A protocol for wide-scope non-target analysis of contaminants in small amounts of biota using bead beating tissuelyser extraction and LC-HRMS.

This work describes a robust and powerful method for wide-scope target and non-target analysis of xenobiotics in biota samples based on bead beating tissuelyser extraction, solid phase extraction (SPE) clean-up and further detection by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Unlike target methodologies, non-target methods usually aim at determining a wide range of still unknown substances with different physicochemical properties. Therefore, losses during the extraction process were minimised. Apart from that, the reduction of possible interferences showed to be necessary to expand the number of compounds that can be detected. This was achieved with an additional SPE clean-up step carried out with mixed-bed multi-layered cartridges. The method was validated with a set of 27 compounds covering a wide range of physicochemical properties, and further applied to the analysis of krill and fish samples.•The bead beating extraction was efficient for a wide range of organic pollutants in small quantities of biota samples.•Multi-layered solid phase extraction clean-up yield a wide xenobiotics coverage reducing matrix effects.•Method validation with 27 compounds led to a suitable method for non-target analysis of organic pollutants in biota.

Contaminants of emerging concern in freshwater fish from four Spanish Rivers.

This study investigated the occurrence of 135 contaminants of emerging concern (CECs) - pharmaceuticals, pesticides, a set of endocrine disrupting compounds (EDCs) (parabens, bisphenols, hormones, triazoles, organophosphorus flame retardants and triclosan), UV-filters, perfluoroalkyl substances (PFASs) and halogenated flame retardants (HFRs) - in 59 fish samples, collected in 2010 in 4 Spanish Rivers (Guadalquivir, Júcar, Ebro and Llobregat). Of the 135 CECs, 76 including 8 pharmaceuticals, 25 pesticides, 10 EDCs, 5 UV-filters, 15 PFASs and 13 HFRs were detected. Pharmaceuticals were the less frequently found and at lower concentrations. Pesticides, EDCs, UV-filters, PFASs and HFRs were detected more frequently (>50% of the samples). The maximum concentrations were 15 ng/g dry weight (dw) for pharmaceuticals (diclofenac), 840 ng/g dw for pesticides (chlorpyrifos), 224 ng/g dw for EDCs (bisphenol A), 242 ng/g dw for UV-filters (EHMC), 1738 ng/g dw for PFASs (PFHxA) and 64 ng/g dw for HFRs (Dec 602). The contaminants detected in fish are commonly detected also in sediments. In light of current knowledge, the risk assessment revealed that there was no risk for humans related to the exposure to CECs via freshwater fish consumption. However, results provide detailed information on the mixtures of CECs accumulated that would be very useful to identify their effects on aquatic biota.

Implementation of a chemical equilibrium constraint in the multivariate curve resolution of voltammograms from systems with successive metal complexes.

A multivariate curve resolution (MCR) method, using a constrained alternating least squares (ALS) procedure with a new chemical equilibrium constraint, was applied to differential-pulse polarograms of successive metal complexes. This new restriction imposes the fulfillment of a chemical model defined by a set of stability constants that are optimised along the iterative ALS procedure. The reliability of the method was tested with simulated data and with polarograms measured for the systems Zn(II) + glutathione and Cd(II) + 1,10-phenanthroline. These systems respectively yield two and three successive and electroactive complexes, which are inert from the electrochemical point of view, that is, the complexes virtually do not dissociate during the measurement. Although the presence of electrode adsorption could induce overestimation of some concentrations and losses of linearity between concentrations and signals, the results showed that the proposed method can yield satisfactory estimations of the stability constants in this kind of system. The performance of the new method is compared with the performances obtained using MCR-ALS without the equilibrium constraint and using traditional curve fitting least-squares approaches.

Soft- and hard-modeling approaches for the determination of stability constants of metal-peptide systems by voltammetry.

The Zn(2+)-glutathione system is studied as a model for metal-peptide systems where some critical factors must be considered when using voltammetric techniques for the determination of stability constants. These factors are the presence of side reactions (in this case, both the protonation of glutathione and the hydrolysis of Zn(2+)), the association-dissociation rates of the complexes compared with the time scales of the measurements (which makes the complexes electrochemically labile or inert), and the electron transfer kinetics on the electrode surface (which makes the metal ion reduction reversible or irreversible). For the study of these factors, three data treatment approaches have been applied: (i) the electrochemical hard-modeling approach (modelization of both chemical equilibrium and electrochemical processes), (ii) a chemical hard-modeling approach (modelization of chemical equilibria only, based on the least-squares curve-fitting program SQUAD), and (iii) a previously developed model-free soft-modeling approach based on multivariate curve resolution with a constrained alternating least-squares optimization. By analyzing differential pulse polarographic data obtained under different experimental conditions, the influence of the mentioned factors on every approach is discussed and, if possible, the corresponding stability constants are computed. The results of this study showed the potential usefulness of voltammetry in combination with hard- and soft-modeling data analysis for the study of peptide complexation equilibria of metal ions such as Zn which have neither relevant spectroscopic properties nor proper isotopes for NMR measurements.

Multivariate curve resolution: a possible tool in the detection of intermediate structures in protein folding.

Different multivariate data analysis techniques based on factor analysis and multivariate curve resolution are shown for the study of biochemical evolutionary processes like conformational changes and protein folding. Several simulated CD spectral data sets describing different hypothetical protein folding pathways are analyzed and discussed in relation to the feasibility of factor analysis techniques to detect and resolve the number of components needed to explain the evolution of the CD spectra corresponding to the process (i.e., to detect the presence of intermediate forms). When more than two components (the native and unordered forms) are needed to explain the evolution of the spectra, an iterative multivariate curve resolution procedure based on an alternating least squares algorithm is proposed to estimate the CD spectrum corresponding to the intermediate form.

Application of multivariate curve resolution to voltammetric data. II. Study of metal-binding properties of the peptides.

The complexation of Cd2+ by glutathione (GSH), in 0.13 m borate buffer at pH 9.5, was studied by differential pulse polarography (DPP) and multivariate curve resolution. The Cd-GSH system has been chosen as a model to check the possibilities of this new polarographic approach to the study of metal ion complexation by peptides. Experimental data obtained by DPP for different Cd2+-to-GSH concentration ratios have been analyzed by a procedure which consists of using several chemometrical techniques based on factor analysis: principal component analysis, evolving factor analysis, and multivariate curve resolution with alternating least-squares (ALS) optimization. The use of different constraints during the ALS optimization process, such as nonnegativity and unimodality constraints, yields the optimal sought solution from a chemical point of view. In the present work, a new constraint has been implemented during ALS optimization to take into account the expected peak-shaped signal of DPP. This data treatment allows us to detect simultaneously the formation of 1:1 and 1:2 Cd:GSH complexes which were very difficult to detect by univariate analysis of DPP data. It is concluded that the described multivariate curve resolution method could be a reliable tool for studying metal-binding properties of peptides.