S pect rof I uo ro metric Measurement of Phe not hia z ines JAMES
B. RAGLAND
and V. JOHN KINROSS-WRIGHT
Houston State Psychiatric lnstitute and Baylor University College of Medicine, Houston, Tex.
b The activation and fluorescence spectra of 31 phenothiazines and three thioxanthenes have been studied. Oxidation with hydrogen peroxide and heat prior to analysis yields more stable solutions and greatly increases fluorescence. Changes in pH affect phenothiazine fluorescence both qualitatively and quantitatively. Fluorescence intensity of phenothiazines varies widely, but all compounds examined could be measured in submicrogram quantities.
I
STUDYIKG the comparative metabolism of phenothiazines, and their derivatives which are used as psychotropic drugs, a highly sensitive method war sought which would be applicable to as many drugs and metabolites as possible. Although many excellent spectrophotometric and colorimetric methods for the determination of phenothiazines have been described ( I , 2, 6, 9, 12), none of them wa,s sufficiently sensitive. I'denfriend et al. (IO) reported that chlorpromazine exhibited fluorescent properties which might be used for quantitation. Recently Mellinger and Keeler ( 5 ) have also reported on fluorescent properties of some phenothiazines. I n the studies reported here the activation and fluorescence maxima of 31 phenothiazines and three thioxanthenes haipe been determined along with the relative yield of fluorescence of each compound. We have also studied the effects of oxidation and p H on fluorescence spectra. On the basis of these data, a widely applicable procedure for the determination of phenothiazines in biological samples has been developed ( 7 , 8).
N
EXPERIMENTAL
Apparatus. All measurements were made using a n .Iminco-l3owman spectrophotofluorometer (American Instrument Co.) coupled to a Houston Instruments X-Y recorder (Model HR-93-1). The instrument was equipped with entrance and exit polarizers which replaced the slits ordinarily used. A1ll of the fluorescence readings were taken directly from the recorder chart as centimeter* of vertical pen deflection from the base line. All spectra or maxima are uncorrected. Procedure. Stock solutions of each
1356
e
ANALYTICAL CHEMfSTRY
compound were prepared ranging in concentration from 25 to I00 @g. per ml., depending upon solubility. Compounds which were available as hydrochloride salts were dissolved in distilled water. The free bases were dissolved in 0.1.Y hydrochloric acid. These solutions were stored in amber glassware and kept refrigerated. Prior t,o fluorometric analysis the stock solutions were diluted with an aqueous buffer ranging from p H 2 to 12 (3) or mixed with an equal volume of glacial acetic acid to give a final concentration of 507, acetic acid. The latter was chosen because it had been reported to be an excellent medium for the osidation of phenothiazines with hydrogen peroxide ( 4 ) . The phenothiazines oxidized in this manner were diluted with the buffers before reading. Readings were also taken on all of the drugs in 50% acetic
Table
Generic name Phenothiazines Chlorphenothiazine Methiophenothiazine Trifluophenothiazine -perazines Thiethylperazine Prochlorperazine Trifluoperazine
I.
acid with and without osidation. Determinations upon oxidized standard> were made with freshly oxidized solutions. Immediately before reading, or extraction, 0.2 nil. of 307, 1)eroside was added to 2.0 ml. of the mlution of phenothiazin~ in 50% acrtir arid. The samples were heated in a boiling water bath for 10 minutes and allowed to cool to room temperature before the fluorescence v a s read. Alllfluorescence values were read directly from the recorder chart. RESULTS A N D DISCUSSION
Representative formulas of t,he compounds investigated are shown in Figure 1. The compounds examined have been subdivided on the ba& of substitution in the 10 position of the ring or
Activation and Fluorescence Maxima of
Ac:tivation Fluorescence Substitution maximum, mp maximum, nifi _ _ _ _ _ ~ . ~-Relative Posi- ITnoxi- Oxi- TJnoxi- Oxi- fluoresPosition 2 tion 5 dized dized dized dized cence" -C1 -S-CH, -CFa
-S-S-
s -
340 310 300
360 390 350
470 340 350
440 385 410
-S-GHs -C1 -CFs
-S-S-S-
285 325 320
360 340 350
470 450 470
445 380 405
71 2 14
0
Trifluoperazine sulfoxide -promazines Promazine Chlorpromazine
1:
-CF,
-S-
360
350
410
405
-H -C1
-S-S-
320 325
340 340
450 455
375 380
11 3
0
Chlorpromazine sulfoxide hlethoxypromazine Trifluopromazine -meprazines Trifluonieprazine Klethiorneprazine -phenazines Fluphenazine Perphenazine
-C1
It -S-
350
340
385
380
-0CH3 -CF3
-S-S-
320 330
340 350
450 475
380 405
5 34
-CFa --S-CH,
-S-S-
32B 330
350 360
480 470
406 440
100
-CF3
-S-
325 330
350 345
47.5 460
40j
20
380
4
4i5
0.8
0 6 4 90
-CI
-S-
30
0
Carphenazine -ridazines Brornridazine Chloridazine Thioridazine
I1
-C-C>Hj
-S-
--Br -CI --8-CH3
-s-S-
-P-
Thioridazine disulfone
0
290 330 330
340 340 360
450 4.55 470
380 380 440
360
360
435
440
0
0
I1 --S-CH, I(
370
-S-
I1 I
0
an
the type of ring. These compounds are given generic names dependent on the type of substitution in the 10 positioni.e., -promazine, -permine, -ridazine, etc. The thioxanthenes in addition are given the suffix, -thixin. Thl? 2 position of the ring is most commonl) substituted with a chloro, thiomethyl, or trifluoromethyl group. I n the compounds containing the phenothiazine ring, the sulfur atom in position 5 may be oxidized to either the sulfoxide or the sulfone level. I n the case of the 2thiomethyl substitution, oxidation may also occur in the sulfur of the substituent group. The substitutions in each individual compound may be seen in Table I. There is some confusion in the literature in regard to the nomenclature of these compounds. I n most cases established generic names have been used, but an attempt has been made to rename some of the compounds according to a more consistent generic nomenclature. The wavelengths at which the maximum activation and fluorescence readings were observed are given in Table I. Usually three to four activation peaks were observed for each com-
At
_ _ _ _ _ _ _ _ _ - _ _ -- - PHENOTHIAZINE
THIOXANTHENE
TYPES OF R ' ( l 0 1
-ridoiln e
-p*railna
SUDSTITUTION
-phrnarln*
F"
-CM2-CIiz-CHz-NGHalr
-Ck-CN2-C~-NICH312
-mrprailnb
--proma x l n *
Figure 1. Type formulas of compounds investigated
pound tested, for both oxidized and unoxidized forms of the drugs. Mellinger and Keeler (5) have suggested that the activation spectra might be used to identify phenothiazines qualitatively; but in this investigation, it was found that the compounds investigated could be as well differentiated
Phenothiazines and Thioxanthenes in 50% Acetic Acid
Generic name
Activation Fluorescence Substitution maximum, mp maximum, mp Relative Posi- Unoxi- Oxi- Unoxi- Oxi- fluoresPosition 2 tion 5 dized dized dized dized cencea 0
Thioridazine-Ssulfoxide
-j-CHa
Thioridazine-Rsulfoxide
-S-CHs
-S-
360
360
500
435
350
360
390
440
360
360
435
435
300
330
350
550
0.6
325
350
470
405
35.0
320
340
450
375
5.6
385
3.8
0
/I
-S-
0
I1
0
/I
Thioridazine di-S-CHa -Ssulfoxide Miscellaneous Pyridyl chlorphenothiazine, 2-chloro-10(4-pyridy1)phenothiazine Methyltrifluoperazine, 2-trifluoromethyi10- [ 2'-methyl-3'-( 1-methyl-4-piperaziny1)propyl]phenothiazine Promethazine, 10-(2'-methyl-2'-dimethylaminoethy1)phenothiazine
Nor'-chlorpromazine, 2-chloro-10-methyl-
350
aminopropylphenotliiazine
iVorl-chlorpromazine aiulfone, 2-rhloro-10methylaminopropyl phenothiazine-5,5dioxide Nor2-chlorpromazine sulfone, 2-chloro-10aminopropylphenothiazine-5,5-dioxide
Acetyl Fluphenazine, 2-trifluoromethyl10-3-11 -( 2-acetoxyethyl)-4-piperazinyl] propyl phenothiazine Thioxanthenes (-thixins) Chlorprothixene, 2-chloro-10-dimethylaminopropylidenyl i hioxanthene Methyldylidenylthixin, lO-(N-methyl-4piperidylidenyl) thioxanthene Thioridothixin, 2-methylmercapto-l0-[2-
340
340
375
375
340
340
380
380
325
350
475
405
25.0
345
...
410
1.9
280
...
370
.. . ...
3.9
300
320
420
395
1.2
(A~-methyl-2-piperidyl)ethylidenyl]
thioxanthene
simply on the basis of fluorescence maxima of the oxidized solutions. S o consistent relationship could be established between fluorescence spectra and the type of substitution of the phenothiazines in the case of the unoxidized solutions. However, if the fluorescence maxima of the oxidized solutions are compared, it can be seen that all of the phenothiazines except pyridyl chlorphenothiazine can be divided into three groups. Oxidized phenothiazines which have no substitution, a methoxygroup, or a halogen in position 2 show fluorescence maxima between 375 and 385 mp. Oxidized phenothiazines substituted in position 2 with a trifluoromethyl group all show fluorescence maxima between 405 and 410 mp. Oxidized phenothiazines substituted in position 2 with a thiomethyl or thioethyl group show fluorescence maxima between 435 and 445 mp. I n every case, the oxidized form of a phenothiazine can be distinguished from its unoxidized form. This is graphically illustrated in the case of trifluoperazine in Figure 2 . With all of the phenothiazines tested oxidation produced a large increase in the amount of fluorescence measured. The multiplication factors in Figure 2 are to be applied to the readings on the ordinate; thus, oxidized trifluoperazine is more than 10 times as fluorescent as the unoxidized solution. I n addition to the increased sensitivity obtained by oxidation, there are greatly increased stability and reproducibility of fluorescence. All of the unoxidized solutions showed minor peaks corresponding to the oxidized form of the phenothiazine after standing for a short time. A small peak at 410 mfi can be seen in Figure 2, indicating that the trifluoperazine has partially oxidized. This apparent autoxidation is further enhanced by exposure to ultraviolet light during the determinations. We have found that phenothiazine solutions oxidized in 50% acetic acid with H2O2 and heat are stable up to 24 hours and show only one fluorescence maximum which is quantitatively reproducible. I t is also extremely important that the solutions be completely cooled after oxidation, since fluorescence is decreased at higher temperatures. We have observed very little change in fluorescence between 2' C. and room temperature (23" C',). At 54' C., however, there was a 50% decrease in the amount of fluorescence observed. From inspection of Table I, it is impossible to determine whether the compounds present in the oxidized solution were the sulfones or sulfoxides. Where such oxidation products were available for testing, further oxidation did not change the spectra. Thioridazine represents a special case, since it has two oxidizable sulfur atoms. VOL. 36, NO. 7 , JUNE 1964
1357
Neither of the monosulfoxides was found to have a spectrum corresponding to the solution of thioridazine oxidized with H2O2. The spectrum of oxidized thioridazine is apparently due to oxidation of the sulfur of both the ring and the side chain, since it is similar to that of thioridazine disulfoxide and thioridazine disulfone. Neither the disulfoxide or the disulfone shows any change upon oxidation. On the other hand, the two monosulfoxides of thioridazine yield spectra identical to the disulfoxide and disulfone upon treatment with HzOz. The fluorescence of the thioxanthenes is almost completely destroyed by oxidation. I n the case of thioridothixin, however, there is a shift in the fluorescence maximum upon oxidation which is presumed to be due to the presence of an oxidizable sulfur in the side chain. The results of our studies of the effect of pH on the fluorescence of four phenothiazines are summarized in Table 11. In addition to the data shown, we have obtained data using other activation maxima; however, no differences were observed that are not illustrated by the data presented. Both chlorpromazine and fluphenazine when unoxidized show considerably greater fluorescence in basic solutions. All four of these phenothiazines when oxidized were found to fluoresce maximally in acid solutions and to show a sharp drop in fluorescence a t pH 12. Chlorpromazine, whether oxidized or not, showed no changes in wavelength of maximum fluorescence associated with changes in pH. The other three showed spectral changes in both the oxidized and un-
Table II.
pH 2 3 5 7 9 10 12
1
650
Figure 2.
*
450 WAVELENGTH
460 460 460 460 460 460 460
470 470 470 490 490 490 480
35 0
the least fluorescent, whereas methiomeprazine was highly fluorescent. Similar studies have also been carried out on most of the other phenothiazines and the relative fluorescences of oxidized solutions are given in Table I. Most compounds can be determined quantitatively a t concentrations of 0 . 5 pg. per ml. and in man3 cases much lower. At high concentrations, the phenomenon of quenching due to self-absorption does not become significant until concentratrations of approximately 25 pg. per ml. are reached, as illustrated by Table 111. One of the problems in the quantitative determination of very IOK concentration of phenothiazines is the interference due to Raman scattering ( 1 1 ) . This type of interference becomes particularly critical in the determination of phenothiazine< which are substituted in the 2 position with either hydrogen or halogen, which give fluorescence maxima ranging from 375 to 380 a t activation maxima of 340 to 350. Lowering the activation
Effect of p H on Fluorescence of Phenothiazines
45 39 36 28 48 69 100 335
385 385 385 385 385 385 385
55 100 98 44 54 99 19 340
470 470 470 470 460 455 450
8 8
8 70 79 70 100 350
410 410 410 410 410 410 420
59 80 75 84 72 80 100 330
440 440 440 440 440 440 475
100 79 71 68 80 71 14 365
Trifluoperazine 73 100 90 73 73 82 9 350
480 490 495 495 500 505 470
100 96 96 93 84 92 93 350
410 415 420 420 420 415 420
59 80 100 91 91 51 45 320
Activation, mp Fluorescence maximum. R.F. = relative fluorescence = fluorescence of sample at pH indicated x 100. fluorescence of sample at pH giving maximum fluorescence
1358
400 my
Fluorescence spectra of oxidized and unoxidized trifluoperazine
oxidized state. Before using a fluometric method for any of the phenothiazines or closely related compounds, the effects of p H on fluorescence yield and spectra should be carefully investigated. For the routine measurement of the phenothiazines, we have chosen to carry out all measurements in 50% acetic acid, since the increase in fluorescence of the oxidized solutions with increasing pH did not appear to be worth adding steps to the procedure. The spectrophotofluorometric method has proved much more sensitive than existing methods and the range of concentrations which can be determined is excellent. iill of the phenothiazines show a linear relationship between concentration and fluorescence. Table I11 illustrates the relationship between concentration and fluorescence for three compounds. Fluphenazine was one of
Fluphenazine
0
500
( 5 vg./ml.) Chlorpromazine Thioridazine Unoxidized Oxidized Unoxidized Oxidized F,,XO, F,,,, F,*X, F,,,, mp R.F.* mp R.F. mp R.F. mp R.F.
Activation, mp 2 3 5 7 9 10 12
ACTIVATION
ANALYTICAL CHEMISTRY
Table 111. Effect of Concentration on Fluorescence. of Phenothiazines Oxidized in 50% Acetic Acid
Compound Fluphen- Prometh- Methipazine azine meprazine 0 0 0 0,0125 0 0 0.2 0 025 0 0 0.4 0 05 0 011 1 0
Concn., pg./ml. 0
0 2.5 0 5 1 0 2 5 5 0 12 5 25 0 50.0 250 0 500 0
ilh,mp F , mp
1 0 024 2 0 0 .i 1 0 4 0 2.9 11 0 23 0 6 0 53 0 13 0 102 0 26 0 l.5
