Fluorometric Determination of Secondary Amines with 2-Methoxy-2,4-diphenyl-3(2H)-f uranone Hlroshl Nakamura,’ Etsuko Tanil, and Zenzo Tamura Department of Analytical Chemistty, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3- 1, Hongo, Bunkyo-ku, Tokyo 1 13, Japan
Reiko Yoda and Yulchl Yamamoto Laboratory for Medicinal Chemlstty, Kyorisu Coliege of Pharmacy, 1-5-30, Shlbakoen, Mingto-ku, Tokyo 105, Japan
2-Methoxy-2,4-diphenyi-3(2H)-furanone (MDPF) was found to react with secondary amines to give nonfiuorescent products (FII‘). F I I ’ was shown to be converted to fluorescent compounds (FI’) which were produced by the reaction of primary amines with MDPF. On the bask of the examinations of the conditions for the formation of F I I ’ and the conversion of F I I ‘ to FI‘ using sarcosine as the model of secondary amine, a spectrofluorometric method has been developed for the determinatlon of secondary amines by using MDPF. Secondary amines were reacted with MDPF at pH 10 and 20 OC for 45 min and then incubated at pH 9 and 45 OC for 10 min with taurine. By measurement of the bluish green fluorescence (Aex 390 nm, A, 480 nm), most secondary amlnes were determined at nanomole levels. The relative standard deviation (n = 4) of the method is 2.48% for the analyses of 25 nmoi of sarcosine.
Scheme I
RNH2
/
Fll
Scheme I1 Fluorescamine (FLA) reacts with primary and secondary amines to produce fluorescent pyrrolinone (FI) and aminodienone-type chromophores (FII), respectively (1-4). Recently, we have found that FII is easily converted by primary amines to FI (Scheme I), and developed a thin-layer chromatographic method for the fluorescence detection of secondary amines (5)and a fluorometric method for the determination of them (6). On the basis of these reactions, we attempted the simultaneous fluorescence detection of primary and secondary amines by high-performance liquid chromatography (HPLC). 2-Methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), an analogous reagent to FLA, is also known (7)to react with primary amines to produce fluorescent adducts (FI’) (Scheme 11). MDPF and FLA are similar in that they and their hydrolysis products are nonfluorescent (8). Weigele et al. (9, 10) reported that l-(dimethylamino)-2,4-diphenyl-l-butene3,4-dione (R, = Rz = CH3 in FII’; Scheme 11) reacted with primary amines to give FI’. Therefore, if MDPF can react with secondary amines to produce FII’ in the similar way as the case of FLA, MDPF may be used as the fluorogenic reagent for secondary amines as well. Furthermore, it is reasonable that FII’ will be more stable than FII, just as FI’ is more stable than FI (8). An additional advantage of FII’ over FII will be the probable ease of the reversed-phase HPLC separation due to the absence of the carboxyl group. This assumption may be supported by the fact that the reversedphase HPLC separation of small peptides failed with FLA derivatives but was successful with MDPF derivatives (11,12). In this paper, the reaction of MDPF with secondary amines (FII’ formation), stability of FII’, and the conversion of FII’ by primary amines to FI’ were investigated by using sarcosine as the representative secondary amine, which led to the development of a fluorometric method for the determination of secondary amines at the nanomole level based on the reactions depicted in Scheme 11. 0003-2700/82/0354-2482$01.25/0
MDPF 9.
Fll’
EXPERIMENTAL SECTION Apparatus. The following were used a Hitachi 650-10 grating spectrophotofluorometer equipped with a 150-W xenon lamp and 1-cm quartz cells; a UVIDEC-505 UV/VIS digital spectrophotometer (Japan Spectroscopic Co., Tokyo, Japan). Fluorescence data are reported without spectral correction. Materials. MDPF and L-epinephrine bitartrate were purchased from Tokyo Kasei (Tokyo, Japan). DL-Metanephrine hydrochloride, L-proline, and L-Chydroxyprolinewere purchased from Nakarai Chemicals (Kyoto,Japan). N-Phenylbenzylamine was purchased from Aldrich (Milwaukee, WI). Acetonitrile was purchased from Kanto Chemical (Tokyo, Japan). Other chemicals and solvents used were of the highest purity commercially available and their sources were described previously (6). Preparation of Stock Solutions of Amines. Ten millimolar stock solutions of the amines were prepared with distilled water whenever possible or with methanol. Distilled water was used to prepare dilute solutions. Standard Assay Procedure for Secondary Amines. A 100-pL aqueous sample solution (containing 2.5-50 nmol of 0 1982 Amerlcan Chemical Society
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Table I. Excitation ( h e x )and Emission ( h e m )Maxima of Primary Amine Induced Fluorescence of FII' Derived
FII'
from Various Secondary Aminesa
10
h,y/h,,b nm taurine ~ a n i l i n e p-toluidine sarcosine 3901480 400/500 4201510 3901480 400/500 4201510 L-proline m orpholine 3901480 400/500 4201510 N-methyl-Dglucamine 3901480 4001500 4201510 2-ethylethanolamine 3 901480 4001500 4 201510 3901480 4001500 4201510 N-methylaniline 3901480 400/500 4201510 benzimidazole 3901480 400/500 4201510 diphenylamine N-methylbenzylamine 3901480 4001500 4201510 N-phenylbenzylamine 3901480 4001500 4201510 ~-
FII' from
WAVELENGTH (nm)
Figure 1. Absorption spectra of the reaction niixture of sarcosine and MDPF after the addition of taurlne. Final concentrations of sarcosine, MDPF, and taurine were 2.5, 0.18, and 125 mM, respectively. The figure indicates the reaction times in minutes. secondary amine) was transferred to a 12 X 75 mm glass test tube and 100 pL of 0.05 M sodium borate buffer at pH 10.0 was added. While the solution was vigorously stirred on a vortex-type mixer, 100 pL of 0.72 mM M I P F solution in acetonitrile was added rapidly. The test tube was further agitated for 10 s and then placed in a water bath equilibrated at 20 "C. After 45 min, the mixture was added to 100 pL of 125mM taurine solution prepared with 0.2 M sodium borate buffer (pH 9.0), incubated immediately at 45 " C for 10 min in a water bath, and then allowed to stand in an ice bath until fluorescence measurement. After addition of 2.0 mL of 0.2 M sodium borate buffer (pH 9.0) and transfer to a quartz cell, fluorescence was measuired at 480 nm using 390-nmexcitation, with 10-nm bandwidth for both monochromators, against a reagent blank containing no secondary amine. Fluorescein dissolved in 0.2 M sodium borate buffer (pH 9.0) was used as a reference for fluorescence measurements to correct for the probable instability of the xenon source. The relative fluorescence intensity was defined as the ratio of the fluorescence intensity (uncorrected) measured at 390/480 nm to that of M fluorescein at 490/510 nm. The amount of secondary amine was calculated from working curves of fluorescence intensity (ordinate) vs. molarity of amine (abscissa).
RESULTS Evidences for the Formation of FIX' and for the Conversion of FII' to FI'. Secondary amines dissolved in buffer did not fluoresce following reaction with MDPF over a pH range from 2 to 12. The reaction mixtures exhibited ahsorption maxima at around 310 nm similar to those of FII (310-330 nm). When the reaction mixture of sarcosine and MDPF was added to taurine, incubated a t p H 9 and 40 O C , and the absorption spectra were measured at appropriate intervals, a new absorption peak appeared at 380 nm and increased with time, with concomitant decrease of the peak a t 310 nm (Figure 1). The final reaction mixture gave identical fluorescence (A,$= 390 nm, hm480 nm) and absorption (Amm 380 nm) characteristics to those of MDPF-labeled taurine. Similar results were obtained when aniline or p-toluidine was used as the primary amine instead of taurine (Table I). From these observations and by analogy with the reactivities of MDPF to FLA witlh primary and probably secondary amines, the intermediates formed in the reaction of secondary amines with MDPF were tentatively considered to be FII', which was convertible to FI' by primary amines. Conditions for Formation of FII' from Secondary Amines and MDPF. The reaction of sarcosine with MDPF, expressed as the ratio of the fluorescence intensity of sample (Fa)to that of blank (Fb), was greatly influenced by p H as shown in Figure 2, and no substantial amounts of FII' were
a 100 pL each of 1 mM secondary amine, 0.05 M borate buffer (pH 10) and 7.2 mM MDPF were mixed together and incubated at 20 "C for 45 min. The mixture was added to 0.1 m L of 0.5 M primary amine dissolved in 0.2 M borate buffer (pH 10) and incubated at 40 "C for 1 0 min. The fluorescence spectra were measured after the addition of 2 m L of 0.2 M borate buffer (pH 10). hex/ hem for authentic MDPF-labeled primary amine: 3901 480 nm (taurine), 400/500 nm (aniline) and 4201510 nm @-toluidine). 4 ,
- 0
2
4
8
6
1 0 1 2
PH
Figure 2. Effect of pH on the reaction of sarcosine with MDPF. Ten millimolar sarcoslne was treated with the standard assay procedure except that the reactlon was conducted at 40 O C .
1' Flgure 3. Effect of MDPF concentratlon on the formation of FII'. One millimolar sarcoslne was treated with the standard assay procedure except that the MDPF concentratlon was variable.
formed in buffers below pH 8 or above p H 12. A pH of 10 (borate buffer) was tentatively chosen for the formation of FII'. The amount of FII' formed increased with increasing concentration of MDPF (Figure 3). However, use of higher concentrations of MDPF resulted in a decrease in F a / F b . The ratio was maximal when 100 p L each of 0.72 mM MDPF, 1
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7-
6-
u"
-
5-
2
4-
32-
MINUTES
Flgure 4. Time courses of the reaction of sarcosine with MDPF at
various temperatures. Ten milllmolar sarcosine was treated with the standard assay procedure except that the temperature was variable.
2ot1Ob
pn:z
33
90
60
120
MINUTES
Flgure 6, Stability of FII' derived from sarcosine at various pHs. One
millimolar sarcosine was treated with the standard assay procedure. The resultant reaction mlxture containing FII' was added to 20 mL of a 0.2 M buffer and allowed to stand at room temperature.
MINUTES
Figure 5. Relationship between the blank fluorescence and the reaction time for the MDPF reactlon at varlous temperatures. Distllled water instead of 10 mM sarcosine was treated as in Figure 4. 2
mM sarcosine, and 0.05 M borate buffer (pH 10.0) were reacted. Therefore, the concentration of MDPF was settled in the following experiments to be 0.72 mM, which coincided with the concentration typically used for the assay of primary amines. Figure 4 depicts the time courses of the reaction of sarcosine with MDPF at pH 10 and various temperatures. Figure 5 shows the relationship between the relative fluorescence intensities of the corresponding blanks and the reaction period under the same conditions as in Figure 4. As can be seen in Figures 4 and 5 at below 10 " C , both the formation of FII' and the hydrolysis of MDPF were incomplete. Whereas the reactions at higher temperatures accelerated both the reaction of sarcosine with MDPF and the hydrolysis of the reagent, it decreased the value of F,/Fb probably because of the decomposition of FII'. Therefore, the incubation at 20 O C for 45 min was chosen for the formation of FII' from sarcosine and MDPF. When monitored at 310 nm, the FII' derived from sarcosine was stable at room temperature for at least 2 h at pH 7-9; however, the adduct was decomposed at above pH 10 with increasing pH and reaction period (Figure 6). Conversion of FII' by Primary Amine to FI'. Taurine was arbitrarily chosen as the primary amine for the conversion of FII' to FI'. The effects of pH and temperature on the conversion reaction with taurine were examined by using the reaction mixture of sarcosine and MDPF. As shown in Figure 7, the efficiency of the conversion expressed as F,/Fb was maximal when the reaction was performed at pH 8-10 and 20-80 "C. The blank fluorescence was increased with increasing temperature, being always maximal when the reaction pH was 10. The fluorescence of the reaction mixture of taurine and FII' was also enhanced as the reaction temperature was increased. However, the optimal pH for the conversion reaction was gradually shifted from 10 to 8 with increasing
4
8
6
1
0
1
2
PH
Flgure 7. Effect of pH on the conversion reaction at various temperatures. Final concentrations of sarcosine, MDPF, and taurine were 0.43, 0.03, and 0.87 mM, respectlvely.
-14
.-. -3
3 ?
z
2
TAURINE ( m M )
Flgure 8. Effect of taurine concentration on the conversion reaction at 45 OC for 60 min. The mixture containing FII' was prepared wlth
10 mM sarcosine under the standard assay procedure and added to 0.1 mL of 0.2 M borate buffer (pH 9.0) containing various concentrations of taurine.
temperature, which seemed to be due to the accelerated decomposition reaction of FII' with OH- ion at elevated temperatures. In view of the stability of FII' and the efficiency of conversion, we decided to perform the conversion reaction at pH 9 and around 40 O C . The effect of the concentration of taurine on the fluorescence yield was investigated next by reacting at 45 " C and pH 9 for 60 min. As shown in Figure 8, the formation of FI' increased with increasing taurine concentration. However, since F,/Fb gradually decreased with
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
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88&
--__I__I _ -
Table 11. Relative Fliorescence Intensities of Various Secondary Amines in the Present Procedure compounda morpholine DL-epinephrine N-methylbenzylamine 2-ethylethanolamine L-proline sarcosine
RFIb 196 186
164 115
compounda N-phenylbenzylamine benzimidazole diphenyl amine N-methyl-D-
Scheme ILK1
RFI
91
LOOc
L-4-hydroxyproline DL-metanephrine
82 77 60 46 27
N-methylaniline 100 A 0.25 mM stock ,solution was assayed. Fluorescence was measured at 390 nm excitation and 480 nm emission. Sarcosine is arbitrarily taken as 100. a
_ I -
C H ~ ~ H
R
NIRl
i ,
p
_-_I-
