Luís M. Magalhães

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).

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.

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.

Rapid assessment of bioactive phenolics and methylxanthines in spent coffee grounds by FT-NIR spectroscopy

Spent coffee grounds (SCGs) are a great source of bioactive compounds with interest to pharmaceutical and cosmetic industries. Phenolics and methylxanthines are the main health related compounds present in SCG samples. Content estimation of these compounds in SCGs is of upmost importance in what concerns their profitable use by waste recovery industries. In the present work, near-infrared spectroscopy (NIRS) was proposed as a rapid and non-destructive technique to assess the content of three main phenolics (caffeic acid, (+)-catechin and chlorogenic acid) and three methylxanthines (caffeine, theobromine and theophylline) in SCG samples obtained from different coffee brands and diverse coffee machines. The content of these compounds was determined for 61 SCG samples by HPLC coupled with diode-array detection. Partial least squares (PLS) regression based models were calibrated to correlate diffuse reflectance NIR spectra against the reference data for the six parameters obtained by HPLC. Spectral wavelength selection and number of latent variables were optimized by minimizing the cross-validation error. PLS models showed good linearity with a coefficient of determination for the prediction set (Rp(2)) of 0.95, 0.92, 0.88, 071 and 0.84 for caffeine, caffeic acid, (+)-catechin, chlorogenic acid and theophylline, respectively. The range error ratio (RER) was higher for caffeine (17.8) when compared to other compounds (12.0, 10.1, 7.6 and 9.2, respectively for caffeic acid, (+)-catechin, chlorogenic acid and theophylline). Moreover, the content of caffeine could be used to predict the antioxidant properties of SCG samples (R=0.808, n=61), despite not presenting this property itself. The results obtained confirmed that NIRS is a suitable technique to screen SCG samples unveiling those with high content of bioactive compounds, which are interesting for subsequent extraction procedures.

Assessing oral bioaccessibility of trace elements in soils under worst-case scenarios by automated in-line dynamic extraction as a front end to inductively coupled plasma atomic emission spectrometry

A novel biomimetic extraction procedure that allows for the in-line handing of ≥400 mg solid substrates is herein proposed for automatic ascertainment of trace element (TE) bioaccessibility in soils under worst-case conditions as per recommendations of ISO norms. A unified bioaccessibility/BARGE method (UBM)-like physiological-based extraction test is evaluated for the first time in a dynamic format for accurate assessment of in-vitro bioaccessibility of Cr, Cu, Ni, Pb and Zn in forest and residential-garden soils by on-line coupling of a hybrid flow set-up to inductively coupled plasma atomic emission spectrometry. Three biologically relevant operational extraction modes mimicking: (i) gastric juice extraction alone; (ii) saliva and gastric juice composite in unidirectional flow extraction format and (iii) saliva and gastric juice composite in a recirculation mode were thoroughly investigated. The extraction profiles of the three configurations using digestive fluids were proven to fit a first order reaction kinetic model for estimating the maximum TE bioaccessibility, that is, the actual worst-case scenario in human risk assessment protocols. A full factorial design, in which the sample amount (400-800 mg), the extractant flow rate (0.5-1.5 mL min(-1)) and the extraction temperature (27-37°C) were selected as variables for the multivariate optimization studies in order to obtain the maximum TE extractability. Two soils of varied physicochemical properties were analysed and no significant differences were found at the 0.05 significance level between the summation of leached concentrations of TE in gastric juice plus the residual fraction and the total concentration of the overall assayed metals determined by microwave digestion. These results showed the reliability and lack of bias (trueness) of the automatic biomimetic extraction approach using digestive juices.

Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions

Total antioxidant capacity assays are recognized as instrumental to establish antioxidant status of biological samples, however the varying experimental conditions result in conclusions that may not be transposable to other settings. After selection of the complexing agent, reagent addition order, buffer type and concentration, copper reducing assays were adapted to a high-throughput scheme and validated using model biological antioxidant compounds of ascorbic acid, Trolox (a soluble analogue of vitamin E), uric acid and glutathione. A critical comparison was made based on real samples including NIST-909c human serum certified sample, and five study samples. The validated method provided linear range up to 100 µM Trolox, (limit of detection 2.3 µM; limit of quantification 7.7 µM) with recovery results above 85% and precision <5%. The validated developed method with an increased sensitivity is a sound choice for assessment of TAC in serum samples.

Automatic flow injection based methodologies for determination of scavenging capacity against biologically relevant reactive species of oxygen and nitrogen

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.

Lab-on-valve combined with a kinetic-matching approach for fast evaluation of total antioxidant capacity in wines

The ABTS˙+ assay, based on reduction of the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation, has been automated using flow injection based methods, but biased results are often obtained because the scavenging reaction is not complete upon detection, providing underestimated antioxidant values for samples containing slow reacting compounds. In this work a mesofluidic lab-on-valve (LOV) system was developed to provide fast determination of total antioxidant capacity in a miniaturized fashion. The sample volume was downscaled to 1 μl, with consumption of 97 μl of ABTS (6.8 nmol) per assay. Total antioxidant capacity values for red wines were attained in <1 min by application of the kinetic matching approach, using tannic acid as standard. Calibration curves were linear between 2.5 and 12.5 μM of tannic acid, with a determination throughput of 36 h−1. No statistical difference was found for Trolox equivalent antioxidant capacity (TEAC) values obtained using either an end-point batch-wise microplate method (reaction time of 300 min) or the proposed LOV method.

Automated Microdialysis-Based System for in Situ Microsampling and Investigation of Lead Bioavailability in Terrestrial Environments under Physiologically Based Extraction Conditions

In situ automatic microdialysis sampling under batch-flow conditions is herein proposed for the first time for expedient assessment of the kinetics of lead bioaccessibility/bioavailability in contaminated and agricultural soils exploiting the harmonized physiologically based extraction test (UBM). Capitalized upon a concentric microdialysis probe immersed in synthetic gut fluids, the miniaturized flow system is harnessed for continuous monitoring of lead transfer across the permselective microdialysis membrane to mimic the diffusive transport of metal species through the epithelium of the stomach and of the small intestine. Besides, the addition of the UBM gastrointestinal fluid surrogates at a specified time frame is fully mechanized. Distinct microdialysis probe configurations and membranes types were investigated in detail to ensure passive sampling under steady-state dialytic conditions for lead. Using a 3-cm-long polysulfone membrane with averaged molecular weight cutoff of 30 kDa in a concentric probe and a perfusate flow rate of 2.0 μL min(-1), microdialysis relative recoveries in the gastric phase were close to 100%, thereby omitting the need for probe calibration. The automatic leaching method was validated in terms of bias in the analysis of four soils with different physicochemical properties and containing a wide range of lead content (16 ± 3 to 1216 ± 42 mg kg(-1)) using mass balance assessment as a quality control tool. No significant differences between the mass balance and the total lead concentration in the suite of analyzed soils were encountered (α = 0.05). Our finding that the extraction of soil-borne lead for merely one hour in the GI phase suffices for assessment of the bioavailable fraction as a result of the fast immobilization of lead species at near-neutral conditions would assist in providing risk assessment data from the UBM test on a short notice.

On-line automated evaluation of lipid nanoparticles transdermal permeation using Franz diffusion cell and low-pressure chromatography.

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.