Determination of the Scavenging Capacity Against Reactive Nitrogen Species by Automatic Flow Injection-Based Methodologies

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1 Chapter 8 Determination of the Scavenging Capacity Against Reactive Nitrogen Species by Automatic Flow Injection-Based Methodologies Marcela A. Segundo, Luís M. Magalhães, Joana P.N. Ribeiro, Marlene Lúcio, and Salette Reis Abstract Automatic flow-based systems have been applied to assay scavenging capacity against reactive oxygen and nitrogen species, providing analytical tools which can cope with different types and large number of samples. In the present chapter, a flow injection analysis procedure is described for the assessment of peroxynitrite scavenging. A sequential injection analysis procedure is also described for determining the scavenging capacity against the nitric oxide radical. For both systems, reaction between putative antioxidants and the reactive species of nitrogen takes place inside the flow conduits before addition of luminol and further detection of remaining reactive species by chemiluminescence. Key words: Flow injection analysis, sequential injection analysis, scavenging capacity, reactive species of nitrogen, nitric oxide, peroxynitrite, chemiluminescence, automation. 1. Introduction Flow injection analysis (FIA) (1) is an automation tool for chemical analysis that allows the performance of assays which are not feasible when carried out manually, by taking advantage of the reproducible timing attained in these systems. The implementation of methods based on transient light formation, generated by bio- and chemiluminescence (2) is only one example that illustrates the advantages of FIA features. During the 1990s the increased availability of computers in the chemical lab fostered the development of sequential injection analysis (SIA) (3). Based on the same principles of FIA, this new H.O. McCarthy, J.A. Coulter (eds.), Nitric Oxide, Methods in Molecular Biology 704, DOI / _8, Springer Science+Business Media, LLC

2 92 Segundo et al. generation of computer-controlled flow systems offers more flexibility concerning the method development and operation (4). Any changes (sample volume, reagent selection, sample dilution, and reagent-to-analyte ratio) are accomplished via flow programming rather than by physical reconfiguration of the flow path. Therefore, the assessment of antioxidant (AO) capacity using these flow-based systems is rather convenient (5). Hence, the evaluation of scavenging capacity against biologically relevant species, such as reactive species of oxygen (ROS) and reactive species of nitrogen (RNS, including nitric oxide and peroxynitrite), has been proposed (6) with a significant improvement in experimental protocol towards mimicking reaction conditions found in vivo (7). In this context, a versatile and simple FIA system based on chemiluminescence (CL) detection was developed by Sariahmetoglu et al. (8) for the determination of scavenging capacity against several reactive species, including peroxynitrite (ONOO ) (8). A three-channel manifold (Fig. 8.1) was assembled to accommodate the chemiluminogenic reagent (luminol), the reactive species (ONOO in this case), and the carrier stream, which transported the injected sample with putative antioxidant properties. Using this manifold configuration, the ONOO produced offline through the reaction between sodium nitrite and hydrogen peroxide (9) was first mixed with the injected sample. After a residence time of about 1.4 s in the reaction coil, this mixture was subsequently merged with luminol, just before entering in the CL-flow cell. Thus, the FIA design exploits the consumption of ONOO by antioxidants, which results in the appearance of negative CL signal proportional to the scavenging ability of the compounds (Fig. 8.2). This manifold was also applied to Fig Schematic representation of FIA system for the chemiluminescence determination of scavenging capacity against peroxynitrite. AO, antioxidant sample; C, MilliQ H 2 O as carrier; ONOO, peroxynitrite standard solution (10 5 M); luminol solution (10 4 M) prepared in carbonate buffer 0.1 M at ph 10; IV, injection valve; P, peristaltic pump; RC, reaction coil (length = 60 cm; volume = 118 μl); CLD, chemiluminescence detector equipped with flow-through cell; PMT, photomultiplier tube; W, waste.

3 Determination of the Scavenging Capacity Against Reactive Nitrogen Species 93 Fig Schematic representation of (a) inhibition signal profile for increasing concentrations (S 1 S 4 ) of test sample with (b) detailed indication of CL signal parameters for calculation of IC 50 value in the FIA method for assessing the scavenging capacity against ONOO. determination against other reactive species (such as superoxide radical ion, hydrogen peroxide, hypochlorite ion, and hydroxyl radical) by changing the solution fed to one of the flow channels. There are also FIA systems for in vivo nitric oxide (NO) detection (10). These include measurements of physiological NO concentrations present in blood or brain tissues (11) and for pathological NO following acute myocardial infarction (12). A flow-based manifold comprising of a microdialysis probe with CL detection of NO has been described for studying the release of this reactive species in the rat brain following traumatic injury (13). However, none of these systems was applied to the scavenging properties of the compound. Recently, Miyamoto et al. (14) proposed a SIA system for CL determination of NO (and also of O 2 ) that accommodates both reactions (14). In this system, sample and NOR1 ((±)-(E)-4-methyl-2-[(E)hydroxyimino]-5- nitro-6-methoxy-3-hexenamide), a NO donor, were sequentially aspirated into the holding coil (Fig. 8.3). After flow reversal, the two overlapped zones were directed towards the detector while NO was scavenged by antioxidants present in the sample. Subsequently, the remaining NO was measured after addition of luminol from a T connector just before the detector (Fig. 8.4). The system was applied to compounds/enzymes with known activity against this radical, and also to commercial vitamin supplements.

4 94 Segundo et al. Fig Schematic representation of SIA system for the chemiluminescence determination of scavenging capacity against nitric oxide radical. AO, antioxidant sample; C, HEPES-NaOH buffer 0.1 M at ph 8.2 as carrier; NOR1, NO donor agent (0.010 μm) prepared in a mixture of DMSO and 0.1 M HCl (50:50, v/v); luminol solution (0.2 mm) prepared in water containing 1% of N,N-dimethylformamide; SV, selection valve; P, peristaltic pump; AP, auxiliary peristaltic pump; HC, holding coil (length = 100 cm; volume = 197 μl); PMT, photomultiplier tube; W, waste. Fig Schematic representation of analytical signal profile for increasing concentrations (S 1 S 3 ) of test sample in the SIA system for the determination of scavenging capacity against NO. 2. Materials 2.1. Automatic Determination of Scavenging Capacity Against Peroxynitrite Flow Injection Apparatus Flow injection systems should contain the following components (see Note 1): 1. Multichannel peristaltic pump, with at least three channels, equipped with propulsion tubes (see Note 2).

5 Determination of the Scavenging Capacity Against Reactive Nitrogen Species Rotary injection valve (6-port) equipped with 20 μl loop. 3. Polytetrafluoroethylene (PTFE) tubing (see Note 3). 4. Y-shaped low-pressure connectors and fittings (see Note 4). 5. Chemiluminescence detector equipped with flow-through cell. 6. Chart strip recorder or signal processing unit (see Note 5) Synthesis of Peroxynitrite Stock Solution 1. Two 10 ml glass syringes. 2. Polytetrafluoroethylene (PTFE) tubing (see Note 3). 3. Y-shaped low-pressure connectors and fittings (see Note 4). 4. Magnetic stirrer ml glass beaker immersed in ice bath. 6. UV/vis spectrophotometer M sodium nitrite (NaNO 2 ) solution: dissolve g of NaNO 2 in 25 ml of MilliQ H 2 O M hydrogen peroxide (H 2 O 2 ) solution: add 1.5 ml of hydrochloric acid (HCl) (37% m/m, density 1.2 g/ml) to 10 ml of MilliQ H 2 O (giving 0.7 M HCl), followed by addition of 1.5 ml of H 2 O 2 (30% m/m, density 1.1 g/ml), and H 2 O to give a final volume of 25 ml. 9. Dissolve 1.2 g of sodium hydroxide (NaOH) in 25 ml of MilliQ H 2 O, producing 1.2 M NaOH Solutions M carbonate buffer (ph 10): dissolve 4.2 g of sodium hydrogen carbonate (NaHCO 3 ) in MilliQ H 2 O(see Note 6), adding enough 2 M NaOH to give the required ph and making up the volume to 500 ml M NaOH solution: dissolve 4 g of NaOH in 50 ml of water M luminol stock solution: dissolve 17.7 mg of luminol in 100 ml of 0.1 M carbonate buffer, ph 10 (see Note 7) M luminol working solution (see Note 8): tenfold dilution of the 10 3 M luminol stock solution using 0.1 M carbonate buffer M ONOO solution: appropriate dilution of stock solution in MilliQ H 2 O M mannitol solution: dissolve 91.1 mg of mannitol in 50 ml M ascorbic acid stock solution: dissolve g of ascorbic acid in 100 ml of MilliQ water , 50, 100, and 500 μm ascorbic acid solutions.

6 96 Segundo et al Automatic Determination of Scavenging Capacity Against NO Sequential Injection Apparatus Sequential injection systems should contain the following components (see Note 1): 1. Single-channel peristaltic pump equipped with propulsion tubing (see Note 9). 2. Auxiliary single-channel pump. 3. Selection valve (8-port). 4. Polytetrafluoroethylene (PTFE) tubing (see Note 3). 5. Y-shaped low-pressure connectors and fittings (see Note 4). 6. Chemiluminescence detector equipped with flow-through cell. 7. Chart strip recorder or signal processing unit (see Note 5). 8. Hardware and software to operate the selection valve and themainpump(see Note 10) Solutions M HEPES buffer (ph 8.2): dissolve g of N-2-hydroxyethyl piperazine-n -2-ethanesulfonic acid in MilliQ H 2 O(see Note 6), adding enough 2 M NaOH to give the required ph and making up the volume to 500 ml MNOR1((±)-(E)-4-methyl-2-[(E)hydroxyimino]- 5-nitro-6-methoxy-3-hexenamide) stock solution: dissolve 1.2 mg of NOR1 in 50 ml of a mixture of DMSO and 10 4 M HCl (50:50, v/v) M NOR1 intermediate solution: 100-fold dilution of 10 4 M NOR1 stock solution using a mixture of DMSO and 10 4 M HCl (50:50, v/v) as diluent M NOR1 working solution: 100-fold dilution of the 10 6 M NOR1 intermediate solution using a mixture of DMSO and 10 4 M HCl (50:50, v/v) as diluent M HCl: dilute 1.64 ml of commercial HCl solution (37% m/m, density 1.2 g/ml) with H 2 Oupto1L M HCl: dilute M HCl solution 200-fold with H 2 O M luminol stock solution: dissolve g of luminol in 50 ml of N,N-dimethylformamide (DMF) (see Note 7) M luminol working solution (see Note 8): 100- fold dilution of the M luminol stock solution using MilliQ H 2 O.

7 Determination of the Scavenging Capacity Against Reactive Nitrogen Species M ascorbic acid stock solution: dissolve g of ascorbic acid in 100 ml of MilliQ H 2 O , 50, 100, 200, and 300 μm ascorbic acid solutions. 3. Methods 3.1. Automatic Determination of Scavenging Capacity Against Peroxynitrite Synthesis of Peroxynitrite In the flow injection system, the scavenging reaction against ONOO takes place at reaction coil RC (Fig. 8.1), where the sample introduced in the system by a rotary valve is mixed with ONOO solution. After a mean residence time of about 1.4 s at RC (calculated from the flow rate and volume contained in RC), the mixture sample/onoo is merged with luminol and directed towards the detector, where a CL signal corresponding to the reaction of the remaining ONOO with luminol is attained. The total analysis time is less than 20 s, providing a determination throughput of at least 180 per hour. Stock sodium ONOO solution is prepared from NaNO 2 and H 2 O 2 by a quenched flow synthesis described by Beckman et al. (9). The original procedure involves a flow network that is simplified. 1. Assemble the system as depicted in Fig. 8.5 (see Note 11). 2. Fill the syringes and the beaker with the respective solutions and immerse all parts in ice. 3. Wait 10 min for temperature stabilization and then press both syringe plungers at the same time as fast as possible (see Note 12), driving NaNO 2 and H 2 O 2 through the synthesis coil into the NaOH solution, where the magnetic stirrer Fig Schematic representation of experimental flow set-up for the synthesis of peroxynitrite. A, 0.6MNaNO 2 ; B, 0.6MH 2 O 2 prepared in 0.7 M HCl; C, 1.2MNaOH; SC, synthesis coil (volume = 250 μl); D, ice bath; E, magnetic stirrer.

8 98 Segundo et al. should be activated. The content of the beaker will turn to yellow and the concentration of ONOO should be in the range of mm (see Note 13). 4. The concentration of ONOO in the stock solution is assayed by measuring its absorbance at 302 nm and by considering an extinction coefficient of 1,670 M 1 cm 1 (15) (see Note 14) Operation of Flow Injection System Analytical Procedure 3.2. Automatic Determination of Scavenging Capacity Against Nitric Oxide 1. Assemble the flow system as depicted in Fig. 8.1 (see Note 15). 2. Introduce the pumping tubes into the solutions and start the pump (see Note 16). 3. Fill all the lines with the appropriate solutions (see Note 17) and check the flow rate in each channel (see Note 18). Flow rates should be 1.25 ml/min in the channels filled with H 2 O and ONOO solution and 2.5 ml/min in the luminol channel (see Note 19). Wait until a stable baseline signal is attained before starting analysis. 4. Perform the analytical procedure (Section 3.1.3) for all samples. 5. After finishing the analytical procedure, wash the system for 15 min, using H 2 O in all channels. Leave all tubing filled with H 2 O if the system is to be used within a week; otherwise empty the tubing and disassemble the system. 1. Fill the loop of the rotary valve (load position) with the solution to be tested (see Note 20). 2. Inject the test solution by changing the injection valve position. Wait until a minimum value of CL is attained before returning the injection valve to the load position. 3. Refill the loop and inject the same test solution once again when CL signal returns to its baseline value. Repeat this procedure until achieving 4 5 replicate measurements for the same test solution. 4. To check if the system is working, use ascorbic acid and mannitol as positive and negative controls, respectively (see Note 21). Under these reaction conditions, the IC 50 (see Section 3.3) value for ascorbic acid is about 63 μm. For the sequential injection system described here, the scavenging reaction against NO takes place at the holding coil HC (Fig. 8.3) as the sample and NOR1 are sequentially aspirated into the tubing. NOR1 solution is prepared in acidic media and it starts to release NO when mixed with a carrier buffer at ph 8.2. Antiox-

9 Determination of the Scavenging Capacity Against Reactive Nitrogen Species 99 idant species present in the sample react with NO formed in the HC. The remaining NO is detected by the reaction with luminol after propelling the content of the HC towards the chemiluminescence detector. The total analysis time is around 60 s, providing a determination throughput of 60 per hour Operation of Sequential Injection System 1. Assemble the sequential injection system as depicted in Fig. 8.3 (see Note 15). 2. Introduce the pumping tube into the carrier solution, select the waste port in the selection valve, and start the pump (see Note 16) at 1.5 ml/min. Fill the HC completely. 3. Fill the port with NOR1 solution by aspirating a small amount (e.g. 100 μl) of this solution into the HC. 4. Select the waste port and wash the HC (e.g., 400 μl), discarding excess NOR1 solution. 5. Execute the last two steps for antioxidant/test sample, selecting the appropriate port. 6. Select the detector port in the selection valve and fill the tubing connecting the selection valve and the chemiluminescence detector with buffer (see Note 22). During this step, the auxiliary pump must also be activated at 1.0 ml/min. Wait until a stable baseline signal is attained before starting analysis. These operations can be carried out manually or through computer control, depending on the equipment applied. 7. Perform the analytical procedure (Section 3.2.2) for all samples. 8. After finishing the analytical procedure, wash the system using H 2 O as a carrier and replace the luminol solution with water. Wash all ports of the selection valve that were used. Leave all the tubing filled with water if the system is to be used within a week; otherwise empty the tubing and disassemble the system Analytical Procedure 1. Start the luminol auxiliary pump at 1.0 ml/min. 2. Aspirate 5 μl of the test sample into the HC by selecting the sample port in the selection valve followed by activation of the pump at 0.3 ml/min. 3. Repeat the same operation for the NOR1 solution, selecting the appropriate port. 4. Select the detector port in the selection valve and, reversing the flow at the main pump, propel the HC content towards the detector at 1.5 ml/min for 24 s. Repeat this procedure to obtain 4 5 replicate measurements for the same test solu-

10 100 Segundo et al. tion (see Note 23). To change the test solution, follow the procedure indicated in Section Use ascorbic acid as a positive control (see Note 21). Under these reaction conditions, the IC 50 value (see Section 3.3) for ascorbic acid is about 54 μm Sample Analysis 1. The flow systems described here can cope with liquid samples. Hence, any liquid sample can be directly introduced into both systems. Extraction procedures should be performed for solid samples, including a filtration step to remove any particle that would block the flow tubing. 2. Tolerance to organic solvents should be tested by injecting the appropriate dilution of the applied solvent into the flow systems. Signal quenching/enhancement should not be observed (see Note 24). 3. Blank measurements should always be taken to assess: (i) direct reaction between sample components and CL reagents. (ii) formation of luminescent products upon reaction of sample components and reactive nitrogen species (RNS). (iii) intrinsic luminescence from sample components. These measurements are performed by assessing the analytical signal for the highest concentration of test sample, replacing some reagent solutions by their solvents. Hence, in the first case, replace the ONOO solution with MilliQ H 2 O (scavenging capacity against ONOO ) or NOR1 solution by DMSO and 10 4 M HCl solution (50:50, v/v) (scavenging capacity against NO). In the second case, replace luminol solution with 0.1 M carbonate buffer, ph 10 (scavenging capacity against ONOO ) or with 1% (v/v) DHF solution (scavenging capacity against NO ). In the third case, replace both RNS and luminol solutions as described before. For all cases, none or negligible luminescence should be attained in order to exclude interferences on CL signal from sample components. 4. Results are generally expressed as IC 50 values defined as the concentration of test sample that provides a 50% inhibitory effect, in this case the 50% decrease of the CL analytical signal attained in the absence of test sample (Figs. 8.2 and 8.4). For the determination of the scavenging capacity against ONOO, the percentage of inhibition is calculated as: ( ) CLbaseline CL sample %inhibition = 100% CL ( baseline ) negative peak height = 100% baseline height

11 Determination of the Scavenging Capacity Against Reactive Nitrogen Species 101 where CL sample is the minimum CL value attained. For the determination of scavenging capacity against NO, as the reactive species is not fed continuously into the detector, the percentage of inhibition is calculated as: ( ) CLblank CL sample %inhibition = 100% CL blank ( ) blank peak height sample peak height = blank peak height 100% where CL blank is the maximum CL value attained for the test sample solvent and CL sample is the maximum CL value attained for the test sample. 5. If liquid sample/extract is introduced undiluted and the CL signal is completely quenched, appropriate dilutions should be taken until partial inhibition of CL signal is attained. Perform at least four different dilution levels to check for linearity in the zone around 50% inhibition (between 20 and 80% inhibition, for instance). If 50% inhibition is not attained, indicate the maximum percentage attained and the respective concentration level (for instance, 35% inhibition for a concentration of 0.2 μg/ml for a given plant extract). 4. Notes 1. Commercial flow injection and sequential injection systems are available from several manufacturers, namely FIAlab Instruments (Bellevue, Washington, USA), Burkard Scientific (Middlesex, UK), Lachat Instruments (Loveland, Colorado, USA), and GlobalFIA (Fox Island, Washington, USA). 2. The flow rate is defined by the rotation speed of the peristaltic pump and by the internal diameter of the pumping tubing. For this particular application, two tubing diameters are necessary in order to provide a given flow rate. This can be attained using Gilson (Villiers-Le-Bel, France) PVC pumping tubes with colour codes black/black (0.76 mm i.d.) and white/white (1.02 mm i.d.). 3. FIA systems usually employ tubing with an internal diameter of 0.8 mm and an external diameter of 1.6 mm. One metre of this tubing contains approximately 502 μl. Tub-

12 102 Segundo et al. ing with an internal diameter of 0.5 mm or 1.0 mm can also be applied, but its length should be adapted, considering that 1 m contains 196 and 785 μl, respectively. 4. These connectors and fittings are also commercially available from Omnifit (Cambridge, UK) and Gilson (Villiers- Le-Bel, France). 5. FIA systems do not require computer control, with analytical data recorded in strip recorders, where peak height is manually measured. However, nowadays most detection systems have already incorporated data processing features. 6. H 2 O used in the preparation of the solutions and buffers should be of high quality, such as H 2 O obtained from MilliQ systems (resistivity > 18 M cm), to avoid contamination and consequent interference by trace metals. 7. This solution should be stored at 4 C and protected from light by a foil wrapper. It should be discarded after 1 week. 8. This solution should be prepared every day and protected from light by a foil wrapper. It can be used for at least 8 h. 9. Piston pumps equipped with syringes or current liquid chromatography pumps are also applicable. 10. Software is commercially available or it can be created from programming tools such as Visual Basic or Lab View R. 11. After assembling the system, check that there are no leaks by performing the procedure using H 2 O instead of reagents. 12. Make sure that the end of the synthesis coil is dipped in the NaOH solution. 13. The ONOO stock solution may be frozen at 80 C. ONOO gradually decomposes with a half-life of 1 2 weeks, yielding nitrite. Storage at 20 C can result in an increased concentration of ONOO as a supernatant layer is formed above the ice crystals. At this layer ONOO concentration can reach up to 1 M. 14. Dilute the ONOO stock solution 100 1,000-fold, using 1.2 M NaOH as the diluent in order to obtain an absorbance value within the linear Lambert Beer relation. 15. The connection between the detector and the confluence where luminol is added must be as short as possible because light production is fast. 16. Check that solutions are aspirated by removing the pumping tubing a few times from the solution, allowing air bubbles to enter the tube. Observe the movement of air bubbles along the system and if flow pulses (short stops of flow) exist, change the pressure exerted from the pump braces so

13 Determination of the Scavenging Capacity Against Reactive Nitrogen Species 103 that no pulses are visible. If any of the solutions are not aspirated, check if the pump braces are correctly adjusted. 17. Discard or filter solutions containing particulates as they can block the flow tubing. 18. Check the flow rate by placing the pump tubing in a measuring cylinder filled with H 2 O. Determine the volume of H 2 O aspirated during 1 min, which will correspond to the flow rate expressed as millilitre per minute. 19. If necessary, adjust the flow rate by changing the rotation speed of the pump. 20. To fill the injection loop, the test solution can be aspirated through extra pump tubing placed in the peristaltic pump or it can be aspirated by an external syringe. 21. For positive controls, CL quenching should be observed in a concentration-dependent manner. For negative controls, no CL quenching is observed. 22. It is necessary to stop the pump while the port position is changed at the selection valve. This can be easily achieved by computer-controlled operation of equipment. 23. It is essential to perform blank measurements (using sample diluent as test solution) at the beginning and end of analysis. Blank signal peak height is necessary to calculate the inhibition percentage. Solution stability can be assessed by the reproducibility of blank measurement peak height over time. 24. If organic solvent interference is verified, replace the H 2 O by a solution with the same composition of the samples (e.g., same organic solvent percentage) as carrier solution. Analyze positive and negative controls to evaluate the performance of the method. References 1. Ruzicka, J., Hansen, E. H. (1975) Flow injection analyses. 1. New concept of fast continuous-flow analysis. Anal Chim Acta 78, Hansen, E. H., Norgaard, L., Pedersen, M. (1991) Optimization of flow-injection systems for determination of substrates by means of enzyme amplification reactions and chemiluminescence detection. Talanta 38, Ruzicka, J., Marshall, G. D. (1990) Sequential injection a new concept for chemical sensors, process analysis and laboratory assays. Anal Chim Acta 237, Segundo, M. A., Magalhães, L. M. (2006) Multisyringe flow injection analysis: stateof-the-art and perspectives. Anal Sci 22, Magalhães, L. M., Santos, M., Segundo, M. A., Reis, S., Lima, J. L. F. C. (2009) Flow injection based methods for fast screening of antioxidant capacity. Talanta 77, Magalhães, L. M., Lucio, M., Segundo, M. A., Reis, S., Lima, J. L. F. C. (2009) Automatic flow injection based methodologies for determination of scavenging capacity against biologically relevant reactive species of oxygen and nitrogen. Talanta 78,

14 104 Segundo et al. 7. Magalhães, L. M., Ribeiro, J. P. N., Segundo, M. A., Reis, S., Lima, J. L. F. C. (2009) Multi-syringe flow-injection systems improve antioxidant assessment. Trend Anal Chem 28, Sariahmetoglu, M., Wheatley, R. A., Cakici, I., Kanzik, I., Townshend, A. (2003) Flow injection analysis for monitoring antioxidant effects on luminol chemiluminescence of reactive oxygen species. Anal Lett 36, Beckman, J. S., Chen, J., Ischiropoulos, H., Crow, J. P. (1996) Oxidative chemistry of peroxynitrite. Method Enzymol 269, Yao, D. C., Vlessidis, A. G., Evmiridis, N. P. (2001) On-line monitoring of nitric oxide complexed with porphyrine-bearing biochemical materials by using flow injection with chemiluminescence detection. Anal Chim Acta 435, Yao, D. C., Vlessidis, A. G., Evmiridis, N. P., Evangelou, A., Karkabounas, S., Tsampalas, S. (2002) Luminol chemiluminescence reaction: a new method for monitoring nitric oxide in vivo. Anal Chim Acta 458, Yao, D. C., Vlessidis, A. G., Evmiridis, N. P., Siminelakis, S., Dimitra, M. (2004) Possible mechanism for nitric oxide and oxidative stress induced pathophysiological variance in acute myocardial infarction development a study by a flow injection-chemiluminescence method. Anal Chim Acta 505, Wang, J. N., Lu, M. Q., Yang, F. Z., Zhang, X. R., Baeyens, W. R. G., Campaña, A. M. G. (2001) Microdialysis with on-line chemiluminescence detection for the study of nitric oxide release in rat brain following traumatic injury. Anal Chim Acta 428, Miyamoto, A., Nakamura, K., Kishikawa, N., Ohba, Y., Nakashima, K., Kuroda, N. (2007) Quasi-simultaneous determination of antioxidative activities against superoxide anion and nitric oxide by a combination of sequential injection analysis and flow injection analysis with chemiluminescence detection. Anal Bioanal Chem 388, Hughes, M. N., Nicklin, H. G. (1968) Chemistry of pernitrites. I. Kinetics of decomposition of pernitrous acid.jchemsoc A 2,