 24.1 19.023 52.726 Combination of solution and instrument settings which yield most intense signal above background. Wavelength and slitwidth of emission monochromator. Time signal integrated, or time to analog peak signal. On separate days with all equipment shut down between runs. Mode refers to digital integration (D) or analog recorder peak (A) readout; both modes used simultaneously for each run. e Linear regression fit of data. Digital: Integral = slope (mV/ppb) X concn (ppb) intercept (V). Analog: Recorder peak signal = slope (wV/ppb) X concn (ppb) intercept (pV). Correlation coefficient also tabulated to indicate linearity. f Concentration resolution (discussed in Ref. 16) is a measure of precision. Values tabulated calculated by dividing the standard error of the estimate (from least-square data reduction, see Ref. 20) by the slope of the calibration line. Note that concentration resolution can be used to compare precision of different runs, regardless of readout mode. g For perphenazine monitored a t 385 nm. Two-min prephotolysis followed by 1-min integration (See text). Thioridazine calibration nonlinear. Note that correlation coefficient for 10-200 ppb indicates good linear approximation. Thioridazine
5.5
421 (2.75)
2.0
I-D I-D 11-D 11-Dh
t 9.787
+
t h a n the lowest reported concentrations for urine (21) and serum (8) phenothiazine levels; application of this method to biological specimens would appear promising. Furthermore, the instrumentation required for the method described here is relatively simple and widely available. T h e precision a n d limits-of-detection listed in Table I for all five drugs are virtually identical when either analog or digital readout is used. This is in contrast to earlier work with naturally fluorescent samples (16) where digital integration was found t o yield lower limits-of-detection and better precision. Fluorescence ingrowth occurs over a 30- t o 60-s time span while decay of native fluorescence begins immediately. Thus, the different analog detection limits obtained in the two different cases are due t o t h e different demands placed on recorder pen response. Data in Table I also show t h a t the fluorescence characteristics for a particular drug depend largely on the nature of the substituent at the 2 position of the phenothiazine ring; this behavior has been previously observed (9).T h e substituent
10-270 270-630 10-200 200-800
+
effects observed for the drugs studied here may be divided into three distinct cases. In the first case, the emission wavelength maxima measured for photooxidation products of t h e trifluoromethyl substituted drugs (fluphenazine and trifluoperazine) agree with published values (13). In t h e second case, photoproduct emission from t h e 2-chloro substituted drugs (perphenazine and chlorpromazine) can be measured a t 385 n m and p H 5.5or a t 469 n m and p H 10. The 385-nm emission is in agreement with previous studies (13), while t h e 469-nm emission has not been reported previously; apparently, photochemical oxidation yields different products from t h e 2chloro substituted drugs than does peroxide (13)or permanganate (9)oxidation. In an attempt to lower the detection limit a t 385 nm, a 2-min prephotolysis was used, followed by a I-min integration of the product fluorescence. However, it was found that a simple 1-min integration of initial fluorescence ingrowth a t 469 n m yielded the required detection limits (compare lines 1-8 of Table I). In the third case, the 2-thiomethyl substituted drug (thioridazine) yielded nonlinear calibration curves a t all ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
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combinations of p H and wavelength. With our apparatus, the most intense photoproduct emission from this drug was obtained a t 421 n m and p H 5.5. Data in Table I indicate t h a t a satisfactory linear approximation can be used for measuring concentrations between 10 and 200 ppb. The photolysis of sulfur-containing compounds is known to be complicated (22) and nonlinear calibration curves have been previously reported for thioridazine (1I ) . This new photochemical-fluorimetric method for phenothiazines represents a novel approach, in which practical application of photochemistry yields rapid and sensitive determinations.
LITERATURE CITED L. S. Goodman and A. Gilman, "The Pharmacologic Basis of Therapeutics," 4th ed., The Macmillan Company, London, 1970,pp 155-168. G. Cimbura, J. Chromatogr.Sci., 10,287 (1972). F. M. Forrest and I. S. Forrest, Clin. Chem. ( Winston-Salem,N.C.), 6, 1 1 ,
362 (1960). W. J. Turner, P. A. Turano, and J.E. March, Clin. Chem. ( Winston-Salem. N.C.), 16, 916 (1970). D. H.Efron, S. R. Harris, A. A. Manian, and L. E. Gaudette, Psychopharmacologia, 19, 207 (1971). J. E. Wallace and J. D. Biggs, J. Pharm. Sci., 60, 1346 (1971). G. W. Christoph, D. E. Schmidt, J. M. Davis, and D.S. Janowsky, Clin. Chim. Acta, 38, 265 (1972). S. L. Tompsett, Acta Pharmacol. Toxicol., 26, 298,303 (1968).
(9)T. J. Mellinger and C. E. Keeler, Anal. Chem., 35, 555 (1963). (IO)T. J. Mellinger and C. E. Keeler, Anal. Chem., 36, 1840 (1964). (1 I) J. B. Ragland, V. J. Kinross-Wright, and R. S. Ragland, Anal. Biochem., 12, 60 (1965). (12)D. H. Manier, J. Sekerke, J. V. Dingell, and M. K. El-Yousef, Clin. Chim.Acta, 57, 225 (1974). (13)J. B.Ragland and V. J. Kinross-Wright, Anal. Cbem., 36, 1356 (1964). (14)R. G. Muusze and J. F. K. Huber, J. Chromatogr.Sci., 12,779 (1974). (15)L. A. Gifford, J. N. Miller, D. L. Phillips, D. T. Burns, and J. W. Bridges, Anal. Chem., 47, 1699 (1975). (16)R. J. Lukasiewicz and J. M. Fitzgerald, Anal. Chem., 45, 51 1 (1973). (17)R. J. Lukasiewicz and J. M. Fitzgerald, Appl. Specfrosc., 28, 151 (1974). (18)J. J. Aaron, J. E. Villafranca, V. R. White, and J. M. Fitzgerald, Appl. Spectrosc., 30, 159 (1976). (19)H.Basinska and K. Nowakowski, Acta Polon. Pharm., 29, 464 (1972). (20)N. M. Bownie and R. W. Heath, "Basic Statistical Methods," 3rd ed. Harper & Row, New York, N.Y., 1970,pp 136-139. (21)A. P. DeLeenheer, J. Pharm. Sci., 63, 389 (1974). (22)J. G. Calvert and J. N. Pitts, "Photochemistry", John Wiley, New York, N.Y., 1967,pp 488-491.
RECEIVEDfor review February 20,1976. Accepted April 30, 1976. Financial support for the segment of this work carried out a t the University of Houston was provided by Grant Number E-384 from The Robert A. Welch Foundation. VRW acknowledges the support of Damon Corporation and Medical Laboratory Associates, in the form of a Postdoctoral Traineeship in Clinical Chemistry.
Determination of Elemental Area Concentration in Ultrathin Specimens by X-ray Microanalysis and Atomic Absorption Spectrophotometry J. A. Chandler* and M. S. Morton Tenovus Institute for Cancer Research, Welsh National School of Medicine, Heath, Cardiff, CF4 4XX, Wales
X-ray microanalysis and atomic absorption spectrophotometry of thin evaporated metal fllms allow the determination of superficial elemental density. The limits of detection are in the order IO-'' g/pm2 with an accuracy of 5 % and a similar degree of precision. Evaporated films on filter paper discs were measured by atomic absorption spectrophotometry using a carbon rod atomizer while the same films were analyzed in the electron microscope microanalyzer (EMMA) by x-ray techniques. The method is of use for the determination of area elemental density and of absolute thickness of thin films.
areas of film, and the destruction of the film during measurement. This paper describes a method of using a combination of atomic absorption spectroscopy and x-ray microanalysis in the electron microscope for the measurement of superficial elemental concentrations. The technique has been developed primarily for the quantitation of elemental concentrations found in ultrathin specimens and is also of use in the determination of specimen thickness.
EXPERIMENTAL X-ray microanalysis of ultrathin specimens in the electron microscope provides relative elemental concentrations when using the method of Hall ( I ) t o compare characteristic and continuous radiation. I t is often necessary t o determine the area (superficial) density of an element in such specimens, especially in subcellular regions of biological thin sections or in areas of ultrathin metallurgical and mineralogical specimens. In nuclear and atomic physics, there sometimes arises the need to measure the thickness of thin metal films such as those used for the scattering of charged particles, or for the measurement of energy loss. A number of methods have been employed to measure film thickness: piezoelectric quartz crystal monitoring (2), cy particle absorption ( 3 ) ,multiple beam interferometry ( 4 ) .T h e main disadvantages of these methods are the need to use large 1316
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
Materials and Methods. A number of carbon-coated electron microscope grids were mounted on 9-cm diameter filter papers (Whatman No. 42,g-cm diameter) and placed in turn in a vacuum coating unit (AEI Vac 12) with a filament-specimen distance of 12 cm. They were coated with various thicknesses of aluminum and iron from lengths of metal wire that had been previously weighed. The coated areas of the filter papers were analyzed for total aluminum and iron using a Varian Techtron AA 1200 atomic absorption spectrophotometer fitted with a standard air-acetylene burner (for iron) or a Model 63 Carbon Rod Atomizer (for aluminum). The instrument was linked to a Smith's Servoscribe IS chart recorder. Working standards for atomic absorption were prepared from commercially available bulk standards (1000 p.p.m. B.D.H.) diluted with de-ionized water. The whole filter papers were first dissolved by refluxing in a mixture of concentrated " 0 3 (Aristar, 5 ml) and concentrated HzS04 (Analar, 2 ml) for 20 min. On cooling, each solution was made up to volume (50 ml) with de-ionized water. Several blank experiments were also conducted.
