Luminescence Determination of Pharmaceuticals of the Tetrahydrocarbazole, Carbazole, and 1,4-Benzodiazepine Class J. Arthur F. de Silva,’ Norman Strojny, and Katherine Stika Department of Biochemistryand Drug Metabolism, Hoffmann-La Roche Inc., Nutley, N.J. 071 10
Luminescence studies were performed on thin layer chromatographic (TLC) plates at 77 K and also with a Farrand Mark I Spectrofluorometer which was modified to accommodate a commericaiiy avaliabie phosphoroscope. The apparatus was used to obtain fluorescence and phosphorescence spectra at 77 K of selected tetrahydrocarbazoles, carbazoles, 1,4-benzodiazepines, and analytlcally useful derivatives of the 1,4-benzodiazepInes. Some of the results were verified on other commerically available phosphorimeters, and the modifled instrument was found to be equal or better in spectral quality, sensitivity, and precision. The slmple modification employed greatly extends the utility of this instrument for cryogenic luminescence research.
Several reviews on low temperature fluorometry and phosphorimetry have appeared in the past decade (1-5), but the practical applications of these techniques have been somewhat sparse because of instrumental limitations. The use of thin layer chromatography as a rapid qualitative procedure for the evaluation of luminescence properties a t low temperatures (77 K) (6-10) significantly facilitates the selection of the analytical parameters for low temperature luminescence studies. The sensitivity and selectivity of detection of complex mixtures of organic compounds is also enhanced. Detection on paper chromatograms a t 77 K (7,8, 11-14) has also been investigated with similar results. Routine instrumental analysis of drugs (15),vitamins (16),and other organic compounds ( 17-20) by phosphorimetric methods is currently possible. The Farrand Mark I spectrofluorometer is not amenable for routine low temperature luminescence studies because of limitation of the available cryogenic accessory, although Neely and Hall (21) have reported using this modification for low temperature absorption spectrometry. The cryogenic attachment supplied by Farrand was found to be unsuitable for routine luminescence analysis a t 77 K because of the long cool-down times required and the severe cracking of solvent glasses due to a large solvent volume (4 ml). This report describes a Farrand Mark I spectrofluorometer modified for use with a commerically available (Aminco) phosphoroscope which was employed to obtain fluorescence and phosphorescence spectra a t 77 K for certain selected tetrahydrocarbazoles, carbazoles, 1,4-benzodiazepines, and the analytically useful derivatives of the 1,4benzodiazepines. The results obtained from the Farrand for some of these compounds were compared to those obtained on other commercially available phosphorimeters and to published data on reference compounds, and were found to be equal or better in spectral resolution, sensitivity, and precision.
EXPERIMENTAL Instrumentation and Equipment. A Farrand Mark I spectrofluorometer (Farrand Optical Co., Inc., Valhalla, N.Y. 10595) equipped with a rotary chopper a t the excitation source and a n optical beam condensing system (Cat. No. 132370) focused onto the micro cell in the sampling area was modified to accommodate a 144
e
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
micro-sample phosphoroscope without any alteration of the main instrument (Figure 1). A 2-inch hole was drilled in the Farrand sample chamber cover (A) to accommodate an Aminco Dewar holder (American Instrument Co., Silver Spring, Md. 20910 (B) which can be either brass welded or fused to it with epoxy cement (C). The phosphoroscope consists of a “micro” Dewar, quartz samples tubes, and the Dewar holder and cover, Figure 1 (D). The complete assembly and its installation in the Farrand spectrofluorometer is shown in Figure 2. (Nitrogen is introduced into the sample chamber area via the side arm of the Dewar holder (Figure 1 (C)) to purge the sample area of moisture and prevent condensation around the Dewar.) The rotating can assembly typically used in other commerically available phosphorimeters is replaced by the rotary chopper a t the excitation source, while built-in electronic circuitry synchronizes the amplifier to the chopper motor. By switching to the phosphorescence function, the amplifier receives a signal input only when the chopper blade is blocking the exciting energy; thus the signal recorded is produced only by phosphorescence. A single-pulse shutter assembly built into the instrument enables the determination of phosphorescence decay lifetimes approaching 1 msec. Phosphorescence lifetime measurements and luminescence spectra were recorded on a Houston Instruments No. 2000 omnigraphic X-Y recorder (Houston Instruments Co., Austin, Texas 78753). An Aminco-Bowman spectrophotofluorometer equipped with an off-axis elipsoidal mirror condensing system and a Lewis-Kasha rotating can phosphoroscope (American Instrument Company) and a Baird-Atomic Fluorispec SF-100, similarly equipped (BairdAtomic, Inc., Bedford, Mass. 01730), were also used t o analyze selected samples to compare results with the Farrand. Commercially available thin layer chromatoplates, E. Merck pre-coated, abrasion resistant, glass backed (Silica gel F-254) marketed by Brinkmann Instruments, Inc., Westbury, L.I., N.Y., were used for luminescence screening. The UV sources for TLC visualization were a short wave ultraviolet lamp, Model No. C-81 (254 nm) and a long wave ultraviolet lamp (Blak Ray) Model No. XX-1500 (366 nm), (Ultraviolet Products, Inc., San Gabriel, Calif.). Reagents. All reagents used were of analytical grade (ACS) purity and were used without further purification. These included EPA (diethyl ether:isopentane:ethanol 5:5:2) (American Instrument Co.), absolute ethanol (Pharmco, Publicker Industries, Inc., Philadelphia, Pa.), concentrated sulfuric acid (36 N ) (J. T . Baker Chemical Co., Phillipsburg, N.J. 08865). All the pharmaceutical compounds examined were of analytical grade (>98% purity), and were found by thin-layer chromatography to show no interfering impurities. Procedure. Ten mg of each compound examined (Tables I, 11, 111) were dissolved in 10 ml of either EPA, ethanol, or 1%(36 N ) H&04 in ethanol. Calibration curves were prepared from serial dilutions of the stock solutions. The stock solutions were stored under refrigeration ( 5 “C) when not in use. A preliminary examination of the luminescence properties of the sample compounds was made on thin layer chromatoplates a t room temperature (298 K) and a t liquid nitrogen temperature (77 K). Samples of 100, 10, 1, and 0.1 fig were applied t o a chromatoplate which was then developed in the solvent mixture of choice for that compound and then air-dried. The plate was then photographed with a Polaroid camera using a Tiffen (Hi-Trans, Yellow 1, Series 7) filter first in visible light, then in short-wave ultraviolet light (254 nm), and finally in long-wave ultraviolet light (366 nm) at room temperature. Any intrinsic UV absorption of fluorescence behavior observed was recorded. The plate was then sprayed with anhydrous ethanol until completely saturated, placed in an aluminum pan, and sufficient liquid nitrogen was poured into the container to completely submerge the plate. The plate was observed again under short-wave (254 nm) ir-
A.
Sample chamber cover to fit Farrand Universal sample chamber Cat. No. L 3 2 2 3 3
6.
C.
7 1
-
I
Nitrogen inlet rube
/
/
Aminco Dewar Holder
I
Brass
Weld
Farrand Cover
Brass welded holder
Dewar holder assembly to fit Aminco Phosphoroscope, Cot. No. C 2 6 - 6 2 1 4 0 (refer to item 6 , pg. 13, bulletin 2 3 9 2 - J - A
D.
Complete
PhoSphorOSCOpe
Assembly For F a r r a n d M a r k
I
+ cover assembly
Spectrofluorometer
Screw C O P pos1tioner
Aminco s a m p l e rube holder
-,
t -
Aminco O u o r l z Dewar
Supporting c o l l a r / a t t a c h e d to dewar
Q u a r t z somple rube
Brass welded dewor holder and sample chamber' cover assembly
IC
I
-
Forrand Spectrofluorometer Model M a r k - I Sample Chamber
-'Farrand
-
I
M a r k - I Somple holder for l c m 2 size cuvetre
'I--.
Figure 1. Component parts of the modificationof a Farrand Mark I Spectrofluorometer for luminescence studies ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
145
- I
02N 2
i
c?
2W I,
I 0
m
m
o m 0 mr-m N N N
0 IC N
0 W
W
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0
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N N
m
N
N
m a
0
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m
40
99
0 0 0 0
8 r
ri
Q
0
r-
m 0 N
m
0
m
m
0
*
i
0 0
m
mm
m
a*
9
0 Lo
m
m- m m G?
m
m
0
rm
0
r-
m
Loom0
mm++ mm*m
.-uE ' m l .-* * I 9I 1 V
m
N
N
NN
N
N
0.1
N
N
P
s m .-U c1
01
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146
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
I
l
radiation, and any cryogenic fluorescence ohserved was photographed. The excitation energy was switched off, and the phosphorescence emission was photographed immediately thereafter. The color and duration of the visihle emission were recorded. To photograph the phosphorescence, multiple exposures at wide aperature settings were necessary. Observations of fluorescence and phosphorescence were made again under long-wave (366 nm) irradiation. Fluorescence and phosphorescence spectra of compounds showing luminescence were obtained with the modified instrument using either the stock solutions or suitahle dilutions. Linear dynamic concentration range curves were also determined for representative compounds. A 10-nm band-pass was utilized for each monochromator except where noted. Spectra were not corrected for energy or other instrumental artifacts. The limit of quantitation is defined as that concentration which gives B sample reading of twice the reagent blank reading. Phosphorescence lifetimes in excess of 0.5 sec were determined using an X-Y recorder with a time base generator driving the X-axis. Spectra and concentration curves were determined on other eommerical instruments for selected compounds to compare the results obtained using the modified Farrand Mark I instrument. RESULTS AND DISCUSSION
Figure 2. Phosphoroscope installed on Farrand Spectrofluorometer
EXCITATION
Amoi=252nm
The utility of screening for the intrinsic luminescence properties of a compound using the thin layer chromatographic (TLC) visualization procedure is apparent from the nature of the information that can be obtained (Table IV). A number of difficulties were experienced with the TLC visualization procedure. The glass plates often shattered when subjected to liquid nitrogen. The thick soft glass plates usually used for TLC plates prepared in house shattered more readily than the thinner commercially available pre-coated plates. Furthermore, when the ethanol saturated gel froze, the ethanol expanded and lifted the gel from the plate; and, upon warming to room temperature, the silica gel flaked off easily. This problem is much worse using aluminum foil-backed TLC plates than with the glass plates which use a gypsum binder. The use of Eastman polyethylene terephthalate hacked chromatogram sheets would alleviate these problems; however, the Rf values oh-
1
n
WAVELENGTH
EMISSION
Amax :375nm
IN N A N O M E T E R S
Figure 3. Ultraviolet absorption and phosphorescence (77 K) excitation and emission spectra of benzene in EPA ANALYTICAL CHEMISTRY, VOL. 48, NO.
I. JANUARY
1976 e 147
d
d
2
2
0
0 0
0, A
d
4
2
8m 83
2
m
A
0
i
m m
*
d
m
8 % r - w
U 0
* m
d
w
2
0 0
0 0
3
3
A
A
0-
m
0
m
O E d *
2
O
N
" 8
rio
m
B 00
*
- 0
0 0
mE-
mm
d
i
m
8
0 E-
IC
m
* E-
Q,
d
OON
BE-
- 0
0 0
~m m
m
m
0-
u?-
0-
m
N
m
W N
m N
2
m
8
m
8m e 84
c-
0-
ri
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o m mtd
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148
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m
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m
0-
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ri
0 0
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WEN M 0-
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0
w
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% t-
8 t-
0 I C
m a
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0"
0-
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m
u-
m
m
m W m
0 W N
mm
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
m
d
N(D
mm
0
d
Q,
YP
o
n 2
CD
0
d i
d
i
m
0
m
o
m
d
i
v
0
d
2 2
EU
m
m
*
- 0
mt(D
U
c-
* Y
m
m
J3
m
d 2
m
8~ E
e
U
m
0
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t-
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0.1
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m W m
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0.1
tained on such sheets are not directly comparable to those obtained from more conventional silica or alumina coated glass plates. While preliminary work showed that dry plates may be used, saturation with ethanol before cooling gave much greater sensitivity and reproducibility. The ethanol supplies a rigid matrix at 77 K which enhances the luminescence through isolation of the excited state molecules from deactivation effects. Also, use of ethanol supplies data which are directly comparable to the data already available on phosphorescence in ethanolic solutions. The TLC luminescence screening technique was useful for non-routine work (such as metabolite identification and toxicological analysis) where chemical sprays cannot be used for characterization. This method may also be used in investigating many solvent effects which are difficult to examine in normal solution spectrometry at low temperatures (Le., in solvents which form cracked glasses, or have important expansion characteristics, such as aqueous media, or concentrated sulfuric acid) (6-9); or in rapid screens to evaluate the feasibility of a more protracted investigation of low temperature luminescence in solution. If proper care is taken, the compounds can eventually be eluted and quantitated. Despite the restrictions of the TLC technique, closely related substances can be selectively identified after thin layer chromatography. The sensitivity and selectivity of visualization is usually increased (often several-fold) at 77 K. Initial efforts using the cold-finger cryogenic attachment in the Farrand Mark I for low temperature (77 K) luminescence studies were disappointing because of the occurrence of cracked solvent-glass caused by the temperature differentials resulting from the large volume (4 ml) cell and the cold-finger mode of cooling the sample. However, the use of the modification employing the Dewar phosphoroscope of the Lewis-Kasha type (as outlined in the instrumental section) resulted in a significant decrease in the incidence of cracked solvent glasses, especially for EPA or ethanol. The cool-down time was reduced significantly by the immersion of the smaller samples so that a concentration curve of a dozen samples could be completed in a relatively short time (30 min, including all transfer and cell clean-up procedures) rather than 1 to 2 hr per sample using the cold-finger mode of cooling as originally supplied by the manufacturer. The wavelengths for fluorescence and for phosphorescence excitation and emission maxima, limits of detection, and phosphorescence lifetimes obtained with the experimental setup described are summarized in Tables I, 11, and 111. The ultraviolet absorption spectrum and the 77 K phosphorescence excitation-emission spectrum of benzene were determined in EPA using 2-nm slits (Figure 3). The spectral details compare well with those reported by Sponer et al. ( 2 2 ) .The phosphorescence lifetime was determined to be 4 sec and is shorter than the value of 7 sec reported by D. S. McClure ( 2 3 ) . The use of the beam condenser arrangement appears to shift the emission peaks 5 to 10 nm to longer wavelengths, otherwise the spectra obtained for the aromatic compounds agree with previous work ( I , 22-25). Although there are some differences in mean phosphorescence lifetime, this may be due to phosphorescence quenching effects caused by the presence of oxygen. The weak phosphorescence exhibited by anthracene a t long wavelengths is in agreement with that reported by McGlynn et al. (25) and the relative intensities of phosphorescence found in these experiments are substantiated by previous workers ( I ) . The phosphorescence lifetime of phenanthrene was 2.6 sec compared to 3.3 sec reported by McClure ( 2 3 ) ,Table I. ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
149
Table 111. Cryogenic (77 K) Fluorescence and Phosphorescence in 1% H,SO, (36N) in Ethanol of the Benzophenones, the 9-Acridanones, and the Quinazolines, All of Which Are Analytically Useful Derivatives of the 1,4-Benzodiazepines Fluorescence
Excitation, No.
Compound
41
Benzophenones 2-Amino-benzophenone
42 43 44
45 46 47 48 49 50 51 52 53 54 55
2-Amino-5-chloro-benzophenone 2-Methylamino-5-chlorobenzophenone 5-Chloro-2-(2-diethylaminoethy1amino)-2’fluorobenzophenoneHC1 5-Chloro-2’-fluoro-2-(2hydroxyethy1)aminobenzophenone N - [4-Chloro-2-(2’-fluorobenzoyl)]glycine 2-Amino-5-chloro-2’fluorobenzophenone 2-Amino-5-nitro-2’chlorobenzophenone 2,5-Diamino-2’-chlorobenzophenone Acridanones 9-(l0H)-Acridanone 2-Chloro-lO-(2-diethylaminoethyl)-g-acridanone.HC1 2-Chloro-10-(2-hydroxyethyl)-9-acridanone 2-Chloro-lO-(carboxymethyl)-g-acridanone 2-Chloro-(lOH)-9-acridanone Quinazolines Quinazoline
nm
265 265, 290b 405 265
270. 415b 275, 405b 265, 290, 398b 375 270, 385b
Emission, nm
415, 445b 480 450
Phosphorescence or delayed fluorescence Limit of quantitation, @g/ml
Excitation, nm
0.05
26kjb
0.3
265b, 290
0.1
N.D.C
415, 445b, 473
0.5
265
425.480b
0.2
477
475 445, 475b
0.5 0.5
265b, 290 275 265b, 290 375
Emission, nm
415,445b, 480 415,445b, 475 N.D.C
Limit of quantitation @ug/ml
Phos. lifetime, sec
0.01
N.D.‘
0.01
N.D.
>mg/ml
N.D.
415, 4456, 473
0.1
N.D.
445b, 475
0.2
N.D.
450
20.
N.D.
420, 4456, 475 515
0.1
N.D.
0.03a
N.D.
515
0.30
450
0.1
265,b 290
450b. 475
0.2
N.D.
300, 380, 390b 265, 390, 405b
500b, 525
0.2
2.5-3
505
0.1
N.D.
515
0.2
0.8
505
0.1
1.0
525
0.2
0.7
1
N.D.
265, 375b, 405 265, 3906, 412
415, 440b
0.002
425, 450b
0.05
265, 395b, 420 270, 394b, 415 265, 390b, 410
430, 4556, 480 425, 4506
0.005
425, 450p, 475
0.005
2706, 390, 410 295,410b
4256,450
0.1
0.01
265, 394b, 418 270, 393, 415b 365, 390, 410b 270b, 390, 410 254, 295b
425b, 450
418,470b 1 N.D. l o 0 pg); 2 = Weak (ca. 100 pg); 3 = Moderate (ca. 10 p g ) ; 4 = High (ca. 1.0 pg); 5 = strong ( < 1 pup).= d:A = Absorbant; F = Fluorescent; P = Phosphorescent. Color: U = UV absorbing, no Color; V = Violet; B = Blue; W = White (Blue-White): G = Green: Y = Yellow: R = Red. Phosphorescence Time o f Duration: 1 = Rapid ( < isec);-2 Intermediate ( < 3 sec); 3 = Long ( > 3 sec); 4 = Very Long ( > l o sec).
Instrument
Phosphorescence intensity Preciunits per pg/mP sion, 7~
Sensitivity, pg/ml
7 84 i 21 0.1 Aminco 316 i23 0.8 Baird-Atomic 777 i 10 0.025 Farrand (without beam condenser) Farrand (with beam 1546 27.4 0.01 condenser) a Compound 14; (d,l)-6-chloro-~-methylcarbazole-2-acetic acid in EPA at 77 K (See Figure 5 ) .
Farrand to be at least as sensitive and precise as the instruments against which it was checked (Table V). The influence of substituent groups in the luminescence of the carbazole nucleus is shown in Figure 6 for 6-methyl9-[2-(6-methyl-3-pyridyl)ethyl]carbazole-2-acetic acid ethyl ester. The aromaticity of the carbazole nucleus is greatly increased because of the electron donating substituents resulting in greatly increased “fine structure” in the excitation-emission spectra seen for its fluorescence at ambient temperature and for its phosphorescence a t 77 K. The luminescence observed for quinoline and quinine sulfate agrees with the findings of Kasha (29) that the fluorescence intensity increases over phosphorescence with increasing polarity of the solvent used. The 1,4-benzodiazepine class of compounds is clinically important as anti-anxiety agents, muscle relaxants, and hypnotics (Table 11). There is no previous work reported on the cryogenic luminescence of these compounds. Luminescence data on a number of 1,4-benzodiazepines (Table 11) were obtained in 1%(36 N)HpS04 in ethanol, because EPA and ethanol gave lower luminescence intensity than did acidified ethanol. The 2,3-dihydro compounds, medazepam and its N-desmethyl metabolite, gave similar spectra but widely differing intensities, whereas compounds having the Na-oxide, such as chlordiazepoxide, showed little luminescence. Diazepam and N-desmethyl diazepam showed the highest phosphorescence intensity and their spectra are charac(Table 11). tekstic of most of the 1,4-benzodiazepin-2-ones The 7-nitro and 7-amino benzodiazepin-2-ones showed distinctive shifts in their maxima. The luminescence observed as “phosphorescence” appears to be delayed fluorescence since no change in maxima occur compared to the maxima recorded for “instantaneous” fluorescence a t 77 K. Although precise determinations of the phosphorescence lifetimes were not made, the 1,4-benzodiazepin-2-ones are relatively short-lived; diazepam, medazepam, bromazepam, and flurazepam showed lifetimes shorter than 0.2 sec, while clonazepam showed a lifetime of between 0.4 and 0.5 sec. The 1,4-benzodiazepines and their 2-ones can be converted to analytically useful luminescent derivatives, such as the o-aminobenzophenone produced by acid hydrolysis, which can be cyclized in base to the 9-acridanone. The parent compound can also be converted to the quinazolinelone by dehydration, Figure 7. The spectrum of 2-amino-5chlorobenzophenone in 1%(36 N)H2SO4 in ethanol, Table 111, is typical of this class of compounds, but the intensities vary significantly, depending upon the substituents in the phenyl rings. The quinazolines are also important luminescent derivatives of the 1,4-benzodiazepines. The luminescence spectrum of 2-hydroxy-4-phenyl-6-chloroquinazoline in 1%(36 N ) H2s0.1 in ethanol (Table 111) is also typical of ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
151
IO
Phosphorescence Excit. 297/Em 455 nm
> k cn
IO'
z W t-
z v)
k
z
3
> a a
Lz
k m a a
10:
W V
z
W
V v)
W Lz
0
I
a
v)
0
I oi
1
a
IO 0.010
0.10 CONCENTRATION &g/ml
I .o I N I% H,SO,
IO
50
100
IN ETHANOL
FLgure 4. Phosphorescence calibration curves of 6-chloro-l,2,3,4-tetrahydrocarbazole-2-carboxylic acid
this class of compounds. The luminescence is probably due to a mixture of the keto-enol tautomers of the quinazoline and quinazolinone derivatives. The luminescence observed for both the benzophenones and the quinazolines appears to be due to delayed fluorescence rather than to true phosphorescence. The 9-acridanones, on the other hand, showed true phosphorescence. The luminescence spectra of 2-chloro-10-(2diethylaminoethyl)-9-acridanone.HCI are shown in Figure 8 and demonstrate the characteristic shift in the maxima to lower energy for phosphorescence. Equivalent spectra were obtained with an Aminco and a Baird-Atomic spectrophosphorimeter. The phosphorescence lifetimes are listed in Table 111. The 9-acridanones are the most useful luminescence derivatives of the 1,4-benzodiazepine class of compounds.
CONCLUSIONS Luminescence spectral data were obtained using a Farrand Mark I spectrofluorometer modified to accommodate 152
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
a phosphoroscope of the Lewis-Kasha type substituting a mechanical chopper at the excitation energy source for the rotating can around the sample chamber. The data substantiate the utility of the modification and demonstrate the applicability of phosphorimetry to the determination of tetrahydrocarbazoles, c,arbazoles, 1,4-benzodiazepines, and to the analytically useful derivatives of the 1,4-benzodiazepines, viz., the benzophenones, 9-acridanones, and quinazolines. The modification is also important, because it will 410w the use of several recent improvements in phosphorimetric techniques, such as the use of the NMR sample tube spinning apparatus of Hollifield and Winefordner (30),and the use of the open-ended capillary cells of Lukasiewicz, et al. (31) which extend the use of phosphorimetry to solvents which form cracked glasses and snowed matrices (32) and to aqueous solutions (33).While low temperature luminescence spectrometry is not as simple a technique as room temperature fluorescence, it can be used on a routine basis to good advantage.
,' /Y EXClT 3 O O / E M 4 5 0 n m
/
/ e
" M o d l f l e d " FARRAND MARK
I
e-4
" M o d i f i e d " FARRAND MARK I
+*-X
AMINCO SPF ( + E F S I
without with
beam condenser
beam condenser
&----A BAlRD ATOMIC FS-100
/ / /
/ '
-
/
-
-,L 'Nm1x
of Quontiialion
I
I
0 01
I
10
0 IO
CONCENTRATION
IO
1
I
!
4
1 1 1 1
I00
( p g / mi I
Figure 5. Phosphorescence calibration curves of ( d , e)-6-chloro-cu-methyIcarbazole-2-aceticacid in EPA at 77 K
I
1 /
I m w '386 48, m p
1
= 110-112 5 O C )
I
FLUORESCENCE IN ETHANOL AT 25OC
I /
! I
I /
!
I I
I
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
153
1.4 - 0 E N Z O D I A Z E P I N - 2 - O N E
I .4-BENZ001AZEPINE
9 - A C R DANONE
0-AMINDBENZOPHENONE
Y
R
REARRANGEMENT IN CONC. ACIDS
R'
-
Group
= H or Alkyl
-H20
R2 = H a l o g e n o r N i t r o
I
f
R3 = H o r H a l o g e n R 4 =
+o
R5 = H o r OH
R6 = H, N H Z , -NHCH3,
Q U I N A Z O L I N E -CARBOXALDEHYDE or QUINAZOLINONE
Figure 7. Chemical reactions of the 1,4-benzodiazepines and their luminescent derivatives
EXCITATION i m a a =260,380.405nrn
FLUORESCENCE EMISSION
PHOSPHORESCENCE EMISSION
Xrnax~425.450nrn Xmaaz5OSnm
&
CH2CH2 N ( C 2 H 5 I 2
CI
0
-Excitation
---- Fluorescence -*-
Phosphorescence
! \
'.
1,
\
'.!
','. '.
200
300
400
500
600
WAVELENGTH IN NANOMETERS
Figure 8. Fluorescence and phosphorescence spectra of 2-chloro-lO-(2-diethylaminoethyl)-9-acridanone (1.O yg/ml) in 1 % H2S04 (36 N) in ethanol at 77 K
154
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
Luminescence analysis is particularly useful in the analysis of drugs in biological fluids where sensitivity and specificity are of paramount importance. The technique has been successfully used in the determination of a number of drug classes, such as the antimalarials (34, 3 5 ) , cannabinols (36), hallucinogens ( 3 7 ) , barbiturates ( 3 8 ) , antihistamines (39), sulfonamides (40), and vitamins ( 4 1 ) .A recent review ( 4 2 ) also shows its utility in Clinical Chemistry; hence the versatility of the technique is well documented.
ACKNOWLEDGMENT The authors thank Carl V. Puglisi for technical assistance in the design and modification of the Farrand Spectrofluorometer to accommodate the phosphoroscope.
LITERATURE CITED M. Zander, "The Application of Phosphorescence to the Analysis of Organic Compounds", Academic Press, New York. N.Y., 1968. J. D. Winefordner, S. G. Schulman, and T. C. O'Haver, "Luminescence Spectrometry in Analytical Chemistry", Vol. 38 in "Chemical Analysis: A Series of Monographs on Analytical Chemistry and Its Applications", P. J. Elving and I. M. Kolthoff, Ed., Wiley-lnterscience. New York, N.Y., 1972. J. D. Winefordner, J. Res. Nat. Bur. Stand., Sect. A, 76, (6) 579 (1972). D. M. Hercules, Ed., "Fluorescence and Phosphorescence Analysis", Interscience Publishers, New York, N.Y., 1966. B. L. Van Duuren and G. Witz, "Phosphorescence Spectroscopy", Chapter 3 in "Methods of Pharmacology" 2, A. Schwartz. Ed., Meredith Corp., New York, N.Y., 1972, pp 63-109. E. Sawicki and H. Johnson, Microchem. J., 8, 85 (1964). J. D. Pfaff and E. Sawicki, Chemist-Analyst, 54, 30 (1965). E. Sawicki and J. D. Pfaff, Anal. Chim. Acta, 32, 521 (1965). J. S. T. Chou and B. M. Lawrence, J. Chromatogr., 27, 279 (1967). H. P. Raaen and L. J. Crist. J. Chromatogr., 39, 515 (1969). A. Szent-Gyorgyi. Science, 126, 751 (1957). M. Zander and U. Schimpf, Angew. Chem., 70, 503 (1958). M. P. Gordon and D. South, J. Chromatogr., 10, 513 (1963). E. Sawicki and J. D. Pfaff, Mikrochim. Acta, 1-2, 322 (1966). J. D. Winefordner and H. W. Latz, Anal. Chem., 35, 1517 (1963). J. D. Winefordner and M. Tin, Anal. Chim. Acta, 31, 239 (1964). H. C. Hollifield and J. D. Winefordner, Anal. Chim. Acta, 36, 352 (1966).
(18) J. J. Aaron, L. B. Sanders, and J. D. Winefordner. Ciin. Chim. Acta, 45, 375 (1973). (19) J. J. Aaron and J. D. Winefordner, Anal. Chem., 44, 2122 (1972). (20) D. R. Venning, J. J. Mousa. R. J. Lukasiewicz, and J. D. Winefordner, Anal. Chem., 44, 2387 (1972). (21) W. C. Neely and T. D. Hall, Appl. Spectrosc., 28, 578 (1974). (22) H. Sponer, Y. Kanda. and L. A. Blackwell. Spectrochim. Acta, 16, 1135 (1960). (23) D. S. McClure, J. Chem. Phys., 17, 905 (1949). (24) S. P. McGlynn, B. T. Neely, and C. Neely, Anal. Chim. Acta, 28, 472 (1963). (25) S. P. McGlynn, M. R. Padhye, and M. Kasha, J. Chem. Phys., 23, 593 (1955). (26) R . C. Heckman, J. Mol. Specfrosc., 2, 27 (1958). (27) S. Freedand W. Salmre. Science, 128, 1341 (1958). (28) A. W. Perry, P. Tidwell, J. J. Cetorelli, and J. D. Winefordner, Anal. Chem., 43, 781 (1971). (29) M. Kasha, Radiat. Res., Suppl. 2, 243 (1960). (30) H. C. Hollifield and J. D. Winefordner, Anal. Chem., 40, 1759 (1968). (31) R. J. Lukasiewicz, P. A. Rozynes. L. B. Sanders, and J. D. Winefordner, Anal. Chem.. 44, 237 (1972). (32) R. Zweidinger and J. D. Winefordner, Anal. Chem., 42, 639 (1970). (33) R. J. Lukasiewicz, J. J. Mousa. and J. D. Winefordner, Anal. Chem., 44, 1339 (1972). (34) S. G. Schulman and L. B. Sanders, Anal. Chim. Acta, 56, 83 (1971). (35) S. G. Schulman, K. Abate, P. J. Kovi, A. C. Capomacchia. and D. Jackman, Anal. Chim. Acta, 65, 59 (1973). (36) A Bowd, P. Byrom, J. B. Hudson, and J. H. Turnbull, Talanta, 18, 697 (1971). (37) D. M. Fabrick and J. D. Winefordner, Talanta, 20, 1220 (1973). (38) L. A. Gifford, W. P. Hayes, L. A. King, J. N. Miiier. D. T. Burns, and J. W. Bridges, Anal. Chem., 46, 94 (1974). (39) D. R . Wirz, D. L. Wilson, and G. H. Schenk, Anal. Chem., 46, 896 (1974). (40) J. W. Bridges, L. A. Gifford, W. P. Hayes, J. N. Miller, and D. Thorburn Burns, Anal. Chem., 46, 1010 (1974). (41) J. J. Aaron and J. D. Winefordner, Talanta, 19, 21 (1974). (42) C. M. O'Donneli and J. D. Winefordner, Clin. Chem., 21, 285 (1975).
RECEIVEDfor review July 31, 1975. Accepted September 18, 1975. Presented at the 26th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 3-7, 1975, Cleveland, Ohio (Paper #139). K.S. is an A.C.S. Summer Research Fellow, Seton Hall University, South Orange, N.J. 07079.
Fluorescence Reactions of Aminophosphonic Acids Jeanne Fourche, Helene Jensen, and Eugene Neuzil" Laboratoire de Biochimie Medicale, Universite de Bordeaux I/, 146, rue Leo-Saignat, 33076-Bordeaux, France
The fluorescence emitted by 21 aminophosphonlc acids upon reaction with o-diacetylbenzene, o-phthaldlaldehyde, and fluorescarnine has been studied and compared to the fluorescence observed with the corresponding carboxylic analogs. A good similarity has been shown in both series for excitation and emission wavelengths; the fluorescence intensities are generally lower In the phosphonic series. The fluorescence spectra are closely related to the molecular structure of the amino compound when o-dlacetylbenzene Is used as the fluorogenic reagent, unsubstituted w-amino acids giving the higher fluorescence yields. o-Phthaldialdehyde and fluorescamine, on the other hand, appear as more general reagents for the unsubstituted primary amino group, allowing detection of the amino acids In the nanomole range. The use of fluorescamine, which yields a very stable fluorophore and thus appears to be a more practical analytical reagent, is limited by a lower reactivity toward the phosphonlc series, especially for the natural compound clliatlne.
In 1959, Horiguchi isolated 2-aminoethylphosphonic acid (AEP or ciliatine) from ruman protozoa ( I ) , introducing in biochemistry the first example of a natural product possessing a C-P covalent bond. AEP has been subsequently detected in a number of lower organisms (2, 3 ) together with its possible metabolic precursor, 2-amino-3-phosphonopropionic acid. More recent papers assigned to AEP a broader biological participation, including several mammalian tissues ( 4 ) . The obvious biological interest of aminophosphonic acids (AAP),some members of which were prepared by Chavanne ( 5 ) as early as 1947, led to the synthesis of numerous compounds possessing the general structure (I). Those nonbiological amino acids may be considered as analogs of natural a-amino acids in which the acid carboxylic group is replaced by the -P03Hz group. R --CH--P03H2
1
"2
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