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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]

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3′,5,5′-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 μg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102–108% and requiring minimal volume (1–15 μL) from serum and saliva. Image 1 • Automatic functionalization of disposable microbeads for immunoaffinity assays. • Fast (<2 min) and repeatable immunoglobulin G immobilization in miniaturized set-up. • In-situ probing of functionalization through on-column colorimetric measurements. • Selective immunoglobulin G quantification in saliva and serum samples. [ABSTRACT FROM AUTHOR]

In this work, an automated system for the study of the interaction of drugs with human serum albumin (HSA) was developed. The methodology was based on the quenching of the intrinsic fluorescence of HSA by binding of the drug to one of its binding sites. The fluorescence quenching assay was implemented in a sequential injection analysis (SIA) system and the optimized assay was applied to ionic liquids based on the association of non-steroidal anti-inflammatory drugs with choline (IL-API). In each cycle, 100 µL of HSA and 100 µL of IL-API (variable concentration) were aspirated at a flow rate of 1 mL min −1 and then sent through the reaction coil to the detector where the fluorescence intensity was measured. In the optimized conditions the effect of increasing concentrations of choline ketoprofenate and choline naproxenate (and respective starting materials: ketoprofen and naproxen) on the intrinsic fluorescence of HSA was studied and the dissociation constants ( K d ) were calculated by means of models of drug–protein binding in the equilibrium. The calculated K d showed that all the compounds bind strongly to HSA ( K d <100 µmol L −1 ) and that the use of the drugs in the IL format does not affect or can even improve their HSA binding. The obtained results were compared with those provided by a conventional batch assay and the relative errors were lower than 4.5%. The developed SIA methodology showed to be robust and exhibited good repeatability in all the assay conditions (rsd<6.5%). [ABSTRACT FROM AUTHOR]

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: Antioxidant capacity of food samples is usually assessed by different analytical methods, however the results attained even for the same method are strongly dependent on the selected reaction time and also on the standard compound used. To tackle this problem, we propose here a kinetic matching approach, associated to the conversion of results into equivalents of a common standard compound, as a universal way for expression of results. The methodology proposed was applied to methods based on different chemistries (Folin–Ciocalteu (F–C), CUPRAC, DPPH• and ABTS•+ assays) and red wines (n=40) were chosen as a model of complex food sample. For implementation of the kinetic matching approach, the standard phenolic mixture (caffeic acid, (+)-catechin, hesperetin, morin and (−)-epigallocatechin gallate) was chosen for calibration in F–C, CUPRAC and DPPH• assays, while tannic acid was suitable for ABTS•+ assay. Results showed that, for all methods, there was no statistical difference between results attained by the kinetic matching approach (after <10min of reaction) and that at endpoint conditions (after 60 to 300min). The repeatability and the reproducibility of the kinetic matching approach was <4.5%, for all antioxidant assays. The sample throughput increases from <18 (endpoint measurements) to >108h−1 using the proposed kinetic approach. Moreover, we have established here a way of converting results to equivalents of a common standard, providing values independent of its kinetic profile, by using the ratio between calibration sensitivities performed at endpoint conditions. [Copyright &y& Elsevier]

Abstract: According to the current demands of environmentally friendly analytical chemistry and with a view to achieving lower reagent consumption with improved analytical performance, an automatic methodology composed of a photoreactor and fluorimetric detection (λ exc =287nm, λ em =378nm) was developed. To this end, a sequential injection analysis (SIA) system was developed for indomethacin determination using ultra-violet (UV) light which promotes an increase in the fluorescence of indomethacin. This increase in sensitivity makes it possible to apply this methodology to a dissolution test and to determine indomethacin in pharmaceutical formulations. The calibration graph for indomethacin was linear between 4.10×10−6 and 9.00×10−5 molL−1and the detection limit was 1.23×10−6 molL−1. The method was proven to be reproducible with a R.S.D.<5% and sampling rate of approximately 20 per hour. The potential effect of several compounds commonly used as excipients on analytical signals was studied and no interfering effect was observed. Statistical evaluation at the 95% confidence level showed good agreement between the results obtained for the pharmaceutical samples with both the SIA system and comparison batch procedures. [Copyright &y& Elsevier]

Abstract: In the present work, a rapid and high-throughput Folin-Ciocalteu (F-C) reducing capacity assay adapted to routine/screening analysis was developed. In order to attain a fast F-C reducing kinetic reaction, the reaction conditions of the classical time-consuming F-C assay were modified and the influence of alkali and F-C reagent concentration was evaluated using gallic acid as standard. The proposed method was performed in a 96-well microplate format and it was applied to several phenolic compounds and food products (wines, beers, infusions and juices) providing F-C reducing capacity results after 3min of reaction similar to those obtained by the time-consuming (120min) conventional method. The additive and synergistic effect of reducing nonphenolic compounds usually found in food samples was also investigated. Ascorbic acid and ferrous sulfate provided an additive effect, while for fructose, glucose and sodium sulfite a synergistic effect was obtained. The detection limit was 0.25mgL−1 (as gallic acid) and the repeatability was <1.6% (n =12). [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: 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 PLA2 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 PLA2 activity and assessment of the inhibitory effect of the non-steroidal anti-inflammatory drugs (NSAIDs) on PLA2 activity. Several studies were performed with the aim of establishing the appropriate flow system configuration: the liposome substrate; PLA2 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 PLA2 is effectively inhibited by the NSAIDs tested (meloxicam, tolmetin and ibuprofen) and by the α-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. [Copyright &y& Elsevier]

Abstract: 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 (HOt oxygen (1O2), hypochlorite anion (OCl−), peroxynitrite anion (ONOO−) and nitric oxide radical (NOasurement 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. [Copyright &y& Elsevier]

Abstract: 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 (DDPHd 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. [Copyright &y& Elsevier]

Abstract: Studies on the complexation of copper(II) by phenolic acids, as ligand models of humic substances were done by potentiometry. The acids under study were: 3,4-dihydroxyhydrocinnamic acid or hydrocaffeic acid (1), 3,4-dihydroxyphenylacetic acid (2) and 3,4-dihydroxybenzoic acid or protocatechuic acid (3). Acidity constants of the ligands and the formation constants of metal–ligand complexes were evaluated by computer programs. The carboxylic group of the phenolic acids has different pK a1 values, being the dissociation constants intrinsically related with the distance between the function and the aromatic nucleus. The results obtained allow concluding that acidity constants of the catechol moiety of the compounds are similar with pK a2 and pK a3 values between 9.47–9.41 and 11.55–11.70. The complexation properties of the three ligands towards copper(II) ion are quite similar, being the species found not different either in nature or stability. Although the model ligands have some structural differences no significant differences were found in their complexation properties towards copper(II). So, it can be postulated that complexation process is intrinsically related with the presence of a catechol group. [Copyright &y& Elsevier]