claimed to be accurate to 0.5y0over the similar complete range of concentration 0.5 to 100%. But this method also requires more time, accurate pressure observations, troublesome manipulation of toxic mercury, temperature stimulation of the reaction, and care in interpreting the end point as well as the
amount of fluorine adsorbed on the product. The direct optical method is therefore the method of choice.
( 2 ) Busch, G. W., Carter, K. C., Mc-
Kenna, F. E., National Suclear Energy Series Div. VIII, Vol. 1, Anal. Chem. of Manhattan Project, McGraw-Hill, 1950. ( 3 )-Simons, J. H., “Fluorine Chemistry,” Vol. 11, p. 56, Academic Press, New LITERATURE CITED York, 1954. ( 1 ) Bodenstein, M,, ~ ~ ~ H,, k ~2, ~ RECEIVED h , for review March 16, 1964. Anorg. Allgem. Chem. 231, 24 (1937). Accepted May 19, 1964.
Factors Influencing Spectrofluorometry of Phenothiazine Drugs THEODORE J. MELLINGER and CLYDE E. KEELER Department of Neuro-Psychopharmacology, Milledgeville State Hospital, Milledgeville, Ga.
b Phenothiazine drugs have a relatively low fluorescence that can b e improved only slightly by using an alkaline instead of an acid medium, or by cooling the solution to as low a temperature as possible. When the drugs are treated with potassium permanganate, their fluorescence reaches a much higher intensity. During this oxidation, the fluorescence intensity curve increases to a maximum, then decreases again with further treatment. At the appropriate pH, this permanganometric fluorescence titration permitted analysis of 0.002 to 0.02 Mg. of drug per ml. of aqueous solution. In biological materials, the lower limit for quantitative determination depends on the presence of fluorescent tissue substances and their degree of interference with the particular fluorescence spectrum of each drug.
I
N A PREVIOUS STUDY ( 8 ) , we described the fluorescence spectra of phenothiazine drugs as a means for their qualitative identification. The purpose of the present paper is to illustrate some of the factors that influence their quantitative analysis during fluorometry, with emphasis upon the procedure of fluorescence titration with potassium permanganate. I n the case of spectrophotometry of phenothiazine drugs in biological materials, the reader of the literature in this particular field will notice that relatively large amounts of these drugs are usually injected into laboratory animals parenterally to get the highest possible concentration into the tissues-a procedure which reflects the unsatisfactory sensitivity of these methods. From our experience with spectrophotometry of phenothiazine compounds in both ultraviolet and visible ranges, we found that spectrofluorometry is not only a more sensitive procedure for these agents but also l e v time consuming than spectro-
1840
ANALYTICAL CHEMISTRY
photometry. Techniques employing radioisotopes have a still higher sensitivity. However, they always require very special and laborious working conditions and are therefore not suitable for a fast routine procedure such as spectrofluorometry. EXPERIMENTAL
Apparatus. The excitation and fluorescence spectra of the compounds under study were obtained with a Bowman-Aminco spectrofluorometer and the absorption spectra with a Beckman DK-2 spectrophotometer. Reagents and Procedures. The 12 phenothiazine substances and one chemically related drug studied were chlorpromazine, perphenazine, prochlorperazine, pipamazine, thiopropazate, fluophenazine, trifluoperazine, trifluopromazine, promazine, mepazine, thioridazine, thiethylperazine, and chlorprothixene. They were used as pure crystalline substances and as commercially available samples from ampoules and tablets. The drugs were dissolved or diluted to various concentrations in triple-distilled water, which was adjusted to different pH’s with sulfuric acid or potassium hydroxide. The method for the fluorescence titration of the phenothiazine drugs is as follows. After measuring the excitation and fluorescence spectra of the unoxidized drug, 0.2-ml. aliquots of potassium permanganate solution were added to the original 2-ml. sample in the cuvette. Thereafter the increase of fluorescence was read with the wavelength set a t the peak of the highest excitation curve and at peak fluorescence of the oxidized drugs, the spectra of which have been described previously ( 8 ) . During successive additions of 0.2 ml. of the reagent a t %minute intervals, the stepwise increase of fluorescence intensity was read on the photomultiplier meter. This operation was continued until the fluorescence intensity reached its peak plateau after which it dropped again to lower levels (see Figure 1). The concentrations of the potassium permanganate
solutions necessary for the titration varied from 1 X 107 to 1 X lo3according to drug concentrations from 0.1 pg. per ml. to 20 Mg. per ml. The reagent was always dissolved in the same p H medium as were the drugs to be analyzed. Because in each particular assay the 2-ml. sample is usually diluted in a varying number of steps with 0.2 ml. of potassium permanganate solution until it reaches the peak plateau fluorescence, the dilution has to be corrected to the original 2-ml. sample as if reagent without wat’erhad been added. The amount of potassium permanganate is noncritical as long as there is no danger of overshooting the plateau on the first step. The height of fluorescence intensity, reached a t peak fluorescence, is proportional to the concentration of the drug in solution. There is no need to measure the reagent consumed during the titration. Xot only would this be laborious, but it might lead to erroneous results because of interference by other reducing agents, which are added to tablets and ampoules as stabilizers by the manufacturers, or by the many reducing substances present in animal tissue fluids. Various factors that influence fluorescence spectra of the phenothiazine compounds were investigated. T o s t u d y the effect of different concentrations upon the excitation and fluorescence curves, each drug was diluted to 1, 10, 100, and 500 pg. per ml. in O.OILVsulfuric acid and their fluorescence spectra were recorded before and during oxidation with potassium permanganate. The influence of the pH medium upon fluorescence was tested after the drugs were diluted in solutions of different pH values to concentrations of 2 pg. of drug per ml. of solution. Their fluorescence was measured before and during treatment with permanganate. The change in fluorescence intensity by differing temperature in both the untreated and oxidized drugs was measured after cooling the 2-pg. per ml. drug solutions in a deep-freeze cabinet to approximately -20’ C. I t was recorded a t rising temperatures, which were continuously checked by a thermistor. So that crystallization below 0” C . could be avoided, the solvent consisted of
equal parts Spectro-G.rade methanol and o.oll$r sulfuric acid. T o compare absorption curves with fluorescence spectra, samples of 10 pg. of drug per ml. of O . l h ' sulfuric acid were treated with potassium permanganate. When peak fluorescence was attained, their fluorescence and ultraviolet absorption curves were examined. I n addition, the absorption and fluorescence spectra of the following drug sulfoxides were recorded: chlorpromazine sulfoxide, trifluoperazine sulfoxide, thioridazine ring monosulfoxide, thioridazine disulfoxide, chlorprothixene sulfoxide and chlorprothixene disulfoxide. These were examined in the same medium and the same concentration. The spectra of propantheline, a structurally related compound, was included in this study. T o analyze the fluorescence of phenothiazine compounds in biological materials, three drugs-chlorpromazine, chlorprothixene, and thioridazine-were tested in three groups of three male rats, each of which weighed 250 grams. T h e animals, fasted overnight, were given 10 mg. of the drug by stomach tube. After 2 hours .they were anesthetized with ether. Following laparotomy and thoracotomy, blood was collected from the portal vein and the aorta. The serum was separated by centrifugation. Two-hundred-milligram organ samples from the animals were frozen and sliced to a thickness of 50 microns, then extracted 3 t.imes after being shaken each time for 10 minutes with 10 ml. of ether. The combined ether extractions were shaken 3 times with 10 ml. of an aqueous phase of p H 3, which was used for spectrofluorometry of chlorpromazine and thioridazine samples. The acid phase of the chlorprothixene organs, however, was first adjusted to p H 10 beciause of the higher fluorescence yield at, this p H medium. All serum and organ samples of each rat were analyzed separately. To determine the degree of tissue fluorescence in control animals, three rats, fasted overnight, were sacrificed and their organs treated as above. The fluorescence was recorded with the same wavelength settings on the spectrofluorometer as were used for the particular fluorescence spectrum of each drug. T o compare the efficiency of the above extraction procedure with radioisotope techniques, three rats of the same weight and sex as above were treated identically after receiving 10 mg. of chlorpromazine t,hat contained radioactive C14-chlorpromazine. Twenty-milligram organ samples and 0.8 ml. of 0.5yoaqueous methyl cellulose were ground in a mortar for 10 minutes. The radioactivity of the homogenized material, after being spread and dried on plsnchets, was counted in a Sharp automatic low background system. T o find out how much of the drug was extracted by the procedure used for fluorometry, another set of 20-mg. organ samples was first extracted, as were the organs for fluorometric analysis. They were homogenized and prepared foir counting of radioactivity as were the above unextracted samples. The ratio of radioactivity to
3000 2800 2600 2400
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2000 1800 1600
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1000
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600 400
PO0 0 TIME+
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Figure 1. Fluorescence titration manganate of chlorpromazine
with
potassium
per-
0.3 to 10 pg. of chlorpromazine per ml. of 0.01 N sulfuric acid. Excitation set, 340 m p ; fluorescence set, 3 8 0 mp. Average time of each step, 1 to 2 min. Left, uncorrected readings; right, corrected for dilution
drug concentrations was obtained for a reference curve by treating mixtures of control tissue, methyl cellulose, and varying concentrations of CI4-chlorpromazine identically as above. The drug concentration was determined fluorometrically before being added to the mixture with a microsyringe. The radioactivity was so adjusted that under identical conditions 1000 counts per minute per planchet corresponded to a drug concentration of 1 pg. per gram of organ. The C14-chlorpromazine organs were further analyzed by ascending paper chromatography to learn how much of the drug is present as unchanged compound or as metabolite. .A Whatman 3MM paperstrip of 6 X 40 cm. was used. Twenty-milligram organ samples were homogenized with 0.3 ml. of water in a mortar and applied in a streak across the paper strip 10 em. above the lower end. It was developed for 16 hours with a solvent system of equal parts of n-butanol, n-propanol, and water. The dried paper was cut crosswise into strips 0.5 cm. in width. These were put on the planchets for counting of the radioactivity of each strip. To get an idea of how many radioactive R , areas are extracted by the procedure used for the fluorometric determinations, 20-mg. organ samples of the rats treated with C14-chlorpromazine were extracted in
the same way as was done for the 200mg. nonradioactive organ samples for fluorometry of the three drugs, except that only of the organs and extraction fluids were used. The extracted tissue was ground with 0.3 ml. of water. The homogenized material was developed by the same chromatographic technique. Counting of the radioactivity was carrkd out' as above. I n addition, each of the 13 drugs listed a t the beginning of this section was tested upon a separate group of three guinea pigs. The compounds were administered intraperitoneally in a dosage of 25 mg. per kg. of body weight. Two hours later, the bile and intestinal contents of the sacrificed animals were collected. To avoid light scattering due to turbidity, the fluids were diluted 50 to 500 times in 0.01A' sulfuric acid before being examined for their fluorescence spectra. To study the possibility of separate examinat,ion of two drugs on the same animal three guinea pigs were treated in the same way, but received simultaneously 10 mg. of chlorpromazine and 10 ma. of chlorprothixene per kg. of body weight. All fluorescence curves, illustrat,ed or cited in the text constitute uncorrected fluorescence spectra. The term oxidation used in this paper refers exclusively to the employment of potassium permanganate as oxidant, VOL. 3 6 , NO. 9 , AUGUST 1 9 6 4
1841
I
I
1 1000
a
250
410
WAVELENGTH,
Figure 2.
rnp
Fluorescence excitation pattern of thioridazine
5 p g , of thioridozine per ml. of 0.01N sulfuric acid, Curve No. 1 , before oxidation with Ruorescence set 4 6 5 m p ; Nos. 2-7, after stepwise addition of 0.2 ml. of potassium permanganate solutions wth fluorescence set 430 my. Above, curves of recorder tracings in lower flgure corrected for dilution
1842
ANALYTICAL CHEMISTRY
RESULTS
Figure 2 shows the changes that occur in the fluorescence excitation curves of thioridazine before and after the stepwise addition of several aliquots of potassium permanganate. The lower part of the figure illustrates the intensity changes, as they are recorded with an X-Y direct writing recorder; the upper part illustrates the curves corrected for dilution. Each addition of a 0.2 ml. of potassium permanganate solution is followed by a n increase of fluorescence intensity until its maximum is reached. Figure 1 shows the changes of intensity readings of different chlorpromazine concentrations during fluorescence titration with potassium permanganate on a corrected and an uncorrected scde. At peak intensity which is proportional to the drug concentration, further oxidation again decreases the fluorescence, a phenomenon which reminds one of the curves of conductivity changes in phenothiazine drugs during treatment with other oxidants as reported by Dusinski (3-6) and Pungor (9). The fluorescence spectra of the unoxidized drugs can still be recorded in 0.2 to 0.4 pg. per ml. However, treatment with potassium permanganate permits both qualitative and quantitative analysis in concentrations as low as 0.001 and 0.02 ug. per ml. a t peak fluorescence. The spectra of t h e oxidized drugs are not only more distinctive with their four-wave crescendo patterns, but also more stable, whereas the untreated compounds in low concentrations, especially in acid medium, decompose rapidly in low concentrations, when exposed to the activating ultraviolet light. Figure 3 shows, with chlorpromazine and thioridazine as examples, how one peak of the excitation pattern becomes smaller in relation to the other when the concentrations of the drugs are altered. Both excitation peaks shift to higher wavelengths with higher concentrations. However, no shifts or only very slight shifts toward higher wavelengths occur in the fluorescence curve with increasing concentrations. These phenomena are less pronounced in the fluorescence of the oxidized phenothiazine drugs, but they become important again on potassium permanganate-treated chlorprothixine. 4 s can be seen in Figure 4, the latter drug shows changes in intensity but no wavelength shift in the fluorescence excitation waves. However, the shift appears in the fluorescence curves not only with higher concentrations, but especially with strong acid media. Another factor that influences the intensity of fluorescence is the temperature of the solution. Figure 5 shows the small change of fluorescence on chlorpromazine, which has a relatively low fluorescence, and the more pronounced change on its higher fluorescent sulf-
250
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350
400
450
500 WAVELENGTH, m r
Figure 3. Excitation and fluorescence patterns with different concentrations of chlorpromazine (left), and thioridazine (right), in 0.1N sulfuric acid Height of curves arbitrarily set for better comparison
oxide when the temperature is varied from -20’ to 60” C. The temperature curves of all phenothrazine drugs before and after oxidation showed that the higher the fluorescencie of the particular compound the larger the change of fluorescence intensity with differing temperatures. Increase of fluorescence intensity with decreasing temperature has been reported by others ( 7 ) . From the above, the advantage of cooling devices for spectrofluorometry becomes evident. It’ might also be useful in cases of overlapping fluorescent impurities in solvents or of interfering fluorescent substances present in biological materials. Because of their different temperatwe quotients, some fluorescence curves could be “filtered out,” and others made more visible in favorable cases. Figure 6 illustrates how the p H of the solution influences the fluorescence. The intensity of the iinoxidized drugs is relatively low and increases only slightly in the alkaline range for all phenothiazine drugs. Meanwhile the oxidized compounds have a imuch higher fluorescence. Three kinds of p H curves of potassium permanganate-treated drugs can be dist,inguished in t,his figure. Thioridazine with a high fluorescence in acid and low intensity in alkaline medium shares t,he pict,ure with t,hiethylperazine because of stheir similar st’ructure-Le., their common additional sulfur atom on position 2 of the phenothiazine nucleus. Oxidized chlorprothixme, however, ha;, its highest. fluore,?cence in the alkaline range. Chlor-
promazine shows a p H curve that is similar to other oxidized phenothiazine drugs with a chlorine or trifluomethyl group on position 2 of the nucleus. Also the unsubstituted promazine and mepazine have this kind of p H curve. These findings emphasize that standard curves, or samples to be analyzed, have to be kept under strictly comparable conditions with regard to temperature, drug concentration, and p H of t h e medium. Figure 7 illustrates absorption and fluorescence excitation spectra of pro-
1
pantheline and chlorpromazine sulfoxide. When the phenothiazine drugs were treated with potassium permanganate, their ultraviolet absorption curves resembled the chlorpromazine sulfoxide pattern shown in this figure. For each fluorescence excitation peak appears an absorption wave. However, with increasing wavelengths, the absorption curves form a decrescendo pattern when the fluorescence excitation waves show a crescendo pattern. This figure illustrates that the highest absorption peak should not be chosen for
WAVELENGTH, rnp
Figure 4. Excitation and fluorescence patterns with different concentrations of chlorprothixene in 0.01 N sulfuric acid (left). Change of fluorescence of chlorprothixene, 1 kg./ml., in varying concentrations of sulfuric acid (right) Height of curves arbitrarily set for better comparison
VOL. 36, NO. 9, AUGUST 1964
1843
getting the best fluorescence excitation, as it is sometimes advised in textbooks. T h a t this procedure can become completely unreliable is shown in the upper part of Figure 7 . There, the absorption curve of propantheline, a phenothiazinelike compound, did not show an absorption at 335 mp, where it had its highest fluorescence excitation peak. When the characteristic four-wave crescendo patterns of potassium permanganate-treated chlorpromazine or trifluoperazine are compared with the spectra of their monosulfoxides, they are identical (8). Thus it is probable that the crescendo excitation and fluorescence curves of other phenothiazine drugs with a chlorine or trifluomethyl group on position 2 are also due to monosulfoxide formation during treatment with potassium permanganate. However, in the case of thioridazine, the ring monosulfoxide of thioridazine produces only a two-wave excitation curve with peaks a t 285 and 335 mp, and fluorescence maximum at 390 mp. The fluorescence spectrum of its disulfoxide, though, resembles the four-wave crescendo excitation and fluorescence of thioridazine during potassium permanganate treatment as shown in Figure 2. Thiethylperazine with its similar structure shows the same fluorescence properties. It follows the same p H curve as thioridazine shown in Figure 5. Therefore, in acid medium, the spectra of both these compounds a t peak fluorescence may be due to the disulfoxide structure. I n the alkaline range only the monosulfoxide pattern can be obtained with potassium permanganate. The monosulfoxide has a relatively low fluorescence compared to the fluorescence of the supposed disulfoxide obtained in the acid range. The ring monosulfoxide spectrum appears in acid medium transiently at the very beginning of the fluorescence titration of thioridazine and thiethylperazine. The structure of potassium permanganate-treated chlorprothixene a t peak fluorescence is still open to question. Neither its monosulfoxide nor its disulfoxide gives the typical fluorescence spectrum of chlorprothixene obtained during fluorescence titration (Figure 7). I n contrast t o the other phenothiazine drugs, it fluoresces as well a t p H 10 as in higher concentrations of sulfuric acid (Figure 4) and has its peak color absorption curve a t the same wavelength (395 mp) where it also retains its main peak of fluorescence excitation in widely different media-that is, from 90% sulfuric acid to an alkaline medium of pH 13.5. When 10 mg. of thioridazine, chlorprothixene, or chlorpromazine was given by stomach tube to rats, the fluorescence spectra of the oxidized drugs could be recorded from all organ extracts 2 hours later. Those from chlorprothixene1844
ANALYTICAL CHEMISTRY
4
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TEMPERATURE, "C.
Figure 5. Fluorescence intensity of chlorpromazine (left) and of chlorpromazine sulfoxide (right) a t different temperatures Concentration of both, 2 pg./ml. in
treated and thioridazine-treated rats developed best. I n the organs of low drug concentration, such as heart, fat, or skeletal muscle, the chlorpromazine samples showed slightly slurred fluorescence spectra because of some interference from fluorescent tissue substances, and also because of some scattering of light which resulted from the nearness of excitation maximum to fluorescence peak in the spectrum of oxidized chlorpromazine ( 8 ) . I n general, the lower limit range for analysis of the drugs in organ extracts was for chlorpromazine 2 to 5 pg. per gram of tissue, and for thioridazine and chlorprothizene 0.3 to 1.0 pg. per gram of tissue. Their fluorescences could still be recorded in two to three times lower concentrations in extracts of blood serum which contains no cellular elements and therefore fewer interfering fluorescent substances. When light scattering was the limiting factor, extracting a larger amount of organ material could improve the sensitivity of the procedure. However, when the fluorescent tissue substances were the interfering factor, using more tissue only increased the interfering fluorescence accordingly. I n all tissues and body fluids, the most frequently interfering substances have a peak fluorescence a t 350 mp and peak excitation a t 285 mp. Since indole derivatives have a similar fluorescence spectrum, compounds with an in ole structure are presumably the interfering
0.1N sulfuric
acid.
agents. Because of their relatively intense fluorescence, they overlap completely the fluorescence spectra of the unoxidized drugs. However, the fluoresence titration technique has here an additional advantage in that the permanganate reagent first quenches this fluorescence of the tissue materials stepwise to a minimum while a t the same time t'he charact.eristic fluorescence curves of the drugs emerge slowly with further additions of the titrant. Table I shows that fluorimetrically determined concentrations and diatribution of each of the three drugs are similar in the organ extracts of the t'hree groups of rats. They resemble the organ distribution of phenothiazine drugs found by others with isotope techniques (6, 10, 12) or by spectrophotomet,ric methods ( I , 2, If). Their drug concentrations are highest in lung, followed by adrenals, liver, kidney, and spleen, whereas brain and blood serum always have a relatively low drug content. By comparing the results of the group of chlorpromazine rats determined by fluorometry wit'h the CI4chlorpromazine group, the organs of the latter showed higher drug levels when determined by radioactivity. When these organs were extracted as in the procedure for fluorometry, they r'etained 15% to 20% as nonextracted radioactivity, which is probably in part due to some adsorption of the drug into
500
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Figure 6. Fluorescence intensity of three drug solutions a t different pH's 0 Before potassium permanganate, treatment
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Thioridazine Chlorprothixene Chlorpromazine Drug concentration, 2 pg./ml.
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Average Drug Concentration in Rat Tissue.
Thioridazine rats Lung Adrenals Liver Kidney Spleen Thyroid Heart Feet Muscle Brain Serurii (portal vein) Serum (aorta)
Drue concn.. un./nrarn of ornan Fluorometry Rrtdioactivi ty ChlorproChlorproC"-Chlr,rpioni,z~:ine razs thixene mazine (ULICS(exrats rats tracted) tmcted)
31.1 22.3 15.9 19.9 17.4 6.3 3.8 4.3 3.2 1.6
26.3 16.9 12.5 15.0 16.4 6.8 2.9 6.5 2.3 4.4
20.3 18.8 17.3 14.8 17.7 5.9 3.3 4.6 3.1 3.8
32.4 16.1 23.0 21.7 22.8 13.2 8.3 6 9 4.5 6.5
3.9 2.1 4.6 4.3 3.9 2.2 1.3 0.9
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0.7
.6
.7
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a Determined by fluorometry or radioactivity in pg./gram organ of 3 rats after a 10-mg. dose.
tissue and in part to a small amount of water-soluble metabolites. Figure 8 illustrates tissue radiograms of C14-chlorpromazine treated rats: in A the radioactivity chromatogram of homogenized liver, when applied to the paper and developed wit'hout previous treatment; and in B , a chromatogram of liver tissue that, before application to the paper, had been extracted in the same way as were the organs of the three rat groups for fluorometric determination. Radiogram B demonstrates the complete ext'raction of the radioactive areas of R, 95 and Rl 80 present in A which correspond to chlorpromazine and its sulfoxide, respectively. There also is a small radioactive ai'ea at approximately R, 25. C shows a chromatogram of unextracted brain, and D of brain that has been extracted prior to chromatography. B and D illustrate the efficiency of the extraction procedure. The lipid soluble chlorpromazine and its sulfoxide have been extracted completely, whereas the small hump at R, 25, which probably represents a compound with high water solubility, has not been extracted In the chromatograms of the other oi'gans, chlorpromazine and its sulfoxide had similar proportions to those in the radiogram of liver tissue. However, the hump at Rl 25 was not clearly visible in the other tissues. Ihain had a relatively higher concentration of chlorpromazine as compared to its sulfoxide. Since these two compounds represent most of the radioactivity found in all organs, the extraction methods used for fluorometry of the phenothiazine drugs seem to be satisfactory. This can be assumed also for other Ijhenothiazine tranquilizers, since they havc, in our experience, a relatively high lii)id solubility. When the fluorescence of the various phenothiazine com1)ounds was examined in guinea pigs, all biles showed immediately, or after treatment wit'h potassium permanganate, the typical crescendo excitation and fluorescence curves of the drugs. After oxidation an increase of their intensity from two to five times greater could be recorded. The same fluorescencc could be easily seen in t,he jejunum content of the animals, \\.her(, the intensity was usually onr t h i d of that of the bile concentration. Two to VOL. 36, NO. 9, AUGUST 1964
1845
10 p l . of these body fluids diluted in 1 ml. of 0.0lX sulfuric acid !%eresufficient for analysis without any extraction or other separation methods. Figure 9 shows the y e c t r a of guinea pig bile from an animal that was treated simultaneously n ith chlorpromazine and chlorprothixene. The fluorescence curves of both drugs could be recorded separately, each a t its corresponding peak of excitation and fluorescence after potassium permanganate treatment. The chlorprothixene fluorescence (lower curve) did not show any overlapping with the chlorpromazine spectrum. Whereas on the recorded chlorpromazine pattern (upper curve) the fluorescence curve of chlorprothixene became visible at higher wavelengths, nevertheless it did so without disturbing the chlorpromazine pattern. DISCUSSION
WAVELENGTH, m r
Figure 7.
Absorption and fluorescence excitation of two drugs
Above, fluorescence set 450 mk; below: 3 8 0 mp. Height of absorption and fluorescence excitation curves arbitrarily set Absorption -Fluorescence excitation A. Propontheline 8. Chlorpromazine sulfoxide Drug concentration, 10 pg./rnl. in 0.2N sulfuric acid
---
BRAIN
LIVER
L
4 Figure 8. Distribution of radioactivity on chromatograms of liver and brain tissue from C14-chlorpromazine-treated rats
C
A, C. Before extraction. 8, D . After extraction. Background, 1.2 counts/rnin., with &,SO% variation
D
FRONT
1846
ANALYTICAL CHEMISTRY
Spectrofluorometry, in our experience, has been a useful method for quantitative analysis of phenothiazine compounds. However, strict standard conditions have to be observed, since several factors influence the fluorescence spectra and their intensity. That the reported wavelength shifts can be overlooked !Then the analysis is not combined with the examination of the wavelength spectrum of the fluorescence points to the advantage of using a spectrofluorometer instead of a filter fluorometer for accurate analysis. The more our basic knowledge in the correlations between molecular structure and fluorescence progresses, the more we may find better methods to increase the intensity of the fluorescence or change the wavelengths of the spectra a t will. This would enable the experimenter to avoid or reduce unfavorable interferences from fluorescent tissue substances, especially when working with very low drug concentrations in biological material. The reported findings on chlorprothixene are especially interesting in this respect. There is still open a wide field of experimentation for further improvements. Except for the quenching phenomenon,
START
FRONT
START
be seen by comparing the fluorescence curves of the sulfoxides of chlorpromazine, chlorprothixene, and thioridazine with the spectra of the corresponding drugs during oxidation with potassium permanganate. Since each of these drugs attains a different state of oxidation at peak fluorescence, as reported in this paper, the close interplay between the oxidized sulfur group and the other substructures of the phenothiazine nucleus becomes evident.
60
40
20
2
ACKNOWLEDGMENT
tc
The drug substances employed in this study were generously supplied by the following companies: Smith Kline & French, Philadelphia, Pa.; Schering Corp., Bloomfield, N. J.; Sandoz Pharmaceuticals, Hanover, N. J., Squibb & Sons, New York, N. Y.; Wyeth Laboratories, Philadelphia, Pa. ; Roche Laboratories, Nut,ley, N. Y.; Searle & Co , Chicago, Ill.; WarnerChilcott Laboratories, Morris Plains, N. J.; Lundbeck & Co., Copenhagen, Denmark.
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1W LITERATURE CITED
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Figure 9 . Fluorescence spectra of permanganated chlorpromazine (above) and of chlorprothixene (below), from guinea pig bile after simultaneous oral administration of both drugs.
(1) Berti, T., Cima, L., Arch. Intern. Pharmacodyn. 98, 452 (1954). (2) Ibid., 100,373 (1955). (3) Dusinski, G., Cesk. Farm. 6 , 302 f 1957). ( 4 j Dusinski, G., Pharmazie 12, 309 (1957). (5) Zbid., 13, 478 (1958). (6) Fyodorov, N. A., Proc. U . N . Intern. Conf. Peaceful Uses At. Enerau, 2nd Genkva, 24,205 (1958). " - I
(7),Green, J., cited by S. C'denfriend in Fluorescence Assay in Biology and Medicine," p. 106, Academic Press, New York, 1962. f 8 ) Mellineer. T. J.. Keeler. C. E. ANAL. CHEM.39, 554 (1963). ' \
little is known about how different solvents, or certain solvent mixtures, or solutes present in the same solutions, influence the fluorescence characteristics of these drugs. Since phenothiazine-like structures, such as imipramine or amitriptyline, have a low fluorescence that cannot be increased by potassium permanganate
treatment, the importance of an oxidizable sulfur atom in the phenothiazine nucleus becomes obvious for the increase of intensity during fluorescence titration (8). However, it seems certain that not only the oxidized sulfur moiety is responsible for the fluorescence phenomena, but actually the phenothiazine nucleus as a whole. This can
-
I
(9) Pungor, E. V., Pharm. Acta Helv. 35, 173 (1960). (10) Walkenstein, S. S., J. Pharmacol. E x p . Therap. 125,283 (1959). (11) Wechsler. M. E.. Roizin., L.,, J. ' ,!ental Sci. '106, 1501 (1960). (12) Zehnder, F. et al., Biochem. Pharmacol. 11,535 (1962).
RECEIVEDfor review May 13, 1963. Resubmitted March 30, 1964. Accepted April 17, 1964.
VOL. 36, NO. 9, AUGUST 1 9 6 4
1847
