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
1 ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
155
Table I. Composition of the Reaction Mixtures o-Diacetylbenzene
Fluoregenic reagent, ml Mercaptoethanol solution, ml Borate buffer,ml Amino acid solution, ml
o-Phthaldialdehyde
Fluores.
camine
0.05
0.05
0.75
0.05
0.05
0.00
3 (pH 10) 0.05
3 (pH 9.5) 0.05
2.35 (pH 9 ) 0.05
Procedure. The fluorometric assay of amino acids were performed following the procedures given by Roth (23)for both o-diacetylbenzene and o-phthalaldehyde,and by Udenfriend et al. (27) for fluorescamine. The composition of the reaction mixtures is shown in Table I. The reaction is initiated by placing the amino acid into a buffer solution of the reagent; the final amino acid concentration is then 8 11M.
RESULTS When the reaction mixtures (Table I) are excited with radiation a t a wavelength of 350 nm, a bluish (DAB) or yellow (OPT or fluorescamine) fluorescence develops quickly and reaches a significant intensity. In a first step, we have determined the excitation and the fluorescence spectra; the Natural and artificial AAP share with amino carboxylic second step was devoted to the kinetics of emission a t seacids very similar analytical properties: color reactions are lected exciting wavelengths, and to the measurement of fluobserved in both series with alloxan (6),o-diacetylbenzene orescence intensities. (7), or ninhydrin; the ninhydrin reaction is currently used Excitation and Fluorescence Spectra. o-Diacetylbenfor paper (8) or column (9) chromatographic detection of zene. The characteristic wavelengths, both for excitation AAP. Some N-substituted derivatives (10) may also repreand for fluorescence, were shown to be identical in the sent valuable tools for the chromatographic identification phosphonic and in the carboxylic series, the wavelengths of AAP (11). for the maxima of the excitation and emission spectra apFor several years, fluorescence reactions have been acpearing, respectively, a t 355 and 445 nm. When the correcttively investigated in the field of the analytical chemistry ed emission spectra accessory is used, the 445-nm value is of amino compounds, especially with regards to amino carslightly shifted toward longer wavelengths, in the range boxylic acids. Dansyl chloride, 5-di-n-butylaminonaph455-460 nm (Figure 1);an increase of the fluorescence inthalene-1-sulfonyl chloride (12) and 7-chloro-4-nitrobenzotensity is concurrently observed. 2-oxa-1,3-diazole (13) are currently used as fluorogenic reSlightly different fluorescences were observed only with agents. Under special conditions, ninhydrin, the classical the two following compounds, both of which possess two amino acid color reagent, can give rise to fluorescence reacprimary amino groups: 1,2-diaminoethylphosphonic acid, tions, as mentioned by Breton (14) and by Lowe (15). InA,, 410 nm, A, 430 nm; 1,3-diaminopropylphosphonic creased fluorescence is observed when n-butyraldehyde is acid, ,A, 335 nm, A,, 445 nm. The corresponding carboxylic added to the reaction medium (16) or when phenylalanine compounds show a more typical behavior: 2,3-diaminoproreacts in the presence of small peptides (17). Studying the pionic acid, A,, 355 nm, A,, 445 nm; 2,4-diaminobutyric mechanism of the fluorescence reaction, Samejima et al. acid, ,A, 360 nm,, ,A 425 nm. (18) and Weigele et al. (19, 20) could identify the active o-Phthaldialdehyde (OPT).The same observations prefluorogenic molecule to be 4-phenylspiro[furan-2(3H),lviously noted with o-diacetylbenzene apply to the fluoresphthalan]-3,3'-dione; this new reagent, now known as fluocence characteristics of AAP reacting with O P T (Aex 340 rescamine (11), allows the assay of amino acids, peptides, = 465 nm). The spectra are nm; A,, 455 nm; corr. A, proteins, and primary amines in the picomole range. Two shown in Figure 2. When the dialdehyde is used as a fluoother carbonyl compounds, o -diacetylbenzene (III), and orogenic agent, the particularities of the emission and fluophthaldialdehyde (IV) are known for their aptitude to give rescence spectra described by Roth (23) for 2,3-diamifluorescence with amino acids and proteins (21, 22). Their nopropionic acid (A,, 395 nm;, ,A 475 nm) are even exaggereactions have been recently investigated by Roth (23-25). rated for its phosphonic analog 1,2-diaminoethylphosphonic acid (A,, 470 nm; A,, 515 nm). FI Fluorescarnine. Completely analogous results (Figure 3) were observed in both phosphonic and carboxylic series 0 0 (Aex 390 nm;, ,A 475 nm; A,, corr. 490 nm). Kinetics of the Fluorescence Reactions. In order to I1 I11 I\' compare the fluorescence reactions given by the three carIn this paper, the fluorescence reactions of fluorescambonyl reagents, the kinetics obtained, using the simplest ine, o-diacetylbenzene (DAB), and o-phthaldialdehyde compounds of both series, aminomethylphosphonic acid (OPT) have been applied to hitherto uninvestigated AAP. and glycine, are illustrated in Figure 4. EXPERIMENTAL The intensity of the fluorescence given by DAB increases gradually to reach a plateau; the intensity corresponding to Apparatus. A MPF 3 Perkin-Elmer fluorescence spectrophothis plateau can be easily measured between 10 and 20 min tometer including two grating monochromators and a corrected (curves 1 and 2). spectra accessory was used throughout this work. Reagents. 0-Diacetylbenzene (Schuchardt, Munich, Germany) The kinetics observed with OPT as the fluorogenic was used as a methanolic solution (35 mg/ml); o-phthaldialdehyde species appear quite different: the intensity is very high as and 2-mercaptoethanol(Fluka, Buchs, Switzerland) were dissolved soon as the reagents are mixed, and then rapidly decreases in absolute ethanol (respectively 10 mg and 5 111 per ml); fluores(curves 3 and 4). With fluorescamine, an immediate fluocamine (Hoffmann-LaRoche, Nutley, N.J.) in anhydrous acetone rescence is also observed, but its intensity remains practi(0.3 mg/ml). cally constant for a t least 1 hr (curves 5 and 6). The 0.2 M borate buffer (pH 9) was prepared from boric acid, 0.05 M borate buffers (pH 9.5 and 10) from sodium tetraborate. With every carbonyl reagent, a lower fluorescence intenAminophosphonic and amino carboxylic acids were purchased sity was recorded with aminomethylphosphonic acid; a from Calbiochem (Los Angeles, Calif.). The phosphonic analogs of comparable lower reactivity of the phosphonic series was citrulline and arginine were synthetized in our laboratory (26). noted with other amino compounds, either for DAB- or The amino acids were used in aqueous 0.5 mM solutions. OPT-induced fluorescence. All other reagents or chemicals were of the highest analytical These kinetics data clearly show that valuable informagrade (Uvasol grade, Merck, Darmstadt, in every possible case). 156
ANALYTICAL CHEMISTRY, VOL. 48, NO. l,JANUARY 1976
L
Figure 1. Reaction of o-diacetylbenzene with aminomethylphosphonic acid (AMPh) and glycine (Gly). Fluorescence excitation (left)and corrected emission (right) spectra.
Figure 3. Reaction of fluorescamine with aminomethylphosphinic acid (AMPh) and glycine (GLy). Fluorescence excitation (left) and corrected emission (right) spectra
I
2
3
4 5
Figure 2. Reaction of o-phthaldialdehyde with aminomethylphospho-
nic acid (AMPh) and glycine (Gly). Fluorescence excitation (left)and corrected emission (right) spectra tion on fluorescence reactions obtained in both series of amino acids does not need a complete kinetics study. The time at which the highest fluorescence intensities are observed can be used. Fluorescence Intensities. According to the kinetics data, the fluorescence intensity values observed with DAB were measured a t 15 min. Because of the rapidly decreasing fluorescence induced by OPT, much shorter times of observation were selected: the maximum value was obtained in every case after 1 or 2 min. The time of observation with fluorescamine has obviously no great importance: 5-min time was routinely chosen. The fluorescence intensities for the two groups of amino acids (final concentration, 8 wLM) are listed in Table 11. The sensitivity of the spectrofluorimeter was fixed at the same level for all measurements, so that the quantitative data given are comparable. Our results are reproducible within the range f 5 % . Fluorescence can also be measured using much more diluted amino acid solutions, since the sensitivity of the spectrofluorimeter may be increased up to 100fold.
DISCUSSION DAB-Induced Fluorescence. In both phosphonic and carboxylic series, the recorded fluorescence intensities are closely related to the molecular structure of the amino acid, in agreement with the results previously obtained by Roth (23) for usual amino acids. Within the group of the simpler amino acids, aminomethylphosphonic acid and glycine are the only molecular species leading to an intense fluorescence; as already pointed out, the fluorescence intensity of the phosphorus-containing compounds is somewhat lower.
L
time (mini
Figure 4. Kinetics of fluorescence development Maximum oeaks, n m
(1) DAB + glycine ( 2 ) DAB + aminomethyl-
phosphonic acid (3) OPT + glycine (4) OPT + aminomethylphosphonic acid (5) Fluorescamine + glycine ( 6 ) Fluorescamine + aminomethylphosphonic acid
Excitation
Emission
355 355
445 445
340 340
455 455
390
47 5
390
475
The results of fluorescence studies then strictly parallel the results obtained when DAB is used as a chromogenic reagent. Polyfunctional Amino Acids. The introduction into the molecular structure of a simple amino acid of a further acidic group (aspartic and glutamic acids, as well as their phosphonic analogs), does not lead to an enhancement of fluorescence intensity. Reversely, the amino acids possessing two amino groups were shown to be highly reactive: with 1,4-diaminobutyl- and 1,5-diaminopentylphosphonic acids, a significant fluorescence was recorded. Furthermore, the fluorescence intensity was somewhat higher with the two carboxylic analogs, ornithine and lysine. The particular behavior of 1,2-diaminoethylphosphonic acid must be emphasized. Compared with the majority of the other amino acids, as we have already mentioned, different excitation and emission maxima were observed; moreover, the fluorescence intensity was about fourfold ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
157
Table 11. Fluorescence of Different Amino Acids with o-Diacetylbenzene, ohhthaldialdehyde, and Fluorescamine (hex Used is Maximum) Fluorescence intensity in arbitrary units at max hem OPT, X =
DAB, X = Amino acid structurea
PO3H2
COOH
Simple &-aminoacids 75 H,NCH,X (Gly) 61 2 H,NCH(X)CH, (Ala) 2 H,NCH(X)CH, CH, 1 1 H; NC ( X )( cH ,jc H ,1.5 3 1.5 1.5 H2NCH(X)(CH2 H,NCH(X)CH(CH,), (Val) 4 2.5 5 3 H~NCH(X)(CH,),CH,’ ’ 2 H,NCH(X)CH,C,H, (Phe) 1 1.5 H,NCH(X)CH,C,H, OH (Tyr) 1.5 Polyfunctional amino acids 1.5 1.5 H,NCH(COOH)CH,X (Asp) 1.5 H,NCH(COOHNCH,),X 1 11.5 1 H;NC€I(X)CH,CH,COOH (Glu) 1 H,NCH(X)CH,CH,X 50 H,NCH(X)CH,NH, 230 H;NCH(X)( CH, ),NH2 6.5 6 49 H,NCH(X)(CH,),NH, (Orn) 32 71 42 H,NCH(X)(CH, (LYs) H,NCH(X)(CH,),NHCONH, 3 2.5 H,NCH(X)(CH,),NHC(NH)NH, (Arg) 2.5 3 w-Amino acids 58 38 H,N(CH,),X @-Ala) 42 63 H,N(CH,),X (GABA) a The usual abbreviation is given only for the carboxylic series.
}
Table 111. Relative Fluorescence of Some Sulfur Aminoacids with DAB, OPT, and Fluorescamine
H,NCH,CH,SO,H (taurine) H,NCH,CH,CH,SO,H (homotaurine) H,NCH(COOH)CH,SO,H .~ icysteic acid)’ H,NCH(COOH)CH,CH,SO,H lhomocvsteic acid)
Fluorescamine
DAB
OPT
58 75
100 90
1
77
7.5
1
94
9
11 11
higher than the data recorded with 2,3-diaminopropionic acid, every experimental condition being the same. Finally, when 1,2-diaminoethylphosphonicacid reacted with DAB in the absence of mercaptoethanol, the emission intensity value was enhanced and rose from 230 (Table 11, experiments with the reducing agent) to 390 arbitrary units. A similar behavior, although to a much lesser degree, was noted with 2,3-diaminopropionic acid (fluorescence intensity when mercaptoethanol was omitted from the reaction medium: 9 arbitrary units). It may be recalled that those two compounds, both possessing two primary amino groups linked to two vicinal carbon atoms, also showed a particular behavior when reacting with DAB in the experimental conditions selected for studying the chromogenic reaction (7); 2,3-diaminopropionic and 1,2-diaminoethylphosphonicacids gave, respectively, orange and yellow colors, very different from the usual purple color observed with most amino compounds. The spacing of the two amino groups by a supplementary carbon atom, a structural characteristic found in the longer-chain 1,3-diaminopropylphosphonicand 2,4-diaminobutyric acids, is associated with a very low fluorescence yield (3 and 1.5 arbitrary units, respectively) when the reaction is conducted without the reducing agent. Addition of rnercaptoethanol increases the fluorescence intensity somewhat, but it is still relatively low (Table 11). 158
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
Fluorescamine, X =
COOH
71 69 79 51 69 66 66 69 65 46
93
7
78
3.5
80
3
78 82 78 79
1
81
71 90 }82
4.5 3 5.5 8 8.5
12 6.5 9 1
14 17
16 14 13
2 5 1.5
68 95 9 25 27 75 81
28 93
5 7 12.5 10.5 4 7.5
66 69
82 74
2 4
1
14 8.5 38 79
5 10
16 15
7 11
9 10
When the two amino groups are more distant (ornithine or lysine, and their phosphonic analogs), a high fluores-
cence is again observed. The influence of the w-amino group is obvious, when ornithine and lysine are compared not only to norvaline or to norleucine, but also to the Nusubstituted derivatives, citrulline and arginine; the same comparisons can be also made in the phosphonic series. w-Amino Acids. When the amino and the acidic group are not linked to the same carbon atom, the fluorescence also reaches significant intensities. The data for ciliatine and 3-aminopropylphosphonic acid are once more lower than in the carboxylic series (P-alanine and GABA), just as in the chromogenic reaction. We have also compared the results given by w-aminophosphonic acids with the experimental data obtained with two sulfur amino acids possessing a sulfonic group as an acidic function (taurine and homotaurine, Table 111). The influence of the -S03H group on fluorescence intensities more closely parallels that of the carboxylic group than the phosphonic group. The introduction of a supplementary carboxylic group in the molecules of taurine and homotaurine changes these amino acids to cysteic and homocysteic acids, and practically eliminates the fluorescence. OPT-Induced Fluorescence. Contrary to the results obtained using DAB as a fluorogenic reagent, OPT-induced fluorescence is poorly influenced by the molecular structure of the amino acid compound: experiments with the simple a-amino acids, acidic amino acids, w-amino acids, and the sulfur amino acids lead to practically identical excitation or emission fluorescence spectra. With this fluorogenic reagent, the lower reactivity of phosphonic derivatives is also observed. However, basic amino acids reacting with OPT show a different behavior. The introduction into the molecule of a free supplementary w-amino group lowers the fluorescence intensity; when this w-amino group is substituted, the fluorescence ability matches that of simple amino acids. The influence of the free w-amino group is specially noticeable
with short chain compounds; there is a progressive decrease of the fluorescence intensity with 1,5-diaminopentylphosphonic, 1,4-diaminobutylphosphonic and especially 1,3diaminopropylphosphonic acids. But this low reactivity ends with the shortest chain compound, 1,2-diaminoethylphosphonic acid, a molecule possessing two vicinal amino groups; this last compound, as in DAB-induced fluorescence, forms a better fluorophore than aminomethylphosphonic acid. The carboxylic analog, 2,3-diaminopropionic acid, curiously, does not form such a good fluorophore. Fluorescamine-Induced Fluorescence. Fluorescamine-induced fluorescence, in the group of simple amino acids, is not very sensitive to molecular structure, as already noted with OPT. Notable exceptions are represented by 1-amino-1-methylethylphosphonicacid and its carboxylic analog. Those two poorly reactive compounds have no hydrogen atom on the carbon atom to which the functional groups are linked; this molecular characteristic is also an unfavorable factor for the production of Ruheman purple, the reaction product of ninhydrin with an amino acid (28). It may be recalled that fluorescamine and ninhydrin are both triketoindane derivatives. In the experiments conducted with fluorescamine, the differences of reactivity between phosphonic and carboxylic compounds are magnified. As in the experiments with DAB, the few sulfur amino acids studied are closer to the carboxylic analogs than to the phosphonic analogs.
CONCLUSIONS The significant fluorescences given by DAB, OPT, and fluorescamine when reacting with the carboxylic amino acids can also be observed when the carboxylic acid group is replaced by the phosphonic group. DAB is especially suitable for the quantitative determination of aminomethylphosphonic acid, diaminophosphonic acids, and also for the natural compound ciliatine, which is poorly reactive with ninhydrin, the classical amino acid reagent. OPT could be considered as a fluorescence reagent covering a larger range of aminophosphonic acids, but its relative insensibility toward molecular structure is restricted by the great instability of the fluorophore. The interest in fluorescamine is linked to the great sta-
bility of the fluorophore; the use of fluorescamine for the determination of ciliatine is limited by the low fluorescence emitted by the chief natural amino phosphonic acid.
ACKNOWLEDGMENT The technical assistance of Mrs. Monique Malgat is gratefully acknowledged.
LITERATURE CITED (1) M. Horiguchi and M. Kandatsu. Nature (London), 184, 901 (1959). (2) M. Kandatsu and M. Horiguchi, Agric. Biol. Chem., 28, 721 (1962). (3) J. S.Kittredge and R. R. Hughes, Biochemistry, 3, 991 (1964). (4) H. Shimizu, Y. Kakimoto, T. Nakajima, A. Kazanawa, and I. Sano, Nature(London), 207, 1197 (1965). (5) V. Chavane, C. R. Acad. Sci. Paris, 224, 406 (1947). (6) M. Labadie and E. Neuzil, in “Composes organiques du phosphore”, Centre National de la Recherche Scientifique, Paris, 1966, p 349. (7) M. Bourhis, H. Jensen, and E. Neuzil, Ann. Pharm. Fr., 28, 561 (1970): 30, 55 (1972). (8) J. S. Kittredge, E. Roberts, and D. G. Simonsen. Biochemistry, 1, 624 (1962). 9. L. Roop and W. E. Roop, Anal. Biochem., 25, 260 (1968). J. Le Pogam, H. Jensen, E. Neuzii, and C. Garrigou-Lagrange, Bull. SOC. Chim. Fr., 12, 3389 (1973). J. Le Pogam, H. Jensen, and E. Neuzii, J. Chromatogr., 87, 179 (1973). N. Seiier, T.Schmidt-Gienewinkel,and H. H. Schneider. J. Chromatogr., 84, 95 (1973). V. H. Ghosh and M. W. Whitehouse, Biochem. J., 108,155 (1968). J. C. Breton, Med. Thesis, Univ. of Bordeaux, 1957, p 194. I. P. Lowe. E. Robins, and G. S.Eyerman, J. Neurochem., 3, 8 (1958). J. Close, quoted by Samejima et ai. (Ref. 18, p 222). M. W. Mc Caman and E. Robins, J. Lab. Clin. Med., 59, 885 (1962). K. Samejima, W. Dairmann, J. Stone, and S. Udenfriend, Anal. Biochem.. 42, 222 (1971): 42, 237 (1971). M. Weigeie, J. F. Blount. J. P. Tengi, R. C. Czajkowski. and W. Leimgruber, J. Am. Chem. SOC.,94,4052 (1972). M. Weigele, S. Debernardo. and W. Leimgruber. Biochem. Biophys. Res. Commun., 50, 352 (1973). G. Hillman, 2.Physiol. Chem., 277, 222 (1943). P. A. Shore, A. Burkhaiter, and V. H. Cohn, J. Pharmacol. Exptl. Therap., 127, 182 (1959). M. Roth, Anal. Chem., 43, 880 (1971). M. Roth and L. Jeanneret, 2. Physiol. Chem., 353, 1607 (1972) M. Roth and A. Hampai, J. Chromatogr., 83, 353 (1973). A. M. Lacoste, A. Cassaigne. and E. Neuzil, C. R. Acad. Sci. Paris, 274, 1418 (1972): 275, 3009 (1972). S.Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M. Weigele, Science, 178, 871 (1972). E. Neuzii, J. C. Breton, and H. Plagnol, Bull, Mem. Ec. Naf. Med. Pharm. Dakar, 8, 169 (1958).
RECEIVEDfor review July 14, 1975: Accepted September 26, 1975. The support granted by the Conseil Scientifique de 1’Universite de Bordeaux I1 is kindly acknowledged.
Fluorescent Detection of Hydrazines via Fluorescamine and Isomeric Phthalaldehydes Robert W. Weeks, Jr.,* Stanley K. Yasuda, and Brenda J. Dean Industrial Hygiene Group, L o s Alamos Scientific Laboratory, University o f California, Los Alamos, N.M. 87545
The use of fluorescarnine, 0-, m-, and p-phthalaldehyde to form fluorescent derivatives Is Illustrated as a class reaction for hydrazine and substituted hydrazines. Results reported herein show that the intensity of product fluorescence and, thus, the likely degree of reaction between a given hydrazine and fluorogen within a few minutes time, is very much a function of the pH of the analyte system. Acid solutions of pH 3-6 showed qualitatively the highest intensity of fluorescence. Using 366 nm wavelength ultraviolet light for irradlatlon of the fluorogenic product, hydrazine was detected at the ng/cm2 level with fluorescamine and with 0- and p
phthalaldehyde. In field survey tests wherein a swipe Is taken, the limit of detection may be even lower because of the effective concentrating of the analyte from a large area onto a relatively small filter paper.
The use of fluorescamine (4-phenylspiro[furan2(3H),l’-phthalan]-3,3’-dione)( I , 2) and of o-phthalaldehyde ( 3 ) for the determination of organic molecules containing amine functionality is well documented. It is the purpose of the present paper to illustrate the utilization of ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
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