Marcela A. Segundo

Rapid microplate high-throughput methodology for assessment of Folin-Ciocalteu reducing capacity

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 3 min of reaction similar to those obtained by the time-consuming (120 min) 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 0.25 mg L(-1) (as gallic acid) and the repeatability was <1.6% (n=12).

Spectrophotometric determination of iron and boron in soil extracts using a multi-syringe flow injection system.

In the last decade, significant advances in flow analysis have been reported, namely the extensive use of computer-controlled devices to enhance the autonomy and performance of analysers. In the present work, computer-controlled multi-syringe flow injection systems are proposed to perform the spectrophotometric determination of available iron and boron in soil extracts. The methodologies were based on the formation of ferroin complex (determination of iron) and azomethine-H reaction (determination of boron). Both determinations were performed in manifolds with similar configurations by changing the reagents present in the different syringes. In the determination of iron, elimination of Schlieren effect in the detection system was achieved through the binary sampling approach, where a three-way valve was actuated to intercalate small slugs of sample and reagent, promoting better mixing conditions for solutions with different values of refractive index. In the determination of boron, in-line sample blank measurement was attained by omitting the introduction of reagent through software control, without manifold reconfiguration. Linear calibration curves were established between 0.50 and 10.0mgFel(-1) and between 0.20 and 4.0mgBl(-1). No systematic difference was found when soil extracts were analysed by the proposed methodologies and compared to the respective reference procedures.

Online Hyphenation of Multimodal Microsolid Phase Extraction Involving Renewable Molecularly Imprinted and Reversed-Phase Sorbents to Liquid Chromatography for Automatic Multiresidue Assays

Molecular imprinted polymers (MIP) have recently drawn much attention as highly selective solid-phase materials for handling and isolation of organic pollutants in complex matrices. Because of the impaired retention capacity for target species as compared with reversed-phase materials and irreversible sorption of interfering compounds by nonspecific interactions, the implementation of MIP-based solid-phase reactors as permanent components in automatic flow-systems has not received widespread acceptance as of yet. To tackle this limitation, a dynamic microscale solid phase extraction (microSPE) method capitalizing on the principle of programmable flow and bead injection analysis is herein proposed as a front end to liquid chromatography for multiresidue assays. It involves in-line renewable tandem-SPE microcolumns composed of molecularly imprinted polymers and copolymeric N-vinylpyrrolidone/divinylbenzene beads integrated within the flow network for multimodal extraction. Chlorotriazine herbicides (namely, atrazine, simazine, propazine) and principal degradation products thereof (namely, deisopropylatrazine and deethylatrazine) were selected as model analytes. The effect of several parameters, including the dimensions and chemical composition of the sorptive microcolumns, the sample loading flow rate, the type and volume of eluent, the interface with liquid chromatography (LC), and the disposable nature of the column on the analytical performance were investigated in detail. The assembled flow setup features appropriate removal of interfering organic species via solvent switch with toluene, the circumvention of analyte band-broadening in LC by in-line merging of the eluate with a water stream, and the transfer of the overall analyte-containing eluate into the LC. For 10-mL sample percolation, limits of detection (S/N = 3) of 0.02-0.04 ng mL(-1), limits of quantification (S/N = 10) of 0.07-0.12 ng mL(-1), absolute recovery percentages >79%, precision within 1.4-5.5%, and enrichment factors of 46-49 were obtained for the suite of assayed herbicides. The multimodal microSPE method with renewable beads was applied to the multiresidue determination of the target herbicides in crude soil extracts and untreated environmental waters at concentration levels below those endorsed by the current EU Water Framework Directives following appropriate sample preconcentration and/or cleanup.

Rapid assessment of endpoint antioxidant capacity of red wines through microchemical methods using a kinetic matching approach

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 <10 min of reaction) and that at endpoint conditions (after 60 to 300 min). 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 >108 h(-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.

On-line renewable solid-phase extraction hyphenated to liquid chromatography for the determination of UV filters using bead injection and multisyringe-lab-on-valve approach

For the first time, an automatic sample pre-treatment/detection method is proposed for the multiclass determination of UV filters (namely, benzophenone-3, ethylhexylmetoxycinnamate, butylmethoxydibenzoylmethane and homosalate) in environmental samples. The new methodology comprises in-line solid-phase extraction (SPE) of the target analytes by exploiting the bead injection (BI) concept in a mesofluidic lab-on-valve (LOV) format, with subsequent determination by liquid chromatography (LC). The proposed microanalytical system, using a multisyringe burette as propulsion unit, automatically performed the overall SPE steps, including the renewal of the sorbent in each analytical cycle to prevent sample cross-contamination and the post-extraction adjustment of the eluate composition to prevent chromatographic band broadening effects. In order to expedite the LC separation, a C(18) monolithic column was applied and an accelerated isocratic elution was carried out by using a cationic surfactant as mobile phase additive. The LOV-BI-LC method was proven reliable for handling and analysis of complex matrices, e.g., spiked swimming pool water and seawater, with limits of detection ranging between 0.45 and 3.2 microg L(-1) for 9 mL sample volume. Linear calibration was attained up to 160 microg L(-1) for homosalate and up to 35 microg L(-1) for the other target analytes, with good reproducibility (RSD<13%, for 5 different SPE columns). The hyphenated scheme is able to process a given sample simultaneously and within the same time frame than the chromatographic separation/determination of the formerly pre-treated sample, providing concentration values every 9 min. Hence, the sample throughput was enhanced up to 33 times when compared with previously reported off-line SPE methods. A drastic reduction in reagent consumption and effluent production was also attained, contributing to the development of an environment-friendly analyzer.

Enzyme based assays in a sequential injection format: a review.

Sequential injection analysis systems have been extensively exploited in the last decades for the implementation of enzyme based assays aiming the evaluation of enzyme activity or the determination of specific analytes. The most prominent aspects of the automation of enzymatic assays in these systems are discussed in this review. Special attention is devoted to the mode of enzyme manipulation in homogeneous or heterogeneous media and to the comparison with batch and flow injection enzyme methodologies. The possibility of implementing strategies for the enhancement of selectivity in specific determinations is also addressed. The more recent trends in this field are discussed focusing mainly on the miniaturization resorting to the lab-on valve platform as well as on the bead injection concept.

Multisyringe flow system: determination of sulfur dioxide in wines

A multisyringe flow system for the spectrophotometric determination of sulfur dioxide in wines is described. The methodology is based on the well-known reaction among SO2, formaldehyde and pararosaniline. The proposed manifold also includes a gas diffusion unit in order to prevent the colour interference of red wines in the spectrophotometric measurement. The proposed method was successfully applied to the determination of free SO2 (2–75 mg l−1) and total SO2 (10–250 mg l−1) in wines, just by changing a few operating parameters in the controlling software. A sampling-rate of 25–30 samples per hour was achieved with good repeatability for 10 consecutive injections of wine samples (RSD < 3.2%). The results obtained from 10 wine samples for each determination were statistically comparable to those obtained by the recommended procedure.

Flow-through Dispersed Carbon Nanofiber-Based Microsolid-Phase Extraction Coupled to Liquid Chromatography for Automatic Determination of Trace Levels of Priority Environmental Pollutants

Handling of carbon nanoparticles as sorptive materials in a flow-through packed-bed mode has been to date hampered by undue pressure drop and deteriorated retention efficiency because of nanoparticle bundling and entanglement. To surmount this limitation, a dedicated stirred-flow sorptive microchamber integrated in a fully automated sequential injection (SI) assembly is herein proposed for expedient handling and reuse of carbon nanoparticles in microsolid-phase extraction (μSPE) procedures. The assembled setup features automatic uptake, preconcentration, and retrieval of target organic species using dispersed nanoparticles as a front-end to liquid chromatographic (LC) assays. Chlorotriazine residues (atrazine, simazine, and propazine) and dealkylated metabolites thereof (deisopropyltriazine (DIA) and deethylatrazine (DEA)) were selected as model compounds because of their electron-poor aromatic structure and potentially strong π-π interactions with electron-rich sorptive materials. The effect of several parameters on the analytical performance including the type and amount of nanoparticles (carbon nanofibers (CNFs), multiwalled carbon nanotubes (MWCNTs) and oxidized carbon nanotubes (MWCNT-COOH), the sample volume (breakthrough volume), the nature and volume of eluent, and the interface between the sample processing module and LC was explored in detail. Using dispersed CNFs at-line coupled to LC, absolute recovery percentages for 10 mL sample percolation were >94% for the overall herbicides with enrichment factors of ca. 20, limits of detection (S/N = 3) of 0.004-0.03 ng mL(-1), limits of quantification (S/N = 10) of 0.01-0.09 ng mL(-1) and repeatability within the range 0.5-1.8%. The SI-CNF-LC hyphenated system was harnessed to the analysis of not merely untreated environmental waters at concentration levels below those endorsed by the current EU Water Framework Directives but to crude soil extracts for which CNF reuse with no loss of retention efficiency was proven feasible by resorting to appropriate automatic regeneration procedures and internal standardization.

Lipid nanoparticles for topical and transdermal application for alopecia treatment: development, physicochemical characterization, and in vitro release and penetration studies

Alopecia is a dermatological disorder, commonly known as hair loss, which affects up to half of the Caucasian male population by middle age, and almost all (95%) Caucasian men by old age. Considering that alopecia affects so many people and that there is currently no scientifically proven treatment with few side effects, new drug-delivery systems able to improve alopecia therapy are urgently required. With this purpose in mind, the present study aimed to develop lipid nanoparticles (nanostructured lipid carriers) with the ability to incorporate and deliver anti-alopecia active compounds (minoxidil and finasteride) into the dermis and hair follicles. Lipid nanoparticles, prepared by ultrasonication method, showed mean particle sizes around 200 nm, which is sufficient for reaching the dermis and hair follicles, and zeta potential values around −30 mV, which indicates good physical stability. Over 28 days of storage, no significant variations in these parameters were observed, which indicates that all nanoformulations are stable in storage over that period. Cryo-scanning electron microscope measurements showed that all the lipid nanoparticles exhibited a spherical shape and a smooth surface regardless of their composition. Differential scanning calorimetry studies allowed the determination of phase transition temperatures and confirmed the recrystallization of the lipid nanoparticles (recrystallization index between 11% and 86%). A high loading efficiency was achieved for finasteride (between 70% and 90%), while less than 30% was achieved for minoxidil nanoparticles, over 28 days. Controlled release assays in physiological conditions demonstrated that nanoparticles loaded with minoxidil yielded a prolonged release, as desired. Penetration assays through pig ear skin demonstrated that nanoparticles loaded with minoxidil and finasteride had low levels of penetration. These results suggest that the proposed novel formulation presents several good characteristics indicating their suitability for dermal delivery of anti-alopecia active compounds.

High-throughput microplate assay for the determination of drug partition coefficients

Partition coefficients (Kp) of drugs between the phospholipid bilayer and the aqueous phase provide useful information in quantitative structure-activity relationship studies. Hexadecylphosphocholine (HePC) micelles, composed of a zwitterionic hydrophilic surface and a hydrophobic core, mimic the biomembranes and have several advantages over other lipid structures to assess Kp values. Their preparation is easy, fast and avoids the use of toxic organic solvents, and the output has fewer spectroscopic interferences. Here, we describe a high-throughput microplate protocol for assessing the Kp of drugs using HePC micelles as membrane models and derivative spectrophotometry as the detection technique. Moreover, the time-consuming data treatment to assess Kp values is easily performed by a dedicated Excel routine developed here and described in detail. The Kp values of nonsteroidal anti-inflammatory drugs (acemetacin, clonixin, diclofenac and indomethacin) were determined to show the simplicity of the method and to validate this protocol, which provides Kp values (n = 3) of two drugs in ∼2 h.