Reactions of superoxide anion radical with antioxidants and their use in voltammetry

Kinetic parameters were calculated for the electrochemical reduction of oxygen at a glassy-carbon electrode with the generation of superoxide radical anions in a 0.05 M solution of (C₂H₅)₄NI in dimethylformamide in the presence of fat-soluble antioxidants, retinol and -tocopherol. A procedure based on the protonation of the radical anion with antioxidant molecules is proposed for the voltammetric determination of antioxidants to determine milligram amounts of retinol and -tocopherol in model solutions (RSD = 1–2%). The calibration graphs for retinol and -tocopherol are linear in the concentration ranges 9.7 × 10^–5–2.3 × 10^–3 and 6.2 × 10^–4–3.1 × 10^–3 M, respectively. The detection limits for retinol and -tocopherol are 4.8 × 10^–5 and 4.1 × 10 ^–4 M, respectively. The procedure was applied to the determination of the active component (retinol and -tocopherol) in pharmaceuticals.Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 1, 2005, pp. 56–59.Original Russian Text Copyright © 2005 by Ziyatdinova, Gilmetdinova, Budnikov.     

On‐line HPLC method for screening of antioxidants against superoxide anion radical from complex mixtures

A practical approach for rapidly screening antioxidants against superoxide anion radicals from complex mixtures was developed based on the quantitative difference in active compounds before and after their reaction. To test the effectiveness of the approach, seven flavonoids with antioxidative properties were investigated both individually and in a mixture. Using the approach, antioxidants could be rapidly separated and screened with a ranked order of activities in the meantime. The radical scavenging activities were in the following order: quercetin > kaempferol > fisetin > puerarin > luteolin > rutin > baicalein. The order of activity was conducted by comparing the scavenging ratio of the antioxidant, which was completely consistent with the results obtained from the traditional electronic spin resonance. Then, the method was successfully applied to black tea extracts. This approach is fast and convenient for screening, isolating, and analyzing potential antioxidants from a mixture with good quantitative and reproducible ability.     

Detection of synergistic effect of superoxide dismutase and jujubosides on scavenging superoxide anion radical by capillary electrophoresis

A new capillary electrophoresis method was developed to study the synergistic effect of superoxide dismutase and jujuboside A or B on scavenging superoxide anion radical in serum matrix respectively, in which superoxide anion radical was generated from pyrogallol autoxidation. The electrophoresis conditions, and the factors affecting the productive rate of purpurogallin, such as pyrogallol autoxidation product and the activity of superoxide dismutase, were optimized. Under optimal conditions, the content of superoxide dismutase in Gibco newborn calf serum was 7.06 mg/L, RSD was 2.01% and the average recovery was 98.4%. The values of IC₅₀ for jujuboside A and B in the serum matrix were 157.67 and 31.60 mg/L respectively, and they both had synergy on scavenging superoxide anion radical with superoxide dismutase, but there was no the dose‐dependency on this synergy.     

分光光度法测定二氢黄酮化合物对超氧阴离子自由基的清除作用

采用分光光度法测定了来源于几种药用植物的天然二氢黄酮类化合物清除超氧阴离子自由基(O-2·)的能力,探讨了二氢黄酮类化合物清除O-2·的活性与结构之间的关系,以期为开发利用天然抗氧化剂提供科学依据.     

Screening for antioxidants in complex matrices using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

The use of high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection to screen for antioxidants in complex plant-derived samples was evaluated in comparison with two conventional post-column radical scavenging assays (2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(+))). In this approach, acidic potassium permanganate can react with readily oxidisable compounds (potential antioxidants), post-column, to produce chemiluminescence. Using flow injection analysis, experimental parameters that afforded the most suitable permanganate chemiluminescence signal for a range of known antioxidants were studied in a univariate approach. Optimum conditions were found to be: 1×10(-3)M potassium permanganate solution containing 1% (w/v) sodium polyphosphates adjusted to pH 2 with sulphuric acid, delivered at a flow rate of 2.5 mL min(-1) per line. Further investigations showed some differences in detection selectivity between HPLC with the optimised post-column permanganate chemiluminescence detection and DPPH and ABTS(+) assays towards antioxidant standards. However, permanganate chemiluminescence detection was more sensitive. Moreover, screening for antioxidants in green tea, cranberry juice and thyme using potassium permanganate chemiluminescence offers several advantages over the traditional DPPH and ABTS(+) assays, such as faster reagent preparation and superior stability; simpler post-column reaction manifold; and greater compatibility with fast chromatographic separations using monolithic columns.     

Determination of polyamines by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the determination of polyamines (PA) by high-performance liquid chromatography with chemiluminescence detection. It is based on the unsaturated complex of PA with Cu(II) which had a strong catalytic effect on the luminol-H₂O₂ chemiluminescence reaction. The separation of PA was carried out on a reveres phase C18 column using methanol/water (25/75, v/v) as a mobile phase. The method was applied to the analysis of putrescine and the total amount of spermine and spermidine in apple leaves and strawberry fruit. The results indicated that the method is practical and useful.     

Optimization of two methods for the analysis of hydrogen peroxide: high performance liquid chromatography with fluorescence detection and high performance liquid chromatography with electrochemical detection in direct current mode

Two complementary methods were optimized for the separation and detection of trace levels of hydrogen peroxide. The first method utilized reversed-phase high-performance liquid chromatography with fluorescence detection (HPLC-FD). With this approach, hydrogen peroxide was detected based upon its participation in the hemin-catalyzed oxidation of p-hydroxyphenylacetic acid to yield the fluorescent dimer. The second method utilized high performance liquid chromatography with electrochemical detection (HPLC-ED). With this approach, hydrogen peroxide was detected based upon its oxidation at a gold working electrode at an applied potential of 400 mV vs. hydrogen reference electrode (Pd/H(2)). Both methods were linear across the range of 15-300 μM, and the electrochemical method was linear across a wider range of 7.4-15,000 μM. The limit of detection for hydrogen peroxide was 6 μM by HPLC/FD, and 0.6 μM by HPLC/ED. A series of organic peroxides and inorganic ions were evaluated for their potential to interfere with the detection of hydrogen peroxide. Studies investigating the recovery of hydrogen peroxide with three different extraction protocols were also performed. Post-blast debris from the detonation of a mixture of concentrated hydrogen peroxide with nitromethane was analyzed on both systems. Hydrogen peroxide residues were successfully detected on this post-blast debris.     

角蛋白金属结合体的制备及清除超氧阴离子自由基性能

本文在制备可溶性羽毛角蛋白溶液的基础上,制备了羽毛角蛋白(FK)结合ZnII、CuII、MnII和NiII的角蛋白金属结合体(FKM,M=Zn,Cu,Mn,Ni)。采用IR、UV-Vis、TGA、CD和SEM等对其结构进行了表征和分析。用核黄素/蛋氨酸-NBT还原法考察了角蛋白金属结合体FKM清除超氧阴离子自由基(O2-)的性能,探讨了其清除机理。结果表明:含不同金属离子的结合体均具有清除O2-的能力。其中,Cu结合体的抗氧化活性最强。     

On-line selective detection of antioxidants free-radical scavenging activity based on Co(II)/EDTA-induced luminol chemiluminescence by flow injection analysis

This study establishes a new method to analyze the radical scavenging activity of antioxidants based on the luminol-H₂O₂-Co(II)/EDTA chemiluminescence and flow injection analysis. The method is based on the catalytic oxidation of hydrogen peroxide by Co(II)/EDTA complex, forming a free radical flux that can produce a stable chemiluminescence signal which is attenuated in the presence of antioxidants. A properly designed FIA manifold and the appropriate regulation of the chemiluminescence-reagent mixture enabled the establishment of a reaction-sensitive analytical procedure that minimizes oxidant-antioxidant interactions while favors the inhibition effect of antioxidants on the free radicals flux. In that manner, the uncontrolled experimental variability induced by side-reactions occurring antagonistically is reduced. The method was examined in-vitro for the continuous monitoring of the generation of oxygen-derived free radicals and antioxidants, which is closer to in-vivo conditions, with three common antioxidants (ascorbic acid, glutathione and uric acid). All three antioxidants were found to inhibit the luminescent signal with strict logarithmic linear mode, yielding calibration curves rectilinear in the range of 5 × 10⁻⁸-5 × 10⁻⁵ mol L⁻¹ and detection limits at the 10⁻⁸ mol L⁻¹ levels. The F-statistic was employed to assess the ability of the method to detect differences in the activity of the examined antioxidants. The results suggest that the proposed method can be used efficiently for the detection of free radical activity in real samples.     

Aryl oxalate chemiluminescence detection for high-performance liquid chromatography

Aryl oxalate chemiluminescence detection for high-performance liquid chromatography offers ultrahigh sensitivity for interactive fluorophores. With optimal analytes, attomole limits of detection can be obtained with conventional instrumentation     

Catalytic action of Mn‐superoxide dismutase in scavenging superoxide radical anion by double hydrogen abstraction from dihydrolipoic acid: A theoretical study

The mechanism of scavenging superoxide radical anion ( ) by dihydrolipoic acid (diLA) in absence and presence of the enzyme Manganese‐superoxide dismutase (Mn‐SOD) has been investigated using density functional theory. Mn‐SOD was modelled by a complex of a manganese cation (Mn²⁺) bonded to three similar molecules having a histidine ring each and a water molecule. It has been shown that the scavenging mechanism involves double hydrogen abstraction by from different pairs of neighboring sites of diLA. It has been found that diLA alone cannot scavenge superoxide radical anions efficiently as the barrier energies involved in the reactions are very high. However, in presence of Mn‐SOD, owing to its catalytic action, the corresponding reactions become barrierless due to which superoxide radical anions would be scavenged highly efficiently. H₂O₂ formed from superoxide radical anion due to double hydrogen abstraction from diLA is scavenged by diLA alone barrierlessly without involving Mn‐SOD or any other catalyst.     

Precolumn derivatization for the determination of fluoropyrimidines by liquid chromatography with chemiluminescence detection

A method for the chemiluminescent detection of fluoropyrimidine compounds with 7-(diethylamino)-3- {4[(iodoacetyl)amino]phenyl}-4-methylcoumarin using peroxyoxalate is described. The procedure is rapid, simple and requires little or no experience in labelling techniques. The amounts of the derivatives are linearly related to the amount of the starting fluoropyrimidine compounds and the procedure can therefore be used in the determination of these solutes. Using reversed-phase liquid chromatography, detection levels of 20–40 fmol could be realized.     

Sensitive detection of oxo-steroids and oxo-bile acids by liquid chromatography with peroxyoxalate chemiluminescence detection

Oxo-steroids and oxo-bile acid ethyl esters were derivatized with 5-N,N-dimethylamino-naphthalene-1-sulphonohydrazide (DNS-hydrazine) to DNS-hydrazone in the presence of hydrochloric acid in ethanol or trifluoroacetic acid in benzene, purified by high-performance gel-permeation chromatography, separated on an ODS column with an eluent containing tetrahydrofuran and detected via a peroxyoxalate chemiluminescence reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO). Sensitive detection (femtomole level) of each oxo-steroid which appeared as a single peak was achieved. The procedure for the isolation of oxo-bile acids developed for GC-MS allowed the detection by this system of an unusual oxo-bile acid, 7α-hydroxy-3-oxo-5β-cholanic acid, at the nanomole l^?1 level in urine from a patient with cholestatic liver disease.     

High-performance liquid chromatography of nitrate esters with chemiluminescence detection

A procedure was developed for the chromatographic determination of nitrate esters with chemiluminescence detection based on the chemiluminescence reaction of 4-dimethylaminophthalhydrazide with the labile products of the alkaline hydrolysis of nitrate esters. The detection limits for nitroglycerin, tetranitropentaerythrite, and isosorbite dinitrate were 0.01, 1, and 9 ng, respectively.     

Quality control of flavonoids in Ginkgo biloba leaves by high-performance liquid chromatography with diode array detection and on-line radical scavenging activity detection

Flavonoids in plants used for the treatment of various cardiovascular, cancer diseases have been reported to possess potential protective effects against oxidative injury. Ginkgo biloba leaves, known for their antioxidant activity, were chosen for this study. In this paper, 12 flavonoids in G. biloba leaves were identified by HPLC-diode array detection (DAD)-electrospray ionization MS. HPLC-DAD coupled with chemiluminescence detection was used to determine free radical scavenging activity of flavonoids. It was found that the flavonol glycosides could markedly inhibit the luminescent signal, which indicated that they are mainly responsible for the antioxidant activities of G. biloba leaves. Total antioxidant activity of these flavonoids was used to evaluate the differences of G. biloba leaves collected in 13 habitats. The combination of chemical and activity analysis can provide a valid method to quantify the bioactive components in G. biloba leaves, and this may be a more rational approach to the quality assessment of G. biloba leaves.     

Low-molecular-mass anion screening for forensic environmental analyses by capillary zone electrophoresis with indirect UV detection

A method for low-molecular-mass anion screening is described using a buffer composed of 5-sulfosalicylate (SS) as a visualizing ion, hexadimethrine bromide as an electroosmotic flow modifier and Tris as a pH buffer component, at pH 8.6. All ions with effective mobility higher than 2610⁻⁹ m² s⁻¹ V⁻¹ can be separated within 7.5 min under −30 kV. By using the moderately mobile SS (5410⁻⁹ m² s⁻¹ V⁻¹), not only the sensitivity of the detection is improved due to its high UV absorptivity, but also a smaller overall overloading effect is achieved. Meanwhile, the resolution of the high mobility ions, which is normally critical, remains almost the same as compared to a chromate buffer. With an electrokinetic injection, the limit of detection (LOD) of the common ions is 2–13 nM and the detection range is linear up to 0.5–3 μM. With a hydrostatic injection the LOD is 0.15–1 μM and the detection range is linear up to 25–200 μM. The identification of ions is performed by comparing the mobility of the ions with that of standards, taking the apparent and effective mobility of HCO₃⁻, which is normally present in the sample solution, as a reference.     

Determination of atmospheric nitrobenzanthrones by high-performance liquid chromatography with chemiluminescence detection.

A method using high-performance liquid chromatography with chemiluminescence detection was developed for analyzing mutagenic nitrobenzanthrone (NBA) isomers in airborne particulates. The method was a modification of our previously described method for analyzing nitropolycyclic aromatic hydrocarbons (NPAHs). The pretreatment and reducing conditions for 1-, 2-, 3- and 10-NBAs were the same as those for NPAHs. In order to separate these NBA isomers, we used a polymeric-type ODS column (Cosmosil 5C-18MS); a mixture of 40% acetonitrile and 60% 10 mM imidazole-HClO4 buffer was employed as the mobile phase at a flow rate of 1 mL/min. The isomers of 1-, 2-, 3- and 10-NBA were determined in chemiluminescence with linear calibration graphs from 0.1 to 4 pmol, from 200 to 4000 pmol, from 1 to 50 pmol and from 10 to 400 pmol, respectively. The detection limits (S/N = 3) of 1-, 2-, 3- and 10-NBA isomers were 0.02 pmol, 35 pmol, 0.3 pmol and 3 pmol, respectively. The method was used to analyze airborne particulates at a heavy traffic site in Kanazawa. 2- and 3-NBAs were detected in the extracts of the particulates, while 1-NBA and 10-NBA were not detected. The atmospheric concentrations of 2- and 3-NBAs were 1.83 pmol/m3 and 24.7 fmol/m3, respectively.     

Determination of polyphenols by high-performance liquid chromatography with inhibited chemiluminescence detection.

A chemiluminescence reaction detector was developed for the detection of polyphenols separated by HPLC based on the inhibition of chemiluminescence from the luminol-potassium hexacyanoferrate(III) reaction by polyphenols. The separation was carried out on a RP-C18 column at 37 degrees C by using stepwise gradient elutions. The detection limits are in the range of 6.8 x 10(-7)-2.0 x 10(-9) g/ml for catechol, protocatechuic acid, chlorogenic acid, rutin, resorcinol, hydroquinone and p-tert.butylpyrocatechol. The method is sensitive, selective, fast and simple. It has been successfully applied to the determination of chlorogenic acid and rutin in real tobacco samples.     

Determination of 1-nitropyrene metabolites by high-performance liquid chromatography with chemiluminescence detection

Three metabolites of 1-nitropyrene, i.e., 3-hydoxy-1-nitropyrene (3-OHNP), 6-hydoxy-1-nitropyrene (6-OHNP) and 8-hydoxy-1-nitropyrene (8-OHNP), were sensitively and selectively detected by HPLC with peroxyoxalate chemiluminescence (CL) detection. In the system, the three OHNPs were reduced to their corresponding amino derivatives through a reduction column packed with Pt/Rh-coated alumina and then concentrated on a concentrator column. By rotating a switching valve, the analytes were eluted into a separator column (ODS), separated and then detected by the bis(2,4,6-trichlorphenyl)oxalate-H(2)O(2) CL system. The detection limits (signal-to-noise ratio=3) were in the range from 5fmol (6-OHNP and 8-OHNP) to 12fmol (3-OHNP) per injection. To demonstrate the proposed method, it was used to determine three compounds in the incubation mixture of 1-nitropyrene and rat S9 mix.