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1,2-Diarylethylenediamines as fluorogenic reagents for catecholamines
Analytica Chimica Acta, 208 (1988) 59-68 Elsevier Science Publishers B.V., Amsterdam
59
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in The Netherlands
1,2-DIARYLETHYLENEDIAMINES AS FLUOROGENIC REAGENTS FOR CATECHOLAMINES
YOSHIHIKO
UMEGAE,
HITOSHI
NOHTA and YOSUKE
OHKURA*
Faculty of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812
(Japan) (Received
15th September
1987)
SUMMARY
Twenty-eight 1,2-diarylethylenediamines were investigated in a search for highly sensitive fluorogenic reagents for catecholamines (norepinephrine, epinephrine and dopamine). They all react with epinephrine usedas a model catecholamine under mild conditions (pH 6.5-6.8,37-50°C) in the presence of potassium hexacyanoferrate (III) to produce fluorescence. Of the compounds tested, 1,2-bis(3,4-dimethoxyphenyl)ethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis(4-ethoxyphenyl)ethylenediamine, all in the meso-form, are the most sensitive for all the catecholamines. 1,2_Diphenylethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis (4-methylphenyl)ethylenediamine in the DL-form are more sensitive to dopamine than the corresponding compounds in the meso-form. Catecholamines can be determined at concentrations as low as lo-15 pmol ml-’ by using those compounds as reagents. The reactions of other catechol compounds are reported. Other types of biologically important compounds do not interfere.
In a previous paper [ 11, it was shown that meso-1,2_diphenylethylenediamine (meso-DPE) reacts in a neutral or slightly acidic medium with catecholamines (norepinephrine, epinephrine and dopamine) and other catechol compounds in the presence of hexacyanoferrate (III) to give strongly fluorescent compounds [ 11. The reaction was applied for pre-column derivatization in high-performance liquid chromatography (HPLC) of free and total (the sum of free and conjugated) catecholamines in human erythrocytes [ 21, platelets [2], plasma [2,3] andurine [4,5]. The purpose of the present study is to find more sensitive reagents for catecholamines by screening of various 1,2_diarylethylenediamines (DAEs). Isoproterenol was also used for the screening, because it was used as an internal standard in the HPLC determination of catecholamines in biological samples [2-61.
0003-2670/88/$03.50
0 1988 Elsevier Science Publishers
B.V.
60 TABLE 1 DAEs as fluorogenic Al-
r
x t’
Ar
NH2
hi
Al.
NH2
Aryl group
(in brackets)
.H
x
I’ NH,
’ *NH, /’
meso-form
I’
Ar
reagents and their abbreviations
DL-
form
H
n Name 1,2_Diphenylethylenediamine
C’” /@ (OCH,),
JQ--
C’43
/@-
a 0 /&
Acronym
[ meso-DPE]
1 l,L-Bis (2-chlorophenyl)ethylenediamine 1,2-Bis (3-chlorophenyl)ethylenediamine 1,2-Bis(4-chlorophenyl)ethylenedamine 2 1,2-Bis(2,6-dichlorophenyl)ethylenediamine 1,2-Bis(3,4-dichlorophenyl)ethylenediamine
[ meso-o-CED] [ meso-m-CED] [ meso-p-CED]
1 l,P-Bis(2-methoxyphenyl)ethylenediamine l,P-Bis(3-methoxyphenyl)ethylenediamine 1,2-Bis(4-methoxyphenyl)ethylenediamine 2 1,2-Bis(3,4-dimethoxyphenyl)ethylenediamine
[meso-o-MOED] [meso-m-MOED] [ meso-p-MOED] [ meso-DMOED ]
[meso-2,6-DCED] [meso-3,4-DCED]
1,2-Bis(2-methylphenyl)ethylenediamine 1,2-Bis(3-methylphenyl)ethylenediamine 1,2-Bis(4-methylphenyl)ethylenediamine
[ meso-o-MED [ meso-n-MED [ meso-p-MED
1,2-Bis(3,4-methylenedioxyphenyl)ethylenediamine
[meso-MDED]
1,2-Diphenylethylenediamine
[DL-DPE]
1,2-Bis( 4-methoxyphenyl)ethylenediamine
[ DL-MOED ]
1,2-Bis(4-methylphenyl)ethylenediamine
[DL-MED ]
] ]
]
EXPERIMENTAL
Reagents, solutions and apparatus Norepinephrine bitartrate and dopamine hydrochloride were purchased from Wake Pure Chemicals (Osaka). Isoproterenol hydrochloride and epinephrine bitartrate were obtained from Nakarai Chemicals (Kyoto) and Sigma Chemicals, respectively. All other chemicals were of reagent grade. Deionized/distilled water was used. Each DAE solution (10 mM, apparent pH 6.7) was prepared in acetonitrile/0.02 M hydrochloric acid (1: 1, v/v). Uncorrected fluorescence spectra and intensities were measured with a Hitachi MPF-4 spectrofluorimeter in 10x10 mm quartz cells; spectral band-
61 TABLE 1 (continued)
Name
Acronym
1,2-Bis(2-ethoxyphenyl)ethylenediamine l,P-Bis(4-ethoxyphenyl)ethylenediamine
[ meso-o-EOED] [ meso-p-EOED]
1,2-Bis( 1-naphthyl)ethylenediamine 1,2-Bis(2-naphthyl)ethylenediamine
[ meso-a-NED] [ meso#-NED ]
1,2-Bis(4fluorophenyl)ethylenediamine
[ meso-FlED ]
l,P-Bis(4-ethylphenyl)ethylenediamine
[ meso-EED ]
1,2-Bis(I-cyanophenyl)ethylenediamine
[ meso-CNED]
1,2-Bis( 2-hydroxyphenyl)ethylenediamine
[ meso-HED]
/cl
1,2-Bis(3-pyridyl)ethylenediamine
[ meso-PED ]
A?
1,2-Bis (2-furyl)ethylenediamine
[ meso-FuED ]
l,P-Bis(4-biphenylyl)ethylenediamine
[ meso-BED ]
Aryl group
H
I \
LF 0
0
widths of 10 nm were used in both the excitation and emission monochromators. Mass spectra (MS) were measured with a JEOL JMSD300 interfaced to a JEOL JNM-3500 data system. ‘H-NMR spectra were obtained with a JEOL JNM-PS-110 spectrometer at 100 MHz; approximately 10% (w/v) solutions in CDC& containing tetramethylsilane served as the internal standard. Ultraviolet (UV) spectra were measured with a Hitachi X0-20 spectrophotometer with quartz cells of 0.4 x l.O-cm optical pathlength, and infrared (IR) spectra with a Nihonbunko DS 710G infrared spectrometer in potassium bromide pellets. The pH was measured with a Hitachi-Horiba M-7 pH meter at 25°C. Melting points are uncorrected. Preparation of 1,2-diarylethylenediamines The twenty-eight DAEs investigated are shown in Table 1 with their abbreviations. The meso-DPE compound was synthesized by the method of Irving
62 TABLE 2 Analytical data for newly synthesized DAEs DAE
’
M.P.
(“C)
Appearance (Recrystallization solvent )
Formula
Elemental data (% ) Calcd. (Found) C
H
N
meso-2,6-DCED
160-164 Colorless small crystals (Petroleum ether )
C,,H,,N,Cl,
48.0 (48.0)
3.4 (3.5)
meso-EED
114
Colorless needles (Petroleum ether)
C,,H,,N,
80.6 (80.4)
9.0 (8.7)
10.45 (10.5)
meso-m-MOED
113
Colorless small crystals (Benzene/petroleum ether, 1:4)
CSH~~N&Z
70.6 (70.7)
7.4 (7.2)
10.3 (10.4)
meso-DMOED
189
Colorless small crystals (Ethyl acetate)
GsH24NJb
65.0 (65.3)
7.3 (7.15)
(::i) 9.3 (9.1)
meso-p-EOED
153-154 Colorless needles (Benzene )
C,sH,,N,O,
72.0 (71.7)
8.05 (8.05)
meso-MDED
146-147 Yellowish needles (Benzene/petroleum ether, 1:4)
C,,H,,N,O,
64.0 (64.0)
5.3 (5.4)
meso-a-NED
168-169 Colorless small crystals (Benzene/petroleum ether, 2:3)
CZHZ~NZ
84.6 (84.6)
6.4 (6.5)
meso+NED
174-175 Yellowish crystalline powder C,,H,,N, (Benzene/petroleum ether, 2:3)
84.6 (84.5)
6.4 (6.45)
(k:)
and Parkins [ 71, and meso-o-CED, meso-m-CED, meso-p-CED, meso-3,4DCED and meso-m-MED were synthesized by the method of Sadler and House [ 81. The compounds meso-p-MOED, meso-o-MED, meso-p-MED, meso-oEOED, meso-FlED, meso-CNED, meso-HED, meso-PED, meso-FuED, mesoBED, DL-DPE, DL-MOED and DL-MED were prepared by the method of Vogtle and Goldschmitt [9]. The new compounds (meso-2,6-DCED, meso-EED, meso-m-MOED, meso-DMOED, meso-p-EOED, meso-MDED, meso-c-w-NED and meso$-NED) were synthesized by the method of Vogtle and Goldschmitt [ 91 with minor modifications and their structures were confirmed by the mass, ‘H-NMR, UV and IR spectral and elemental (CHN) data shown in Tables 2 and 3. Procedures Screening of DAEs with epinephrine. To 2.0 ml of DAE solution, 0.1 ml of 10 nmol ml-l epinephrine, 2.0 ml of aqueous 50% acetonitrile and 0.1 ml of 5 mM potassium hexacyanoferrate (III ) were added successively. The mixture was
3.89
633-I) 3.86 (&X-I) 3.86 (s,2H)
3.83 (s,2H) 4.24 (s2I-I) 5.21 (s,2H)
1.48 (s.4H) 1.42 (s,4H) 1.46 (s,4H)
1.36 (s,4H) 1.00-4.12 (sb4I-I) 1.52 (s,4H)
273(M++l)’
332(M+)’
301(M++l)’
301(M++l)’
313(M+ +l)’
312(M+)e
meso-m-MOED
meso-DMOED
meso-p-EOED
meso-MDED
meso-ar-NED
meso+NED
5.94 (s,4H,20CHz)
4.03 (q,4H,20CH,) 1.41 (t,6H,2CH,)
3.88 (s,12H,40CH,)
3,80 (s,6H,20CH,)
284
276
287
275
279
274
272
17 830
10 300
11664
3410
6600
5390
1540
740
emaX
3330, 3400
3330, 3360
3330, 3390
3300, 3370
3240, 3330
3270, 3360
3270, 3340
3330, 3390
LH
1200-1250
1240
1230
1250
ZO-C
IR’ v,,, (cm-‘)
1020-1050
1040
1030
1040
:-o-c
“Chemical shifts are given with peak patterns single (s), triplet (t), quartet (q), multiplet (m), broad (b) and proton numbers in parentheses. bin methanol. ‘KBr pellets. dThe peaks disappeared on adding D,O. “Electron-impact ionization. fField-desorption ionization.
7.21-7.88 (mJ4H)
7.20-7.88 (mJ4H)
W3-I)
6.70-6.93
6.82-7.32 (m8H)
6.79-6.99 (m6H)
6.80-7.34 (m8I-I)
7.08-7.36 (m8H)
2.66 (q,4H,2CH,) 1.24 (t,6H,2CH,)
3.87 (s,2H)
1.32-2.12 (s,bPH)
269(M++l)’
meso-EED
h6I-I)
6.90-7.52
273
5.56 (s6I-I)
1.88 (s,4H)
351(M++l)”
meso-2,6-DCED
1 mar (nm)
Subst.-H
CH-N
NHzd
Arom.-H
UVb
‘H-NMR (in CDC13)’ 6 (ppm)
MS(mIz)
DAE
Spectral data for the new DAEs
TABLE 3
%
64 TABLE 4 Excitation and emission maxima (A,,, &.,) of the fluorescences obtained from epinephrine with DAEs by the screening procedure, and their relative fluorescence intensities” (RFI) DAE
a BI (run)
I *In (nm)
RF1
DAE
1 ex (nm)
I em (nm)
RF1
meso-DPE meso-HED meso-o-CED meso-m-CED meso-p-CED meso-3,4-DCED meso-2,6-DCED meso-FlED meso-o-MED meso-m-MED meso-p-MED meso-EED meso-o-MOED meso-m-MOED
360 360 355 360 355 370 370 360 355 355 355 355 345 355
480 480 495 485 495 505 500 475 415 410 475 475 470 480
100 115 76 105 110 109 7 96 123 127 152 148 110 138
meso-p-MOED meso-DMOED meso-o-EOED meso-p-EOED meso-CNED meso-MDED meso-PED meso-a-NED meso-B-NED meso-FuED meso-BED DL-DPE DL-MOED DL-MED
355 355 355 355 400 355 390 365 370 350 360 360 355 355
470 470 475 470 560 470 515 510 530 470 510 480 470 475
158 176 122 178 32 166 30 70 52 85 4 112 158 144
“The fluorescence intensity obtained with meso-DPE was taken as 100.
TABLE 5 Excitation and emission maxima of the fluorescences from catecholamines and isoproterenol with the selected DAEs, and their relative fluorescence intensities” DAE
meso-DPE meso-p-CED meso-FlED meso-p-MED meso-EED meso-p-MOED meso-p-EOED meso-DMOED meso-a-NED meso-p-NED meso-MDED DL-DPE DL-MOED DL-MED
Norepinephrine
Epinephrine
Dopamine
Isoproterenol
1 ex (nm)
I,, (nm)
RFI A,, (nm)
A.,,, (nm)
RFI 1,. (nm)
A,,,, (nm)
RFI a,, I,, (run) (nm)
350 360 355 360 355 350 350 355 365 360 350 350 350 360
475 486 410 465 470 460 460 470 505 525 475 475 460 465
100 132 125 136 110 147 142 155 59 44 138 117 139 122
480 490 415 470 475 475 470 470 510 530 485 480 475 470
78 82 88 84 73 92 96 92 38 27 88 79 87 82
465 500 470 475 465 470 465 470 505 530 490 465 470 475
53 31 42 57 54 68 70 71 12 8 49 67 18 69
360 360 355 355 355 355 355 355 360 370 365 360 355 355
360 370 355 350 360 350 355 355 365 365 360 360 350 350
365 375 365 370 360 365 365 365 375 375 365 365 365 370
475 495 465 470 415 470 415 470 515 530 495 475 470 470
“The fluorescence intensity of meso-DPE derivative of norepinephrine was taken as 100.
RF1 132 102 138 158 134 172 166 176 72 41 148 155 167 134
65
allowed to stand at 37°C for 45 min to develop fluorescence. A reagent blank was prepared in the same way except that the 0.1 ml of epinephrine solution was replaced with 0.1 ml of water. The fluorescence intensities of the test and blank were measured at respective excitation and emission maxima (see Table 4). Fluorimetric determination of catecholumines with DAEs. To 0.1 ml of aqueous test solution, 2.0 ml each of DAE solution and aqueous 50% (v/v) acetonitrile, and 0.1 ml of 25 mM potassium hexacyanoferrate (III) were successively added. The mixture was maintained at 37’ C for 45 min. To prepare the reagent blank, the same procedure was applied except that 0.1 ml of the test solution was replaced by 0.1 ml of water. The fluorescence intensities were measured at the respective excitation and emission maxima (see Table 5). RESULTS AND DISCUSSION
Screening of DAEs as fluorogenic reagents for catecholamines For the screening of the DAEs, epinephrine was used as the model compound, because this amine occurs at very low concentrations in urine [lo] and blood [ 11,121. The fluorescence excitation and emission spectra of the DAE derivatives of epinephrine were very similar in shape to those from the mesoDPE derivative of epinephrine [ 41, but the excitation and emission maxima differed depending on the DAE derivative used (Table 4). The maxima for the meso-3,4-DCED, meso-2,6-DCED, meso-CNED, meso-PED, meso-a-NED, meso-P-NED and meso-BED derivatives were at longer wavelengths. These reagents may therefore offer higher selectivity in the determinations of catecholamines in biological samples, because weaker native fluorescences were generally observed in the samples when they were measured at longer emission wavelengths. The DL-DAEs gave the same fluorescence spectra as those obtained with the corresponding meso-DAEs. Electron-donating groups introduced to the 4-position of the phenyl groups of DAEs caused higher fluorescence intensities from epinephrine; meso-DMOED, meso-p-MOED and meso-pEOED gave stronger fluorescences in that order. By considering the above observations, 14 compounds (Table 5) were selected for further investigations. Reaction of catecholamines and isoproterenol with the selected DAEs and optimal conditions The selected DAEs did not give very great differences in the excitation and emission maxima among the catecholamines and isoproterenol (Table 4). The meso-DMOED, meso-p-MOED and meso-p-EOED derivatives gave higher fluorescence intensities in that order for all the catecholamines and isoproterenol. The DAEs in the DL-form were more sensitive for dopamine than the corresponding DAEs in the meso-form, and provided the same excitation and emis-
66 TABLE 6 Optimal conditions for the fluorescence reactions of the selected DAEs with norepinephrine, epinephrine andisoproterenol. Optimal conditions for dopamine are given in parentheses, if different. DAE
&Fe(CNh meso-DPE meso-p-CED meso-FlED meso-p-MED meso-EED meso-p-MOED meso-p-EOED meso-DMOED meso-a-NED meso&NED meso-MDED DL-DPE DL-MOED DL-MED
pH of DAE solution
Concentration
(mM)
DAE (mM)
25-63 25 25-63 25 25-63 2.5-63 25 25 25 25-63 25-63 25 25 25-63
10-15 10 5-10 10 5 5-15 10 5-10 10-20 10 15 20 10 5-15
(2.5-63) (25-63)
(25)
6.5-7.0 (5)
(10) (5)
K-7.0 7.0 6.5-7.0 6.5-7.0 7.0 6.5 7.0 6.5 6.5 6.5-7.0 7.0 7.0
(6.5) (6.0) (6.0-6.5) (6.0) (6.5) (6.5) (6.0) (6.5) (6.0) (6.0) (6.5) (6.0) (6.0)
Reaction time (min) 37°C
50°C
45 ii:
30 30 30 30 30 30 30 30 30 30 45 30 30 30
45 45 45 45 45 30 45 45 45 60 45
sion maxima for all the catecholamines and isoproterenol as those given by the corresponding DAEs in the meso-form. Optimal conditions for the fluorescence reactions of DAEs with the three catecholamines and isoproterenol are listed in Table 6. Hexacyanoferrate (III) caused almost maximum fluorescence intensity at 2.5-63 mM for the meso-pMOED derivatives but at 25-63 mM for the others; 25 mM was selected as optimum. Concentrations of DAE ranging from 5 to 20 mM in the solution added gave almost maximum fluorescence intensity for each amine; 10 mM was used as an appropriate concentration. Optimum pH values of DAE solution were 6.5-7.0 for norepinephrine, epinephrine and isoproterenol but 6.0-6.5 for dopamine; pH 6.5 was selected. With all the DAEs investigated, the fluorescence reaction occurred above 0’ C; higher temperature allowed the fluorescence to develop more rapidly. However, temperatures higher than 70’ C caused reduction of the fluorescence, probably because of decomposition of the produced fluorophores. In most cases the fluoresence intensites at 37 ‘C and 50’ C reached their maxima after standing for 45 min and 30 min, respectively. Warming at 37” C for 45 min was selected for reproducible results. Stability of the fluorescences and precision of the procedure The fluorescences developed from the amines under the recommended procedure did not change on irradiation for 10 min at their excitation maxima,
81 81 99 153 142 20 60 137 54 20 24
69 93
92
400 395 400 405 409 400 400 400 410 395 400 400 400 400
520 515 515 515 510 530 525 530 520 525 530 520 520 520
6 28 4 4 6 4 4 4 13 14 5 5 2 3
450 460 450 450 450 455 450 450 470 500 450 450 450 450
335 345 340 340 340 345 345 340 350 355 345 340 335 355
17 10 46 28 43 42 0 0 24 21 81 0 0 0
355 365 355 360 355 350 355 360 365 375 360 355 355 355 500 485 480 480 475 480 475 485 550 480 490 475 485
490
N-Methyldopamin
I em (nm)
1.. bun)
RF1
3,4-Dihydroxybenzylamine
74 14 62 93 123 115 117 109 49 35 63 98 99 135
124 62 44 24
66 94 92 a2 94 89 167 141 ::
RFI
360 360 370 350 355 355 350 350 360 375 360 360 360 360
L-DOPA
350 350 350 360 355 340 345 355 360 370 350 350 350 460
A,, (run)
460 415 490 455 460 450 465 450 490 465 465 460 460 460
460 460 460 480 475 445 455 470 490 510 460 460 460 460
I em (nm)
3,4-Dihydroxymandelic acid
5 9 1 7 I 7 4 12 4 12 4 14 9 21
1 2 5 1 2 1 1 1 1 3 0 8 6 8
RFI
450 460 455 450 450 450 445 455 465 505 455 455 435 450
I em (run)
360 365 355 350 360 355 365 360 375 365 360 365 360 360
470 490 480 480 455 465 465 480 480 490 470 470 470 470
cr-methyldopamine
340 355 350 350 350 345 350 350 360 360 350 355 345 340
A,, (run)
16 14 19 15 6 12 4 15 9 13 5 11 7 7
82 88 106 103 106 124 211 120 55 69 97 62 63 94
RF1
3,4_Dihydroxyphenylacetic acid
“Portions (0.1 ml) of 10 nmol ml-’ solutions of the catechol compound were treated as in the procedure. The fluorescence intensity of the meso-DPE derivative of epinephrine was taken as 100.
455 470 455 450 455 445 450 450 475 510 455 450 455 455
355 345 340 340 340 340 345 340 350 360 345 350 335 335
meso-DPE meso-p-CED meso-FIED meso-p-MED meso-EED meso-p-MOED meso-p-EOED meso-DMOED meso-cr-NED meso+NED meso-MOED DL-DPE DL-MOED DL-MED
Adrenalone
3,4-Dihydroxyphenylethyleneglycol
DAE
DL-h4ED
460 470 460 450 460 450 460 460 465 515 455 460 460 460
350 360 350 355 355 355 350 350 355 360 355 350 350 350
450 465 470 450 460 450 450 450 465 505 455 450 450 450
350 350 345 350 350 345 345 340 350 360 345 350 350 350
meso-DPE meso-p-CED meso-FlED meso-p-MED meso-EED meso-MOED meso-p-EOED meso-DMOED meso-a-NED meso-/I-NED meso-MDED DL-DPE DL-MOED
5 13 8 6 14 4 32 22 12 13 15 4 10 0
I,, (run)
1.. (run)
Aem (run)
I ex (nm)
RF1
Protocatechuic aldehyde
Pyrocatechol
DEA
Fluorescence excitation and emission maxima and relative fluorescence intensities (RFI) from catechol compounds with the selected DAEs”
TABLE I
68
and were stable for at least 2 h in daylight. The calibration graphs for the amines prepared by using the fourteen DAEs were linear in the range 15pmol ml-l-20 nmol ml-‘. The precision of the procedures for the three catecholamines and isoproterenol was established on 1 nmol ml-’ solutions. With all the selected DAEs, the relative standard deviation (n= 10) for each amine was less than 2.5%. Fluorescences from other catechol compounds All other catechol compounds tested fluoresced under the recommended conditions. The fluorescence excitation and emission maxima, and the relative fluorescence intensities are shown in Table 7. The excitation and emission spectra of the fluorescences from those compounds other than adrenalone were very similar in shapes and maxima to those from the catecholamines. The maxima for adrenalone were shifted to longer wavelengths; the reason remains unknown. Reaction of biologically important substances other than catechol compounds None of the other biologically important substances examined fluoresced under the recommended conditions at a concentration of 10 nmol ml-l. The compounds tested were seventeen different L-a-amino acids, tyramine, histamine, serotonin, octopamine, creatine, creatinine, uric acid, putrescine, spermidine, spermine, acetone, acetaldehyde, a-ketoglutaric acid, phenylpyruvic acid, oxalic acid, homovanillic acid, acetic acid, D-glucose, D-fructose, D-galactose, D-ribose, D-glucosamine, maltose, sucrose, L-ascorbic acid, guanine, cholesterol and citrate. This suggests that the recommended procedure with DAEs is selective for catechol compounds. In conclusion, meso-DMOED, meso-p-MOED and meso-p-EOED give strong fluorescence to all the catecholamines; the intensities were ca. 1.5times higher than those given by meso-DPE. They should be useful in the determination of catecholamines in small amounts of sample.
REFERENCES 1 H. Nohta, A. Mitsui and Y. Ohkura, Anal. Chim. Acta, 165 (1984) 171. 2 H. Nohta, A. Mitsui, Y. Umegae and Y. Ohkura, Anal. Sci., 2 (1986) 303. 3 A. Mitsui, H. Nohta and Y. Ohkura, J. Chromatogr., 344 (1985) 61. 4 H. Nohta, A. Mitsui and Y. Ohkura, Bunseki Kagaku, 33 (1984) E263. 5 H. Nohta, A. Mitsui, Y. Umegae and Y. Ohkura, Biomed. Chromatogr., 2 (1987) 9. 6 L. M. Nelson and M. Carruthers, J. Chromatogr., 183 (1980) 295. 7 M. N. H. Irving and R. M. Parkins, J. Inorg. Nucl. Chem., 27 (1965) 271. 8 W. A. Sadler and D. A. House, J. Chem. Sot, Dalton Trans. (1973) 1937. 9 F. Vogtle and E. Goldschmitt, Chem. Ber., 109 (1976) 1. 10 S. J. Soldin and J. G. Hill, Clin. Chem., 26 (1980) 291. 11 M. Hamaji and T. Seki, J. Chromatogr., 163 (1979) 329. 12 Y. Yui, T. Yamamoto, Y. Itokawa and C. Kawai, Clin. Chem., 26 (1980) 194.
59
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1,2-DIARYLETHYLENEDIAMINES AS FLUOROGENIC REAGENTS FOR CATECHOLAMINES
YOSHIHIKO
UMEGAE,
HITOSHI
NOHTA and YOSUKE
OHKURA*
Faculty of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812
(Japan) (Received
15th September
1987)
SUMMARY
Twenty-eight 1,2-diarylethylenediamines were investigated in a search for highly sensitive fluorogenic reagents for catecholamines (norepinephrine, epinephrine and dopamine). They all react with epinephrine usedas a model catecholamine under mild conditions (pH 6.5-6.8,37-50°C) in the presence of potassium hexacyanoferrate (III) to produce fluorescence. Of the compounds tested, 1,2-bis(3,4-dimethoxyphenyl)ethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis(4-ethoxyphenyl)ethylenediamine, all in the meso-form, are the most sensitive for all the catecholamines. 1,2_Diphenylethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis (4-methylphenyl)ethylenediamine in the DL-form are more sensitive to dopamine than the corresponding compounds in the meso-form. Catecholamines can be determined at concentrations as low as lo-15 pmol ml-’ by using those compounds as reagents. The reactions of other catechol compounds are reported. Other types of biologically important compounds do not interfere.
In a previous paper [ 11, it was shown that meso-1,2_diphenylethylenediamine (meso-DPE) reacts in a neutral or slightly acidic medium with catecholamines (norepinephrine, epinephrine and dopamine) and other catechol compounds in the presence of hexacyanoferrate (III) to give strongly fluorescent compounds [ 11. The reaction was applied for pre-column derivatization in high-performance liquid chromatography (HPLC) of free and total (the sum of free and conjugated) catecholamines in human erythrocytes [ 21, platelets [2], plasma [2,3] andurine [4,5]. The purpose of the present study is to find more sensitive reagents for catecholamines by screening of various 1,2_diarylethylenediamines (DAEs). Isoproterenol was also used for the screening, because it was used as an internal standard in the HPLC determination of catecholamines in biological samples [2-61.
0003-2670/88/$03.50
0 1988 Elsevier Science Publishers
B.V.
60 TABLE 1 DAEs as fluorogenic Al-
r
x t’
Ar
NH2
hi
Al.
NH2
Aryl group
(in brackets)
.H
x
I’ NH,
’ *NH, /’
meso-form
I’
Ar
reagents and their abbreviations
DL-
form
H
n Name 1,2_Diphenylethylenediamine
C’” /@ (OCH,),
JQ--
C’43
/@-
a 0 /&
Acronym
[ meso-DPE]
1 l,L-Bis (2-chlorophenyl)ethylenediamine 1,2-Bis (3-chlorophenyl)ethylenediamine 1,2-Bis(4-chlorophenyl)ethylenedamine 2 1,2-Bis(2,6-dichlorophenyl)ethylenediamine 1,2-Bis(3,4-dichlorophenyl)ethylenediamine
[ meso-o-CED] [ meso-m-CED] [ meso-p-CED]
1 l,P-Bis(2-methoxyphenyl)ethylenediamine l,P-Bis(3-methoxyphenyl)ethylenediamine 1,2-Bis(4-methoxyphenyl)ethylenediamine 2 1,2-Bis(3,4-dimethoxyphenyl)ethylenediamine
[meso-o-MOED] [meso-m-MOED] [ meso-p-MOED] [ meso-DMOED ]
[meso-2,6-DCED] [meso-3,4-DCED]
1,2-Bis(2-methylphenyl)ethylenediamine 1,2-Bis(3-methylphenyl)ethylenediamine 1,2-Bis(4-methylphenyl)ethylenediamine
[ meso-o-MED [ meso-n-MED [ meso-p-MED
1,2-Bis(3,4-methylenedioxyphenyl)ethylenediamine
[meso-MDED]
1,2-Diphenylethylenediamine
[DL-DPE]
1,2-Bis( 4-methoxyphenyl)ethylenediamine
[ DL-MOED ]
1,2-Bis(4-methylphenyl)ethylenediamine
[DL-MED ]
] ]
]
EXPERIMENTAL
Reagents, solutions and apparatus Norepinephrine bitartrate and dopamine hydrochloride were purchased from Wake Pure Chemicals (Osaka). Isoproterenol hydrochloride and epinephrine bitartrate were obtained from Nakarai Chemicals (Kyoto) and Sigma Chemicals, respectively. All other chemicals were of reagent grade. Deionized/distilled water was used. Each DAE solution (10 mM, apparent pH 6.7) was prepared in acetonitrile/0.02 M hydrochloric acid (1: 1, v/v). Uncorrected fluorescence spectra and intensities were measured with a Hitachi MPF-4 spectrofluorimeter in 10x10 mm quartz cells; spectral band-
61 TABLE 1 (continued)
Name
Acronym
1,2-Bis(2-ethoxyphenyl)ethylenediamine l,P-Bis(4-ethoxyphenyl)ethylenediamine
[ meso-o-EOED] [ meso-p-EOED]
1,2-Bis( 1-naphthyl)ethylenediamine 1,2-Bis(2-naphthyl)ethylenediamine
[ meso-a-NED] [ meso#-NED ]
1,2-Bis(4fluorophenyl)ethylenediamine
[ meso-FlED ]
l,P-Bis(4-ethylphenyl)ethylenediamine
[ meso-EED ]
1,2-Bis(I-cyanophenyl)ethylenediamine
[ meso-CNED]
1,2-Bis( 2-hydroxyphenyl)ethylenediamine
[ meso-HED]
/cl
1,2-Bis(3-pyridyl)ethylenediamine
[ meso-PED ]
A?
1,2-Bis (2-furyl)ethylenediamine
[ meso-FuED ]
l,P-Bis(4-biphenylyl)ethylenediamine
[ meso-BED ]
Aryl group
H
I \
LF 0
0
widths of 10 nm were used in both the excitation and emission monochromators. Mass spectra (MS) were measured with a JEOL JMSD300 interfaced to a JEOL JNM-3500 data system. ‘H-NMR spectra were obtained with a JEOL JNM-PS-110 spectrometer at 100 MHz; approximately 10% (w/v) solutions in CDC& containing tetramethylsilane served as the internal standard. Ultraviolet (UV) spectra were measured with a Hitachi X0-20 spectrophotometer with quartz cells of 0.4 x l.O-cm optical pathlength, and infrared (IR) spectra with a Nihonbunko DS 710G infrared spectrometer in potassium bromide pellets. The pH was measured with a Hitachi-Horiba M-7 pH meter at 25°C. Melting points are uncorrected. Preparation of 1,2-diarylethylenediamines The twenty-eight DAEs investigated are shown in Table 1 with their abbreviations. The meso-DPE compound was synthesized by the method of Irving
62 TABLE 2 Analytical data for newly synthesized DAEs DAE
’
M.P.
(“C)
Appearance (Recrystallization solvent )
Formula
Elemental data (% ) Calcd. (Found) C
H
N
meso-2,6-DCED
160-164 Colorless small crystals (Petroleum ether )
C,,H,,N,Cl,
48.0 (48.0)
3.4 (3.5)
meso-EED
114
Colorless needles (Petroleum ether)
C,,H,,N,
80.6 (80.4)
9.0 (8.7)
10.45 (10.5)
meso-m-MOED
113
Colorless small crystals (Benzene/petroleum ether, 1:4)
CSH~~N&Z
70.6 (70.7)
7.4 (7.2)
10.3 (10.4)
meso-DMOED
189
Colorless small crystals (Ethyl acetate)
GsH24NJb
65.0 (65.3)
7.3 (7.15)
(::i) 9.3 (9.1)
meso-p-EOED
153-154 Colorless needles (Benzene )
C,sH,,N,O,
72.0 (71.7)
8.05 (8.05)
meso-MDED
146-147 Yellowish needles (Benzene/petroleum ether, 1:4)
C,,H,,N,O,
64.0 (64.0)
5.3 (5.4)
meso-a-NED
168-169 Colorless small crystals (Benzene/petroleum ether, 2:3)
CZHZ~NZ
84.6 (84.6)
6.4 (6.5)
meso+NED
174-175 Yellowish crystalline powder C,,H,,N, (Benzene/petroleum ether, 2:3)
84.6 (84.5)
6.4 (6.45)
(k:)
and Parkins [ 71, and meso-o-CED, meso-m-CED, meso-p-CED, meso-3,4DCED and meso-m-MED were synthesized by the method of Sadler and House [ 81. The compounds meso-p-MOED, meso-o-MED, meso-p-MED, meso-oEOED, meso-FlED, meso-CNED, meso-HED, meso-PED, meso-FuED, mesoBED, DL-DPE, DL-MOED and DL-MED were prepared by the method of Vogtle and Goldschmitt [9]. The new compounds (meso-2,6-DCED, meso-EED, meso-m-MOED, meso-DMOED, meso-p-EOED, meso-MDED, meso-c-w-NED and meso$-NED) were synthesized by the method of Vogtle and Goldschmitt [ 91 with minor modifications and their structures were confirmed by the mass, ‘H-NMR, UV and IR spectral and elemental (CHN) data shown in Tables 2 and 3. Procedures Screening of DAEs with epinephrine. To 2.0 ml of DAE solution, 0.1 ml of 10 nmol ml-l epinephrine, 2.0 ml of aqueous 50% acetonitrile and 0.1 ml of 5 mM potassium hexacyanoferrate (III ) were added successively. The mixture was
3.89
633-I) 3.86 (&X-I) 3.86 (s,2H)
3.83 (s,2H) 4.24 (s2I-I) 5.21 (s,2H)
1.48 (s.4H) 1.42 (s,4H) 1.46 (s,4H)
1.36 (s,4H) 1.00-4.12 (sb4I-I) 1.52 (s,4H)
273(M++l)’
332(M+)’
301(M++l)’
301(M++l)’
313(M+ +l)’
312(M+)e
meso-m-MOED
meso-DMOED
meso-p-EOED
meso-MDED
meso-ar-NED
meso+NED
5.94 (s,4H,20CHz)
4.03 (q,4H,20CH,) 1.41 (t,6H,2CH,)
3.88 (s,12H,40CH,)
3,80 (s,6H,20CH,)
284
276
287
275
279
274
272
17 830
10 300
11664
3410
6600
5390
1540
740
emaX
3330, 3400
3330, 3360
3330, 3390
3300, 3370
3240, 3330
3270, 3360
3270, 3340
3330, 3390
LH
1200-1250
1240
1230
1250
ZO-C
IR’ v,,, (cm-‘)
1020-1050
1040
1030
1040
:-o-c
“Chemical shifts are given with peak patterns single (s), triplet (t), quartet (q), multiplet (m), broad (b) and proton numbers in parentheses. bin methanol. ‘KBr pellets. dThe peaks disappeared on adding D,O. “Electron-impact ionization. fField-desorption ionization.
7.21-7.88 (mJ4H)
7.20-7.88 (mJ4H)
W3-I)
6.70-6.93
6.82-7.32 (m8H)
6.79-6.99 (m6H)
6.80-7.34 (m8I-I)
7.08-7.36 (m8H)
2.66 (q,4H,2CH,) 1.24 (t,6H,2CH,)
3.87 (s,2H)
1.32-2.12 (s,bPH)
269(M++l)’
meso-EED
h6I-I)
6.90-7.52
273
5.56 (s6I-I)
1.88 (s,4H)
351(M++l)”
meso-2,6-DCED
1 mar (nm)
Subst.-H
CH-N
NHzd
Arom.-H
UVb
‘H-NMR (in CDC13)’ 6 (ppm)
MS(mIz)
DAE
Spectral data for the new DAEs
TABLE 3
%
64 TABLE 4 Excitation and emission maxima (A,,, &.,) of the fluorescences obtained from epinephrine with DAEs by the screening procedure, and their relative fluorescence intensities” (RFI) DAE
a BI (run)
I *In (nm)
RF1
DAE
1 ex (nm)
I em (nm)
RF1
meso-DPE meso-HED meso-o-CED meso-m-CED meso-p-CED meso-3,4-DCED meso-2,6-DCED meso-FlED meso-o-MED meso-m-MED meso-p-MED meso-EED meso-o-MOED meso-m-MOED
360 360 355 360 355 370 370 360 355 355 355 355 345 355
480 480 495 485 495 505 500 475 415 410 475 475 470 480
100 115 76 105 110 109 7 96 123 127 152 148 110 138
meso-p-MOED meso-DMOED meso-o-EOED meso-p-EOED meso-CNED meso-MDED meso-PED meso-a-NED meso-B-NED meso-FuED meso-BED DL-DPE DL-MOED DL-MED
355 355 355 355 400 355 390 365 370 350 360 360 355 355
470 470 475 470 560 470 515 510 530 470 510 480 470 475
158 176 122 178 32 166 30 70 52 85 4 112 158 144
“The fluorescence intensity obtained with meso-DPE was taken as 100.
TABLE 5 Excitation and emission maxima of the fluorescences from catecholamines and isoproterenol with the selected DAEs, and their relative fluorescence intensities” DAE
meso-DPE meso-p-CED meso-FlED meso-p-MED meso-EED meso-p-MOED meso-p-EOED meso-DMOED meso-a-NED meso-p-NED meso-MDED DL-DPE DL-MOED DL-MED
Norepinephrine
Epinephrine
Dopamine
Isoproterenol
1 ex (nm)
I,, (nm)
RFI A,, (nm)
A.,,, (nm)
RFI 1,. (nm)
A,,,, (nm)
RFI a,, I,, (run) (nm)
350 360 355 360 355 350 350 355 365 360 350 350 350 360
475 486 410 465 470 460 460 470 505 525 475 475 460 465
100 132 125 136 110 147 142 155 59 44 138 117 139 122
480 490 415 470 475 475 470 470 510 530 485 480 475 470
78 82 88 84 73 92 96 92 38 27 88 79 87 82
465 500 470 475 465 470 465 470 505 530 490 465 470 475
53 31 42 57 54 68 70 71 12 8 49 67 18 69
360 360 355 355 355 355 355 355 360 370 365 360 355 355
360 370 355 350 360 350 355 355 365 365 360 360 350 350
365 375 365 370 360 365 365 365 375 375 365 365 365 370
475 495 465 470 415 470 415 470 515 530 495 475 470 470
“The fluorescence intensity of meso-DPE derivative of norepinephrine was taken as 100.
RF1 132 102 138 158 134 172 166 176 72 41 148 155 167 134
65
allowed to stand at 37°C for 45 min to develop fluorescence. A reagent blank was prepared in the same way except that the 0.1 ml of epinephrine solution was replaced with 0.1 ml of water. The fluorescence intensities of the test and blank were measured at respective excitation and emission maxima (see Table 4). Fluorimetric determination of catecholumines with DAEs. To 0.1 ml of aqueous test solution, 2.0 ml each of DAE solution and aqueous 50% (v/v) acetonitrile, and 0.1 ml of 25 mM potassium hexacyanoferrate (III) were successively added. The mixture was maintained at 37’ C for 45 min. To prepare the reagent blank, the same procedure was applied except that 0.1 ml of the test solution was replaced by 0.1 ml of water. The fluorescence intensities were measured at the respective excitation and emission maxima (see Table 5). RESULTS AND DISCUSSION
Screening of DAEs as fluorogenic reagents for catecholamines For the screening of the DAEs, epinephrine was used as the model compound, because this amine occurs at very low concentrations in urine [lo] and blood [ 11,121. The fluorescence excitation and emission spectra of the DAE derivatives of epinephrine were very similar in shape to those from the mesoDPE derivative of epinephrine [ 41, but the excitation and emission maxima differed depending on the DAE derivative used (Table 4). The maxima for the meso-3,4-DCED, meso-2,6-DCED, meso-CNED, meso-PED, meso-a-NED, meso-P-NED and meso-BED derivatives were at longer wavelengths. These reagents may therefore offer higher selectivity in the determinations of catecholamines in biological samples, because weaker native fluorescences were generally observed in the samples when they were measured at longer emission wavelengths. The DL-DAEs gave the same fluorescence spectra as those obtained with the corresponding meso-DAEs. Electron-donating groups introduced to the 4-position of the phenyl groups of DAEs caused higher fluorescence intensities from epinephrine; meso-DMOED, meso-p-MOED and meso-pEOED gave stronger fluorescences in that order. By considering the above observations, 14 compounds (Table 5) were selected for further investigations. Reaction of catecholamines and isoproterenol with the selected DAEs and optimal conditions The selected DAEs did not give very great differences in the excitation and emission maxima among the catecholamines and isoproterenol (Table 4). The meso-DMOED, meso-p-MOED and meso-p-EOED derivatives gave higher fluorescence intensities in that order for all the catecholamines and isoproterenol. The DAEs in the DL-form were more sensitive for dopamine than the corresponding DAEs in the meso-form, and provided the same excitation and emis-
66 TABLE 6 Optimal conditions for the fluorescence reactions of the selected DAEs with norepinephrine, epinephrine andisoproterenol. Optimal conditions for dopamine are given in parentheses, if different. DAE
&Fe(CNh meso-DPE meso-p-CED meso-FlED meso-p-MED meso-EED meso-p-MOED meso-p-EOED meso-DMOED meso-a-NED meso&NED meso-MDED DL-DPE DL-MOED DL-MED
pH of DAE solution
Concentration
(mM)
DAE (mM)
25-63 25 25-63 25 25-63 2.5-63 25 25 25 25-63 25-63 25 25 25-63
10-15 10 5-10 10 5 5-15 10 5-10 10-20 10 15 20 10 5-15
(2.5-63) (25-63)
(25)
6.5-7.0 (5)
(10) (5)
K-7.0 7.0 6.5-7.0 6.5-7.0 7.0 6.5 7.0 6.5 6.5 6.5-7.0 7.0 7.0
(6.5) (6.0) (6.0-6.5) (6.0) (6.5) (6.5) (6.0) (6.5) (6.0) (6.0) (6.5) (6.0) (6.0)
Reaction time (min) 37°C
50°C
45 ii:
30 30 30 30 30 30 30 30 30 30 45 30 30 30
45 45 45 45 45 30 45 45 45 60 45
sion maxima for all the catecholamines and isoproterenol as those given by the corresponding DAEs in the meso-form. Optimal conditions for the fluorescence reactions of DAEs with the three catecholamines and isoproterenol are listed in Table 6. Hexacyanoferrate (III) caused almost maximum fluorescence intensity at 2.5-63 mM for the meso-pMOED derivatives but at 25-63 mM for the others; 25 mM was selected as optimum. Concentrations of DAE ranging from 5 to 20 mM in the solution added gave almost maximum fluorescence intensity for each amine; 10 mM was used as an appropriate concentration. Optimum pH values of DAE solution were 6.5-7.0 for norepinephrine, epinephrine and isoproterenol but 6.0-6.5 for dopamine; pH 6.5 was selected. With all the DAEs investigated, the fluorescence reaction occurred above 0’ C; higher temperature allowed the fluorescence to develop more rapidly. However, temperatures higher than 70’ C caused reduction of the fluorescence, probably because of decomposition of the produced fluorophores. In most cases the fluoresence intensites at 37 ‘C and 50’ C reached their maxima after standing for 45 min and 30 min, respectively. Warming at 37” C for 45 min was selected for reproducible results. Stability of the fluorescences and precision of the procedure The fluorescences developed from the amines under the recommended procedure did not change on irradiation for 10 min at their excitation maxima,
81 81 99 153 142 20 60 137 54 20 24
69 93
92
400 395 400 405 409 400 400 400 410 395 400 400 400 400
520 515 515 515 510 530 525 530 520 525 530 520 520 520
6 28 4 4 6 4 4 4 13 14 5 5 2 3
450 460 450 450 450 455 450 450 470 500 450 450 450 450
335 345 340 340 340 345 345 340 350 355 345 340 335 355
17 10 46 28 43 42 0 0 24 21 81 0 0 0
355 365 355 360 355 350 355 360 365 375 360 355 355 355 500 485 480 480 475 480 475 485 550 480 490 475 485
490
N-Methyldopamin
I em (nm)
1.. bun)
RF1
3,4-Dihydroxybenzylamine
74 14 62 93 123 115 117 109 49 35 63 98 99 135
124 62 44 24
66 94 92 a2 94 89 167 141 ::
RFI
360 360 370 350 355 355 350 350 360 375 360 360 360 360
L-DOPA
350 350 350 360 355 340 345 355 360 370 350 350 350 460
A,, (run)
460 415 490 455 460 450 465 450 490 465 465 460 460 460
460 460 460 480 475 445 455 470 490 510 460 460 460 460
I em (nm)
3,4-Dihydroxymandelic acid
5 9 1 7 I 7 4 12 4 12 4 14 9 21
1 2 5 1 2 1 1 1 1 3 0 8 6 8
RFI
450 460 455 450 450 450 445 455 465 505 455 455 435 450
I em (run)
360 365 355 350 360 355 365 360 375 365 360 365 360 360
470 490 480 480 455 465 465 480 480 490 470 470 470 470
cr-methyldopamine
340 355 350 350 350 345 350 350 360 360 350 355 345 340
A,, (run)
16 14 19 15 6 12 4 15 9 13 5 11 7 7
82 88 106 103 106 124 211 120 55 69 97 62 63 94
RF1
3,4_Dihydroxyphenylacetic acid
“Portions (0.1 ml) of 10 nmol ml-’ solutions of the catechol compound were treated as in the procedure. The fluorescence intensity of the meso-DPE derivative of epinephrine was taken as 100.
455 470 455 450 455 445 450 450 475 510 455 450 455 455
355 345 340 340 340 340 345 340 350 360 345 350 335 335
meso-DPE meso-p-CED meso-FIED meso-p-MED meso-EED meso-p-MOED meso-p-EOED meso-DMOED meso-cr-NED meso+NED meso-MOED DL-DPE DL-MOED DL-MED
Adrenalone
3,4-Dihydroxyphenylethyleneglycol
DAE
DL-h4ED
460 470 460 450 460 450 460 460 465 515 455 460 460 460
350 360 350 355 355 355 350 350 355 360 355 350 350 350
450 465 470 450 460 450 450 450 465 505 455 450 450 450
350 350 345 350 350 345 345 340 350 360 345 350 350 350
meso-DPE meso-p-CED meso-FlED meso-p-MED meso-EED meso-MOED meso-p-EOED meso-DMOED meso-a-NED meso-/I-NED meso-MDED DL-DPE DL-MOED
5 13 8 6 14 4 32 22 12 13 15 4 10 0
I,, (run)
1.. (run)
Aem (run)
I ex (nm)
RF1
Protocatechuic aldehyde
Pyrocatechol
DEA
Fluorescence excitation and emission maxima and relative fluorescence intensities (RFI) from catechol compounds with the selected DAEs”
TABLE I
68
and were stable for at least 2 h in daylight. The calibration graphs for the amines prepared by using the fourteen DAEs were linear in the range 15pmol ml-l-20 nmol ml-‘. The precision of the procedures for the three catecholamines and isoproterenol was established on 1 nmol ml-’ solutions. With all the selected DAEs, the relative standard deviation (n= 10) for each amine was less than 2.5%. Fluorescences from other catechol compounds All other catechol compounds tested fluoresced under the recommended conditions. The fluorescence excitation and emission maxima, and the relative fluorescence intensities are shown in Table 7. The excitation and emission spectra of the fluorescences from those compounds other than adrenalone were very similar in shapes and maxima to those from the catecholamines. The maxima for adrenalone were shifted to longer wavelengths; the reason remains unknown. Reaction of biologically important substances other than catechol compounds None of the other biologically important substances examined fluoresced under the recommended conditions at a concentration of 10 nmol ml-l. The compounds tested were seventeen different L-a-amino acids, tyramine, histamine, serotonin, octopamine, creatine, creatinine, uric acid, putrescine, spermidine, spermine, acetone, acetaldehyde, a-ketoglutaric acid, phenylpyruvic acid, oxalic acid, homovanillic acid, acetic acid, D-glucose, D-fructose, D-galactose, D-ribose, D-glucosamine, maltose, sucrose, L-ascorbic acid, guanine, cholesterol and citrate. This suggests that the recommended procedure with DAEs is selective for catechol compounds. In conclusion, meso-DMOED, meso-p-MOED and meso-p-EOED give strong fluorescence to all the catecholamines; the intensities were ca. 1.5times higher than those given by meso-DPE. They should be useful in the determination of catecholamines in small amounts of sample.
REFERENCES 1 H. Nohta, A. Mitsui and Y. Ohkura, Anal. Chim. Acta, 165 (1984) 171. 2 H. Nohta, A. Mitsui, Y. Umegae and Y. Ohkura, Anal. Sci., 2 (1986) 303. 3 A. Mitsui, H. Nohta and Y. Ohkura, J. Chromatogr., 344 (1985) 61. 4 H. Nohta, A. Mitsui and Y. Ohkura, Bunseki Kagaku, 33 (1984) E263. 5 H. Nohta, A. Mitsui, Y. Umegae and Y. Ohkura, Biomed. Chromatogr., 2 (1987) 9. 6 L. M. Nelson and M. Carruthers, J. Chromatogr., 183 (1980) 295. 7 M. N. H. Irving and R. M. Parkins, J. Inorg. Nucl. Chem., 27 (1965) 271. 8 W. A. Sadler and D. A. House, J. Chem. Sot, Dalton Trans. (1973) 1937. 9 F. Vogtle and E. Goldschmitt, Chem. Ber., 109 (1976) 1. 10 S. J. Soldin and J. G. Hill, Clin. Chem., 26 (1980) 291. 11 M. Hamaji and T. Seki, J. Chromatogr., 163 (1979) 329. 12 Y. Yui, T. Yamamoto, Y. Itokawa and C. Kawai, Clin. Chem., 26 (1980) 194.