Anal. Chem. 2000, 72, 4064-4072
Simultaneous Derivatization and Quantification of the Nitric Oxide Metabolites Nitrite and Nitrate in Biological Fluids by Gas Chromatography/Mass Spectrometry Dimitrios Tsikas*
Institute of Clinical Pharmacology, Hannover Medical School, D-30623 Hannover, Germany
Simultaneous quantification of nitrite and nitrate, the major oxidative metabolites of L-arginine-derived nitric oxide (NO), in biological fluids by GC or GC/MS methods is currently impossible. The separate analysis of these anions is associated with severe methodological problems. Therefore, a GC/MS method was developed which allows, for the first time, simultaneous quantification of nitrite and nitrate in various biological fluids. The method involves a single derivatization procedure, by which endogenous nitrite and nitrate and their externally added 15N-labeled analogues are simultaneously converted in aqueous acetone by pentafluorobenzyl bromide to the nitro and nitric acid ester pentafluorobenzyl derivatives, respectively, and a single GC/MS analysis. Nitrite and nitrate concentrations measured in plasma and urine of humans by this method correlated excellently with those from quantification of nitrite and nitrate in these matrixes using a previously reported GC/MS method that, however, requires reduction of nitrate to nitrite. Also, the present method enables discrimination between S-nitro- and S-nitroso-glutathione, which have identical chromatographic and spectrophotometric properties. The method is very useful to routinely study metabolism and reactions of NO and its metabolites in vitro and in vivo. It is accurate, interference-free, sensitives50 fmol of [15N]nitrite and [15N]nitrate were detected at signal-to-noise ratios of 870:1 and 95:1, respectivelysand should be a reference method for nitrite and nitrate measurements. The emergence of nitric oxide (NO) as a cell signalling agent is one of the most important and exciting discoveries of the last two decades. Interest in NO was heightened when it emerged that the L-arginine/NO biosynthetic pathway is involved in many physiological and pathophysiological processes. These processes include vasodilation, inhibition of platelet aggregation and adhesion, immune function, neurotransmission, inflammation, atherosclerosis, cytotoxicity, cell proliferation, and apoptosis. Consequently, NO and NO-releasing drugs are targets of many therapeutic interventions. * Corresponding author. Phone: 49 511 532 3959. Fax: 49 511 532 2750. E-mail:
[email protected].
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To understand the role of NO in these conditions, sensitive and specific detection of NO and its many metabolites in complex biological fluids is required. The variety of NO metabolites includes nitrite and nitrate, which are the most abundant circulating and excretory NO metabolites in humans, peroxynitrite, nitrosonium and nitroxide ions, and S-nitroso and S-nitro compounds.1 The latter NO metabolites are unstable and degrade to form again nitrite and nitrate and other reaction products such as 3-nitrotyrosine, hydroxylamine, and ammonia.2 Interest in the measurement of nitrite, nitrate, and other NO metabolites in various body fluids has greatly increased in recent years.3 The assays currently available for the measurement of nitrite and nitrate in body fluids and their clinical applications have been recently reviewed.3 Among them, GC/MS methods form the basis for reference methods for the assessment of nitrite and nitrate in biological fluids. Non-GC/MS methods include spectrophotometric assays based on the Griess reaction,4 chemiluminescence,5 capillary electrophoresis,6 and high-perfromance liquid chromatography.7 Without any exception, GC and GC/MS methods require conversion of nitrite and nitrate to volatile and thermally stable derivatives prior to detection. In principle, two derivatization reactions are useful for nitrite and nitrate: The first reaction involves concentrated sulfuric acid or trifluoroacetic anhydride catalyzed nitration of aromatic reactants such as benzene and 1,3,5trimethoxybenzene8,9 and toluene,10 respectively. Analysis of nitrite by these assays requires preceding oxidation of nitrite to nitrate. The second reaction is the nucleophilic substitution of bromide in R-bromo-2,3,4,5,6-pentafluorotoluene (PFB-Br) by nitrite to form (1) Butler, A. R.; Flitney, F. W.; Williams, D. L. H. Trends in Pharmaceutical Science 1995, 16, 18-22. (2) Gaston, B. Biochim. Biophys. Acta 1999, 1411, 323-333. (3) Ellis, G.; Adatia, I.; Yazdanpanah, M.; Makela, S. K. Clin. Biochem. 1998, 31, 195-220. (4) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131-138. (5) Farell, A. J.; Blake, D. R.; Palmer, R. M. J.; Moncada, S. Ann. Rheum. Dis. 1992, 51, 1219-1222. (6) Leone, A. M.; Francis, P. L.; Rhodes, P.; Moncada, S. Biochem. Biophys. Res. Commun. 1994, 200, 951-957. (7) Preik-Steinhoff, H.; Kelm, M. J. Chromatogr., B 1996, 685, 348-352. (8) Tesch, J. W.; Rehg, W. R.; Sievers, R. E. J. Chromatogr. 1976, 126, 743755. (9) Gutzki, F.-M.; Tsikas, D.; Alheid, U.; Fro¨lich, J. C. Biol. Mass Spectrom. 1992, 21, 97-102. (10) Smythe, G. A.; Matanovic, G.; Yi, D.; Duncan, M. W. Nitric Oxide 1999, 3, 67-74. 10.1021/ac9913255 CCC: $19.00
© 2000 American Chemical Society Published on Web 07/19/2000
R-nitro-2,3,4,5,6-pentafluorotoluene (PFB-NO2).11 The former reaction has been utilized to specifically and accurately quantify nitrite and nitrate in human plasma and urine by GC/MS and GC/ tandem MS.12-14 However, in this method, nitrate has to be reduced to nitrite prior to derivatization. In the present paper, this method is referred to as the old method. From all the GC and GC/MS assays currently available, there is no method allowing simultaneous quantification of nitrite and nitrate using a single derivatization step. Such a method would be greatly superior over existing methods not only because it would enable simultaneous analysis of nitrite and nitrate within a single run, but it would also eliminate serious problems arising from oxidation of nitrite to nitrate and reduction of nitrate to nitrite. These problems include variations of recovery due to incomplete reduction4 or oxidation8 and lack of suitable internal standards,4,8 interferences by nitro- or nitroso-group-containing compounds that may artifactually produce nitrate and nitrite during oxidation15,16 or reduction,15,17,18 respectively, and inability to accurately measure nitrite in the presence of a high excess of nitrate.8,9 In the present work, we describe a specific and accurate GC/MS method (referred to as the new method) that allows simultaneous derivatization and quantification of nitrite and nitrate in relevant human biological fluids such as plasma, urine, and saliva, which does not suffer from the severe problems and disadvantages of previous methods mentioned above,8,9,15-18 and is, furthermore, useful for routine analysis. Therefore, the new method is preferable to older methods and should be a reference method for the measurement of nitrite and nitrate. EXPERIMENTAL SECTION Instrumentation. A Hewlett-Packard MS engine 5890A connected directly to a gas chromatograph 5890 series II equipped with an autosampler (sample tray capacity for 100 vials), HewlettPackard model 7673 (Waldbronn, Germany) was used for GC/ MS analyses. GC/tandem MS was carried out on a Thermoquest TSQ 7000 apparatus (San Jose, CA) connected directly to a Thermoquest Carlo Erba Instruments gas chromatograph Trace 2000 equipped with an autosampler, model AS 2000. Three fusedsilica capillary columns were used: An Optima 17 (15 m × 0.25mm i.d., 0.25-µm film thickness; referred to as column 1) for method validation, an Optima 17 (12 m × 0.2-mm i.d., 0.2-µm film thickness; referred to as column 2) for routine analyses, both from Macherey-Nagel (Du¨ren, Germany), and a DB-5 MS (30 m × 0.25mm i.d., 0.25-µm film thickness) from J & W Scientific (Rancho Cordova, CA). HPLC analyses were performed on an LKB solvent delivery system, model 2249 (Freiburg, Germany), coupled with (11) Wu, H.-L.; Chen, S. H.; Funazo, K.; Tanaka, M.; Shono, T. J. Chromatogr. 1984, 291, 409-415. (12) Tsikas, D.; Bo ¨ger, R. H.; Bode-Bo ¨ger, S. M.; Gutzki, F.-M.; Fro ¨lich, J. C. J. Chromatogr., B 1994, 661, 185-191. (13) Tsikas, D.; Gutzki, F.-M.; Rossa, S.; Bauer, H.; Neumann, C.; Dockendorff, K.; Sandmann, J.; Fro ¨lich, J. C. Anal. Biochem. 1997, 244, 208-220. (14) Tsikas, D.; Gutzki, F.-M.; Sandmann, J.; Schwedhelm, E.; Fro¨lich, J. C. J. Chromatogr., B 1999, 731, 285-291. (15) Tsikas, D.; Fuchs, I.; Gutzki, F.-M.; Fro¨lich, J. C. J. Chromatogr., B 1998, 715, 441-444. (16) Rhodes, P.; Leone, A. M.; Francis, P. L.; Struthers, A. D.; Moncada, S. Biochem. Biophys. Res. Commun. 1995, 209, 590-596. (17) Greenberg, S. S.; Xie, J.; Spitzer, J. J.; Wang, J.; Lancaster, J.; Grishamn, M. B.; Powers, D. R.; Giles, T. D. Life Sci. 1995, 57, 1949-1961. (18) Tsikas, D.; Sandmann, J.; Gutzki, F.-M.; Fro ¨lich, J. C. J. Chromatogr., B 1999, 729, 375-378.
a variable UV-vis LDC Spectromonitor, model 1204D (LDC Analytical, Gelnhausen, Germany), connected with a Shimadzu integrator, model C-R3A (Kyoto, Japan). The analytical column (250 × 4.6-mm i.d.) was packed with 5-µm-particle-size Nucleosil 100-5C18 material from Macherey-Nagel (Du¨ren, Germany). The mobile phase consisted of acetonitrile/water (1:1, v/v) and was pumped at a flow rate of 2 mL/min. The effluent was monitored at 205 nm. Reagents. Sodium [15N]nitrate (declared as 98+ at. % at 15N) was obtained from Sigma (Munich, Germany). Sodium [15N]nitrite (declared as 98 at. % at 15N) was bought from Cambridge Isotope Laboratories (Andover, MA). For quantitative measurements, stock solutions (each 8 mM) of unlabeled nitrate and nitrite and [15N]nitrate and [15N]nitrite were prepared in distilled water and diluted with distilled water, appropriately, when needed. 2,3,4,5,6Pentafluorobenzyl bromide (PFB-Br) and nitronium tetrafluoroborate were obtained from Aldrich (Steinheim, Germany). Safety Considerations. PFB-Br is corrosive and an eye irritant. Cd and HgCl2 are highly toxic. Inhalation and contact with skin and eyes should be avoided. All work should be performed in a well-ventilated fume hood. Reduction and Derivatization Procedures. For reduction of nitrate to nitrite, solutions of nitrate and [15N]nitrate in aqueous phosphate buffer (50 mM, pH 7.4), plasma, urine, or saliva (each 100 µL) were diluted with ammonium chloride buffer (1 M, pH 8.8; each 900 µL), spiked with cadmium powder (100 mesh; each 10 mg), and samples were shaken for 90 min at room temperature.13 In quantitative analyses, derivatization with PFB-Br was performed by mixing a nitrite-12 and/or nitrate-containing matrix (100 µL) with acetone (400 µL) and PFB-Br (10 µL) and incubating either at 50 or 25 °C, each for 60 min. For quantitative measurements, biological fluids (1 mL) were spiked with the 15N-labeled nitrite and/or nitrate at relevant final concentrations prior to the reduction and/or derivatization procedures. After derivatization, samples were cooled to room temperature, acetone was evaporated under a nitrogen stream, reaction products were extracted from the remaining aqueous phase by vortex-mixing with toluene (1 mL) for 1 min, and a 800-µL aliquot of the organic phase was transferred into a clean 1.5-mL autosample glass vial. Gas Chromatography/Mass Spectrometry. Aliquots (0.5 µL) were injected in the splitless mode using the following temperature program in both instruments: columns were held at 70 °C for 1 min, and then the temperature was increased to 280 °C at a rate of 30 °C/min. Helium (70 kPa for column 1 and 50 kPa for column 2) and methane (200 Pa) were used as the carrier and the reagent gas, respectively, for negative-ion chemical ionization (NICI) in the Hewlett-Packard instrument. In some experiments, isothermal (105 °C) analyses were performed using column 2. Electron energy and electron current were set to 230 eV and 300 µA, respectively, for NICI. Electron energy was 70 eV in electron impact (EI) ionization. In GC/tandem MS analyses, helium (at a constant pressure of 70 kPa) and methane (530 Pa) were used as carrier and reagent gases, respectively. For collisioninduced dissociation (CID), argon (0.13 Pa) was used at a collision energy of 25 eV. Electron energy and electron current were 230 eV and 300 µA, respectively. Constant temperatures of 180, 280, and 200 °C were kept at the ion sources, interfaces, and injectors of both instruments, respectively. Analytical Chemistry, Vol. 72, No. 17, September 1, 2000
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Figure 1. HPLC-UV chromatograms from analyses of separate reaction mixtures of [15N]nitrite (A) and [15N]nitrate (B) with PFB-Br. Aqueous solutions of [15N]nitrite and [15N]nitrate (each 100 µL of 100 mM solutions in 50 mM potassium phophate buffer, pH 7.4) were treated with acetone (400 µL) and PFB-Br (10 µL) and incubated at 50 °C for 60 min. After cooling to room temperature, each 20-µL aliquot was injected onto the HPLC column. Label indicates identified peaks.
Statistical Analysis. Quantitative analyses in the present study were performed in duplicate. Values are given as the mean ( SD. RESULTS Identity of Reaction Products. Direct HPLC analysis of reaction mixtures of [15N]nitrite and [15N]nitrate with PFB-Br in
aqueous acetone (Figure 1) and GC/MS analysis of toluene extracts (not shown) showed formation of various reaction products. GC/MS analysis of the reaction product eluting at 6.2 min on HPLC (Figure 1, A) and emerging from the GC column 1 as a single peak at 3.24 min was identified as R-[15N]nitro2,3,4,5,6-pentafluorotoluene, i.e., PFB-15NO2. The EI mass spectrum of this compound showed a very weak molecular ion at m/z 228 ([PFB-15NO2]+,
