María Dolores Gil García

Detection of unintended stress effects based on a metabonomic study in tomato fruits after treatment with carbofuran pesticide. Capabilities of MCR-ALS applied to LC-MS three-way data arrays.

A chemometric strategy based on multivariate curve resolution and alternating least-squares (MCR-ALS) applied to LC-MS three-way data arrays has been developed to perform a metabonomic study in tomato (Lycopersicon esculentum) fruits (cultivar Rambo) following treatment with carbofuran. This methodology has proved to be adequate for the detection of unintended stress effects due to the previous treatment with this pesticide. MCR-ALS was performed on augmented matrices built with the LC-MS three-way data obtained from treated and nontreated samples through the sampling time. The strategy allowed us to obtain the concentration and spectra profiles of the main components (previously estimated with the SVD algorithm) from samples treated with pesticide as well as from blank samples, showing how they vary with time after plants treatment with the pesticide. In addition, a simple resolved mass spectrum was obtained corresponding to the peaks of a particular component in all matrices, thus avoiding ambiguity in the compound identity assignment. Different time profiles were found for some metabolites in treated and nontreated samples, which demonstrate that the presence of pesticide causes changes thorough time in the behavior of certain endogenous tomato metabolites as a result of physiological stress.

Multivariate curve resolution modeling of liquid chromatography–mass spectrometry data in a comparative study of the different endogenous metabolites behavior in two tomato cultivars treated with carbofuran pesticide

A metabonomic study based on the application of multivariate curve resolution and alternating least squares (MCR-ALS) to three-way data sets obtained by liquid chromatography coupled to mass spectrometry detection (LC-MS) was carried out for Rambo and Raf tomato cultivars treated with carbofuran pesticide. Samples were picked up during a 21 days period after treatment and analyzed by LC-MS in scan mode, along with the corresponding blank samples. Then, MCR-ALS was applied to the three-way data sets using column wise augmented matrices, and the evolutionary profiles as a function of the time after treatment were estimated for the metabolites present in both cultivars, as well as their corresponding pure spectra estimations. A comparative study using those estimations showed that some of these metabolites followed different behavior for the different cultivars after treatment. Since all treated and untreated Rambo and Raf samples were picked up according to the same sampling protocol and in a similar state of maturation, any difference in the behavior between profiles can be interpreted as an effect due to the presence of pesticide and to the kind of cultivar. Based on this hypothesis, several PLS-DA approaches were tested to check if it would be possible to classify samples by using the metabolites MCR estimations. Results showed that PLS-DA models for classification of treated or non-treated (blank) samples were the best ones obtained (98.44% of correct classifications for the validation set), which supports the stress effects related to carbofuran treatment. In addition, excellent discrimination among the four groups could be attained (89.06% of correct classifications for the validation set).

Determination of (fluoro)quinolones in environmental water using online preconcentration with column switching linked to large sample volumes and fluorescence detection

An analytical method based on online enrichment using coupled-column liquid chromatography with fluorescence detection has been developed to determine marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, norfloxacin, lomefloxacin, oxolinic acid, and nalidixic acid at trace levels in surface water. The sample containing the pharmaceuticals was pumped through a short C18 column in such a way that the analytes were retained on the column, whereas polar interferences, eluting at the first of the chromatogram, were discarded to waste. Then, the analytes were transferred by the chromatographic mobile phase to a second C18 analytical column, where they were separated following a conventional chromatography. The optimized approach allowed to preconcentrate 15 mL of sample volume adjusted at acid pH with phosphoric acid and modified with 5% of methanol, at a flow rate of 1.5 mL/min in 10 min. R(2) values were between 0.994 and 0.998, detection and quantitation limits ranged between 0.001 and 0.080 and between 0.002 and 0.100 μg/L, respectively, and the interday precision was below 9.8%. Recoveries in three different surface water samples, spiked at concentration levels between 0.002 and 0.500 μg/L (n = 3 for each spiking level), ranged from 82.1 to 125.8% with the relative standard deviation lower than 12%.

Enhanced high-performance liquid chromatography method for the determination of retinoic acid in plasma. Development, optimization and validation

When determining endogenous compounds in biological samples, the lack of blank or analyte-free matrix samples involves the use of alternative strategies for calibration and quantitation. This article deals with the development, optimization and validation of a high performance liquid chromatography method for the determination of retinoic acid in plasma, obtaining at the same time information about its isomers, taking into account the basal concentration of these endobiotica. An experimental design was used for the optimization of three variables: mobile phase composition, flow rate and column temperature through a central composite design. Four responses were selected for optimization purposes (area under the peaks, quantity of peaks, analysis time and resolution between the first principal peak and the following one). The optimum conditions resulted in a mobile phase consisting of methanol 83.4% (v/v), acetonitrile 0.6% (v/v) and acid aqueous solution 16.0% (v/v); flow rate of 0.68 mL min(-1) and an column temperature of 37.10 °C. Detection was performed at 350 nm by a diode array detector. The method was validated following a holistic approach that included not only the classical parameters related to method performance but also the robustness and the expected proportion of acceptable results lying inside predefined acceptability intervals, i.e., the uncertainty of measurements. The method validation results indicated a high selectivity and good precision characteristics that were studied at four concentration levels, with RSD less than 5.0% for retinoic acid (less than 7.5% for the LOQ concentration level), in intra and inter-assay precision studies. Linearity was proved for a range from 0.00489 to 15.109 ng mL(-1) of retinoic acid and the recovery, which was studied at four different fortification levels in phuman plasma samples, varied from 99.5% to 106.5% for retinoic acid. The applicability of the method was demonstrated by determining retinoic acid and obtaining information about its isomers in human and frog plasma samples from different origins.

Analysis of β‐blockers in groundwater using large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection and liquid chromatography time‐of‐flight mass spectrometry

Atenolol, nadolol, metoprolol, bisoprolol and betaxolol were simultaneously determined in groundwater samples by large-volume injection coupled-column reversed-phase liquid chromatography with fluorescence detection (LVI-LC-LC-FD) and liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS). The LVI-LC-LC-FD method combines analyte isolation, preconcentration and determination into a single step. Significant reductions in costs for sample pre-treatment (solvent and solid phases for clean up) and method development times are also achieved. Using LC-TOF-MS, accurate mass measurements within 3 ppm error were obtained for all of the β-blockers studied. Empirical formula information can be obtained by this method, allowing the unequivocal identification of the target compounds in the samples. To increase the sensitivity, a solid-phase extraction step with Oasis MCX cartridge was carried out yielding recoveries of 79-114% (n=5) with RSD 2-7% for the LC-TOF-MS method. SPE gives a high purification of β-blockers compared with the existing methods. A 100% methanol wash was allowed for these compounds with no loss of analytes. Limit of quantification was 1-7 ng/L for LVI-LC-LC-FD and 0.25-5 ng/L for LC-TOF-MS. As a result of selective extraction and effective removal of coextractives, no matrix effect was observed in LVI-LC-LC-FD and LC-TOF-MS analyses. The methods were applied to detect and quantify β-blockers in groundwater samples of Almería (Spain).

Benzimidazole and imidazole fungicide analysis in grape and wine samples using a competitive enzyme-linked immunosorbent assay

This manuscript reports a quantitative pesticide residue method analysis employing an enzyme-linked immunosorbent assay (ELISA) for detecting carbendazim, imazalil and thiabendazole pesticide residues in vegetable samples. The results have been compared with those obtained from a liquid chromatography system coupled to a triple quadrupole mass spectrometer (LC-ESI-QqQ-MS). A validation study was undertaken to establish this methods performance characteristics on the 3 pesticide residues in two commodities (table grapes and wine). The carbendazim, imazalil and thiabendazole ELISA showed an IC50 reporting limit ranging from 0.5 to 13.8 µg kg−1. The limit of quantification of the developed assay was 5 µg kg−1 for both grape and wine matrices. The optimised assay was compared with a reference procedure based on liquid chromatography using samples acquired from various supermarkets, and a good correlation between both methods was found.

Analysis and evaluation of (neuro)peptides in honey bees exposed to pesticides in field conditions

During the last years, declines in honey bee colonies are being registered worldwide. Cholinergic pesticides and their extensive use have been correlated to the decline of pollinators and there is evidence that pesticides act as neuroendocrine disruptors affecting the metabolism of neuropeptides. However, there is a big absence of studies with quantitative results correlating the effect of pesticide exposure with changes on neuropeptides insects, and most of them are conducted under laboratory conditions, typically with individual active ingredients. In this study, we present an analytical workflow to evaluate pesticide effects on honey bees through the analysis of (neuro)peptides. The workflow consists of a rapid extraction method and liquid chromatography with triple quadrupole for preselected neuropeptides. For non-target analysis, high resolution mass spectrometry, multivariate analysis and automatic identification of discriminated peptides using a specific software and protein sequence databases. The analytical method was applied to the analysis of target and non-target (neuro)peptides in honey bees with low and high content of a wide range of pesticides to which have been exposed in field conditions. Our findings show that the identification frequency of target neuropeptides decreases significantly in honey bees with high concentration of pesticides (pesticide concentrations ≥ 500 μg kg-1) in comparison with the honey bees with low content of pesticides (pesticide concentrations ≤ 20 μg kg-1). Moreover, the principal component analysis in non-target search shows a clear distinction between peptide concentration in honey bees with high level of pesticides and honey bees with low level. The use of high resolution mass spectrometry has allowed the identification of 25 non-redundant peptides responsible for discrimination between the two groups, derived from 18 precursor proteins.

Diseño de un video tutorial para la mejora del aprendizaje de conceptos y procedimientos difíciles en el Área de Química Analítica

The study presented here examines the incorporation of a video tutorial as a supplement to learning in an advanced Instrumental Analytical Chemistry course. The video tutorial was based on “matrix effect” problem and it was designed by the instructor using audio narration from Powerpoint in the “screencast” mode. It was uploaded to the courses Web site portal (Blackboard Learn) at the University of Almería. The video has been shown to serve as a suitable learning that prepares students for laboratory more effectively, with an average of 70% more students answering questions correctly after watching the video than after receiving only teaching assistant instruction.