Anal. Chem. 1980, 52, 2087-2092
29). Such lasers exceed the present laser in performance of the pulse repetition rate and in average power. However, the transverse gas flow, TEA nitrogen laser and its pumped dye laser have distinct advantages with respect to the pulse energy per pulse, simplicity and a low cost for the construction. We should stress that a tunable wavelength region of the present system extends over from the ultraviolet to visible region while the mode-locked, cavity-dumped system is limited to rhodamine 6G dye region practically. In the ultratrace analysis, lasers are very useful as the exciting sources for the fluorometric analysis, since the fluorescence intensity is proportional to the intensity of the exciting source. However, laser fluorometry is so sensitive that the detection limit is not always determined by the sensitivity of the apparatus but determined by the background signal from contaminant fluorescence ( 4 ) . In order to reduce this solution blank, the temporal separation of the components has been achieved for the samples with a long fluorescence lifetime such as pyrene (30) and polycyclic aromatic hydrocarbons ( 3 I ) ,since the emission lifetime of the background signal is relatively short. The TEA nitrogen laser developed in this study s e e m to be promising for the temporal separation of the components with nanosecond fluorescence lifetimes.
ACKNOWLEDGMENT The authors wish to thank M. Ichishima for his assistance in the preparation of the sample solution. The authors also wish to thank F. E. Lytle for his helpful suggestions about the high-speed photomultiplier. LITERATURE CITED (1) Bradley, A. 6.; Zare, R. N. J . Am. Chem. SOC.1976, 98,620-621. (2) Geel, T. F. V.; Winefordner, J. D. Anal. Chem. 1976, 48,335-338. (3) Richardson, J. H.; George, S. M. Anal. Chem. 1978, 50, 616-620.
2087
(4) Ishibashi, N.; Ogawa, T.; Imasaka, T.; Kunitake, M. Anal. Chem. 1979, 57,2096-2099. (5) Lin, C.; Shank, C. V. Appl. f h y s . Lett. 1975,26, 389-391. (6) Cubeddu, R.; Polloni, R.; Sacchi, C. A. Appl. Phys. 1977, 73, 109-110. (7) Wyatt, R. Opt. Commun. 1978,26, 429-431. (8) Bor, Zs.;Opt. Commun. 1979, 29, 103-108. (9) Strohwald, H.; Salzman, H. Appl. Phys. Lett. 1976, 28, 272-274. (10) Bauer, R. K.; Kowalczyk, K. Opt. Commun. 1977,23, 169-170. (11) Bergmann, E. E. Appl. fhys. Lett. 1977,37, 661-663. (12) Hugenschmidt, M.; Vollrath, K. Opt. Commun. 1978, 26, 415-418. (13) Patel. B. S. Rev. Sci. Instrum. 1976, 49, 1361-1363. (14) Maeda, M.; Yamashita, T.; Miyazoe, Y. Jpn. J . Appl. Phys. 1976, 17, 239-240. (15) Hasson, V.; von Bergmann, H. M. Rev. Scl. Instrum. 1979,5 0 , 59-63. (16) Hugenschmidt, M.; Wey, J. Opt. Commun. 1979, 29, 191-196. (17) Cubeddu, R.; De Slkestri, S. Opt. Quantum Electron. 1979, 7 7 , 276-277. (18) Kobayashi, T.; Sugimura, K.; Inaba, H. Opt. Quantum Electron. 1979, 1 7 , 373-376. (19) Imasaka, T.; Ogawa, T.; Ishibashi, N. Anal. Chem. I97B,5 1 , 502-504. (20) Targ, R. IEEE J . Quantum Electron. 1972, QE-8, 726-728. (21) Gcdard, 0. IEEE J . Quantum Electron. 1974, QE- 70, 147-153. (22) Beck, G. Rev. Sci. Instrum. 1976, 4 7 , 537-541. (23) Kina, K.; Tamura, K.; Ishibashi, N. Bunseki Kagaku 1974, 23, 1404- 1406. (24) Wright, J. C., University of Wisconsin, personal communication. (25) Hiraki, K.;Morishige, K.; Nlshikawa, Y. Anal. Chim. Acta 1978, 97, 121-128. (26) Leskovar, 6.; Lo, C. C.; Hartig, P. R.; Sauer, K. Rev. Sci. Insfrum. 1978, 4 7 , 1113-1121. (27) Aussenegg, F.; Leitner, A,; Opt. Commun. 1980, 32, 121-122. (28) Spears, K. G.; Cramer. L. E.; Hoffland, L. D.Rev. Sc!. Insfrum. 1978, 49,255-262. (29) Koester, V. J.; Dowben, R. M. Rev. Sci. Instrum. 1978,49,1186-1191. (30) Kunitake, M.; Imasaka, T.; Ishibashi, N., submitted for publication in Nippon Kagaku Kaishi. (31) Dickinson, R. B., Jr.; Wehry, E. L. Anal. Chem. 1979, 5 7 , 778-780.
RECEIVED for review April 8, 1980. Accepted July 25, 1980. This research is supported by a Grant-in-Aid for Scientific Research (Grant No. 347054,575501) and for Environmental Science (Grant No 303046) from the Ministry of Education of Japan.
Fluorometric Determination of Secondary Amines Based on Their Reaction with Fluorescamine Hiroshi Nakamura * and Zenzo Tamura Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3- 1, Hongo, Bunkyo-ku,
A spectrophotofluorometric method has been developed for the determination of secondary amines by using fluorescamlne. When compounds having a secondary amino group were reacted with fluorescamlne at pH 12 and then heated at 70 OC for 10 min with a primary amine (L-Leu-L-Ala), they gave a bluish green fluorescence. By measurement of the fluorescence, most secondary amines could be determined in 0.5 nmol quantities. Relative standard deviations of 4.4 and 5.2 YO are observed for the analyses of 5 nmol of N-methylanlllne and sarcosine, respectively.
Scheme I
", 1
FI
11
0
Fluorescamine is known to react almost instantaneously with primary and secondary amines to give fluorescent pyrrolinones (FI) ( I , 2) and nonfluorescent aminoenones (FII) ( 3 , 4 ) respectively , (Scheme I). Fluorescamine itself is rapidly hydrolyzed to nonfluorescent products under reaction conditions ( I ) . We have previously reported both general (5-7) and selective (8-12) methods for the analysis of primary amino 0003-2700/80/0352-2087$01 .OO/O
Tokyo 1 13, Japan
Fluorescami n e
R'>" R2
aq?J COOH
FI I
groups with fluorescamine. In this paper, we plan to utilize fluorescamine for the fluorometric determination of secondary amines. We found that FII is readily converted by the reaction with primary amines to FI (Scheme 11). The reaction of 0 1980 American Chemical Society
2088
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
Scheme I1
the net fluorescence intensity ( F J obtained by method A represents the sum of their fluorescence intensities. Therefore, the amount of secondary amine was estimated by subtracting Fpfrom F,, where Fpwas the expected fluorescence intensity in method A corresponding to the amount of primary amine determined in advance by the method B procedure for the determination of primary amines. FI
secondary amines with fluorescamine (aminoenone formation reaction) and the reaction of FII with primary amines (conversion reaction) were therefore examined in detail. This led to the development of a fluorometric method for the determination of secondary amines on the basis of their reaction with fluorescamine. EXPERIMENTAL SECTION Apparatus. The following were used: a Hitachi MPF-2A grating spectrophotofluorometer equipped with a xenon lamp and 1-cm quartz cells; a water bath circulator (Model BT-35, Yamato Scientific, Tokyo, Japan); a Toa HM-5 pH meter (Toa Denpa Kogyo, Tokyo, Japan). Fluorescence data are reported without spectral correction. Materials. Fluorescamine (Fluram) was purchased from Nippon Roche (Tokyo, Japan). L-Proline, skatole (grade I), LLeu-L-Ala, DL-metanephrine hydrochloride, DL-epinephrine, N-acetyl-L-tryptophan, N-methyltryptamine, N,N-dimethyltryptamine, melatonin, tryptamine hydrochloride, imidazoleacetic acid hydrochloride, tyramine hydrochloride, DL-normetanephrine hydrochloride, and spermine tetrahydrochloride were purchased from Sigma (St. Louis, MO). N-Methylaniline, sarcosine, N acetyl-D-P-phenylalanine,taurine, diphenylamine, acetaminophen, sulfanilic acid, metanilic acid, p-aminosalicylic acid sodium salt dihydrate, benzylamine, @-indoleaceticacid potassium salt, N methyl-D-glucamine, N-ethylaniline, diethanolamine hydrochloride, imidazole, benzimidazole, carbazole, N-ethylethanolamine, N-methyltaurine sodium salt, N-methylformamide, N methylbenzylamine, pyrrole, N-acetyl-DL-a-alanine,morpholine, and hippuric acid sodium salt were purchased from Tokyo Kasei (Tokyo, Japan). y-Amino-n-butyric acid, histamine (free base) (Nakarai Chemicals, Kyoto, Japan), monoethanolamine and p-dimethylaminobenzaldehyde (Kanto Chemical, Tokyo, Japan) were used. Other chemicals and solvents used were of the highest purity commercially available. Preparation of Stock Solutions of Amines. Ten-millimolar stock solutions of the amines were prepared with distilled water whenever possible or with methanol. One-tenth-millimolar stock solutions were prepared by adding a 100-pLaliquot of the 10 mM solution to 9.99 mL of distilled water. Distilled water was used to prepare dilute solutions. Assay Procedure for Secondary Amines Alone (Method A). A 50-pL aqueous sample solution (containing 0.5-5 nmol of secondary amine) was transferred to a 12 x 75 mm glass test tube and 50 FL of 0.05 M Kolthoff s buffer (NaOH-Na2HP04) at pH 12.0 was added. While the test tube was vigorously stirred on a vortex-type mixer, 100 pL of acetone solution of fluorescamine (20 mg/100 mL) was added rapidly. The test tube was further stirred for 10 s and then placed in an ice bath. After 5 min, the mixture was mixed with 1.5 mL of 0.67 mM L-Leu-L-Aladissolved in 0.2 M sodium phosphate buffer, pH 6.60 (hereafter designated as converting agent), heated immediately at 70 "C for 10 min in a water bath, and then allowed to stand in an ice bath until the fluorescence measurement. The fluorescence intensity was measured at 390 nm excitation and 480 nm emission, with 10-nm bandwidth for both monochromators, against a reagent blank containing no secondary amine. The amounts of secondary amines were calculated from working curves of fluorescence intensity (ordinate) vs. nanomoles of amine (abscissa). Assay Procedure for Secondary Amines in the Presence of Primary Amine (Method B). Determination of Primary Amines. The amount of primary amine in the sample was determined with the procedure described above (method A) except for the omission of L-Leu-L-Alafrom the converting agent. Determination of Secondary Amines. When the sample solution contains primary amine in addition to secondary amine
RESULTS P r e l i m i n a r y Examination of the Fluorescence Reaction of Secondary Amines. Secondary amines dissolved in buffers did not fluoresce following reaction with fluorescamine over a p H range from 5 to 11. However, the nonfluorescent reaction mixtures which contained aminoenone-type chromophores (FII)fluoresced bluish green under a long-wave (365 nm) UV lamp immediately after the addition of a primary amine. The blank without secondary amine also yielded a similar color of fluorescence, especially when acidic buffers were used. This was thought to be obviously due to the reaction of the primary amines added with nonhydrolyzed fluorescamine since its hydrolysis rate (13) was reported to be almost proportional to the hydroxide ion concentration in media. However, even in that case, the fluorescence intensity of the sample was much greater than that of the blank. The present reaction was shown t o be positive with compounds having a RIRzNH functional group (R, = alkyl or allyl, R2= alkyl or allyl) including N-monomethylamino acids. Compounds with a secondary amino group in the ring system such as derivatives of indole, imidazole, and morpholine also showed positive responses. Although some N-acetyl compounds could be detected by the present fluorogenic reaction, general amides of carboxylic acid, peptide bonds, tertiary and quaternary amines, and other compounds not containing the nitrogen atom were negative. Upon reaction with aniline, the FII derived from various secondary amines gave the same color of fluorescence with spectral characteristics identical with those of fluorescamine-labeled aniline, though different fluorescence intensities were obtained depending on the secondary amines. This was shown to hold true when glycine or spermine was used as the primary amine instead of aniline (Table I). These data strongly suggested the conversion of FII by primary amine into FI. Table I also shows that the fluorescence intensity induced from FII depends on which primary amine was used. In this connection, some aromatic primary amines such as p-aminoacetophenone, p-aminobenzoic acid, and 2-aminopyridine which gave no fluorescence by the reaction with fluorescamine could not induce fluorescence from FII. Furthermore, tert-butylamine which gave weak fluorescence on reaction with fluorescamine, due obviously t o the steric hindrance of the primary amino group, induced very weak fluorescence from the FII tested. Including these typical results, the primary amines whose reaction mixture with fluorescamine fluoresced intensely also generally induced intense fluorescence from FII. Accordingly, the primary amines which gave intense fluorescence on reacting with fluorescamine were first tested for use as the converting agent of FII to FI. Screening of P r i m a r y Amine Candidates f o r the Conversion Reaction. Over 50 primary amines including amino acids, biogenic amines, and aromatic amines were reacted with fluorescamine at p H 7 , 8, 9, and 10, and their fluorescence characteristics were examined. In phosphate (pH 7 and 8) or borate (pH 9 and 10) buffer solutions containing 5.9% acetone, pyrrolinone fluorophores (FI) of the amines showed excitation maxima a t 388-408 nm and emission maxima at 480-516 nm and their spectral characteristics, except for intensities, were not significantly altered over this p H range. Of these primary amines L-Leu-L-Ala,taurine, rn-aminobenzoic acid, sulfanilic acid, and metanilic acid which gave intense
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
2089
TAURINE(Tau)
L-Leu - L - A l a
7 W
W
0
.2
W 0
W
W LJ I n W
8 NAcPhe Prc Sar
5
6
7
8
[L
0
0
3 LL
3
3 LL
3
z
W 0 m W LL
M W
W
?,L
[L
3
V W
z
Z
LL
NMeA
9 1 0 1 1
6
5
7
Pr
8
5
9 1 0 1 1
6
8
7
PH
9
5
1011
6
8
7
9
1011
PH
P i
METANILIC ACID (MA)
SULFANILIC ACID ( S A )
1
W U
z
W
0 Lo W
U E
[L
0
0
NMeA
3
LL
5
6
7
8 PH
9
1011
5
6
8
7
9
1
1 0 "
5
6
7
8
9
1
0
I
PH
?H
m-AMINOBENZOIC ACID(m-ABA)
1 7 I
W U Z W In 0 W
1
Lo
iIL
C Y
~
0
0
3 LL
3
Sar NAcPhe
4
5
6
7 Pr
8
9
'
0
L
5
6
7
8
9
DH
Figure 1. Effect of pH on the conversion of FII to F I by L-Leu+-Ala, taurine, sulfanilic acid, metanilic acid, and rn-aminobenzoic acid. The primary amine induced fluorescence was measured by using the following excitation and emission maxima: 390/480 nm (L-Leu+-Ala and taurine), 398/496 nm (sulfanilic acid), 400/494 nm (metanilic acid), and 401/502 nm (rn-aminobenzoic acid). Buffers used were 0.2 M sodium phosphate (open symbols) and 0.2 M sodium borate (closed symbols). Abbreviations used are as follows: Sar, sarcosine; Pro, L-proline; NAcPhe, N-acetyl+@phenylalanine; Imd, imidazole; NMeA, N-methylaniline; AAP, acetaminophen; SK, skatole.
fluorescence were selected as tentative candidates for the converting agent of FII to FI. Conversion of FII by the Primary Amine Candidates. The effect of pH on the conversion of FII to FI by the primary amine candidates was examined by using FII derived from sarcosine, L-proline, N-acetyl-D-6-phenylalanine, N-methylaniline, acetaminophen, skatole, and imidazole. One milliliter each of 0.1 mM secondary amine and 0.05 M sodium borate buffer (pH 7.0) were mixed together and then rapidly mixed with 2.0 mL of fluorescamine in acetone (20 mg/100 mL) with vigorous stirring. After 5 min, a 200-fiL aliquot of the reaction mixture was added to 1.5mL of 0.2 M sodium phosphate or 0.2 M sodium borate buffer with various pHs and 100 pL of 10 mM primary amine, and the mixture was heated a t 90 "C for 5 min. As shown in Figure 1,the conversion a t 90 "C for 5 min was optimal between pH 5.5 and pH 9 depending on
Figure 2. Effect of temperature on the conversion of F I I to FI by (a) L-Leu-L-Ala or (b) sulfanilic acid.
2090
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
Table I. Fluorescence Characteristics of Primary Amine Induced Fluorescence of Aminoenones (FII)" re1 fluorescence primary amine
aminoenone from
excitation max, nm
emission max, nm
intens
20.6 387 (388)b 480 (480)b 11.2 392 (392)b 482 (483)b 6.4 504 ( 5 0 5 ) b 401 (402)b 53.5 N-acetyl-D -p -phenylalanine 389 479 10.6 482 39 2 19.9 505 403 N-methy lan iline glycine 481 100 382 spermine 19.7 484 391 aniline 504 29.7 40 2 skatole glycine 78.5 480 387 spermine 17.8 483 392 aniline 3.3 505 402 a 50 FL each of 0.1 mM secondary amine and 0.2 M sodium borate buffer (pH 9.0) were mixed together and then rapidly mixed with 100 pL of fluorescamine (20 mg/100mL) in acetone with vigorous stirring. After standing at room temperature f o r 1 0 min, the mixture was mixed with 1.5 mL of 0.2 M sodium borate buffer (pH 9.0) and 100 pL of 1 0 niM primary Excitation or emission maxima for authentic pyrrolinone amine and incubated at 90 "C for 5 min to induce fluorescence. fluorophores (FI). Values are corrected for respective blanks and are expressed in arbitrary units. sarcosine
I
glycine spermine aniline glycine spermine aniline
Sar
Pq
L-Leu-L-Ala ( m M )
Figure 3. Effect of concentration of L-Leu-L-Ala on the conversion reactions.
the combination of both FII and primary amine. The temperature of the conversion reaction markedly influenced the fluorescence yield. Figure 2 shows typical results obtained with L-Leu-L-Ala and sulfanilic acid. When various FIIs were reacted with L-Leu-L-Ala a t p H 6.6, a temperature of 70 "C was optimal in all cases (Figure 2a). In contrast, in the conversion reaction with sulfanilic acid at pH 6.0 the temperature which gave maximal fluorescence ranged from 30 to 70 "C depending on individual FII (Figure 2b). At this stage, LLeu-L-Ala was considered the best as the converting agent based on the following criteria: (i) it induced maximal fluorescence at the same temperature (70 "C) from all FII tested; (ii) of primary amine candidates it gave the largest signal-to-noise (S/N) ratio in conversion reactions; (iii) the fluorescence intensity of the fluorophore (fluorescamine-labeled L-Leu-L-Ala) it induced from FII was independent of p H over a wide range (pH 5.5-10.5). The effect of the concentration of the converting agent on the fluorescence yield was investigated next by reacting several FII at 70 "C and p H 6.6 for 5 min. As shown in Figure 3, the formation of fluorophores increased with increasing concentration of L-LeuL-Ala up to 100 mM. However, since the S/N ratio gradually decreased with increasing L-Leu-L-Alaconcentration, a concentration of 10 mM was chosen for these experiments. The conversion reactions of FII with both 10 and 100 mM LLeu-L-Ala were completed within 10 min a t 70 "C and pH 6.6. There was a tendency toward decreased fluorescence with prolonged incubation at 70 "C. However, the final fluorophore induced by L-Leu-L-Ala was quite stable when the reaction mixture was stored at 0 "C, with no decrease in fluorescence intensity for a t least 3 h. F o r m a t i o n of FII f r o m S e c o n d a r y A m i n e s a n d Fluorescarnine. The optimal pH for the reaction of secondary amines with fluorescamine differed greatly depending
Figure 4. Effect of pH on the formation of FII. One-tenth millimolar solution of secondary amine was treated by method A procedure except that (a) 0.05 M sodium phosphate, (b) 0.05 M sodium borate, or (c) 0.05 M sodium hydroxide solution was used for the formation of F I I instead of 0.05 M Kolthoff's buffer (pH 12.0).
'OI ~
-; I ' I
Figure 5. Effect of fluorescarnine concentration on the formation of FII. The concentration of secondary amines assayed was (a) 0.1 mM and (b) 0.01 mM.
on the nature of the amines, as shown in Figure 4, and no substantial amounts of FII were formed in buffers below pH 5 or above pH 13.5. A pH of 12 (Kolthoffs buffer) was selected for the FII formation reaction since the reaction of primary amines with fluorescarnine was markedly suppressed a t p H 12. The rate formation of FII at two concentrations of secondary amines (0.1 and 0.01 mM) at pH 1 2 increased with increasing concentration of fluorescamine (Figure 5). However, use of higher levels of fluorescamine decreased the S / N ratio, especially in the case of sarcosine. Therefore, the concentration of fluorescamine typically used for the assay of primary amines, 20 mg/100 mL (0.72 mM), was chosen for these experiments. Under the conditions determined above, i.e., the labeling of secondary amines in pH 12 Kolthoffs buffer with 0.72 mM fluorescamine reagent, FII formation became
7 1 -
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
Table 11. Relative Fluorescence Intensities of Various Secondary Amines in the Present Procedure
NAcPhe
-
compound"
RFIb
N-methyltryptaniine skatole diphenylamine N-phenylbenzylamine N-methyl-
171 167 153 146
142
benzylamine
melatonin benzimidazole
142
PH Flgure 6. Effect of pH on the stabili of FII. Buffers used are (a)0.05 M sodium phosphate and (b) 0.05 M sodium borate. DH
w
0
z
0
w
0
*
E
L
m
5
6.60
DH 12.0
Sar NAcPhe NMeA
SK AAP
'li I\
compound" N-methyltaurine DL-meta-
50 47
sarcosine
35 35
134 133
morpholine imidazole
pyrrole
115
N-methylanthranilic
106
37
35 31 6
1 02
N-acetyl-DL-aalanine iriiidazoleacetic acid N-acetylglycine N-acetyl-DL-
100d
N-methyl-
0
fornianiide hippuric acid
0
acid
N-acetyl-D-0phenylalanine N-inethylaniline acetaminophen
53
DL-epinephrine diethanolamine
N,N-dimethyltryptamine N-e thylaniline N-acetyl-Ltryptophan
130
RFIb
nep hrine
N-methyl-DJucamine N-ethylethanolamine
140 140
carbazole
0-
2091
4
2
0 0
valine
98
" A 0.1 mM stock solution was assayed. Fluorescence was measured at 390 nm excitation and 480 nm emission. Native fluorescence (excitation maximum at 338 nia and emission maximum at 413 n m ) was also observed. N-Methylaniline is arbitrarily taken as 100.
\
B, sarcosine was successfully determined in the presence of benzylamine: the mean values of duplicate assays of 1 nmol of sarcosine were 0.98 and 1.03 nmol in the presence of 2 and 4 nmol of the primary amine, respectively. 0204060K
DISCUSSION Flgure 7. Effect of temperature on the stability of FII at various pHs. Buffers used are 0.05 M sodium phosphate (pH 6.60),0.05 M sodium borate (pH 10.0), and 0.05 M Kolthoff's (pH 12.0).
constant within 5 min a t both 0 and 30 "C. Stability of FII. T h e effect of p H on the stability of FII was examined by incubating them a t 30 "C for 15 min in buffers of various pHs. As shown in Figure 6, none of the FII tested was stable in acidic media. With the exception of FII derived from sarcosine, they were most stable a t p H 9.5-11. Figure 7 depicts the effect of temperature on the stability of FII a t p H 6.6, 10.0, and 12.0, indicating the susceptibility of FII to temperature. Although FII was stable a t 0 "C and p H 12.0 for a t least 30 min, FII formed from aromatic secondary amines gradually degradated at p H 6.60 even when they were kept a t 0 "C.It was therefore necessary to start the conversion reaction at 70 "C immediately after adding the converting agent in buffer a t p H 6.60. Quantitation of Secondary Amines. On the basis of the above findings, the recommended procedure described in the Experimental Section was established. With skatole, Nmethylaniline, and sarcosine, a plot of fluorescence intensity vs. amount of compound was linear with 0.5 to 5 nmol. T h e standard deviations (n = 10) were 0.22 nmol for 5.0 nmol of N-methylaniline and 0.26 nmol for 5.0 nmol of sarcosine. Table I1 summarizes the relative fluorescence intensities of various secondary amines obtained with the standard procedure (method A). Although the procedure adopted p H 12.0, some primary amines still fluoresced considerably; the relative fluorescence intensities of benzylamine, tyramine, normetanephrine, taurine, sulfanilic acid and L-Leu-L-Ala were 86,20, 20,19,12, and 10, respectively, when that of N-methylaniline was arbitrarily taken as 100. Therefore, in the presence of primary amines, method B should be used. By use of method
Two methods are available for the analysis of secondary amines by their reaction with fluorescamine. One is the colorimetric method consisting of measurement of the absorption of the aminoenone chromophores (FII) which have an absorption maximum a t 310-330 nm (e 16ooCrl8000) ( 4 ) . T h e other is the fluorometric method which involves the conversion of secondary amines with N-chlorosuccinimide to primary amines followed by reaction with fluorescamine, permitting the determination of ca. 4 nmol (14). The present fluorometric assay permits the determination of 0.5 nmol of secondary amines and is more sensitive than the previous methods (4, 14). The conversion reaction of FII to FI by primary amines on which the present assay is based is similar to the reaction reported by Weigele e t al. (15, 16) on the synthesis of fluoroscamine. They reported that l-dimethylamino-2,4-diphenyl-l-butene-3,4-dione (FII') reacted with primary amines to give fluorescent pyrrolinones (FI'). However, they also reported that the reaction was restricted to nonaqueous systems because of the rapid hydrolysis of FII' (16). On the other hand, FII is fairly stable a t p H 9.5-11 and reacts with primary amines to yield F I quantitatively even in aqueous solutions if heated a t p H 6.60. According to Weigele et al. (16, 17), FII is converted to fluorescamine upon acidification a t p H 3.5-4.0. Therefore, there is a possibility that fluorescamine is produced from FII in the present assay by the addition of the acidic converting agent (pH 6.60) to react with L-Leu-L-Ala. However, our data show that no appreciable amount of fluorescent products was formed by the reaction of FII with the converting agent a t pH 6.60 without heating (Figure 2), whereas fluorescamine was able to react quickly with L-Leu-L-Ala to give fluorescence a t this pH. Thus, the conversion of FII by primary amines
2092
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
Scheme I11
h?
/’
I1
m
FI
elevated temperature produces fluorescent pyrrolinone (FI) as shown in Scheme IV. In fact, in the course of development of a thin-layer chromatographic (TLC) method (20) for the detection of secondary amines based on the present fluorogenic reaction, we found that the hydrolysis product of fluorescamine separated on a TLC plate fluoresced after spraying with primary amines followed by heating. In the course of the preparation of our manuscript, Castell et al. (21) also reported a similar observation. The probable occurrence of this reaction (Scheme IV) may be disadvantageous in the present assay; however, this drawback may be eliminated in methods involving a separation step. A high-performance liquid chromatographic method for the determination of secondary amines consisting of the separation of FII, postcolumn conversion to FI, and fluorometric detection is under investigation in our laboratory.
LITERATURE CITED
to FI a t elevated temperature seems to take place directly rather than via fluorescamine, as shown in Scheme 111, in which the pathways of formation and degradation of FII postulated by Weigele et al. ( I 7, 18) are also included. The degradation of FII via carbinolamine (CA) to zwitterionic 2,4-dioxopyrrolidine derivatives (Z) is probably a main source of the instability of FII observed in this work. The incomplete conversion of secondary amines to FI via FII seems to be due to the presence of a series of equilibrium reactions (Scheme 111) as well as the incomplete labeling of primary amines with fluorescamine as stressed by Chen et al. (19). As with most other fluorometric assays, the fluorescence in the blank limited the sensitivity of the present assay. The excitation and emission spectra of the blank fluorescence were always identical with those of the FI derived from the primary amine which was added. The fluorescence of the blank increased with increasing concentration of fluorescamine or of the primary amine. Therefore, the relatively high blank (equivalent to ca. 50 pmol of N-methylaniline) obtained in the present assay seems to be due to the reaction of unhydrolyzed fluorescamine with a large amount of L-Leu-L-Ala. However, our data show that fluorescamine was completely hydrolyzed after a 5-min incubation in the p H 12 buffer for the aminoenone formation and that the blank fluorescence increased with increasing temperature of the conversion reaction while it was negligible without heating. These data strongly suggest that the reaction of the hydrolyzed product of fluorescarnine with a large excess of primary amine a t
(1) Udenfriend, S.; Stein, S.; Bohlen, P.: Dairman. W.; Leimgruber, W.; Weigele, M. Science 1972, 778, 871-872. (2) DeBernardo, S.; Weigele, M.; Toome, V.; Manhart, K.; Leimgruber, W. Arch. Biochem. Blophys. 1974, 763, 390-399. (3) Felix, A. M.; Toome, V.; DeBernardo, S.;Weigele. M. Arch. Bbchem. Biophys. 1975, 768, 601-608. (4) Toome, V.; Manhart, K. Anal. Let?. 1975, 8 , 441-448. (5) Nakamura, H.;Pisano, J. J. J. Chromatogr. 1978, 127, 33-40. (6) Nakamura, H.;Pisano. J. J. J. Chromatogr. 1078, 721, 79-81. (7) Nakamura, H.; Pisano, J. J. Arch. Biochem. Blophys. 1976, 772,
102-105.
H.; Pisano, J. J. Arch. Biochem. Blophys. 1978, 772, 98-101. Nakamura, H.;Pisano, J. J. J. Chromatogr. 1978, 752, 153-165. Nakamura, H.; Pisano, J. J. Arch. Bbchem. Blophys. 1978, 777, 334-335. Nakamura, H. J . Chromatogr. 1977, 737, 215-222. Nakamura, H.;Pisano. J. J. J. Chromatogr. 1978, 754, 51-59. Stein, S.; Bohlen, P.; Udentriend, S. Arch. Blochem. Blophys. 1974, 763, 400-403. Weigele, M.; DeBernardo, S.; Leimgruber, W. Biochem. Biophys. Res. Common. 1973, 50, 352-356. Weigele, M.; Biount, J. F.; Tengi, J. P.; Czajkowski, R. C.; Leimgruber, W. J . Am. Chem. SOC. 1972, 94, 4052-4054. Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber, W. J . Am. Chem. Soc. 1972, 94, 5927-5928. Weigele, M.; Tengi, J. P.; DeBernardo, S.; Czajkowski, R.; Leimgruber, W. J . Org. Chem. 1978, 41, 388-389. Weigele, M.; Czajkowski, R.; Blount, J. F.; DeBernardo, S.; Tengi, J. P.; Leimgruber, W. J. Org. Chem. 1978, 47, 390-392. Chen, R. F.; Smnh, P. D.; Maw, M. Arch. Biochem. Biophys. 1978, 789, 241-250. Nakamura, H.;Tsuzuki, S.; Tarnura, 2.; Ycda, R.; Yamamoto, Y. J . Chromatogr., in press. Castell, J. V.; Cervera, M.; Marco, R. Anal. Biochem. 1979, 99, 379-39 1.
(8) Nakamura,
(9) (10) (11) (12) (13) (14) 15) 16) 17) 18) 19) (20) (21)
RECEIVED for review May 27, 1980. Accepted July 22, 1980. Presented in part a t the 100th Annual Meeting of the Pharmaceutical Society of Japan, Tokyo, April 2-5, 1980.
