Determinazione di ritardanti di fiamma bromurati contaminanti matrici acquose mediante LC/APPI-MS/MS

I ritardanti di fiamma sono composti chimici utilizzati come additivi, aggiunti durante o al termine della lavorazione di un materiale, per ritardare la fase iniziale del processo di combustione, per ridurre l’infiammabilità di materiali polimerici nel rispetto delle norme antincendio, per impedire o rallentare lo sviluppo di incendi. La tipologia dei ritardanti di fiamma copre un ampio spettro di composti che soddisfano le più svariate esigenze. Il gruppo dei ritardanti di fiamma a base di bromo comprende diversi composti organobromurati utilizzati per impedire la combustione e/o per ritardare la diffusione delle fiamme in diversi tipi di plastica, prodotti tessili ed altri materiali. Sono tre i gruppi chimici impiegati più comunemente: i difenileteri polibromurati (PBDE), l’esabromociclododecano (HBCD), usati come additivi, ed i bisfenoli bromurati (in particolare il TBBP-A) più comunemente usati come reagenti data la loro maggiore capacità di legarsi con i polimeri in cui vengono incorporati. Il problema ambientale dei ritardanti di fiamma nasce negli anni novanta, quando nei sedimenti del Mar Baltico è stato rilevato un aumento sensibile delle concentrazioni di PBB (difenilipolibromurati) e PBDE. Studi successivi condotti in Svezia, hanno dimostrato che tali composti sono persistenti e soggetti a notevole bioaccumulo nella catena alimentare. Infine, la Direttiva 2003/11/CE ha posto limitazioni alla produzione ed utilizzo di questo tipo di composti per le ragioni sinteticamente esposte, sebbene in presenza di caratteristiche di tossicità acuta non preoccupanti. I metodi analitici finora sviluppati per la determinazione di queste sostanze comprendono l’estrazione e la micro-estrazione in fase solida (SPE e SPME), l’estrazioni liquido-liquido con membrana microporosa (MMLLE) e la mirco-estrazione liquido-liquido (DLLME) utilizzate su campioni liquidi; l’estrazione Soxhlet utilizzata su campioni atmosferici di particolato. Le tecniche analitiche sin qui utilizzate sono: cromatografia liquida ad alte prestazioni accoppiata a rivelatore ultravioletto (HPLC-VWD), gascromatografia con rivelatore a cattura di elettroni (GC-ECD) e gascromatografia con ionizzazione chimica (CI) o elettronica (EI) e rivelazione mass-spettrometrica (GC-CI–MS, GC-EI–MS, GC-NCI-MS). Lo scopo di questo lavoro è la messa a punto di un nuovo metodo di conferma, rapido e robusto, basato sulla HPLC APPI*-MS/MS per la determinazione di basse concentrazioni di ritardanti di fiamma polibromurati in campioni di acqua; in tabella I sono riportati i composti investigati, caratteristici della classe

HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum

A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 +/- 12%, while analytical recovery was 81 +/- 10%. For the eight samples analyzed, the matrix effect was found to be only -14 +/- 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%)

ORGANOPHOSPHORUS FLAME RETARDANTS OCCURRENCE IN ENVIRONMENTAL WATERS

This chapter gives a survey of recent literature on the analysis of organophosphorus flame retardants (OPs) and plasticizers in water. The main literature sources are reviewed from the last eight years, taking into account research articles reporting new analytical developments. Only few new techniques, such as solid-phase microextraction, solid-phase extraction or microporous membrane liquid liquid extraction, have been investigated for their ability of combining the extraction and clean-up steps. Commonest analytical procedures used for the identification of OPs are based on gas chromatography coupled with nitrogen-phosphorus detector or mass spectrometry; as an alternative, liquid chromatography followed by (tandem) mass spectrometry detection is employed

Liquid chromatography–negative ion atmospheric pressure photoionization tandem mass spectrometry for the determination of brominated flame retardants in environmental water and industrial effluents

We describe the development of a liquid chromatography with negative-ion atmospheric pressure photoionization tandem mass spectrometric (LC/NI-APPI/MS/MS) method for the simultaneous determination of tetrabromobisphenol A (TBBP-A) and five polybrominated diphenyl ethers (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154) in water. A mobile phase methanol/acetone/water was used, where acetone acts also as dopant. NI-APPI produced precursor ions corresponding to [M?H]? for TBBP-A, [M?Br+O]?, and [M?2Br+O]? for the BDE congeners studied. Each compound was quantified operating in multiple reaction monitoring mode. Linearity was observed in the range 0.025–10 ng injected for all compounds. Coefficients of determination R2 ranged from0.9934 to 0.9982. BDEswere poorly retained by solid-phase extraction (SPE) from river water and sewage treatment plant effluent, thus liquid–liquid extraction (LLE) by n-hexane should be used for these samples. The recoveries of TBBP-A and PBDEs from tap water (SPE), river water and industrial wastewater (LLE) were in the range of 81–88%, 78–92%, and 43–99%, respectively, with relative standard deviations below 17%. The limits of detection, based on signal-to-noise ratio of 3, ranged from 0.004 to 0.1 ng injected, and method quantification limits were 0.2–3.3 ng L?1 but BDE47 (20.3 ng L?1). Only TBBP-A was found in a treated industrial sewage at 4 ng L?1, while BDE-99 and BDE-100 were detected on suspended solids

Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol-DOPE/DNA lipoplexes in serum

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a 'protein corona'. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications. (C) 2009 Elsevier B.V. All rights reserved

Evaluation of different two-dimensional chromatographic techniques for proteomic analysis of mouse cardiac tissue

In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large-scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide-level fractionation, the first dimension of separation was performed with high-pH reversed-phase chromatography (pH-RP) and strong cation exchange chromatography (SCX). Regarding protein-level fractionation, C(8) protein reversed-phase (C(8)-RP Prot) and high-recovery protein reversed-phase (hr-RP Prot) were used instead. The second dimension consisted of a reversed-phase nano-HPLC on-Chip coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide-level pH-RP and the hr-RP Prot protein-level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide-level SCX, with 509 proteins identified, was the worst method. Copyright (C) 2010 John Wiley & Sons, Ltd

Immunoprecipitation on magnetic beads and LC/MS/MS for carbonic anhydrase II quantification in human serum.

In this study, a magnetic bead-based platform amenable to high-throughput protein carbonic anhydrase II (CA II) capture is presented. The key steps in this approach involved immunoaffinity purification of the target protein from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) operating in multiple reaction monitoring acquisition mode. Using a synthetic peptide standard and a structural analogue free-labeled internal standard, the resulting concentration was stoichiometrically converted to CA II serum concentration. The analytical steps, such as preparation of immunobeads, protein capture, proteolysis, and calibration, were optimized. The method was validated in terms of recovery (77%), reproducibility (relative standard deviation [RSD]<12%), and method detection limit (0.5pmolml-1). The developed method was applied to determining the CA II in eight healthy subjects, and the concentration measured was 27.3pmolml-1 (RSD=65%)

A proteomic study of microgravity cardiac effects: feature maps of label-free LC-MALDI data for differential expression analysis

We present a computational analysis of Mass Spectrometry (MS) data based on a proteomic study of mice cardiac tissue samples whose measured changes in peptide and protein abundance were obtained under normal (control or CTRL) and simulated microgravity conditions (hind-limb unloading or HLU). A pipeline consisting of experimental and computational steps has been designed to achieve, respectively, pre-fractionation to simplify mouse heart protein extracts and data synthesis by feature consensus maps. Both acid and neutral protein fractions obtained from differential solubility have been digested, and peptide mixtures have been resolved by LC-MALDI. The computational tools employed to analyze the MS data and reduce their complex dimensionality have included spectra alignment, denoising and normalization to obtain good-quality peak detection performance. In turn, features could be identified and further refined by searching patterns across repeated measurements obtained under differential conditions (HLU-CTRL, acid-neutral protein extracts). The assessment of reproducibility aspects for evaluating the efficacy of label-free comparative proteomic analysis, combined with the goal of identifying from HLU-CTRL consensus maps candidate proteins with differential expression, led to a computationally efficient approach delivering proteins related to the basic heart functions, cardiac stress and energy supply

Surface adsorption of protein corona controls the cell uptake mechanism in efficient cationic liposome/DNA complexes in serum

Serum has often been reported as a barrier to efficient lipidmediated transfection. Here we found that the transfection efficiency of DC-Chol–DOPE/DNA lipoplexes increases in serum. Our results suggest that ‘protein corona’ can promote large aggregation of intact lipoplexes resulting in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost