Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals
In the present work, a fluorimetric automatic method based on multisyringe flow injection analysis (MSFIA) was developed for in vitro evaluation of scavenging capacity against nitric oxide (NO) using 4,5-diaminofluorescein (DAF-2) as an NO-selective fluorogenic probe. The MSFIA manifold was assemble...
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| Veröffentlicht in: | Analytical and bioanalytical chemistry 2010-08, Vol.397 (7), p.3005-3014 |
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| container_title | Analytical and bioanalytical chemistry |
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| creator | Ribeiro, Joana P. N Magalhães, Luís M Segundo, Marcela A Reis, Salette Lima, José L. F. C |
| description | In the present work, a fluorimetric automatic method based on multisyringe flow injection analysis (MSFIA) was developed for in vitro evaluation of scavenging capacity against nitric oxide (NO) using 4,5-diaminofluorescein (DAF-2) as an NO-selective fluorogenic probe. The MSFIA manifold was assembled to perform the in-line generation of NO and the competitive reaction of putative scavenger molecules and DAF-2 with NO at conditions close to those found in vivo regarding temperature (37°C), pH (7.4), and concentration of NO (less than 1 μM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC₅₀ values were calculated for rutin (1.30 ± 0.02 μM, positive control), cysteine (321 ± 8 μM), reduced glutathione (1106 ± 93 μM), uric acid (134 ± 12 μM), dipyrone (1.36 ± 0.06 μM), and captopril (363 ± 28 μM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC₅₀ assessment was performed automatically, in a dilution chamber placed in the flow system. A determination throughput of 16 h⁻¹ and a good precision were attained (relative standard deviation between 1.6 and 9.0%), fostering the application of the proposed method to routine/screening analysis of scavenging capacity against NO. [graphic removed] |
| doi_str_mv | 10.1007/s00216-010-3883-5 |
| format | Article |
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N ; Magalhães, Luís M ; Segundo, Marcela A ; Reis, Salette ; Lima, José L. F. C</creator><creatorcontrib>Ribeiro, Joana P. N ; Magalhães, Luís M ; Segundo, Marcela A ; Reis, Salette ; Lima, José L. F. C</creatorcontrib><description>In the present work, a fluorimetric automatic method based on multisyringe flow injection analysis (MSFIA) was developed for in vitro evaluation of scavenging capacity against nitric oxide (NO) using 4,5-diaminofluorescein (DAF-2) as an NO-selective fluorogenic probe. The MSFIA manifold was assembled to perform the in-line generation of NO and the competitive reaction of putative scavenger molecules and DAF-2 with NO at conditions close to those found in vivo regarding temperature (37°C), pH (7.4), and concentration of NO (less than 1 μM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC₅₀ values were calculated for rutin (1.30 ± 0.02 μM, positive control), cysteine (321 ± 8 μM), reduced glutathione (1106 ± 93 μM), uric acid (134 ± 12 μM), dipyrone (1.36 ± 0.06 μM), and captopril (363 ± 28 μM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC₅₀ assessment was performed automatically, in a dilution chamber placed in the flow system. A determination throughput of 16 h⁻¹ and a good precision were attained (relative standard deviation between 1.6 and 9.0%), fostering the application of the proposed method to routine/screening analysis of scavenging capacity against NO. 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The MSFIA manifold was assembled to perform the in-line generation of NO and the competitive reaction of putative scavenger molecules and DAF-2 with NO at conditions close to those found in vivo regarding temperature (37°C), pH (7.4), and concentration of NO (less than 1 μM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC₅₀ values were calculated for rutin (1.30 ± 0.02 μM, positive control), cysteine (321 ± 8 μM), reduced glutathione (1106 ± 93 μM), uric acid (134 ± 12 μM), dipyrone (1.36 ± 0.06 μM), and captopril (363 ± 28 μM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC₅₀ assessment was performed automatically, in a dilution chamber placed in the flow system. A determination throughput of 16 h⁻¹ and a good precision were attained (relative standard deviation between 1.6 and 9.0%), fostering the application of the proposed method to routine/screening analysis of scavenging capacity against NO. 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| ispartof | Analytical and bioanalytical chemistry, 2010-08, Vol.397 (7), p.3005-3014 |
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| subjects | Analysis Analytical Chemistry Antioxidants Automation Biochemistry Characterization and Evaluation of Materials Chemistry Chemistry and Materials Science Cysteine Exact sciences and technology Flow Injection Analysis - instrumentation Flow Injection Analysis - methods Fluorimetry Food Science Free Radical Scavengers - analysis Free Radical Scavengers - blood Human plasma Humans Laboratory Medicine Mathematical analysis Methods Monitoring/Environmental Analysis Multisyringe flow injection analysis Nitric oxide Nitric Oxide - analysis Original Paper Radicals Scavenging Scavenging capacity Spectrometric and optical methods Uric acid |
| title | Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals |
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