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Development of an Analytical Method for Nitric Oxide Radical Determination in Natural Waters Emmanuel F. Olasehinde, Kazuhiko Takeda, and Hiroshi Sakugawa* Graduate School of Biosphere Science, Department of Environmental Dynamics and Management, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan The measurement of photochemically generated nitric oxide radicals (NO) in natural waters has long been an arduous task because of a lack of simple analytical techniques, even though the environmental significance of this radical is paramount. We have developed a simple analytical method for the determination of photochemically generated NO in natural waters using 4,5-diaminofluorescein (DAF-2) as a probe compound. This method is based on the reaction of photoformed NO with DAF-2 in air-saturated solution to produce a highly fluorescent triazolofluorescein (DAF-2T) product. DAF-2T was determined by using reversed-phase HPLC with fluorescence detection, with excitation and emission wavelengths of 495 and 515 nm, respectively. Under optimum conditions, the calibration curve exhibited linearity in the range of 0.025-10 nM DAF-2T. The coefficients of variance for the measurements of the signal intensities of DAF-2T (from the photolysis of 0.5 µM and 5 µM NO2- with DAF2) were less than 5% and 3%, respectively. For a total irradiation time of 30 min, the detection limit of the photoformation rate of NO was 1.65 × 10-13 M s-1, defined as 3σ of the lowest measured DAF-2T concentration (0.025 nM). The proposed method is relatively unaffected by potential interferents in seawater. The method was employed to determine the photoformation rate of NO in the Seto Inland Sea and the Kurose River in Hiroshima Prefecture, Japan. The measured NO photoformation rates in seawater and river water samples ranged from (5.3-32) × 10-12 M s-1 and (9.4-300) × 10-12 M s-1, respectively. Numerous studies have provided unequivocal evidence that the surface layers of natural waters contain many reduced inorganic and organic compounds which undergo photochemical transformation to generate transient species. These include but * To whom correspondence should be addressed. E-mail: hsakuga@ hiroshima-u.ac.jp. Phone: +81 82 424 6504. Fax: + 81 82 424 6504. (1) Cooper, W. J.; Zika, R. G.; Petasne, R. G.; Plane, J. M. C. Environ. Sci. Technol. 1988, 22, 1156–1160. (2) Wilson, C. L.; Hinman, N. W.; Sheridan, R. P. Photochem. Photobiol. 2000, 71, 691–699. (3) Zepp, R. G.; Schlotzhauer, P. F.; Sink, R. M Environ. Sci. Technol. 1985, 19, 74–81. (4) Zepp, R. G.; Braun, A. M.; Hoigne, J.; Leenheer, J. A. Environ. Sci. Technol. 1987a, 21, 485–490. (5) Fischer, A. M.; Kliger, D. S.; Winterle, J. S.; Mill, T. Chemosphere 1985, 14, 1299–1306. 10.1021/ac901128y CCC: $40.75 2009 American Chemical Society Published on Web 07/27/2009
are not limited to H2O2,1,2 singlet oxygen,3 hydrated electrons,4,5 superoxide ions,6 organo peroxyl radicals,7,8 hydroxyl radicals,9-15 and nitric oxide radicals.16-18 The nitrite ion (NO2-), a trace compound in natural waters, is known to be chemically stable but photochemically unstable because of its large absorption in the sea-level uv region.19 Although NO2- is usually present at low concentrations in natural waters, its photolysis to produce mainly nitric oxide radicals (NO) and O- radicals, which are rapidly protonated and converted to hydroxyl radicals ( · OH) at pH e 11.9 (the pKa value for OH/O-), has been well documented.16-21 NO2- + H2O + hv f NO + ·OH + -OH
(1)
Nitric oxide radicals can also be produced through biological activities, primarily controlled by nitrification and denitrification processes in natural waters.22 In recent years, studies have focused on the photochemical formation of · OH in natural waters, ignoring the analogous NO radical. Meanwhile, it has been proposed that the free radical reaction of alkyl peroxy radicals and NO could be one of the sources of alkyl nitrates in natural waters.23 Moreover, Zafiriou et al.17 have reported that oceans could be a potential source of atmospheric NO. As recognized by Jacob and Hilker,24 NO could be transferred to the gas phase because of its low solubility in aqueous solution. Thus, information on the photochemical production rates of NO is critical to determining the NO steady-state (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
Petasne, R. G.; Zika, R. G. Nature 1987, 325, 516–518. Mill, T.; Hendry, D. G.; Richardson, H. Science 1980, 207, 886–887. Kieber, D. J.; Blough, N. V. Anal. Chem. 1990, 62, 2275–2283. Zhou, X. L.; Mopper, K. Mar. Chem. 1990, 30, 71–88. Nakatani, N.; Hashimoto, N.; Shindo, H.; Yamamoto, M.; Kikkawa, M.; Sakugawa, H. Anal. Chim. Acta 2007, 581, 260–267. Takeda, K.; Takedoi, H.; Yamaji, S.; Ohta, K.; Sakugawa, H. Anal. Sci. 2004, 20, 153–158. Takeda, K.; Shindo, H.; Nakatani, N.; Sakugawa, H. J. Jpn. Soc. Water Environ. 2005, 28, 509–513. Vaughan, P. P.; Blough, N. V. Environ. Sci. Technol. 1998, 32, 2947–2953. Zafiriou, O. C. Mar. Chem. 1977, 5, 497–522. Zafiriou, O. C. J. Geophys. Res. 1974, 79, 4491–4497. Zafiriou, O. C.; McFarland, M. J. Geophys. Res. 1981, 86, 3173–3182. Zafiriou, O. C.; McFarland, M.; Bromund, R. H. Science 1980, 207, 637– 639. Zafiriou, O. C.; McFarland, M. Anal. Chem. 1980, 52, 1662–1667. Zafiriou, O. C.; True, M. B. Mar. Chem. 1979, 8, 33–42. Fischer, M.; Warneck, P. J. Phys. Chem. 1996, 100, 18749–18756. Mack, J.; Bolton, J. R. J. Photochem. Photobiol., A 1999, 128, 1–13. Ward, B. B.; Zafiriou, O. C. Deep-Sea Res. 1988, 35, 1127–1142. Dahl, E. E.; Saltzman, E. S. Mar. Chem. 2008, 112, 137–141. Jacobi, H.-W.; Hilker, B. J. Photochem. Photobiol., A 2007, 185, 371–382.
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concentrations in natural waters. The assessment of the steady state concentrations of NO in natural waters is therefore very important for the determination of the flux of NO from natural waters to the atmosphere and the reaction rate of NO with various dissolved compounds in natural waters. However, despite its environmental significance, the measurement of photochemically generated NO in natural waters has been difficult because of a lack of simple analytical techniques. One of the techniques used to measure NO in natural waters is electrochemical methods; however, this technique has low sensitivity (LOD 1.4 × 10-8M).25,26 Zafiriou et al.18 developed an elegant method for the quantification of NO in seawater and also hypothesized that this radical may be generated in other natural waters. The basic approach of their method involves equilibrating the highly insoluble NOaq in solution with a flowing gas stream (“stripping”) and measuring the NOg with a sensitive and stable chemiluminescence detector. To date, this is the only available method for the determination of photochemically generated nitric oxide radicals in seawater. Hence it is important to develop a more versatile and simpler analytical method which can be widely applied for routine determination of NO in natural waters. Gomes et al.27 reported that since the development of diaminofluoresceins (DAFs) in 1998 by Kojima et al.,28 they have been widely used for NO detection and imaging in biological systems, being employed in more than 100 scientific reports in the last 2 years. However, DAFs do not react directly with NO but rather with the oxidized form of NO. In fact, it has been proposed that the reaction mechanism of DAFs with NO involves N2O3.29 N2O3 is generated according to the following scheme: 2NO + O2 f 2NO2
(2)
2NO2 + 2NO a 2N2O3
(3)
According to this reaction scheme, the formation of the Nnitrosation species, N2O3, requires two molecules of NO. Therefore, the stoichiometry of the reaction between DAFs and NO is 1:2. We employed DAF-2, a derivative of DAFs, for the determination of NO in natural waters. Our approach is based on the reaction of photoformed NO with DAF-2 in air-saturated solution to produce a highly fluorescent DAF-2T (see Figure 1). The product DAF-2T is determined by reversed-phase HPLC equipped with a fluorescence detector operated at excitation and emission wavelengths of 495 and 515 nm, respectively. This allows NO quantification. The proposed method has been successfully applied to the determination of photochemical formation rates of nitric oxide radicals in seawater and other natural water samples. EXPERIMENTAL SECTION Reagents, Chemicals and Water Samples. All reagents were of reagent grade and used as received unless otherwise stated. (25) Zhang, Z. B.; Xing, L.; Jiang, L. Q.; Wang, Y. C.; Ren, C. Y.; Cai, W. J. Sensors 2003, 3, 304–313. (26) Zhang, Z. B.; Xing, L.; Wu, Z.; Liu, C.; Lin, C.; Liu, L. Sci. China Ser. B: Chem. 2006, 49, 475–480. (27) Gomes, A.; Fernandes, E.; Lima, J. L. F. C. J. Fluoresc. 2006, 16, 119–139. (28) Kojima, H.; Sakurai, K.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Chem. Pharm. Bull. 1998, 46, 373–375. (29) Nakatsubo, N.; Kojima, H.; Kikuchi, K.; Nagoshi, H.; Hirata, Y.; Maeda, D.; Imai, Y.; Irimura, T.; Nagano, T. FEBS Lett. 1998, 427, 263–266.
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Figure 1. Structures of DAF-2 and DAF-2T.
All solutions were prepared with ultrapure water obtained from a Milli-Q Plus system (Millipore; g 18.2 MΩ cm). Acetonitrile (HPLC grade) and disodium hydrogen phosphate heptahydrate (guaranteed reagent) were obtained from Nacalai Tesque (Tokyo, Japan). Sodium phosphate monobasic monohydrate was obtained from Sigma-Aldrich Japan. DAF-2 was obtained from Daiichi Pure Chemicals (Japan) and DAF-2T from Alex Biochemicals. NO2standard solution (1000 mg L-1) was obtained from Kanto Chemical Co. Inc. A detailed description of the seawater and river water used in this study has been given elsewhere.30,31 Briefly, seawater samples from various depths and locations were collected from the Seto Inland Sea using Niskin sampling bottles and a CTD carousel multisampling system (General Oceanic Inc., U.S.A) during the cruise of the R/V Toyoshio Maru belonging to Hiroshima University on October 6-10, 2008. Samples were immediately transferred to clean amber 1 L glass bottles. River water samples were collected from the Kurose River, Hiroshima Prefecture, by polyethylene buckets. All water samples were filtered through a precleaned glass fiber filter (Advantech, 0.45 µm nominal rating) and stored in the dark at 4 °C until analysis. Analyses were completed within 2 weeks of sample collection. Equipment. An HPLC system consisting of a PU-2089 plus pump (Jasco, Japan), a Rheodyne injection valve (Cotati, CA, U.S.A.) with a 100 µ` L sample loop, and an FP-2020 plus intelligent fluorescence detector (Jasco, Japan) interfaced with a C-R6A Chromatopac integrator (Shimadzu, Japan) were used. The separations were carried out on an RP-18 GP column (150 × 4.6 mm I.D., 5 µm) from Kanto Kagaku (Japan). The eluent was 10 mM phosphate buffer solution (PBS) at pH 7.4 with 6% acetonitrile at a flow rate of 1 mL min-1. Under these conditions, the retention times of DAF-2 and DAF-2T were 4.2 and 4.8 min, respectively. The detector was operated at wavelengths of 495 and 515 nm for excitation and emission, respectively. UV-vis absorbance spectra were acquired using a Shimadzu UV-2400 PC UV/vis scanning spectrophotometer. Preparation of Oxy-Hemoglobin. To prepare oxy-hemoglobin (HbO2) from commercial bovine hemoglobin, 1 mM sodium dithionite was added to a solution of bovine hemoglobin in 100 mM potassium phosphate at pH 7.0 to reduce any methemoglobin present in the solution. The mixture was dialyzed overnight at 4 °C in 100 mM potassium phosphate, pH 7.0, to remove any residual dithionite. The concentration of HbO2 was (30) Olasehinde, E. F.; Makino, S.; Kondo, H.; Takeda, K.; Sakugawa, H. Anal. Chim. Acta 2008, 627, 270–276. (31) Derbalah, A. S. H.; Nakatani, N.; Sakugawa, H. Geochem. J. 2003, 37, 217– 232.
determined by measuring the absorbance of the dialyzed solution at 415 nm.32 Irradiation Experiment. In the irradiation experiments, a solar simulator (Oriel model 81160-1000, Oriel Corp.) equipped with a 300 W Xe lamp (ozone free, model 6258, Oriel Corp.) was used. To simulate actual solar irradiance, wavelengths less than 300 nm were filtered out by optical filters (Oriel AM 0 and AM1.0, Oriel Instruments).33 The solar simulator employed in this study has been demonstrated to produce spectra which mimic that of the solar radiation. The wavelength range represents radiation very close to natural sunlight, from 300 to 700 nm.34 The photochemical reactions were performed in a custom-made quartz glass cell (Workshop for Advanced Techniques, Hiroshima University). The quartz photochemical reaction cell was 3 cm in diameter, 1.5 cm in length, and had a 6.5 mL capacity. The solution inside the cell was gently stirred with a Teflon stirring bar and maintained at about 20 °C using a Neslab RTE 111 recirculating water bath. The daily actinic flux was determined by chemical actinometry (2-nitrobenzaldehyde, 2-NB) using the same quartz cell that was used for the photochemical experiments.35,36 The apparent firstorder photolysis rate constant for the degradation of 2-nitrobenzaldehyde (J2NB) was determined by HPLC with a UV detector set at an absorbance wavelength of 260 nm. The separations were carried out on the same column as that used for the NO determination, with an acetonitrile-water mixture (40/60, v/v) as eluent at a flow rate of 1 mL min-1. The J2NB values for the solar simulator employed in this study ranged from 0.0057 to 0.0064 s-1. However, all data relating to photochemical reactions were normalized to a 2-NB degradation rate of 0.0093 s-1 which was determined at noon under clear sky conditions in Higashi-Hiroshima city (34° 25′ N) on May 1, 1998.37 This suggests that the solar simulator employed in this study was about two-thirds as powerful as natural sunlight. Moreover, it must be recognized that photochemical reaction rates including NO photoformation rates depend on the wavelength of the light source. Hence, the normalization using the 2-NB photolysis rate constant enables comparison among data obtained. Analytical Procedure. A 50 µM DAF-2 solution was prepared from 500 µM stock solution in phosphate buffer (0.1 M phosphate, pH 7.4) just before use. An aliquot of 50 µM DAF-2 solution was added to 5.0 mL of water sample in an airtight 6.5 mL quartz glass cell, giving a final concentration of 5 µM DAF-2 for NO determination. In all experiments, water samples were diluted by
