Anal. Chem. 1999, 71, 3003-3007
Determination of Nitrite in Waters by Microplate Fluorescence Spectroscopy and HPLC with Fluorescence Detection Andrea Bu 1 ldt and Uwe Karst*
Anorganisch-Chemisches Institut, Abteilung Analytische Chemie, Westfa¨lische WilhelmssUniversita¨t Mu¨nster, Wilhelm-Klemm-Strasse 8, D-48149 Mu¨nster, Germany
A selective and versatile fluorescence spectroscopic method for the determination of nitrite in waters has been developed. Nitrite reacts in the presence of mineral acids with the nonfluorescent N-methyl-4-hydrazino-7-nitrobenzofurazan forming N-methyl-4-amino-7-nitrobenzofurazan, which can be detected by fluorescence spectroscopy with an excitation maximum at λ ) 468 nm and an emission maximum at λ ) 537 nm in acetonitrile. Three new methods based on this reaction have been developed: Direct fluorescence spectroscopy, HPLC/fluorescence, or HPLC with UV/vis detector may be selected as detection techniques. On microplates, high-throughput fluorescence spectroscopy is achieved, while HPLC/fluorescence provides lower limits of detection, and HPLC with UV/ vis detection enables evaluation of the reaction with standard instrumentation. Different water samples were investigated using all detection modes, and a photometric standard procedure was successfully employed to validate the new methods with an independent technique. Due to the importance of nitrite in the environmental sciences and in food chemistry, a large number of analytical methods has been developed in recent years. For the simultaneous determination of nitrite and other anions in waters, ion chromatography with conductometric detection1 is a suitable technique, but problems arise due to the elution of nitrite in the tailing of the chloride peak and the high limit of detection at approximately 2 µmol/L. Enrichment techniques in combination with ion chromatography have also been described with the goal to achieve lower limits of detection.2,3 Another method described for the determination of nitrite is based on two-dimensional capillary isotachophoresis4 or on on-line coupled capillary isotachophoresis with capillary zone electrophoresis,5 both with conductivity detection. The acid-catalyzed diazotation of p-aminobenzenesulfonamide with subsequent coupling to N-(1-naphthyl)-1,2-diaminoethane yields an azo dye which is quantified by means of spectrophotometry at a wavelength of 540 nm. This method has been (1) German standard methods for the examination of water, wastewater and sludge; anions (group D). 1988, DIN 38405, part 19. (2) Haddad, P. R.; Jackson, P. E. J. Chromatogr. 1987, 407, 121-132. (3) Cassidy, R. M.; Elchuk, S. J. Chromatogr. 1983, 262, 311-315. (4) Meissner, T.; Eisenbeiss, F.; Jastorff, B. Fresenius J. Anal. Chem. 1998, 361, 459-464. (5) Kaniansky, D.; Zelensky´, I.; Hybenova´, A.; Onuska, F. Anal. Chem. 1994, 66, 4258-4264. 10.1021/ac981330t CCC: $18.00 Published on Web 06/25/1999
© 1999 American Chemical Society
established as a European standard.6 Its major drawbacks are interferences from colored samples and insufficient limits of detection for ultratrace determination of nitrite. Several fluorescence spectroscopic methods have also been published in recent years.7-10 They are based on the reaction of nitrite with organic fluorogens to form highly fluorescent compounds. Some of these methods are based on a direct fluorescence reading;7-9 others require HPLC separation of the product and the reagents.10 Lee and Field11 published a method for postcolumn derivatization with fluorescence detection. A major advantage of the fluorescence methods is the low limit of detection, but their robustness cannot compete with photometry for concentrations in the lower to midppb range which is typical for surface waters in agriculturally used areas. Kieber and Seaton12 developed a HPLC method with UV/vis detection based on the reaction of nitrite in acidic media with 2,4dinitrophenylhydrazine (DNPH), a well-known reagent for the determination of aldehydes and ketones.13-15 2,4-Dinitrophenyl azide (DNPA) is formed as a reaction product which is easily separated from the hydrazone of formaldehyde. The NO+ cation is the active species in this reaction, as demonstrated before during investigations on chemical interferences by nitrogen dioxide in the determination of carbonyls using the DNPH method.13 Gro¨mping et al. previously published a method, based on this reaction, for the determination of nitrogen dioxide in air samples.16 The method of Kieber and Seaton is characterized by a low limit of detection at 0.1 µmol/L (4.6 ppb) nitrite and a rapid derivatization. The same group has recently reported on the determination of 15N nitrate and nitrite using the same reaction with subsequent FT-IR determination.17 (6) European Standard, Water qualitysDetermination of nitritesMolecular absorption spectrometric method. 1993, EN 26777. (7) Ohta, T.; Arai, Y.; Takitani, S. Anal. Chem. 1986, 58, 3132-3135. (8) Damiani, P.; Burini, G. Talanta 1986, 33 (8), 649-652. (9) Axelrod, H. D.; Engel, N. A. Anal. Chem. 1975, 47, 922-924. (10) Zhou, J. Y.; Prognon, P.; Dauphin, C.; Hamon, M. Chromatographia 1993, 36, 57-60. (11) Lee, S. H.; Field, L. R. Anal. Chem. 1984, 56, 2647-2653. (12) Kieber, R. J.; Seaton, P. J. Anal. Chem. 1995, 67, 3261-3264. (13) Karst, U.; Binding, N.; Cammann, K.; Witting; U. Fresenius J. Anal. Chem. 1993, 345, 48-52. (14) Po ¨tter, W.; Karst, U. Anal. Chem. 1996, 68, 3354-3358. (15) Beasley, R. H.; Hoffmann, C. E.; Rueppel, M. L.; Worley, J. W. Anal. Chem. 1980, 52, 1110-1114. (16) Gro¨mping, A. H. J.; Karst, U.; Cammann, K. J. Chromatogr. 1993, 653, 341347.
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We have described a new group of hydrazine reagents for aldehyde and ketone determination which are characterized by an R-methyl hydrazine function.18 These reagents are less susceptible to interferences by oxidants, as only one defined byproduct with both nitrogen dioxide and ozone is formed. N-Methyl2,4-dinitrophenylhydrazine (MDNPH) is a reagent that reacts with these oxidants with formation of N-methyl-2,4-dinitroaniline.18 The product is easily separated from the hydrazones and detected by UV/vis spectroscopy. N-Methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH), a nonfluorescent reagent, however, yields N-methyl4-amino-7-nitrobenzofurazan (MNBDA) as a highly fluorescent product in the reaction with NO+. We have used this new reagent to develop an array of methods for the determination of nitrite using microplate fluorescence spectroscopy, HPLC with fluorescence detection, and HPLC with UV/vis detection. EXPERIMENTAL SECTION Chemicals. All chemicals were purchased from Aldrich Chemie (Steinheim, Germany) in the highest quality available, except the following: Sodium nitrite and triethylamine were from Fluka (Neu-Ulm, Germany). Acids were Merck (Darmstadt, Germany) analytical grade. Acetonitrile for HPLC was Merck gradient grade. Synthesis and Characterization of the Reagent. The synthesis and characterization of MNBDH are described in refs 19 and 20. Synthesis and Identification of the Reaction Product. The synthesis, based on a literature procedure by Clusius and Schwarzenbach,21 was carried out for the synthesis of 2,4dinitrophenyl azide and modified as follows: 200 mg of N-methyl4-hydrazino-7-nitrobenzofurazan (9.4 × 10-4 mol) was dissolved in 30 mL of ethanol and 5 mL of concentrated hydrochlorid acid. While cooling with ice, a solution of 70 mg of sodium nitrite (1 × 10-3 mol) in 5 mL of distilled water was added. After leaving the reaction solution for 0.5 h at room temperature, 50 mL of distilled water was added. Afterward the solution was left at room temperature again for 1 h. The product precipitated as a yellow material. This was filtered off and washed with ice/water. The yield was 67%. Product characterization (for peak assignment compare to the structure below): 1H NMR δ 3.24 (d, 3H, N-CH3, J ) 5.4 Hz), 6.18 (d, 1H, Hb, J ) 8.5 Hz), 8.54 (d, 1H, Ha, J ) 8.6 Hz); MS m/z 194 (M+, 100), 164 (194 - NO, 27), 118 (29), 103 (26), 91 (31), 76 (29); IR 3040, 2718, 2677, 1650, 1621, 1540, 1312, 1269, 1161 cm-1. Anal. Calcd for C7H6N4O3: C, 43.30; H, 3.12; N, 28.87. Found: C, 43.18; H, 3.43; N, 28.55. UV/vis Spectrometer. The HP 8453 diode array spectrophotometer (Hewlett-Packard, Waldbronn, Germany) with software HP Chem Station 845x biochemical UV/vis system was used. Spectrofluorophotometer. The RF-5301 PC spectrofluorophotometer (Shimadzu, Duisburg, Germany) with software version 1.10 was employed for fluorescence spectroscopy in cuvettes. Microplate Spectrophotometer. The microplate spectrophotometer Spectra Max 250 with software Soft Max Pro Version 1.1 (Molecular Devices, Sunnyvale, CA) was used. (17) Kieber, R. J.; Bullard, L.; Seaton, P. J. Anal. Chem. 1998, 70, 3969-3973. (18) Bu ¨ ldt, A.; Karst, U. Anal. Chem. 1997, 69, 3617-3622. (19) Bu ¨ ldt, A.; Karst, U. German Patent, DE 198 00 537.7, 1998. (20) Bu ¨ ldt, A.; Karst, U. Anal. Chem. 1999, 71, 1893-1898. (21) Clusius, K.; Schwarzenbach, K. Helv. Chim. Acta 1958, 41, 1413-1416.
3004 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999
Microplate Fluorophotometer. The microplate fluorophotometer Fluostar (BMG LabTechnologies, Offenburg, Germany) with software version 2.10-0 was used. Excitation filter, 470 nm; emission filter, 538 nm. HPLC Instrumentation. A high-performance liquid chromatograph consisting of the following components was used: two LC-10AS pumps (Shimadzu), an SIL-10A autosampler (Shimadzu), a SPD-M10Avp diode array detector (Shimadzu), a RF-10Axl fluorescence detector (Shimadzu), Class LC-10 Version 1.6 software (Shimadzu), and a CBM-10A controller unit (Shimadzu). The injection volume was 10 µL. The column material was Merck LiChroSpher RP-18 (Merck) in ChromCart cartridges (MachereyNagel, Du¨ren, Germany): particle size, 5 µm; pore size, 100 Å; column dimensions, 250 mm × 3 mm; guard column, 8 mm × 3 mm. HPLC Analysis. For separation, a binary gradient consisting of acetonitrile and a mixture of water/triethylamine/acetic acid (preparation: 500 mL water with 2415 µL of triethylamine and 975 µL of acetic acid, pH ∼ 7.5) was chosen with the following profile and a flow rate of 0.62 mL/min:
time (min) c (CH3CN) (%)
0 45
1.5 45
8.5 100
10.5 100
11.5 45
12.5 (stop) 45
Nitrite Solutions for the Calibration. A total of 495 mg of sodium nitrite was dissolved in 1 L of deionized water (7.17 × 10-3 mol/L). This solution was diluted 1:100 (7.17 × 10-5 mol/L) in water. The following concentrations were used for the calibrations: 2.15 × 10-7, 3.59 × 10-7, 7.17 × 10-7, 1.44 × 10-6, 2.15 × 10-6, 2.87 × 10-6, 3.59 × 10-6, 7.17 × 10-6, 1.08 × 10-5, and 1.44 × 10-5 mol/L. Microplate Fluorophotometer Method. A total of 2.5 mg of MNBDH was dissolved in 25 mL of acetonitrile (4.8 × 10-4 mol/ L). A 200-µL aliquot of the corresponding nitrite solutions or deionized water, respectively (blank solution), was pipetted into each well of the microplate. A 7-µL sample of the MNBDH solution was added to each solution, and afterward, 15 µL of concentrated phosphoric acid was pipetted into each well. After a reaction time of 30 min, the fluorescence was read with an excitation wavelength of 470 nm and an emission wavelength of 538 nm. HPLC Method. A 0.9-mL sample of the corresponding nitrite solutions or deionized water, respectively (blank solution), was pipetted into a vial. A 33-µL sample of the MNBDH solution described in the last paragraph and 67 µL of concentrated phosphoric acid were added. After 30 min, the solutions were injected into the HPLC system. The substances were detected with a diode array detector and a fluorescence detector (excitation wavelength, 468 nm; emission wavelength, 537 nm). Comparative Measurements on Microplates Using a Reference Method with Microplate Spectrophotometry. This method6 is a European Standard method for the spectrophotometric determination of nitrite. The original procedure in cuvettes has been modified in this work for microplate spectrophotometry as stated below. Preparation of the Reagent Solution. Sulfanilamide (1 g) was dissolved in 10 mL of concentrated phosphoric acid and 50 mL of deionized water. After 100 mg of N-(1-naphthyl)ethylenediamine dihydrochloride was added, the resultant solution
was transferred into a 100-mL volumetric flask and filled to volume with water. Determination of Nitrite. A 5-µL aliquot of the reagent solution was pipetted into the wells of the microplate. A 200-µL sample of the corresponding nitrite solution or deionized water, respectively (blank solution), was added. Afterward, 45 µL water was added to adjust the pH to 1.9. After a reaction time of at least 20 min, the absorbance was read at a wavelength of 540 nm. RESULTS AND DISCUSSION Structure Elucidation of the Reaction Product. MNBDH has previously been described as reagent for the determination of aldehydes and ketones. The reaction is shown below. In acidic media, MNBDH reacts with nitrite under formation of MNBDA as follows:
The structure of the reaction product was confirmed by 1H NMR, IR, UV/vis, and fluorescence spectroscopy, mass spectrometry, and elementary analysis. The respective analysis data are listed in the Experimental Section. For a positive identification, the substance was synthesized as described above. The synthesized product exhibited identical elution properties on different columns in HPLC and identical UV/vis spectra compared to the reaction product. The fluorescence spectrum of MNBDA in a solution of phosphoric acid and water is depicted in Figure 1. Under these conditions, the excitation maximum is located at λ ) 468 nm and the emission maximum at λ ) 548 nm. DNPA, the reaction product of DNPH with nitrite, exhibits long-term stability only in the freezer, but not at room temperature.14 In contrast to this, MNBDA is a very stable substance and can be stored for several weeks at room temperature without decomposition. This is valid for the solid derivative as well as for solutions in water and organic solvents, e.g., acetonitrile. Derivatization. The derivatization reaction requires the presence of strong acids to achieve complete derivatization within a reasonable time scale. The development of the fluorescence for two nitrite concentrations (1.29 × 10-5 and 1.29 × 10-6 mol/L, respectively) when reacting with 1.6 × 10-5 mol/L MNBDH in the presence of 1 mol/L phosphoric acid was recorded on a cuvette spectrofluorometer and is presented in Figure 2. The relative fluorescence (compared to the fluorescence at a reaction time at 30 min defined as 100%) with the excitation wavelength of 468 nm and the emission wavelength of 548 nm is plotted versus time. It is obvious that the reaction is completed after ap-
Figure 1. Excitation spectrum (solid) and emission spectrum (dotted) of MNBDA. The MNBDA concentration was 1.29 × 10-5 mol/L in water and phosphoric acid corresponding to the conditions chosen for the microplate fluorescence and HPLC methods. Excitation wavelength, λ ) 468 nm; emission wavelength, λ ) 548 nm.
Figure 2. Fluorescence/time curves for the formation MNBDA at the emission wavelength of 548 nm. Excitation wavelength, 468 nm. The concentrations of the nitrite solutions were 1.29 × 10-5 (square) and 1.29 × 10-6 mol/L (circle) when reacting with 1.6 × 10-5 mol/L MNBDH in the presence of 1 mol/L phosphoric acid.
proximately 25 min for both analyte concentrations, which were selected as representative for the real samples to be analyzed. As no significant decay of fluorescence was observed within 1 h after completed derivatization, 30 min was selected as reaction time for all methods described below. Microplate Fluorescence Method. To achieve low limits of detection, a large volume of the sample was reacted with small volumes of acid and reagent as described in the Experimental Section. The limit of detection of the microplate as well as the HPLC fluorescence methods is limited by the triple RSD of the blank, not by the signal-to-noise ratio to 1.5 × 10-8 mol/L. The respective limit of quantification is 5 × 10-8 mol/L. In contrast, the instrumental limit of detection for a calibration experiment with pure MNBDA in acetonitrile is 5 × 10-9 mol/L. Further purification of the reagent results in lower blanks, but highly purified reagent is easily oxidized by nitrogen dioxide from ambient air, thus leading to large standard deviations of the blank. However, the degree of purity used in this study is sufficient for most real samples of nitrite taken from waters in agriculturally used areas. To cover the concentration range expected for the Analytical Chemistry, Vol. 71, No. 15, August 1, 1999
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Table 1. Cross Selectivities of Different Substances Determined by Means of the Microplate Fluorophotometer without nitrite
c (nitrite) ) 10-6 mol/L
interference (10-4 mol/L)
found valuea (%)
RSDb (%)
recovery of nitrite (%)
RSDb (%)
NaCl NaNO3 NaHCO3 K2CO3 MgSO4 Na2SO4 KBr CaCl2 NaI FeCl3 H2O2 glucose formaldehyde acetaldehyde acetone acrolein benzaldehyde
102.0 98.4 97.1 91.5 109.1 95.9 100.2 100.5 126.9 85.7 106.7 89.6 145.8 110.3 97.7 98.8 91.8
7.1 10.7 5.7 5.0 4.5 5.1 6.0 4.2 7.8 8.8 6.7 9.4 7.0 12.7 7.8 8.6 7.4
103.9 103.9 106.1 97.9 111.9 94.0 116.1 115.5 128.8 103.5 82.9 93.2 107.7 100.0 99.0 90.2 101.2
2.1 1.3 1.3 4.3 7.2 2.0 2.3 2.3 3.4 2.8 4.1 0.5 3.3 3.1 2.7 1.9 3.7
a
Blank ) 100%. b n ) 4.
real samples, linear calibration curves have been recorded for nitrite in the concentration range from 3.59 × 10-7 to 1.44 × 10-5 mol/L. The relative standard deviation for selected concentrations was 3.5% for 1.44 × 10-6 mol/L and 2.5% for 1.44 × 10-5 mol/L. The microplate method is well suited to analyze up to 96 samples on one standard microplate, but a more favorable arrangement includes 8 nitrite standard concentrations and up to 16 samples, with each standard and sample in 4 wells of the microplate. This way, high-throughput screening is combined with some statistical evaluation. Compared to the cuvette format, the microplate analysis saves more than 90% of reagent, as the total volume in a well is below 250 µL, in contrast to 3 mL in a standard cuvette with a 1-cm optical path length. Due to the convenience of microplate fluorescence spectroscopy, all interference studies were carried out with this method. As shown in Table 1, a series of inorganic salts, oxidants, and organic substances including the most important carbonyl compounds were added as 0.1 mmol/L solutions to a reagent solution with phosphoric acid, but without nitrite to investigate the influences in the absence of nitrite (left columns). The same experiment was carried out in the presence of 1µmol/L nitrite, thus simulating a 100-fold excess of the interfering compound (right columns). For the experiments with and without nitrite, the blank (water added instead of the interferent solution) was defined as 100%. Without nitrite, the RSDs are relatively large due to the low absolute values. Only iodide and formaldehyde exhibit significantly increased values. In the case of formaldehyde, this may be traced back to the formation of the respective hydrazone, which is slightly fluorescent. As the absolute fluorescence signal in the presence of nitrite is far larger, the formation of the hydrazone can no longer be observed. Therefore, formaldehyde is only slightly interfering in the microplate method, while the separation in the HPLC methods excludes an interference from the aldehydes, except due to consumption of the reagent. The effect of sodium iodide may be due to the catalytic properties of the iodide ion, which may increase the rate of formation of MNBDA with nitrite and nitrogen dioxide from 3006 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999
Figure 3. Chromatogram of a nitrite solution (7.17 × 10-6 mol/L) after reaction with MNBDH and HPLC with UV/vis detection (474 nm).
ambient air. In the presence of nitrite, hydrogen peroxide causes a reduction of the fluorescence signal which may be due to the saturation of the solution with oxygen from the peroxide decomposition. All substances that have been identified as potential interferences do not occur in these high concentrations in any nitrite samples known to the authors. Therefore, interferences from the investigated substances may be neglected. HPLC Methods with Fluorescence and UV/vis Detection. As stated above, the limit of detection is limited by the blank. However, due to the lower RSDs and the separation from the other components of the reaction solution, limits of detection are lower for the HPLC fluorescence method compared to the microplate method, while the HPLC method with UV/vis detection exhibits a similar LOD compared to the microplate method. The extremely strong fluorescence of MNBDA is obvious from two chromatograms of a derivatized sample (1.6 × 10-5 mol/L reagent, 7.17 × 10-6 mol/L nitrite, 1 mol/L phosphoric acid). Figure 3 shows the chromatogram with UV/vis detection at λ ) 474 nm, Figure 4 shows the same chromatogram with fluorescence detection at an excitation wavelength of λ ) 468 nm and an emission wavelength of λ ) 537 nm. The delay of the MNBDA peak (8 s) between the detectors is due to the void volume of the detector cells and lines, as both detectors are connected in series. The peak at 1.6 min in the UV/vis chromatogram is caused by the phosphoric acid. The chromatograms show that there is almost no fluorescence from the MNBDH reagent, thus enabling a direct fluorescence spectroscopic reading as performed in the microplate method. For the HPLC method with UV/vis detection, the blank is almost not detectable. For HPLC with fluorescence detection, the limit of detection is limited by the triple RSD of the blank, not by the signal-to-noise ratio. Linear calibration curves were recorded from
Table 2. Comparison of Water Samples Analyzed with Four Different Methods reference method source of water sample cattle watering place, Papenburg, Germany ditch in agriculturally used area close to Papenburg, Germany lake Aasee, Mu ¨ nster, Germany tap water (Mu ¨ nster, Germany) a
microplate fluorophotometer method
HPLC with fluorescence detection
HPLC with UV/vis detection
concn (mol/L)
RSDa (%)
concn (mol/L)
RSDa (%)
concn (mol/L)
RSDa (%)
concn (mol/L)
RSDa (%)
2.6 × 10-5 5.9 × 10-6
1.6 2.0
2.510-5 6.0 × 10-6
5.8 6.6
2.610-5 6.4 × 10-6
1.0 0.9
2.6 × 10-5 6.4 × 10-6
1.0 0.5
5.7 × 10-6 nqb
4.2
5.1 × 10-6 4.3 × 10-7
3.8 11.3
5.7 × 10-6 4.1 × 10-7
1.5 1.5
5.7 × 10-6 3.9 × 10-7
1.2 3.5
n ) 3. b Below the limit of quantification.
2.15 × 10-7 to 7.17 × 10-6 mol/L for HPLC with fluorescence and 2.15 × 10-7 to 1.44 × 10-5 mol/L for HPLC with UV/vis detection, respectively. RSDs for triple determination of nitrite standards ranged from 1 to 3% for both fluorescence and UV/vis detection. The limit of detection is 10-8 mol/L for HPLC with fluorescence detection and 3 × 10-8 mol/L for HPLC with UV/ vis detection. The respective limits of quantification are 3 × 10-8 and 10-7 mol/L, respectively. Reference Method. The established reference method based on the following reaction
•
was converted to a microplate method for more convenient measurements of large numbers of samples. The procedure for the analysis is stated in the Experimental Section. Linear calibration curves were recorded from 7.17 × 10-7 to 1.44 × 10-5 mol/ L. The limit of detection for this method on microplates is 2 × 10-7 mol/L and the respective limit of quantification is 6 × 10-7 mol/L. Comparative Measurements of Real Samples. Four water samples of different origin have been investigated by all four methods. The respective data are listed in Table 2. It is obvious that all methods are well-suited for the analysis of the four samples, except the photometric reference method, which does not exhibit a sufficiently low limit of detection for the tap water sample. All analyses were carried out in triplicate. The HPLC data are not for triple injection of the same reaction mixture, but for triple reaction and chromatographic analysis. The respective RSDs of all methods are listed in Table 2. The RSDs for the HPLC methods are excellent, with RSDs not exceeding 3.5% even for the lowest concentrations. The RSDs for the microplate methods are higher, as could be expected from pipetting small volumes and from the
Figure 4. Chromatogram of a nitrite solution (7.17 × 10-6 mol/L) after reaction with MNBDH and HPLC with fluorescence detection (excitation, 468 nm; emission, 537 nm).
fact that the reaction products are not separated from the reaction mixture. All four methods correlate well for the water samples. Discussion. MNBDH is well suited as a new reagent for the determination of nitrite in waters. A major advantage of the reaction is the versatility, thus allowing one to quantify nitrite by fluorescence spectroscopy without or after HPLC separation as well as by HPLC with UV/vis detection. The first microplate fluorometric method for nitrite determination has been developed and allows for high-throughput screening of a very large number of nitrite samples within a short time scale. Received for review December 1, 1998. Accepted April 27, 1999. AC981330T
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