LITERATURE CITED
No. 6 fuel oil measured a t 80 and 20 K are compared in Figure 5. The major differences are increases in the intensities of the bands a t 720 and 725 cm-I a t the lower temperature. These changes provide additional data for identifying petroleum; however, we do not feel that a couple of additional data values warrant the time and effort it takes to measure spectra a t 20 K. Thus, we are confining our analyses to liquid N2 temperature. Results from this study indicate that low temperature infrared spectrometry can be used subsequent to room temperature infrared analysis for identifying petroleum products and petroleum pollution sources. The low temperature technique is only slightly more time consuming than room temperature analysis. Collaborating data from other analytical techniques will undoubtedly be needed in court cases; however, in spills where several similar oils are involved, infrared spectra measured a t room temperature followed by measurements a t 80 K provide convincing evidence for identifying the source.
(1) P. F. Lynch and C. W. Brown, Environ. Scl. Techno/., 7, 1123 (1973). (2) C. W. Brown, P. F. Lynch, and M. Ahmadjian. Environ. Sci. Techno/., 8, 699 (1974). (3) C. W. Brown, P. F. Lynch, and M. Ahmadjian, Anal. Chem., 46, 183 (1974). (4) C. W. Brown, P. F. Lynch, and M. Ahmadjian, Appl. Spectrosc. Rev.. in
press.
(5) M. Ahmadjian. P. F. Lynch and C. W. Brown, J. Water Po//ut. Control Fed., submitted for publication. (6) E. R. Adlard, J. lnst. Petrol., 58, 63 (1972). (7) R . E. Baier, J. Geophys. Res., 77, 5062 (1972). (8) R . D. Cole, J. lnst. Petrol., 54, 288 (1968). (9) F. K. Kawahara, J. Environ. Sci. Techno/., 3, 150 (1969). (10) F. K. Kawahara and D. G. Ballinger. /nd. Eng. Chem., Prod. Res. Dev., 9,
553 (1970). (11) F. K. Kawahara, J. Chromafogr. Sci., 10, 629 (1972). (12) F. K. Kawahara, J. F. Santner, and E. C. Julian. Anal. Chem., 46, 266 119741. (13) H. G. Mark, T. C. Yu, J. S. Mattson. and R. L. Kolpack. Environ. Sci. Techno/., 6, 833 (1972). (14) J. S. Mattson, H. B. Mark, R. L. Kolpack, and C. E. Schutt, Anal. Chem.. 42, 234 (1970). (15) J. S. Mattson, Anal. Chem., 43, 1872 (1971). (16) R . G. Snyder, J. Mol. Spectrsc., 7, 116 (1961).
ACKNOWLEDGMENT We express our appreciation to D. Eastwood for suggesting this study, and to A. P. Bentz, J. J. Elliott, and J. s. Mattson for helpful advice. We also express appreciation to M. Ahmadjian for collecting the spill samples and to C. D. Baer for help in measuring the room temperature spectra.
RECEIVEDfor review February 3, 1975. Accepted April 4, 1975. This research was partially supported by a U S . Coast Guard Contract (DOT-CG-81-74-1099)and partially by the National Sea Grant Program, National Oceanic and Atmospheric Administration.
Phosphorimetric Analysis of Phenothiazine Derivatives L. A. Gifford, J. N. Miller, D. L. Phillipps, and D. Thorburn Burns Department of Chemistry, University of Technology, Loughborough, LE 1 7 3TU, Leicestershire, U.K.
J. W. Bridges Department of Biochemistry, University of Surrey, Guildford, Surrey, U.K.
The phenothiazine derivatives are a large and important family of compounds from a medical point of view. Over 3000 have been synthesized and a t least 100 have been used clinically ( I ) , mainly as antihistamines or major tranquillizers. Since their introduction into clinical practice, several methods for their rapid and accurate determination a t normal plasma levels have been studied. A comparison of the methods used before 1972 has been given by Usdin ( 2 ) (for chlorpromazine and its metabolites only) and by Cimbura ( 3 ) . Current methods with sufficient sensitivity include mass fragmentography ( 4 ) , electron capture-GLC (5-7), and fluorescent derivatization (8-10). All these methods have some disadvantages; mass fragmentography is far too complex and the equipment too expensive to be considered for general purposes, electron capture-GLC can only be used when halogen atoms are present, and fluorescent derivatization only permits the detection of metabolites containing primary amine or hydroxyl groups. The room temperature fluorescence of phenothiazines in both oxidized and unoxidized states has been well studied (11-15) and their UV adsorption characteristics have been recorded ( 1 6 ) .However, luminescence data a t low temperatures are sparse. Winefordner and Tin ( 1 7 ) described an analytical curve for chlorpromazine phosphorescence but did not give a detection limit. Thiery e t al. (18) described the low temperature phosphorescence of 13 phenothiazine derivatives, but less than half of them are in general medi-
cal use and neither detection limits nor phosphorescence lifetimes were given. This paper provides such data for some commonly-used phenothiazines. EXPERIMENTAL The phenothiazine derivatives studied are shown in Table I. All the compounds melted within 2 'C of the literature value, except for the chlorpromazine HC1 which was therefore purified by precipitation from ethanol using diethyl ether. After the chlorpromazine hydrochloride was dried in a vacuum oven, its melting point was in agreement with literature values (19). The solvents used were water, triply distilled from an all glass apparatus, and ethanol (A.R. grade, James Burroughs Ltd.). Uncorrected luminescence spectra were obtained using a Baird-Atomic SF lOOE spectrofluorimeter fitted with a rotating cylinder phosphoroscope and connected to a Bryans 26000 X-Y recorder. The sample tube, a piece of open ended Spectrosil tubing (1-mm i.d. X 4-mm o.d., Thermal Syndicate Ltd., Wallsend) was rotated a t 320 rpm using a small electric motor and cooled using a partially silvered Dewar flask containing liquid nitrogen. Phosphorescence lifetimes were measured using an Advance O S . 200 storage oscilloscope in conjunction with the spectrofluorimeter. A micro-switch was placed on the light shutter so that when the shutter was raised the oscilloscope was triggered. The trace obtained was photographed on Kodak Tri X film using a Shackman A.C. 2/25 oscilloscope camera. The negatives were then projected onto graph paper (30 cm X 20 cm) and the lifetimes calculated from these enlargements. In the case of methotrimeprazine maleate, six lifetime values obtained on three separate occasions showed a coefficient of variation of 4%. The detection limit for each compound was taken as being the concentration a t which the emission was equivalent to two stanANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
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Table I. Phenothiazines Studied and Sources of Samples
R R
Same
Supplier'
R'
*
c1
May and B a k e r Ltd.
CF3
B. P. C o m m i s s i o n
OCH,
May and Baker Ltd. *
CN
May and B a k e r Ltd.*
c1
Allan and Hanburys*
H
Hopkin and Williams *
Prochlorperazine maleate
c1
B. P. C o m m i s s i o n
P r o m a z i n e HC1
H
B. P. C o m m i s s i o n
ICH I \ I C H
Chlorpromazine HC1
)
n
Fluphenazine HC1
tCH I 2
2 CH C H OH
v
CHCHCH \ [ i H
I
Methotrimeprazine maleate
I
CH
Pericyazine
( H ,\>OH
Perphenazine
!(HI\
Phenothiazine
H
A
\ (HCHOH
v
Sandoz P r o d u c t s Ltd.*
Thiethylperazine m a l e a t e
n
Thiopropazate HC1
a
!CH I 1
\CH CH 0 CO
CH
U
G.D. Searle'
C1
Thioproperazine m e s y l a t e
May and B a k e r Ltd. *
Thioridazine HC1
B. P. C o m m i s s i o n
T r i f l u o r p e r a z i n e HC1
B.P. C o m m i s s i o n
Trif lupr omazine HC 1
E.R. Squibb and Sons
All compounds marked with an asterisk were generously donated by the companies named.
Table 11. Low Temperature Luminescence Data for the Phenothiazines Rel. peak height
kMax,
Chlorpromazine HC1 Fluphenazine HC1 Methotrimeprazine Pericyazine Pherphenazine Phenothiazine Prochlorperazine maleate P r o m a z i n e HC1 Thie thylpe r azine maleate Thioproperazine mesylate Thioridazine HC1 Trifluoperazine HC1 T r i f l u p r o m a z i n e HC1
ex, nm
255,310 260,315 255,305 270,315,350 255,310 260,325 250.305 250,305 265,315 255,310
AMax,
i, nm
... 450
...
470
...
...
... ...
... ...
260.330 265,315 255,305 255,305
445 455
...
Detection l i m i t
r , ms
Equimolar
Equi. g l l .
ngiml
nmol
490 505 485 540 495 505,535 490
72 59 58 43 77 56 78
0.61 0.46 0.82 0.16 0.71 0.43 0.29
0.68 0.36 1.006 0.18 0.71 0.87 0.19
30 55 20 45 30 25 100
85 110 60 125 75 125 165
495 495 490
78 60 75
0.59 0.72
l.ooa
0.74 0.46 0.77
30 45 25
95 70 50
520
57
0.35
0.22
100
155
495 505 510
66 65 68
0.89 0.47 0.49
0.88 0.39 0.51
25 50 40
60 105 105
A ~ a xp,, nm
a Assumed t o be 1.00 for equimolar concentrations, * Assumed to be 1.00 for equal weight concentrations. Fluorescence maxima only given if peak >2% of phosphorescence peak.
1700
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
F
A
!!
I
\
! I
\,
\
I 0
30
60
90
120
150
imondr
Flgure 1. Effect of UV irradiation (320 nm) on the phosphorescence
emission of chlorpromazine HCI at 77 K, ( A ) in ethanol and ( B ) in water
dard deviations of the background luminescence. Readings were taken in triplicate and six blank readings were taken for each compound studied. The detection limits were determined directly for methotrimeprazine maleate, thioproperazine mesylate, and pericyazine. Since the slopes of the analytical curves for these compounds were equal, within the limits of experimental error, the detection limits of the other compounds were estimated from their relative phosphorescence intensities at equal weight concentrations.
RESULTS
It was originally intended to measure the detection limits of the compounds using water as the solvent because, having such a low background luminescence, very good detection limits might be obtained with it (20). However, the compounds studied photodecomposed in water even a t 77 K. Flushing the solution with oxygen-free nitrogen (obtained by passing commercial oxygen-free nitrogen through Mercury/Vanadium I1 solution) for 15 minutes reduced the rate of decomposition, but did not prevent it (Figure 1). Therefore all the results in this paper were obtained using ethanol as the solvent. The excitation maxima, emission maxima, phosphorescence lifetimes, relative phosphorescence intensities for equimolar and equal weight concentrations, and detection limits are given in Table 11. Most of the compounds studied show characteristic double excitation and single phosphorescence maxima a t 250265, 305-315, and 485-505 nm. (Figure 2). When a fluorescence band is also present (-450 nm), the phosphorescence undergoes a red shift. Phenothiazine itself has a pronounced shoulder in the phosphorescence spectrum at 535 nm. Pericyazine has an excitation spectrum with three maxima at 270, 315, and 350 nm and a more intense fluorescence (amounting to approximately 30% of the total apparent luminescence) than any of the other compounds. The presence or absence of the rotating cylinder phosphoroscope had no significant effect on the wavelengths of any of the phosphorescence maxima. The phosphorescence lifetimes ( 7 ) are all in the range 60-80 msec, except in the case of pericyazine (43 msec), and there is a fivefold variation of the phosphorescence intensities of the compounds. DISCUSSION Table I1 shows that, except for pericyazine, all the compounds studied have rather similar luminescence properties. The absence of the shoulder at 535 nm in the phosphorescence spectra of all the compounds except phenothiozine itself suggests that the substituent at position 10 may cause this effect. Otherwise, the nature of the substituent a t this position has little effect on the spectra: this is not
Figure 2. Luminescence characteristics at 77 K of methotrimeprazine maleate (--), thioproperazine mesylate (-) and pericyazine - ) . ( A ) excitation; ( F ) fluorescence; and ( P ) phosphores-
--
( - a
-
a
cence spectra. Fluorescence is not detectable in the case of methotrimeprazine maleate
surprising since substituents in all the phenothiazines studied have three carbon atoms separating the ring system from the nitrogen atom. The groups a t position 2 which have the largest effects are -CF3, -S02N(CH&, and -CN, all of which produce a bathochromic shift of phosphorescence (10-45 nm compared with promazine) and also cause detectable fluorescence. All these groups are strongly electron-withdrawing but further studies of compounds with electron-donating substituents would be required to verify that this type of effect is involved. The detection limit for pericyazine is lower than would be expected from its relative phosphorescence intensity because the luminescence background of the solvent is greatest at about 380 nm and decreases with increasing wavelength. As pericyazine has a phosphorescence maximum a t a greater wavelength than the rest of the compounds, there is a lower background to contend with when it is being studied. The detection limits obtained may allow phenothiazines to be determined at levels found in blood plasma. A major problem, however, is the large number of metabolites obtained in vivo. Turano and Turner (21)identified 35 chlorpromazine metabolites and found another 42 which could not be identified. In this respect, the use of a scanning thin layer phosphorimeter, as described by Gifford et al. (22), to separate and identify metabolites, would be advantageous and work is proceeding using this technique for the analysis of phenothiazines and their derivatives. LITERATURE C I T E D (1)“Lewis’s Pharmacology,” 4th ed., J. Crossland, Ed., Livingstone, London, 1970. (2)E. Usdin, Crit. Rev. Clin. Lab. Sci., 2, 347 (1971). (3)G.Cimbura, J. Chromatogr. Sci., I O , 287 (1972). (4)C. G. Hammar, B. Holmstedt, and R . Rhyage, Anal. Biochem., 25, 532 (1968). (5)S.H. Curry, Agressologie, 9, 115 (1968). (6) S.H. Curry, Anal. Chem., 40, 1251 (1968). (7)G. W. Christoph, D. E. Schmidt, J. M. Davies, and D. S. Janowsky, Clin. Chim. Acta, 38, 265 (1972). (8)P. N. Kaul, M. W. Conway. M. L. Clark, and J. Huffine, J. Pharm. Sci.. 59, 1745 (1970). (9)P. N. Kaul, M. W. Conway. M. K. Ticku, and M. L. Clark, J. Pharm. Sci.. 61, 581 (1972). (10)P. N. Kaul, M. W. Conway, M. K. Ticku, and M. L. Clark, J. Lab. Clin. Med., 81, 467 (1973). (11) T. J. Mellinger and C. E. Keeler, Anal. Chem., 35, 554 (1963). (12)T. J. Mellinger and C. E. Keeler, Anal. Chem., 36, 1840 (1964). (13)J. 8.Ragland and V. J. Kinross-Weight. Anal. Chem., 36, 1356 (1964). (14)J. B. Ragland, V. J. Kinross-Wright, and R. S. Ragland, Anal. Biochem., 12, 60 (1965). (15)E. A. Martin, Can. J. Chem., 44, 1783 (1966). (16)A. De Leenheer, J. Assoc. Off. Anal. Chem., 56, 105 (1973)
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
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(17)J. D. Winefordner and M. Tin, Anal. Chim. Acta, 32, 64 (1965). (18)C. Thiery, J. Capette, J. Meunier, and F. Leterrier, J. Chim. fhys. Physicochim. Biol., 66, 134 (1969). (19)“British Pharmacopaeia.” 1973. (20)R. J. Lukasiewicz, P. A. Rozynes. L. B. Sanders, and J. D. Winefordner, Anal. Chem., 44,237 (1972). (21) P. Turano and W. J. Turner, J. Chrornatogr., 75, 277 (1973).
(22)L. A. Gifford, J. N. Miller, D.T. Burns, and J. W. Bridges, J. Chromatogr., 103, 15 (1975).
RECEIVEDfor review November 7, 1974. Accepted April 28, 1975. We thank the Medical Research Council for the award of Project Grants in support of this work.
Determination of Anomeric Configuration of D-Ribofuranosyl Nucleosides from Nuclear Magnetic Resonance Spectra by a Pattern Recognition Technique Jure Zupan, Joie Kobe, and Dugan Had% Chemical Institute Boris Kidriz and Faculty of Natural Sciences, University of Ljubljana. Hajdrihova 19, 6 1000 Ljubljana, Yugoslavia
The difficulty of obtaining an unambiguous answer as to the anomeric configuration of ribosides prompted us to apply the pattern recognition method to this problem. The recently developed criterion ( I ) based on the differences of the NMR chemical shifts of the two methyl groups of the 2,2-dimethyl dioxolane ring has certainly facilitated decisions in the nucleosides series. We felt that the extension of this problem to the riboside series in general, independently of the anisotropic influence of the anomeric substituents to the methyl groups, might be successful if the following parameters were used: the chemical shifts of the l’,2’, and 3’ ribose protons and the corresponding 3 J H H vicinal coupling constants 3J1,2,and 3 5 2 , 3 , (Table I). It might be argued that the parameters used are not independent of the ribose ring puckering and the possible conformational state (syn-anti). Therefore, the chemical shifts within the limits given in Table I were chosen and the values of the coupling constants cover the almost complete number of predicted 3 5 H H for various compositions of conformational equilibrium mixtures of N and S conformers (2, 3 ) . To the best of our knowledge, the chemical shifts limits should be satisfied by the requirements, because neither the difference A6 due to the anisotropic effect of the base and/or substituents nor the effect of the electronegativity of the substituents fall out of our frame. Even the dependence of the constants on the nature of the substituents in nucleosides as well as theoretical predictions do not prevent this application (4).A pattern recognition technique was recently applied to various kinds of structural problems based on different data collections, e.g., mass (5, 6),IR (7-9), NMR (10-12) spectra, search for possible anticancer drugs ( I 3 ) , etc. Table I shows the organization of our data base which
consists of 552 computer simulated spectra, representing R, or Rg patterns. The simulation was carried out using the LAOCN3 computer program for the NMR spectra simulation, developed by A. A. Bothner-By and S. M. Castellano. All 552 simulated NMR spectra were digitalized using digital resolution R of 1 Hz and then transformed using the formula_ suggested by Kowalski and Reilly (IO) into the vector A (0, R, 2R, 3 R , . . .):
and F ( f ) is the digitalized NMR spectrogram as a function of frequency f. The summation over f was cut oftat 750 Hz. The problem was to defin? a decision vector W in such a way that a dot product (W-A) will give positive or negative values for A representing R, or Rb configuration, respectively. About 100 vectors, randomly chosen out of the set of 552 vectors A, were selected as the training set. The training begag with an arbitrary vector W of the same dimension as 4. In order to, improve \rtT, each incorrect answer (giving (W-A) < 0 for A represzntins R, or vice versa) causes a feedback of the form (5):W = W cA
+
so that the new vector W gives a correct classification. The “learning“ process was stopped at such \rtT that all training vectors were classified correctly. After the training proce-
Table I. Representative Chemical Shifts and Coupling Constant Intervals for R,, and R,j Patterns R, Interval, Hz
61
62 63 512 52 3
1702
650-700 450-600 550-600 4-5 6-7 All together
Step, Hz
R0 Interval, Hz
No. of cases
25 3 25 7 25 3 1 2 1 2 3.7.3.2-2 = 252
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
51 62 63 J12 J2 3
600-700 525-625 550-650 0-1 4-5 A l l together
Step, Hz
No. of cases
25 5 25 5 50 3 1 2 1 2 5.5.3.2’2 = 3 0 0
