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LITERATURE CITED (1) O'Reilly, J. E.;Hale, M. A. Anal. Lett. 1977, 10, 1095-1104. (2) O'Reilly, J. E.; Hicks, D. G. Anal. Chem.1979, 5 1 , 1905-1915. (3) Mohamd, N.; McCurdy, D. L.; Wichman, M. D.; Fry, R. C.; O'Reilly,J. E. Appl. Spectrosc. 1985, 3 9 , 979-983. (4) Fuller, C. W.; Hutton, R. C.; Preston, B. Analyst (London) 1981. 106, 9 13-920. (5) Dick, W. A,; Page, J. R.; Jewell, K. E. SollSci. 1985, 139, 211-218. (6) McCurdy, D. L.; Wichman, M. D.; Fry, R. C. Appl. Spectrosc. 1985, 3 9 , 984-988. (7) Mohamed, N.; Fry, R. C.; Wetzel, D. L. Anal. Chem. 1981, 5 3 , 639-645.
(8) Skogerboe, R. K.; Olson, K. W. Appl. Spectrosc. 1978, 32, 181-187. (9) Browner, R. F.; Boorn, A. W.; Smith, D. D. Anal. Chem. 1982, 5 4 , 1411-1419.
RECEIVED for review December 24, 1985. Resubmitted June 30, 1986. Accepted July 7, 1986. This project was supported in part by Applied Research Laboratories, Sunland, CA. The work was presented in part at the 1985 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New Orleans, LA (Paper No. 400B).
Fluorometric Determination of Nitrite with 4-Hydroxycoumarin Takafumi Ohta,* Youichi Arai, and Shoji Takitani Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12, Ichigaya-Funagawara-Machi,Shinjuku-ku, Tokyo 162, Japan
A simple, sensitive, and reproducible fluorometric method for determination of nitrite has been developed. Thls method is based on the nitrosation of 4-hydroxycoumarin in acidic medium and subsequent reduction to 3-amino-4-hydroxycoumarin, which is fluorescent in alkaline medium. The fluorescence intensity is proportional to the nitrite concentration in the range of 3 ng/mL to 1 pg/mL in the sample solution, with a relative standard deviation of 0.5% (50 ng/ mL). The method has been applied to the determination of nitrite in saliva.
Nitrite ion is of importance not only as an intermediate in the nitrogen cycle, in which its concentration indicates the quality of water, but also as a precursor that forms carcinogenic and/or mutagenic nitrosamines by the reaction with various amines. Recent studies showed the presence of several N-nitroso amino acids in normal human urine, supporting the endogenous reaction of nitrite with amino acids ( I , 2). Thus, a simple, sensitive, and specific determination of nitrite present in foods and biological fluids such as saliva is highly desired. Various fluorometric methods have been developed for the determination of nitrite. However, these methods have several disadvantages. The method with 2,6-diaminopyridine ( 3 ) , 2,3-diaminonaphthalene (4),or resorcinol (5)is tedious because of solvent extraction or prolonged heating. The method with 5-aminofluorescein (6) is liable to variation probably due to the fact that the measurement is based on an increase in fluorescence intensity a t the emission maximum of its reagent: the sensitivity was largely affected depending on the difference in batches and brands of hydrochloric acid used as a reagent. The method with benzidine (7) has drawbacks of serious interferences and toxicity of the reagent used. The method with pyridoxal 5-phosphate 2-pyridylhydrazone (8) lacks sensitivity. Nitrite reacts rapidly with phenolic compounds in acidic solution t,o give their nitroso derivatives (9),which are generally nonfluorescent because of the electron-withdrawing effect of the nitroso group. These nitrosation reactions, therefore, have been used in spectrophotometric methods for the determination of nitrite ( 1 0 , l I ) . On the other hand, amino groups increase the fluorescence intensity of aromatic compounds (12). Therefore, nitrosation of a phenolic compound and subsequent reduction to an aromatic amine seems to
enable a fluorometric determination of nitrite. The present paper describes a simple, sensitive, and reproducible method for the determination of nitrite based on the nitrosation of 4-hydroxycoumarin and subsequent reduction to 3-amino-4hydroxycoumarin, which is fluorescent in alkaline medium.
EXPERIMENTAL SECTION Apparatus. Fluorescence measurements were made with a Shimadzu RF-510 spectrofluorometerequipped with a xenon lamp (Kyoto, Japan) using a 10 x 10 mm quartz cell at room temperature. The slit widths in terms of wavelength were 5 nm (excitation) and 10 nm (emission). All fluorescence excitation and emission spectra were uncorrected. Chemicals. All reagents used were of analytical grade except for sodium hydrosulfite (75% purity). A standard nitrite solution was prepared by drying sodium nitrite (Kanto Chemical Co.) at 110 "C for 4 h and dissolving it in deionized water (MilliporeRO-Q system) to give a 1 mg/mL solution. This standard solution was prepared weekly and kept in a refrigerator, and further dilution was made daily as required. 4-Hydroxycoumarin (Nakarai Chemical Co.) was dissoloved in acetonitrile2 M HCl (1:l) to give 0.04% solution. 3-Amino-4-hydroxycoumarinhydrocholoride was synthesized by the method of Heubner and Link ( 1 3 ) . Procedure. To 2.0 mL of sample solution in a 10-mL glassstoppered test tube was added 1.0 mL of 0.04% 4-hydroxycoumarin reagent. The tube was left in an ice bath for 5 min. To the reaction mixture was added 0.1 mL of 8% sodium thiosulfate, and this was left for 5 min at room temperature. The reduced mixture was made alkaline with 1.0 mL of 1.5 M NaOH and left for 10 min at room temperature. The fluorescence intensity was measured with excitation at 347 nm and emission at 453 nm. Each value in the figures and tables, except Table IV, represents the mean of duplicate runs. RESULTS AND DISCUSSION Fluorescence Spectrum. 4-Hydroxycoumarin, 7hydroxycoumarin, 7-hydroxy-4-methylcoumarin, 6,7-dihydroxycoumarin, 1-naphthol, and 2-naphthol were tested as fluorometric reagents for the determination of nitrite. Satisfactory results were obtained with 4-hydroxycoumarin; the other compounds were themselves highly fluorescent and therefore not usable. The fluorescence of the final reaction mixture resulting from nitrite and 4-hydroxycoumarin showed an excitation maximum a t 347 nm and emission maximum a t 453 nm (Figure 1). 3-Amino-4-hydroxycoumarin dissolved in a blank solution gave the same excitation and emission spectrum. In addition, this amino derivative was found to be present in the reaction mixture by means of thin-layer chromatography (data not
0003-2700/86/0358-3132$01.50/0@ 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
I
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4
* NaOH Corcentrdion(M)
6
4
Figure 2. Effects of hydrochloric acid and sodium hydroxide concentrations: HCI concentrations are as follows: (0)0.5 M, (A)1 M, (0) 2 M, ( 0 ) 4 M, ( 0 )6 M, (A)8 M, (W) 10 M; nitrite, 1 pg/mL. Figure 1. Fluorescence excitation and emission spectra of the final reaction mixture: nitrite, 1 pg/mL; broken-lined spectrum, reagent blank (X4).
Table I. Effect of Acids and Basesn acids 2 M HC1 2 M HC104 1 M HzS04 2/3
M H3P04
RFIb using the given bases (1.5 M) NaOH ",OH piperidine 100 (231)c 43 (111) 32 (84)
82 (20) 31 (8) 19 (10)
111 (44)
7 (26)
2 (2)
9 (9)
54 (18) 36 (13)
"Nitrite (1 pg/mL) was determined by the recommended procedure using a set of each acid and base. * RFI, relative fluorescence intensity; the intensity with HC1 and NaOH is arbitrarily taken as 100. cThe figures in Darentheses are test-to-blank ratios. shown). Therefore, 3-amino-4-hydroxycoumarin is probably a fluorophore in the final reaction mixture. The recovery of nitrite as 3-amino-4-hydroxycoumarinwas 87% when 1pg/mL of nitrite was analyzed by the present method. Effect of Acids and Bases and Their Concentrations. Since an acid was necessary for the reaction of nitrite with 4-hydroxycoumarin and a base was necessary for fluorescence development of the reduced mixture, the most adequate combination of an acid and a base was examined. The effects of acids and bases on fluorescence intensity of the final reaction mixture are shown in Table I. Hydrochloric acid gave the highest fluorescence intensity when used as an acid. Piperidine gave the highest fluorescence intensity when used as a base in combination with hydrochloric acid, but gave a higher reagent blank than sodium hydroxide. Consequently, hydrochloric acid and sodium hydroxide were used. Figure 2 shows the effect of concentration of hydrochloric acid and sodium hydroxide on the fluorescence intensity. 4-Hydroxycoumarin reagent was prepared by dissolving it in a mixture of acetonitrile and various concentrations of hydrochloric acid (l:l,v/v). The reduced mixture after addition of sodium thiosulfate was made alkaline with various concentrations of sodium hydroxide. The highest fluorescence intensity decreased with an increase in sodium hydroxide concentration. No fluorescence, however, was observed unless the final reaction mixture was made alkaline. In order to clarify the optimal pH of the final reaction mixture, 3amino-4-hydroxycoumarin was dissolved in Britton-Robinson's buffer (pH 5-13), and the fluorescence intensity was measured. The highest fluorescence intensity was observed a t pH 11-13, but the fluorescence decreased rapidly below pH 11. Finally, 2 M HC1 and 1.5 M NaOH were used: the pH of the resulting final reaction mixture was about 13. Effect of Organic Solvents for 4-Hydroxycoumarin Reagent. An organic solvent was necessary to prepare 4-
0.1
0.2
4-Hydroxycoumarin Concentration (%)
Figure 3. Effect of 4-hydroxycoumarin concentration. Various concentrations of dhydroxycoumarin were prepared in a mixture of CH,CN and 2 M HCI (l:l, vlv). Other conditions are the same as in the recommended procedure: nitrite, 1 pg/mL.
Table 11. Effect of Organic Solvents" solvents
acetonitrile methanol ethanol 1-propanol
dioxane dimethylformamide dimethylsulfoxide acetone
RFIb
test, blank
100
219 2
9 30 30
8 30
I
1
120
169
28 3
91 24
4-Hydroxycoumarin reagent (0.04%) was prepared by dissolving it in a mixture of 2 M HC1 and each organic solvent (l:l,v/v). RFI, relative fluorescence intensity; acetonitrile is arbitrarily taken as 100. Nitrite (1 pg/mL) was determined by the recommended procedure. hydroxycoumarin reagent because of its low solubility in water. The effect of various organic solvents on the fluorescence intensity is shown in Table 11. Alcoholic solvents, acetone, dimethyl sulfoxide, and dioxane gave only low fluorescence intensity, while acetonitrile and N,N-dimethylformamide gave satisfactory high fluorescence intensity. Acetonitrile was employed because of its higher test-to-blank ratio. Effect of 4-Hydroxycoumarin Concentration. Figure 3 shows the effect of 4-hydroxycoumarin concentrations on the fluorescence intensity. Almost constant intensity was obtained at concentrations over 0.04%. Because the reagent blank increased with an increase in the reagent concentration, 0.04% was used in the present study. Effect of Reaction Time with 4-Hydroxycoumarin. Figure 4 shows the effect of reaction time with 4-hydroxycoumarin on the fluorescence intensity. When the reaction was carried out a t room temperature, the fluorescence in-
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Table 111. Effect of Various Ions and Compounds on Nitrite Determination" ion or compd; concn, pg/mL
recovery,
none
so,2-;1000 Po:-; 1000 co,*-;1000
$ 1
,
c
,
,
,
20 Time ( m i n l
,
3c
NO;; 1000 SCN-; 1000 F-; 1000 I-; 100 I-; 10 NH,'; 1000
. A n 7"
Figure 4. Effect of reaction time: ( 0 )room temperature, (0)0 'C. Other conditions are the same as in the recommended procedure: nitrite, 1 kg/mL.
?&
ion or compd; concn, fig/mL
recovery. 7%
100 101 98 101 98 99 101 67 100 103
Ca2+;1000 Mg2+; 1000 K+; 1000 cu2+; 10 cuz+; 1 Fe3+; 10 FeS+;1 phenol; 1000 aniline; 100 aniline; 10
102 98 100 0 93 79 97 99 58 92
Nitrite (0.1 pg/mL) was determined by the recommended procedure in the presence of each ion or compound.
Table IV. Analytical Results with Saliva Sample" sample
NO2- added, fig/mL
found,b pg/mL
1
0 5 10 0 10
5.98 f 0.09 11.06 f 0.20 15.53 f 0.05 8.35 f 0.03 18.17 f 0.21
2
recovery, 7~ 101.6 95.5 98.2
"Saliva (1 g) was diluted with water to 100 mL, and insoluble matter was removed by centrifugation before analysis. Mean *standard deviation ( n = 3).
Figure 5. Effect of reducing agents: ( 0 )NalS,O,
(0)Na2S203,(0) NaSH. Other conditions are the same as in the recommended procedure: nitrite, 1 pg/mL.
tensity reached a maximum within a few minutes, but decreased greatly on further standing. On the other hand, the fluorescence intensity reached a maximum in about 5 min and was relatively stable when the reaction was carried out at 0 O C . The reaction time of 5 min a t 0 "C was used. Effect of Reducing Agents and Reduction Time. In preliminary experiments, sodium hydrosulfite, sodium sulfide, sodium sulfite, sodium thiosulfate, sodium hydrosulfide, ascorbic acid, and hydrazine were tested for their reducing ability at room temperature. Only sodium hydrosulfide, sodium hydrosulfite, and sodium thiosulfate were effective. Figure 5 shows the effect of concentration of these three reducing agents. The highest fluorescence intensity was obtained with sodium thiosulfate at a concentration over 8% and with sodium hydrosulfite at a concentration of about 3-7%. The fluorescence intensity reached a plateau 5 min after the addition of reducing agent when sodium thiosulfate was used. Although a shorter reduction time was obtained with sodium hydrosulfite, we used 8% sodium thiosulfate because of its stability: the relative fluorescence intensity was lowered below 5% with aged 3% sodium hydrosulfite that had been left 30 h after preparation. Stability of the Fluorophore. The final reaction mixture became slightly turbid on addition of 1.5 M NaOH and gradually became clear within 7-8 min. Because of this, the fluorescence measurement was made 10 min after the addition of alkali. The fluorescence developed under the present conditions decreased gradually on standing: about 7 70 loss was observed after 1 h at room temperature in the dark. Calibration Curve. A calibration curve was constructed according to the recommended procedure. The fluorescence intensity was proportional to the nitrite concentration in the range of 3 ng/mL to 1 pg/mL ( r = 0.9998). The relative standard deviations (n = 10) were 0.5% and 0.4% at 50 ng/mL and 1 Fg/mL, respectively.
Effect of Foreign Species. The effect of foreign species on the determination of nitrite is shown in Table 111. Ions that are present in human saliva ( 1 4 ) and several other ions and compounds that are suspected to affect the nitrite determination were tested. Most of the ions and compounds examined did not interfere in the determination. However, cupric ion, ferric ion, iodide, and aniline interfered with the determination seriously when present in concentrations over 10-100 kg/mL. The interference by these two metal ions may be attributed to chelation, because various hydroxycoumarin derivatives have been used as analytical reagents for metal ions based on their chelating characteristics ( 1 5 ) . The interference by cupric ion could be reduced somewhat by using 4-hydroxycoumarin reagent containing 6 mM EDTA: 89% recovery was obtained in the presence of 10 pg/mL of cupric ion. The cupric and ferric ion levels in biological fluids, however, are lower than their interfering concentrations (14). The nitrite content in saliva and recovery of nitrite added to saliva were determined by the present method. Table IV shows that recoveries are between 95.5% and 101.6% and that the present method is applicable to saliva samples. The sensitivity of the present method in terms of nitrite concentration in the sample solution (3 ng/mL) was lower than that of the fluorometric methods with 2,3-diaminonaphthalene (
