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Automation of total antioxidant capacity assessment can substantially increase the determination throughput, allowing large scale studies and screening experiments. Total reducing capacity evaluation can be implemented under different chemistries, including the CUPRAC-Cupric Ion Reducing Antioxidant Capacity -assay. This assay is based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex by reducing (antioxidant) components of biological samples. In this chapter, we propose an automatic flow injection method for evaluation of total reducing capacity in serum and urine samples, attaining end-point data within 4 min using a kinetic matching strategy.

High-throughput cupric ion reducing antioxidant capacity (CUPRAC) methods were developed for assessment of total antioxidant capacity (TAC) in urine and serum, based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex, measured spectrophotometrically at 450 nm. The reaction time was significantly reduced from 30 to 4 min by application of a calibration compound (uric acid) with kinetic behavior similar to that shown by urine samples. The method was implemented in a microformat (96 well plates) and also in an automatic fashion (flow injection analysis, FIA). A determination throughput value of 288 h(-1) (microplate method) or of 15 h(-1) (automatic FIA) was attained. Application of both methods to human serum (SRM 909b, level I) and urines (n = 9) provided TAC values in agreement with those of the end-point batch method.

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.

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 degrees C), pH (7.4), and concentration of NO (less than 1 microM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC(50) values were calculated for rutin (1.30 +/- 0.02 microM, positive control), cysteine (321 +/- 8 microM), reduced glutathione (1106 +/- 93 microM), uric acid (134 +/- 12 microM), dipyrone (1.36 +/- 0.06 microM), and captopril (363 +/- 28 microM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC(50) 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.

This work reports the development of an automatic methodology based on the use of 1-anilinonaphthalene-8-sulfonate (ANS) as an interfacial fluorescent probe for detecting the hydrophobic environment shift around the probe, caused by the hydrolytic action of PLA(2) on the liposomes. The implementation of this reaction in a sequential injection analysis (SIA) system along with the use of the mixing chambers permitted the evaluation of PLA(2) activity and assessment of the inhibitory effect of the non-steroidal anti-inflammatory drugs (NSAIDs) on PLA(2) activity. Several studies were performed with the aim of establishing the appropriate flow system configuration: the liposome substrate; PLA(2) and ANS optimum concentrations and incubation times before and after the enzyme addition. Based on these studies, the optimum reaction conditions were selected. It was shown that PLA(2) is effectively inhibited by the NSAIDs tested (meloxicam, tolmetin and ibuprofen) and by the alpha-lipoic acid, used as a positive control. Results obtained from the flow system are in agreement with those provided by the comparison batch procedures. The proposed methodology is in fact more efficient and rapid than the comparison batch experiments, enabling the exact timing of fluidic manipulations and precise control of the reaction conditions.

Redox reactions are the heart of numerous biochemical pathways found in cellular chemistry, generating reactive oxygen species (ROS) and reactive nitrogen species (RNS), that includes superoxide anion radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (HO), singlet oxygen ((1)O2), hypochlorite anion (OCl-), peroxynitrite anion (ONOO-) and nitric oxide radical (NO). The measurement of scavenging capacity against these reactive species presents new challenges, which can be met by flow injection analysis (FIA). In the present review several methods based on FIA and also on its predecessors computer-controlled techniques (sequential injection analysis, multisyringe flow injection analysis, multicommutated and multipumping flow systems) are critically discussed. The selectivity and applicability of the methodology, the generation and detection of the target reactive species, the benefits and limitations of automation when compared to batch methods are some of the issues addressed.

The role and importance of antioxidants in different fields, ranging from physiology to food technology, have become evident in the past years, requiring adequate analytical methodologies. Therefore, the determination of antioxidant capacity as a routine or screening analysis fosters its automation. In this context, several flow injection methods based on scavenging of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS(+)) or 2,2-diphenyl-1-picrylhydrazyl radical (DDPH) or based on the determination of total reducing capacity have been proposed. The objective of the present review is to critically compare the different approaches, regarding their degree of automation, their performance vs. the respective batch procedure and its applicability to real samples.

In the present work, an automatic flow procedure based on multi-syringe flow injection analysis was developed for the assessment of Folin-Ciocalteu reagent (FCR) reducing capacity in several types of food products using gallic acid as the standard. Different strategies for mixing of sample and reagent were tested (continuous flow of FCR, merging zones, and intercalated zones approaches); lower reagent consumption and higher determination throughput were attained for the merging zones approach (100 microL of sample+100 microL of FCR). The application of the proposed method to compounds with known antioxidant activity (both phenolic and nonphenolic) and to samples (wines, beers, teas, soft drinks, and fruit juices) provided results similar to those obtained by the conventional batch method. The detection limit was 0.6 mg L-1, and the determination frequency was about 12 h-1. Good repeatability was attained (RSD<1.3%, n=10).

A multi-syringe flow injection system for the determination of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) scavenging capacity using methanol or ethanolic solutions as reaction media is presented. A stopped flow approach was implemented in order to monitor the DPPH• consumption using spectrophotometry at 517 nm. The kinetic profile for the first 3 min of reaction was determined for several antioxidant compounds and the number of DPPH• molecules reduced by one molecule of antioxidant was calculated whenever the absorbance value was stable within the period of measurement. For ascorbic acid, trolox, isoeugenol and quercetin in methanol, the values obtained were similar to those reported in the literature. It was possible to perform 14 determinations per hour, consuming 0.34 µmol of DPPH• per determination, which accounts for the application of this system as a screening tool for fast scavenger compounds. [ABSTRACT FROM AUTHOR]

A multi-syringe flow injection system for the determination of total phenolics based on the 4-aminoantipyrine reaction is proposed. In-line preconcentration using solid-phase extraction was implemented, offering an environmentally friendly alternative to organic solvent extraction performed in the batch procedure and in previously described flow methodologies. Therefore, phenolic compounds were retained in Amberlite XAD4 resin using acidic conditions. The elution was carried out using 0.10?mol?L-1 NaOH solution, promoting the ionisation of the retained phenolic compounds, and thus their desorption.Since different elution profiles were obtained for several phenol derivatives, the calibration curves were established using the peak area as analytical signal. Moreover, a dynamic calibration range was applied by using a variable sample volume (0.500-6.00?mL) and calibration curves based on the mass of phenol retained in the pre-concentration column. A methodology based on a fixed sample volume (24.0?mL) was also proposed for determination at concentration levels below 50?µg?L-1. A limit of detection of 52?ng of phenol (9 or 2?µg?L-1) was achieved with a sampling frequency of 10-16?h-1 for the variable volume method and 4?h-1 for the fixed volume method. The application of the proposed methodology to reference material provided results that were within the certified acceptance limits. [ABSTRACT FROM AUTHOR]

Abstract: The determination of hydrogen peroxide (H2O2) and the evaluation of scavenging capacity against this species were performed using five colorimetric reactions, which were adapted to flow injection analysis. The reactions chosen were based on the oxidation of iodide (I− method), on the formation of titanium-peroxide complex (TiP method), on the formation of titanium-xylenol orange-peroxide complex (TiXoP method), on the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB method) and on the co-oxidation of phenol-4-sulfonic acid and 4-aminoantipyrine (PSA/4-AAP method). The operational conditions were studied in order to improve the sensitivity of each method. Concerning to the method sensitivity, the ranking order was TMB method>I− method>TiXoP method ∼PSA/4-AAP method>TiP method. All methods showed an excellent repeatability (RSD<2%) and, except for I− method, relative deviations from the reference method were <1.9%. The FIA manifolds were adapted to perform the determination of scavenging capacity against H2O2 and glutathione (GSH) was applied as model compound. TiP and TiXoP methods were not suitable as no inhibition or an increase of analytical signal was attained. PSA/4-AAP method was chosen for further application to dietary phenolics and pharmaceutical compounds, providing IC50 values for those compounds that are fast reacting antioxidants. [Copyright &y& Elsevier]

Abstract: In the present work, an automatic flow procedure for the sequential spectrophotometric determination of Folin-Ciocalteu reducing capacity (FC assay) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS +) scavenging capacity expressed as the trolox equivalent (TEAC assay) is proposed for a comparative study of antioxidant properties in food products. Exploiting the flexibility of flow management associated to the computer control offered by multisyringe flow injection analysis, both methodologies were carried out in the same manifold using gallic acid and trolox as standard compounds. The proposed system configuration allowed the performance of each method separately or in tandem, providing 24 determinations per hour, which accounts for its application in routine analysis. The present methodology was applied to a large number of beverages (n =72), namely red and white wines, herbal and tea infusions, juices and beers. The results obtained showed that FC reducing capacity and TEAC values of red wines were significantly different from those obtained for the other beverages, while tea infusions provided significantly higher TEAC values when compared to white wines, herbal infusions, juices and beers. A good correlation was found between TEAC and FC reducing capacity (R >0.9) for red wines, herbal and tea infusions, and beers. For these beverages, similar slope values were found (106–140mgL−1 of gallic acid per mM of Trolox), except for beers that showed a higher response for FC assay. These results provided evidence that the correlation between these assays vary according to the type of sample, reinforcing the idea that more than one method should be used for evaluation of antioxidant capacity. [Copyright &y& Elsevier]

An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90±5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 μM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h −1 ), low operating costs and low generation of waste. [ABSTRACT FROM AUTHOR]

A low-pressure liquid chromatography system for the on-line quantification of caffeine loaded into lipid nanoparticles that permeates pig skin was developed. The apparatus includes a Franz diffusion cell with computer-controlled sampling that allows collection of acceptor solution with automatic compensation for sample withdrawing, and a C-18 reversed-phase monolithic column integrated in a typical Flow Injection Analysis (FIA) set-up where separation between caffeine and other matrix elements is performed before spectrophotometric quantification at 273 nm. Several parameters regarding chromatographic analysis (propulsion element, column length, mobile phase composition, and flow rate) were studied along with the establishment of the sampling procedure. Under the selected conditions (monolithic column Chromolith® RP-18 15 mm×4.6 mm i.d., acetonitrile:water 10:90 (v/v), flow rate 0.45 mL min −1 ) a detection limit of 4 μM and RSD values for caffeine concentration <2% were achieved. High recovery values were obtained when Hepes buffer incubated as acceptor solution in presence of pig skin for 8 h was spiked with caffeine (103±5%). The developed system also accounts for low organic solvent consumption, low operating costs, low generation of waste and high sample throughput (24 h −1 ). Due to the real time automated sampling and high throughput, transdermal permeation profiles of nanoformulations can be established within a time frame seldom observed by conventional techniques. [ABSTRACT FROM AUTHOR]

Abstract: In the present work, an automatic method based on multisyringe flow injection analysis (MSFIA) was developed for determination of scavenging capacity against H2O2 using a non-enzymatic fluorimetric assay for H2O2 detection based on the formation of europium-tetracycline-H2O2 complex. The MSFIA-fluorimetric methodology was developed to perform in-line the scavenging reaction of H2O2 prior to reaction of the remaining H2O2 with the fluorescent probe, using conditions close to those found in vivo regarding pH (6.9), temperature (37°C) and H2O2 concentration (25μM). This approach allowed the evaluation of scavenging capacity against H2O2 in a non-competitive (antioxidant+H2O2) or a competitive (antioxidant+H2O2 +biological target) scheme. Using the first strategy, IC50 values determined for the antioxidant compounds glutathione reduced (1191±46μM) and pyruvate (446±49μM) were lower than those obtained for biological targets such as cysteine (2616±182μM), taurine (359±38mM) and adenine (2224±214μM), indicating that reactivity towards H2O2 was higher for antioxidant compounds than for biological targets. However, when a competitive scheme was applied, the scavenging effectiveness against H2O2 depended on the biological molecule present, showing that antioxidant assessment should also take into consideration the concomitant reactivity of biological molecules or structures that are prone to oxidative damage. [Copyright &y& Elsevier]

Abstract: Conventionally, measuring antioxidant capacity is time consuming, laborious and costly, especially in routine work. These limitations can be overcome using multi-syringe flow-injection analysis (MSFIA), which has succeeded in automating the most widely applied antioxidant-capacity assays that use chromogen radicals or reactive species found in vivo. We highlight the features of MSFIA and improved methodologies in using MSFIA to measure antioxidant capacity. [Copyright &y& Elsevier]