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The global prevalence of obesity more than doubled between 1990 and 2022. By 2022, 2.5 billion adults aged 18 and older were overweight, with over 890 million of them living with obesity. The urgent need for understanding the impact of high-fat diet, together with the demanding of analytical methods with low energy/chemicals consumption, can be fulfilled by rapid, high-throughput spectroscopic techniques. To understand the impact of high-fat diet on the metabolic signatures of mouse cecal contents, we characterized metabolite variations in two diet-groups (standard vs high-fat diet) using FTIR spectroscopy and multivariate analysis. Their cecal content showed distinct spectral features corresponding to high- and low-molecular-weight metabolites. Further quantification of 13 low-molecular-weight metabolites using liquid chromatography showed significant reduction in the production of short chain fatty acids and amino acids associated with high-fat diet samples. These findings demonstrated the potential of spectroscopy to follow changes in gut metabolites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Residual nonvisible bladder cancer after proper treatment caused by technological and therapeutic limitations is responsible for tumor relapse and progression. This study aimed to demonstrate the feasibility of a solution for simultaneous detection and treatment of bladder cancer lesions smaller than one millimeter. The α5β1 integrin was identified as a specific marker in 81% of human high-grade nonmuscle invasive bladder cancers and used as a target for the delivery of targeted gold nanorods (GNRs). In a preclinical model of orthotopic bladder cancer expressing the α5β1 integrin, the photoacoustic imaging of targeted GNRs visualized lesions smaller than one millimeter, and their irradiation with continuous laser was used to induce GNR-assisted hyperthermia. Necrosis of the tumor mass, improved survival, and computational modeling were applied to demonstrate the efficacy and safety of this solution. Our study highlights the potential of the GNR-assisted theranostic strategy as a complementary solution in clinical practice to reduce the risk of nonvisible residual bladder cancer after current treatment. Further validation through clinical studies will support the findings of the present study., Competing Interests: Competing interests statement:F.C., A.C., M.A., I.L., E.A., M. Maturi, E.L., and M.C.F. are inventors of patents regarding GNRs functionalized with peptide Iso4.

A novel analytical method for the simultaneous gas chromatography-mass spectrometry (GC-MS) determination of methionine and selenomethionine in food samples is described. Samples were digested with methanesulfonic acid in a closed vessel without the need for reflux. A single step derivatization using triethyloxonium tetrafluoroborate was optimized for the conversion of the analytes into their ethyl derivatives, followed by their extraction with hexane and GC-MS analysis.. This derivatization approach was simpler and/or safer with respect to current methods based on alkyl chloroformate or silylating reagents and it yielded very clean chromatography. A design of experiment approach, based on an open source chemometric software, was used to optimize the experimental conditions. When analysis of a 1 mL volume of aqueous standard was performed, detection limits of 1 ng/g methionine and 10 ng/g for selenomethionine were obtained. The method was validated by analysis of a selenized yeast Certified Reference Material NRC SELM-1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Pyrolysis in an inert atmosphere is a widely applied route to convert tannery wastes into reusable materials. In the present study, the Cr(III) conversion into the toxic hexavalent form in the pyrolyzed tannery waste referred to as KEU was investigated. Ageing experiments and leaching tests demonstrated that the Cr(III)-Cr(VI) inter-conversion occurs in the presence of air at ambient temperature, enhanced by wet environmental conditions. Microstructural analysis revealed that the Cr-primary mineral assemblage formed during pyrolysis (Cr-bearing srebrodolskite and Cr-magnetite spinel) destabilized upon spray water cooling in the last stage of the process. In the evolution from the higher to the lower temperature mineralogy, Cr is incorporated into newly formed CrOOH flakes which likely react in air forming extractable Cr(VI) species. This property transforms KEU from an inert waste to a hazardous material when exposed to ordinary ambient conditions., (© 2023. Springer Nature Limited.)

The mechanisms controlling the generation of PbH 4 by reaction of inorganic Pb(II) with aqueous NaBH 4 were investigated both in the presence and in the absence of the additive K 3 Fe(CN) 6 . For the first time PbH 4 has been identified in analytical chemical vapor generation (CVG) by using gas chromatographic mass spectrometry (GC-MS), which allows the use of deuterium labelled experiments. In the absence of the additive, under reaction conditions typically employed for trace lead determination by CVG, Pb(II) is converted to solid species and no volatile lead species can be detected by either atomic or mass spectrometry for Pb(II) concentration up to 100 mg L -1 . In alkaline conditions Pb(II) substrates are unreactive towards NaBH 4 . In the presence of K 3 Fe(CN) 6 , deuterium labelled experiments clearly indicated that the generated PbH 4 is formed by the direct transfer of hydride from borane to lead atoms. Kinetic experiments were carried out to evaluate the rate of reduction of K 3 Fe(CN) 6 by NaBH 4 , the rate of hydrolysis of NaBH 4 both in the presence and in the absence of K 3 Fe(CN) 6 , and the rate of dihydrogen evolution following NaBH 4 hydrolysis. The effect of delayed addition of Pb(II) to NaBH 4 -HCl- K 3 Fe(CN) 6 , and K 3 Fe(CN) 6 to NaBH 4 -HCl-Pb(II) reaction mixtures on the efficiency of plumbane generation was investigated by continuous flow CVG coupled with atomic fluorescence spectrometry. The collected evidences, complemented with thermodynamic considerations and literature data, have made it possible to clarify long-standing controversial aspects related to the mechanism of plumbane generation and the role of K 3 Fe(CN) 6 additive., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Saliva is an easily sampled matrix containing a variety of biochemical information, which can be correlated with the individual health status. The fast, straightforward analysis of saliva by vibrational (ATR-FTIR and Raman) spectroscopy is a good premise for large-scale preclinical studies to aid translation into clinics. In this work, the effects of saliva collection (spitting/swab) and processing (two different deproteinization procedures) were explored by principal component analysis (PCA) of ATR-FTIR and Raman data and by investigating the effects on the main saliva metabolites by reversed-phase chromatography (RPC-HPLC-DAD). Our results show that, depending on the bioanalytical information needed, special care must be taken when saliva is collected with swabs because the polymeric material significantly interacts with some saliva components. Moreover, the analysis of saliva before and after deproteinization by FTIR and Raman spectroscopy allows to obtain complementary biological information.

Background . Salivary metabolomics is garnering increasing attention in the health field because of easy, minimally invasive saliva sampling. Dihydrouracil (DHU) is a metabolite of pyrimidine metabolism present in urine, plasma, and saliva and of fluoropyrimidines-based chemotherapeutics. Its fast quantification would help in the identification of patients with higher risk of fluoropyrimidine-induced toxicity and inborn errors of pyrimidine metabolism. Few studies consider DHU as the main salivary metabolite, but reports of its concentration levels in saliva are scarce. We propose the direct determination of DHU in saliva by reversed-phase high-performance liquid chromatography (RP-HPLC-UV detector) as a simple, rapid procedure for non-invasive screening. Methods . The method used was validated and applied to 176 saliva samples collected from 21 nominally healthy volunteers and 4 saliva samples from metastatic colorectal cancer patients before and after receiving 5-fluorouracil chemotherapy. Results. DHU levels in all samples analyzed were in the μmol L -1 range or below proving that DHU is not the main metabolite in saliva and confirming the results found in the literature with LC-MS/MS instrumentation. Any increase of DHU due to metabolism dysfunctions can be suggestive of disease and easily monitored in saliva using common, low-cost instrumentation available also for population screening.

Mitochondria are organelles of bacterial origin historically identified as the cell power plant. In addition to energy, mitochondria produce reactive oxygen species and they have been found to have a key role in cell defense regulation, cell stress and damage. All the investigations regarding the nature of the molecules mediating these processes include compounds from mammalian cell metabolism. We hypothesize that the bacterial origin of mitochondria brings them to produce small fermentation products when cell is subjected to stress. In this work we studied the effect of hyperglycemia on the metabolome of hippocampal HN9.10e neurons, an in vitro model of one of the most vulnerable regions of central nervous system. Targeted metabolites were analyzed in the cell culture medium by liquid chromatography - diode array detection and headspace - gas chromatography - mass spectrometry. Twenty-two low molecular weight metabolites were identified and quantified in the growth medium of the cells, treated with 25, 50 or 75 mM glucose, sampled along 8 days to mimic a prolonged hyperglycemia. The results of statistical analysis showed the clear impairment of neuronal metabolism already after 48 h, represented by a significant reduction of the metabolic activity, together with the production of typical fermentative compounds., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Metabolomic profiling of cell lines has shown many potential applications and advantages compared to animal models and human subjects, and an accurate cellular metabolite analysis is critical to understanding both the intracellular and extracellular environments in cell culture. This study provides a fast protocol to investigate in vitro metabolites of immortalized hippocampal neurons HN9.10e with minimal perturbation of the cell system using a targeted approach. HN9.10e neurons represent a reliable model of one of the most vulnerable regions of the central nervous system. Here, the assessment of their extracellular metabolic profile was performed by studying the cell culture medium before and after cell growth under standard conditions. The targeted analysis was performed by a direct, easy, high-throughput reversed-phase liquid chromatography with diode array detector (RP-HPLC-DAD) method and by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) for the study of volatile organic compounds (VOCs). The analysis of six different batches of cells has allowed to investigate the metabolic reproducibility of neuronal cells and to describe the metabolic "starting" conditions that are mandatory for a well-grounded interpretation of the results of any following cellular treatment. An accurate study of the metabolic profile of the HN9.10e cell line has never been performed before, and it could represent a quality parameter before any other targeting assay or further exploration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Campanella, Colombaioni, Nieri, Benedetti, Onor and Bramanti.)

The aqueous-phase reaction of dimethylarsinc acid (DMAs(V)) with NaBH 4 (THB) was studied under non-analytical conditions (1000 μg/mL As, 0.1 M HCl, 1% NaBH 4 ) with the aim of identifying intermediates and reaction products. The use of direct analysis in real time (DART) with high-resolution mass spectrometry (HRMS), in combination with two different chemical vapor generation systems, allowed the identification of some species not detected by GC-MS such as Me 2 As-AsMe-AsMe 2 and the arsonium species [Me 3 As-AsMe 2 ] + and [Me 2 As-AsMe 2 -AsMe 2 ] + . Many other methylated species of arsenic containing up to four arsenic atoms have been observed. Unfortunately, the oxidation mechanism that took place in the DART source interfered with the identification of some of those species formed in solution following THB reduction. The species identified by DART-HRMS, together with those previously identified by GC-MS (Me 2 AsH, Me 2 AsOH, Me 3 As, Me 3 AsO, Me 2 AsAsMeH, Me 2 AsAsMe 2 , and Me 2 As-O-AsMe 2 )' enabled the formulation of hypotheses on the possible reaction pathways and revealed an aqueous-phase reactivity of DMAs(V) which could not be explained on the basis of current knowledge. Graphical Abstract.

Saliva is an interesting, non-conventional, valuable diagnostic fluid. It can be collected using standardized sampling device; thus, its sampling is easy and non-invasive, it contains a variety of organic metabolites that reflect blood composition. The aim of this study was to validate a user-friendly method for the simultaneous determination of low molecular weight metabolites in saliva. We have optimized and validated a high throughput, direct, low-cost reversed phase liquid chromatographic method with diode array detection method without any pre- or post-column derivatization. We indexed salivary biomolecules in 35 whole non-stimulated saliva samples collected in 8 individuals in different days, including organic acids and amino acids and other carbonyl compounds. Among these, 16 whole saliva samples were collected by a single individual over three weeks before, during and after treatment with antibiotic in order to investigate the dynamics of metabolites. The concentrations of the metabolites were compared with the literature data. The multianalyte method here proposed requires a minimal sample handling and it is cost-effectiveness as it makes possible to analyze a high number of samples with basic instrumentation. The identification and quantitation of salivary metabolites may allow the definition of potential biomarkers for non-invasive "personal monitoring" during drug treatments, work out, or life habits over time.

The bio-engineering technologies of medical devices through nano-structuring and coating was recently proposed to improve biocompatibility and to reduce microbial adhesion in the prevention of implantable device-related infections. Our aim was to evaluate the ability of new nano-structured and coated materials to prevent the adhesion and biofilm formation, according to the American Standard Test Method ASTM-E2647-13. The materials composition was determined by X-ray Fluorescence and Laser Induced Breakdown Spectroscopy. Silver release was evaluated by Inductively Coupled Plasma Mass Spectrometry analysis. The gene expression levels of the Quorum Sensing Las and Rhl system were evaluated by the ΔΔCt method. The Log bacterial density (Log CFU/cm 2 ) on TiAl6V4 was 4.41 ± 0.76 and 4.63 ± 1.01 on TiAl6V4-AgNPs compared to 2.57 ± 0.70 on CoCr and 2.73 ± 0.61 on CoCr-AgNPs (P < 0.0001, A.N.O.V.A.- one way test). The silver release was found to be equal to 17.8 ± 0.2 µg/L after the batch phase and 1.3 ± 0.1 µg/L during continuous flow. The rhlR gene resulted in a 2.70-fold increased expression in biofilm growth on the silver nanoparticles (AgNPs) coating. In conclusion, CoCr showed a greater ability to reduce microbial adhesion, independently of the AgNPs coating. The silver release resulted in promoting the up-regulation of the Rhl system. Further investigation should be conducted to optimize the effectiveness of the coating., Competing Interests: The authors declare no conflicts of interest.

A mini review of the toxicity of Thallium (Tl) at low doses is herein presented. Thallium has severe toxicity. Although its acute biological effects have been widely investigated and are well known, its biological effects on human health and in cell cultures at low doses (<100 μg/L) due, for example, to Tl chronic exposure via consumption of contaminated water or foods, have often been overlooked or underestimated. Relatively few papers have been published on this topic and are herein reviewed to provide a focused scientific opinion in the light of current worldwide regulatory issues.

In this work, a straightforward analytical approach based on headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was developed for the analysis of salivary volatile organic compounds without any prior derivatization step. With a sample volume of 500 μL, optimal conditions were achieved by allowing the sample to equilibrate for 10 min at 50 °C and then extracting the samples for 10 min at the same temperature, using a carboxen/polydimethylsiloxane fibre. The method allowed the simultaneous identification and quantification of 20 compounds in sample headspace, including short-chain fatty acids and their derivatives which are commonly analysed after analyte derivatization. The proof of applicability of the methodology was performed with a case study regarding the analysis of the dynamics of volatile metabolites in saliva of a single subject undergoing 5-day treatment with rifaximin antibiotic. Non-stimulated saliva samples were collected over 3 weeks from a nominally healthy volunteer before, during, and after antibiotic treatment. The variations of some metabolites, known to be produced by the microbiota and by bacteria that are susceptible to antibiotics, suggest that the study of the dynamics of salivary metabolites can be an excellent indirect method for analysing the gut microbiota. This approach is novel from an analytical standpoint, and it encourages further studies combining saliva metabolite profiles and gut microbiota dynamics. Graphical abstract.

Anthropogenic impact over the Pasvik River (Arctic Norway) is mainly caused by emissions from runoff from smelter and mine wastes, as well as by domestic sewage from the Russian, Norwegian, and Finnish settlements situated on its catchment area. In this study, sediment samples from sites within the Pasvik River area with different histories of metal input were analyzed for metal contamination and occurrence of metal-resistant bacteria in late spring and summer of 2014. The major differences in microbial and chemical parameters were mostly dependent on local inputs than seasonality. Higher concentrations of metals were generally detected in July rather than May, with inner stations that became particularly enriched in Cr, Ni, Cu, and Zn, but without significant differences. Bacterial resistance to metals, which resulted from viable counts on amended agar plates, was in the order Ni 2+ >Pb 2+ >Co 2+ >Zn 2+ >Cu 2+ >Cd 2+ >Hg 2+ , with higher values that were generally determined at inner stations. Among a total of 286 bacterial isolates (mainly achieved from Ni- and Pb-amended plates), the 7.2% showed multiresistance at increasing metal concentration (up to 10,000 ppm). Selected multiresistant isolates belonged to the genera Stenotrophomonas, Arthrobacter, and Serratia. Results highlighted that bacteria, rapidly responding to changing conditions, could be considered as true indicators of the harmful effect caused by contaminants on human health and environment and suggested their potential application in bioremediation processes of metal-polluted cold sites.

When working with humid gaseous samples, the amount of water vapour collected in a needle trap along with volatile analytes may vary from sample to sample and decrease during the storage. This has a major impact on desorption efficiency and recovery. We propose the addition of a labelled internal standards to nullify the effect of variable humidity on the analytical performance of needle trap micro-extraction combined with gas chromatography mass spectrometry. Triple-bed (Divinylbenzene/Carbopack X/Carboxen 1000) and single-bed (Tenax GR) needles were tested with standard gaseous mixtures prepared at different relative humidity levels (85%, 50% and 10%). The standard mixtures contained twenty-five analytes representative of breath and ambient air constituents, including hydrocarbons, ketones, aldehydes, aromatics, and sulphurs, in the concentration range 0.1-700 ppbv. The two needles showed different behaviours, as recovery was independent of humidity for single-beds, whereas a low recovery (10-20%) was observed when triple-beds trapped very volatile compounds at low humidity (e.g. pentane and ethanol, 10% relative humidity. Triple-beds showed an almost quantitative recovery (>90%) of all the analytes at 50% and 85% relative humidity. This big difference was probably due to the reduced action of water vapour pressure during the desorption step. The addition of 6 D-acetone and 8 D-toluene to the sorbent material before gas sampling and the normalization of raw data nullified this effect, thereby lowering the variations of analyte recovery at different humidity levels down to 20%. Internal standards were also exploited to limit within 10-20% alterations in peak areas of very volatile compounds during needle storage at room temperature. This variation may results from a loss of water vapour either retained from the sorbent material and/or condensed on triple-bed needle walls. After normalization, the inter- and intra-day precision were halved to 5% and 10% in the case of single-beds, respectively, and to 15% and 20% with three-beds. The addition of an internal standard to the sorbent helps to keep the overall analytical procedure under control and improves the reliability of needle trap micro-extraction for the analysis of volatile organic compounds at ultra-trace levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A high-precision exact-matching quadruple isotope dilution method (ID 4 MS) was employed for the quantitation of nitrate in an air-dried spinach powder Certified Reference Material (CRM). The analyte was extracted in hot water following addition of 15 NO[Formula: see text] internal standard. The blend was then treated with sulfamic acid to remove nitrite and with triethyloxonium tetrafluoroborate to promote aqueous conversion of nitrate into volatile EtONO 2 . The derivative was analyzed by headspace GC-MS with 3-min elution time. The method performance was validated with a series of tests which demonstrated adequate selectivity and ruggedness. This method supported the development of novel SPIN-1 CRM giving a modest contribution to its uncertainty (u char = 0.85%). With respect to previous attempts, the SPIN-1 was proven stable, homogeneous (u hom = 0.44%), and suitable for spinach monitoring under EU regulations. On dried basis, the nitrate content of SPIN-1 was found to be 22.53 ± 0.43 mg/g (U c = 1.9%, k = 2). The material was also used in an inter-laboratory study where four laboratories employed a total of ten measurement methods. Graphical Abstract SPIN-1 Certified Reference Material for nitrate in spinach powder.

Heavy metals (HMs) and polychlorobiphenyls (PCBs) enter the Arctic environment through a variety of anthropogenic sources with deleterious effects towards biota and public health. Bacteria first transfer toxic compounds to higher trophic levels and, due to the tight link existing between prokaryotic community functions and the type and concentration of contaminants, they may be useful indicator of pollution events and potential toxicity to other forms of life. The occurrence and abundance of HM-tolerant and PCB-oxidizing bacteria in the sub-Arctic Pasvik river area, heavily impacted by anthropogenic modifications, was related to HM and PCB contamination. This latter more likely derived from local inputs rather than a global contamination with higher PCB and HM amounts (and higher bacterial viable counts) that were determined in inner and middle sections of the River. Finally, a panel of bacteria with potential applications in the bioremediation of cold environments were selected and phylogenetically identified., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In order to elucidate controversial results emerging in chemical vapor generation (CVG) for trace element determination, we conducted a series of experiments devoted to the identification of intermediates formed by acid hydrolysis of amine-boranes. For the first time, direct analysis in real time coupled with high-resolution mass spectrometry (DART-Orbitrap) was applied for detection of this class of compounds. Mass spectra of both solid amine-boranes and their aqueous solutions (pH ~ 8, no hydrolysis) were acquired for understanding their ionization pathway. Mass spectra of aqueous solutions of t-BuNH 2 ·BH 3 and Me 2 NH·BH 3 were acquired under conditions that are employed in CVG (0.017-4.0 mol L -1 HCl, 0.167-0.2 mol L -1 borane reagent). The results disclose a reactivity driven by pH of amine-boranes undergoing hydrolysis. At low acidity, the hydrolysis proceeds according to the currently accepted displacement mechanisms (i.e., R 3 N·BH 3 + H 3 O + → R 3 NH + + H 2 OBH 3 ). At higher acidity, N-tert-butyl, cyclotriborazane, and bis(dimethylamino)boronium were identified, for the first time, during the hydrolysis of t-BuNH 2 ·BH 3 and Me 2 NH·BH 3 , respectively. Formation of these intermediates was ascribed to a hydrolysis pathway starting with the ionization of the amine-borane, (i.e., R 3 N·BH 3 + H 3 O + → [(H 2 O)R 3 NBH 2 ] + + H 2 ). The new evidence explains the anomalous behavior observed in CVG by amine-borane derivatization, and updates the currently accepted mechanisms for the acid hydrolysis of amine-boranes. Graphical Abstract.

The heavy metal thallium is an emerging pollutant among the most potentially toxic species to which human populations are exposed. Its harmful effects on living organisms are well-known at high doses, typical of acute intoxication. Its harmful effects at low doses are by far less known. In a previous paper, we reported a TlCl-induced metabolic shift to lactate and ethanol production in living hippocampal HN9.10e neurons that appeared after a single short exposure (48 h) at low doses (1-100 μg/L). This metabolic shift to lactate and ethanol suggests a marked impairment of cell bioenergetics. In this work, we provide detailed evidence for TlCl-induced changes of neuronal morphology and mitochondrial activity. Confocal microscopy and fluorescent probes were used to qualitatively and quantitatively analyze, at the subcellular level, living HN9.10e neurons during and after TlCl exposure. An early onset mitochondrial dysfunction appeared, associated with signs of cellular deregulation such as neurite shortening, loss of substrate adhesion, and increase of cytoplasmic calcium. The dose-dependent alteration of mitochondrial ROS (mtROS) level and of transmembrane mitochondrial potential (ΔΨ m ) has been observed also for very low TlCl doses (1 μg/L). The treatment with the ATP synthase inhibitor oligomycin revealed a severe impairment of the mitochondrial function, more significant than that measured by the simple quantification of the tetramethylrhodamine methyl ester (TMRM) fluorescence. These results highlight that mitochondria are a key subcellular target of TlCl neurotoxicity. The transmembrane mitochondrial potential was significantly correlated with the ethanol concentration in cell culture medium ( P < 0.001, r = -0.817), suggesting that ethanol could be potentially used as a biomarker of mitochondrial impairment.

In this study, we propose a novel approach for the determination of total dissolved nitrogen (TDN) in seawater combining high-precision isotope dilution GC-MS with persulfate digestion. A 2 mL sample aliquot was digested with an alkaline solution of persulfate to convert nitrogen containing compounds to nitrate. Digested samples were spiked with 15 NO 3 - internal standard and treated with aqueous triethyloxonium to convert the analyte into volatile EtONO 2 . This derivative was readily separated from the matrix under gaseous form and could be sampled from the headspace before GC-MS analysis. The resulting chromatograms showed a stable flat baseline with EtONO 2 as the only eluting peak (retention time 2.75 min on a DB 5.625 column). Such an approach provides specificity and obviates the shortcomings of current detection methods employed to analyze seawater samples after digestion with persulfate. In negative chemical ionization mode, the method reached a detection limit of 0.5 μmol/kg TDN (7 ng/g N) and could be applied to quantify seawater samples with 1-25 μmol/kg TDN. On the upper end of the range, quantitation could be repeated within 1%, whereas on a 6 μmol/kg TDN sample repeatability was 2.3% on eight measurements. The method was employed in two proficiency testing exercises providing results in agreement with consensus values. We investigated the impact of reagent blank and we implemented a blank-matching optimal design to account for such contribution. Finally, we performed a study on the yield of persulfate oxidation for organic and inorganic nitrogen compounds typically present in seawater. Whilst nitrite and ammonium are fully converted to nitrate, more complex organic molecules showed recoveries varying from 70% to 100%., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

The abandoned mining area of Valdicastello Carducci (Tuscany, Italy) is characterized by the massive presence of thallium in the acid mine drainages and in the valley stream crossing the region. We previously found that Tl(III), generally considered the less stable oxidation state of thallium, is present both in the stream and in tap water distributed in the area, whereas acid mine drainages only contain Tl(I). These findings posed some concern related to the reactivity and dispersion of this toxic element in the environment. Since the valence state of thallium determines its toxicity, distribution and mobility, the study of thallium redox speciation appears crucial to understand its environmental behaviour. In this work, water samples collected from the mine drainages and the contaminated stream were adopted as model to study the distribution of aqueous Tl(I)/Tl(III) as a function of light exposure and solution properties and composition. The influence of three light sources and organic acids was evaluated. Thallium speciation was also assessed in tap water after treatment with common oxidizing agents, and in the rust crust collected from the public waterworks., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The behavior of NaBH 4 (THB) and the amine boranes, NH 3 BH 3 (AB), tertbutylNH 2 BH 3 (TBAB), Me 2 NHBH 3 (DMAB) was investigated in continuous flow chemical vapor generation of H 2 Se from aqueous Se IV coupled with atomic absorption spectrometry. Unexpected higher efficiency of H 2 Se generation was obtained with amine boranes compared to THB (TBAB > AB > THB) using millimolar concentration of reductant (0.001-0.1 mol L -1 ) under strongly acidic conditions (HCl, HClO 4 , H 2 SO 4 , HNO 3 , 0.5-5 mol L -1  H + ). Analytical applicability of the CVG system was tested by the determination of Se IV in natural water samples certified reference materials, using 0.01 mol L -1 TBAB in 0.5 M H 2 SO 4 . In order to explain this unexpected higher efficiency of amine boranes with respect of THB, the kinetic of hydrolysis of AB, TBAB and DMAB was investigated in acid media typically employed in chemical vapor generation for trace element determination. The kinetic was investigated by monitoring the rate the hydrogen gas evolved during hydrolysis, using a laboratory made thermostated reaction cell. Kinetics were measured for AB, TBAB and DMAB in 0.1, 0.5, 5 mol L -1 HCl or HClO 4 reaction media and in 0.1 mol L -1 cysteine +0.1 mol L -1 HCl or HClO 4 buffer, for reaction times from 0 to 30 min. Under strongly acidic conditions, the rates of hydrogen evolution produced by amine boranes hydrolysis appear to be much slower than those predicted by a pseudo-first order reaction and using the rate constants reported in the literature. This suggests that, at elevated acidities (5 mol L -1 HCl or HClO 4 ), the hydrolysis of amine boranes takes place in two steps, generating a first amount of H 2 (0.67-1.15 mol) much faster than the remaining about 2 mol. This evidence indicates a different mechanism of hydrolysis to the one accepted in the literature for amine boranes. The relatively high efficiencies of H 2 Se observed with amine borane reduction of inorganic Se IV at elevated acidities can be addressed to the action of borane intermediates, most probably amine borane cations, formed during amine borane hydrolysis in the same reaction conditions., (Copyright © 2017. Published by Elsevier B.V.)

Lactate and ethanol (EtOH) were determined in cell culture medium (CCM) of immortalized hippocampal neurons (HN9.10e cell line) before and after incubation with Thallium (Tl). This cell line is a reliable, in vitro model of one of the most vulnerable regions of central nervous system. Cells were incubated for 48 h with three different single Tl doses: 1, 10, 100 μg/L (corresponding to 4.9, 49 and 490 nM, respectively). After 48 h, neurons were "reperfused" with fresh CCM every 24/48 h until 7 days after the treatment and the removed CCM was collected and analysed. Confocal microscopy was employed to observe morphological changes. EtOH was determined by head space-solid phase microextraction -gas chromatography -mass spectrometry (HS-SPME-GCMS), lactate by RP-HPLC with UV detection. Tl exposure had significant effects on neuronal growth rate and morphology. The damage degree was dose-dependent. In not exposed cells, EtOH concentration was 0.18 ± 0.013 mM, which represents about 5% of lactate concentration (3.4 ± 0.10 mM). After Tl exposure lactate and EtOH increased. In CCM of 100 and 10 μg/L Tl-treated cells, lactate increased 24 h after reperfusion up to 2 and 3.3 times the control value, respectively. In CCM of 10 and 100 μg/L Tl-treated cells 24 h after reperfusion, EtOH increased up to 0.3 and 0.58 mmol/L. respectively. These results are consistent with significant alterations in energy metabolism, despite the low doses of Tl employed and the relatively short incubation time.

The present work describes a non invasive lactate sensing in sweat during workout. The sensing system is based on a non-equilibrium potentiometric measure performed using disposable, chemically modified, screen printed carbon electrodes (SPCEs) that can be wetted with sweat during the exercise. The potentiometric signal, which is proportional to lactate concentration in sweat, is produced by a redox reaction activated by UV radiation, as opposed to the enzymatic reaction employed in traditional, blood-based measuring devices. The sensing system exhibits chemical selectivity toward lactate with linearity from 1 mM up to 180 mM. The dynamic linear range is suitable for measurement of lactate in sweat, which is more than 10 times concentrated than hematic lactate and reaches more than 100 mM in sweat during workout. The noninvasive measure can be repeated many times during exercise and during the recovery time in order to get personal information on the physiological and training status as well as on the physical performance. The device was successfully applied to several human subjects for the measurement of sweat lactate during prolonged cycling exercise. During the exercise sweat was simultaneously sampled on filter paper and extracted in water, and the lactate was determined by HPLC for method validation. The lactate concentration changes during the exercise reflected the intensity of physical effort. This method has perspectives in many sport disciplines as well as in health care and biomedical area., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this work we present an advantageous method for the simultaneous separation and detection of Tl(I) and Tl(III) species through ion chromatography coupled with on-line inductively coupled plasma - mass spectrometry. Chromatographic separation between Tl(III) and Tl(I) was achieved in less than two minutes. The method was validated by recovery experiments on real samples, and by comparing the sum of the concentrations of individual Tl species with total thallium values obtained from continuous flow ICP-MS. The experimental procedure offers an accurate, sensitive and interference-free method for Tl speciation at trace levels in environmental samples. This allowed us to investigate the Tl speciation in acid mine drainages (AMD), surface waters and springs in a mining catchment in Valdicastello Carducci (Tuscany, Italy), where severe Tl contamination ad been evidenced previously. This study shows for the first time that Tl(III), in addition to Tl(I), is present in considerable amounts in water samples affected by acid mining outflow, raising the question of the origin of this thermodynamically unstable species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study we present a novel isotope dilution gas chromatography method for the determination of nitrate in vegetables. The analyte was extracted in water at 70 ∘ C and mixed with 15 N isotopically enriched nitrate internal standard. The sample was centrifuged and the supernatant reacted with sulfamic acid for removal of nitrite, and with triethyloxonium tetrafluoroborate for converting nitrate into volatile EtONO 2 . This simple aqueous chemistry allowed for separation of analyte from sample matrix in the form of a gaseous derivative which could be sampled in the headspace before GC-MS analysis. This key-feature of the method made possible the collection of clean chromatograms within an elution time of only 1.8 min. Detection of EtONO 2 could be performed using electron impact ionization with a standard GC-MS setup. The method was optimized and validated for the analysis of nitrate in fresh vegetables in the 10-10,000 μg/g range with a detection limit of 2 μg/g. Due to the use of primary isotope dilution quantitation, traceable results of high-precision were attained., (Copyright © 2017. Published by Elsevier B.V.)

The high toxicity of cyanide, along with its widespread industrial use, has fuelled interest in the development of analytical methods for its determination in complex matrices. In this study, we propose a novel approach for the measurement of total cyanide in soil samples based on single-step derivatization with pentafluorobenzyl bromide (F 5 Bn-Br) followed by quantitation with gas chromatography mass spectrometry in negative chemical ionization mode. The reaction between CN - and F 5 Bn-Br resulted in the identification of several derivatives such as F 5 Bn-CN, (F 5 Bn)(F 5 Ph)CH-CN, and (F 5 Bn) 2 (F 5 Ph)C-CN. The relative proportion between such compounds was dependent on experimental conditions. When a 100 μL aliquot of an alkaline-aqueous extract was reacted with 700 μL of 1.3% F 5 Bn-Br in acetone, the tri-alkylated derivative was the most abundant. In such conditions a detection limit of 0.5 ng/g of CN - was attained. Soil samples were initially spiked with an alkaline solution of K 13 C 15 N internal standard and suspended in 7.5% aqueous NaOH. Determination of total cyanide was achieved by digestion of the alkaline extract with H 3 PO 4 to produce HCN which was then trapped in 0.1% NaOH in a sealed double vial system, followed by reaction with F 5 Bn-Br. Isotope dilution calibration was chosen for quantitation, and the validity of the novel method was demonstrated by analysis of soil Certified Reference Materials (CRMs) and by spike recovery tests., (Copyright © 2017. Published by Elsevier B.V.)

The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called "indole-3-acetamide pathway" to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis. Here, two knockout mutants, ΔiaaL and ΔiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that ΔiaaL and ΔiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of ΔiaaL and ΔiaaM, as well as growth of the non-pathogenic mutant ΔhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a concerted regulatory network in this phytopathogen among virulence, fitness and drug efflux., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

A geological study evidenced the presence of thallium (Tl) at concentrations of concern in groundwaters near Valdicastello Carducci (Tuscany, Italy). The source of contamination has been identified in the Tl-bearing pyrite ores occurring in the abandoned mining sites of the area. The strongly acidic internal waters flowing in the mining tunnels can reach exceptional Tl concentrations, up to 9000μg/L. In September 2014 Tl contamination was also found in the tap water distributed in the same area (from 2 to 10μg/L). On October 3, 2014 the local authorities imposed a Do Not Drink order to the population. Here we report the results of the exposure study carried out from October 2014 to October 2015, and aimed at quantifying Tl levels in 150 urine and 318 hair samples from the population of Valdicastello Carducci and Pietrasanta. Thallium was quantified by inductively coupled plasma - mass spectrometry (ICP-MS). Urine and hair were chosen as model matrices indicative of different time periods of exposure (short-term and long-term, respectively). Thallium values found in biological samples were correlated with Tl concentrations found in tap water in the living area of each citizen, and with his/her habits. Thallium concentration range found in hair and urine was 1-498ng/g (values in unexposed subjects 0.1-6ng/g) and 0.046-5.44μg/L (reference value for the European population 0.006μg/L), respectively. Results show that Tl levels in biological samples were significantly associated with residency in zones containing elevated water Tl levels. The kinetics of decay of Tl concentration in urine samples was also investigated. At the best of our knowledge, this is the first study on human contamination by Tl through water involving such a high number of samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 molL(-1) urea, 3 molL(-1) guanidinium thiocyanate, 6 molL(-1) guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS). The number of -SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of -SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique. We also investigated the labeling kinetics for denatured OVA and pHMB, finding that the 4 thiolic groups of OVA have a very different reactivity toward mercury labeling, in agreement with previous studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A novel method for the determination of salivary thiocyanate is presented. Thiocyanate was converted into ethyl thiocyanate by single-step aqueous derivatization based on triethyloxonium tetrafluoroborate and measured by gas chromatography-mass spectrometry (15 min runtime). The ethyl thiocyanate derivative is volatile and can be sampled from the headspace. The derivatization chemistry proposed allows for separation of the analyte from saliva matrix whose introduction in the measurement system is avoided. Quantitation of the analyte was obtained by isotope dilution, employing a (13)C-enriched thiocyanate as internal standard. Technical details and fundamental aspects of derivatization chemistry and calibration strategy are presented. The method was validated by comparison with a standard method based on ion chromatography. The two independent methodologies produced results in agreement within 3%. Also a three level spike recovery test was carried out for validation purpose and quantitative recoveries were attained. The method is fast, simple, safe, and sensitive. Measurement of a 1 mL volume 50 ng/g of thiocyanate standard produced a signal-to-noise ratio of 250 for the analytical peak. This method is therefore suitable for ultra-trace determination of thiocyanate (low part-per-billion range). For the application described the full detection potential of the method was not required and the sample preparation presented has been designed for quantitation of saliva samples containing 1-400 μg/g of thiocyanate with a combined standard uncertainty of 2% relative for saliva samples containing 25 μg/g of thiocyanate. This method was applied for the determination of thiocyanate in human saliva samples., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Volatile anaesthetics and disinfection chemicals pose ubiquitous inhalation and dermal exposure risks in hospital and clinic environments. This work demonstrates specific non-invasive breath biomonitoring methodology for assessing staff exposures to sevoflurane (SEV) anaesthetic, documenting its metabolite hexafluoroisopropanol (HFIP) and measuring exposures to isopropanol (IPA) dermal disinfection fluid. Methods are based on breath sample collection in Nalophan bags, followed by an aliquot transfer to adsorption tube, and subsequent analysis by thermal desorption gas chromatography-mass spectrometry (TD-GC-MS). Ambient levels of IPA were also monitored. These methods could be generalized to other common volatile chemicals found in medical environments. Calibration curves were linear (r(2)=0.999) in the investigated ranges: 0.01-1000 ppbv for SEV, 0.02-1700 ppbv for IPA, and 0.001-0.1 ppbv for HFIP. The instrumental detection limit was 10 pptv for IPA and 5 pptv for SEV, both estimated by extracted ion-TIC chromatograms, whereas the HFIP minimum detectable concentration was 0.5 pptv as estimated in SIM acquisition mode. The methods were applied to hospital staff working in operating rooms and clinics for blood draws. SEV and HFIP were present in all subjects at concentrations in the range of 0.7-18, and 0.002-0.024 ppbv for SEV and HFIP respectively. Correlation between IPA ambient air and breath concentration confirmed the inhalation pathway of exposure (r=0.95, p<0.001) and breath-borne IPA was measured as high as 1500 ppbv. The methodology is easy to implement and valuable for screening exposures to common hospital chemicals. Although the overall exposures documented were generally below levels of health concern in this limited study, outliers were observed that indicate potential for acute exposures., (Copyright © 2015. Published by Elsevier B.V.)

Background and Objective: The determination of warfarin, RS/SR- and RR/SS-warfarin alcohols in oral fluid may offer additional information to the INR assay. This study aimed to establish an optimized sampling technique providing the best correlation between the oral fluid and the unbound plasma concentrations of these compounds., Materials and Methods: Samples of non-stimulated and stimulated oral fluid, and blood were collected from 14 patients undergoing warfarin therapy. After acidification, analytes were extracted with a dichloromethane/hexane mixture and determined by HPLC with fluorescence detection. Plasma samples were also ultrafiltered for the determination of the unbound fraction. The chromatographic separation was carried out in isocratic conditions with a phosphate buffer/methanol mobile phase on a C-18 reversed-phase column. The absence of interfering compounds was verified by HPLC-ESI-Q-TOF., Results: Stimulation generally increased the oral fluid pH to values close to blood pH in about 6 minutes. The concentration of warfarin and RS/SR-warfarin alcohols in oral fluid followed the same trend, whereas the concentration of RR/SS-warfarin alcohols was not affected. Six minute stimulation with chewing gum followed by collection with a polyester swab was the best sampling procedure, with a good repeatability (RSD < 10%) and relatively low inter-subject variability (RSD  = 30%) of the oral fluid to plasma ratio. This procedure provided strong correlations between the measured oral fluid and unbound plasma concentration of warfarin (r  =  0.92, p < 0.001) and RS/SR-warfarin alcohols (r  =  0.84, p < 0.001), as well as between stimulated oral fluid and total plasma concentration of warfarin (r  =  0.78, p < 0.001) and RS/SR-warfarin alcohols (r  =  0.81, p < 0.001)., Conclusion: The very good correlation between oral fluid and unbound plasma concentration of warfarin and RS/SR-warfarin alcohols suggests that oral fluid analysis could provide clinically useful information for the monitoring of anticoagulant therapy, complementary to the INR assay.

We developed a novel and straightforward derivatization method for the determination of lactate by reversed phase high-performance liquid chromatography (RP-HPLC) with fluorescence and UV detection in biological matrices as urine and saliva. The derivatization of lactate was achieved employing 9-chloromethyl anthracene (9-CMA) as fluorescence reagent, which has never been previously used to obtain a lactate derivative. Lactate reacts with 9-CMA with high selectivity in a very short time, without requiring extraction procedures from the aqueous solution, and the reaction reaches 70% completion in 30 min. The ester derivative obtained can be easily determined by RP-HPLC with fluorescence detection at 410 nm (λ ex=365 nm) and UV detection at 365 nm. The method was also optimized in order to allow for the simultaneous determination of lactate and creatinine for the application to urine samples. The lactate calibration curve was linear in the investigated range 2 × 10(-4)-3 × 10(-2)mM and the limit of detection, calculated as three times the standard deviation of the blank divided by the calibration curve slope, was 50 nM for both fluorescence and UV detection. Intra-day and inter-day repeatability were lower than 5% and 6%, respectively. The method proposed was successfully applied to the analysis of urine and saliva samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein-pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC-CVG-AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75-100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced SH groups before mercury labelling. We obtained a detection limit of 100 nmol L(-1) based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L(-1), depending on the number of cysteines in the protein sequence., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Ultraviolet (UV) absorbance of spent dialysate has been proposed as a method for monitoring hemodialysis efficiency. The contribution of the various uremic toxins to UV absorption, however, needs clarifying., Methods: Urea, creatinine and uric acid were measured in blood and dialysate before and during dialysis in 22 maintenance hemodialysis patients. Absorbance was measured in dialysate., Results: High-performance liquid chromatography (HPLC) analyses of dialysate revealed uric acid as the predominant peak. Spent dialysate absorbance decreased, during dialysis, similarly to serum and dialysate urea, creatinine and uric acid. Dialysate urea correlated closely with absorbance, though urea did not contribute to absorbance, which was determined mostly by uric acid. Uric acid and urea removals were very similar. The spectrophotometric Kt/V correlated with spKt/V urea, with slight but significant differences between the two measurements., Conclusions: UV absorbance is determined mostly by uric acid. Absorbance measurements seem suitable as a method for monitoring dialysis efficiency.

We optimized a hyphenated system based on size exclusion chromatography coupled to a microwave/UV mercury oxidation system and an atomic fluorescence detector (SEC-CVG-AFS) for the online oxidation of free and protein-complexed p-hydroxymercuribenzoic acid (pHMB) without the employment of chemical oxidizing agents. This system has been applied to the study of labeling of thiolic groups of native ovalbumin (OVA) as a function of protein concentration. We found that the protein concentration strongly affects the species distribution of OVA, the number of thiolic groups titrated in each species, and thus, the accuracy in the determination of the total number of thiolic groups. The amount of titrated sulfhydryl groups in the protein concentration range investigated (5-100 μmol L(-1)) varied from 2.40 ± 0.01 to 1.85 ± 0.05 for the monomeric form of OVA and from 4.63 ± 0.01 to 5.63 ± 0.05 for the total OVA, which represents more than four theoretical number of reduced Cys. This information is important from the analytical point of view because it suggests that, unless to operate with diluted concentration of protein, the number of titrated thiolic groups results from both the aspecific interaction of the probe with aggregates species and to the specific bond of the probe with the accessible -SH groups.

The efficiency of chemical generation of arsanes from inorganic arsenic, monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA), to arsane, AsH3, monomethylarsane, CH3AsH2 (MMA), and dimethylarsane, (CH3)2AsH (DMA), has been investigated in different reaction media with the aim to better elucidate the mechanisms controlling their generation process and to find the experimental conditions to implement a nonchromatographic arsenic speciation analytical method, which is based on the selective determination of some arsenic species. Studies were performed by continuous flow hydride generation coupled with atomic spectrometry (CF-HG-AS), using different reductants such as borane-ammonia (AB), borane-tert-butylamine (TBAB), and sodium tetrahydridoborate (THB) in HCl and HClO4 media, in the presence or absence of L-cysteine (Cys). The efficiency of HG processes for MMA and DMA is mainly controlled by the reactivity of the substrates with the borane, which could be strongly influenced by the formation of ion couples. The protonation of arsane did not play a significant role in the employed reaction system. By taking advantage of the different reactivity pattern of As species in selected generation conditions, DMAA and MMAA could be selectively determined in 0.5 and 10 M HClO4 solutions, respectively, in the presence of Cys, with AB as the reducing agent. The presence of Cys as a masking agent and the peculiar reducing properties of AB ensured a good control of interferences, as far as it has been observed for Co(II), Ni(II), Cu(II), Fe(II), Fe(III). The overall time needed to complete the prereduction step has been verified for MMAA and DMAA at different acidities in order to achieve the best selectivity. The selective determination of DMAA with AB/Cys in HClO4 has been optimized and applied to certified reference materials (CRMs) of natural waters CASS-4, SLRS-4, and NASS-4 (NRCC). The estimation of DMAA concentration allows us to correct the concentration of As(III) for the interference of DMAA in the selective determination of As(III) according to a selective HG method recently reported.

A novel method is presented for the characterization and determination of thiolic proteins. After the labeling with p-hydroxymercurybenzoate, the pHMB-labeled proteins underwent on-line oxidation with a novel microwave (MW)/UV photochemical reactor, followed by cold vapor generation-atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the conversion of pHMB to Hg(II) with a yield of 89.0 ± 0.5% without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. Hg(II) was reduced to Hg(0) in a knotted reaction coil with NaBH4 solution, stripped from the solution by an argon flow and detected. The chromatographic method for labeled thiolic peptides was linear in the 0.2-100 μmol L(-1) range, with a LOD as mercury of 57 nmol L(-1). This system has proven to be a useful interface for liquid chromatography coupled with CVG-AFS in the determination and characterization of thiolic proteins. This method has been applied to the determination of thiolic peptides after tryptic digestion of serum albumins from different species (human, bovine, rat, horse, and sheep).

We developed a flow injection (FI) method for the determination of thiomersal (sodium ethylmercurithiosalicylate, C9H9HgNaO2S) based on the UV/microwave (MW) photochemical, online oxidation of organic mercury, followed by cold vapor generation atomic fluorescence spectrometry (CVG-AFS) detection. Thiomersal was quantitatively converted in the MW/UV process to Hg(II), with a yield of 97±3%. This reaction was followed by the reduction of Hg(II) to Hg(0) performed in a knotted reaction coil with NaBH4 solution, and AFS detection in an Ar/H2 miniaturized flame. The method was linear in the 0.01-2 μg mL(-1) range, with a LOD of 0.003 μg mL(-1). This method has been applied to the determination of thiomersal in ophthalmic solutions, with recoveries ranging between 97% and 101%. We found a mercury concentration in commercial ophthalmic solutions ranging between 7.5 and 59.0 μg mL(-1)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Two analytical procedures are presented for the determination of the total content and unbound fraction of both warfarin and warfarin alcohols in human plasma. Chromatographic separation was carried out in isocratic conditions at 25°C on a C-18 reversed-phase column with a mobile phase consisting of a 70% buffer phosphate 25mM at pH=7, 25% methanol and 5% acetonitrile at a flow rate of 1.2mL/min. Fluorescence detection was performed at 390nm (excitation wavelength 310nm). Neither method showed any detectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcohols at a concentration level of 1000ng/mL was 89±3% and 73±3%, respectively, whereas for their unbound fraction (at a concentration level of 10ng/mL) was 66±8% and 90±7%, respectively. The intra- and inter-day precision (assessed as relative standard deviation) was <10% for both methods. The limits of detection were 0.4 and 0.2ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfully applied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The binding of chlorosulphonated paraffins to collagen triple helices is studied by means of classical molecular dynamics simulations and experimental spectroscopic techniques in order to disclose the principal characteristics of their interaction during the leather fattening process. Indeed, collagen is the main target to develop new leather modifying agents with specific characteristics, and an accurate design of the collagen binders, supported by predictive computational strategies, could be a successful tool to obtain new effective eco-compatible compounds able to impart to the leather the required functionalities and distinctive mechanical properties. Possible effects caused by the tanning agents on the collagen matrix have been identified from both experimental and theoretical points of view. Computational data in agreement with experiment have revealed that chlorosulphonated paraffins can interact favorably with the collagen residues having amine groups in their side chains (Arg, Lys, Asn and Gln) and reduce the tendency of the solvated collagen matrix to swell. However, the interference of chlorosulphonated paraffins with the unfolding process, which is operated mainly by the action of water, can be due both to covalent cross-linking of the collagen chains and intermolecular hydrogen bonding interactions involving also the hydroxyl groups of Hyp, Ser and Thr residues.

The simultaneous determination of lactate and pyruvate in sweat has been performed using reversed phase high-performance liquid chromatography (RP-HPLC) with UV detection at 220 nm. The calibration curves were linear in the investigated range 0.3 - 350 mm of lactate, 0.003- 1 mm of pyruvate. The sensitivity was good with a limit of detection of 0.03 mm for lactate and 0.001 mm for pyruvate. Recoveries evaluated for the entire procedure were 102 ± 0.1 and 96 ± 0.1 for lactate and pyruvate, respectively. The method was successfully applied to analysis of sweat in 8 athletes at rest (pilocarpine sweating) and during physical exercise., (Copyright © 2012 John Wiley & Sons, Ltd.)

In this work, we characterized paint reconstructions using ovalbumin and casein as binders, and cinnabar (HgS) as a pigment, before and after artificial ageing. Egg and casein are common paint binders that were used historically in the technique of tempera painting. Despite extensive research on the identification of proteinaceous binders in paintings, there is a substantial lack of knowledge regarding the ageing pathway of their protein content, and their chemical interaction with inorganic pigments. Thermogravimetric analysis, infrared spectroscopy and size-exclusion chromatography (SEC) were used to reveal the physico-chemical processes involved in the ageing of proteins in paintings. Taken together, the three techniques highlighted that proteins are subject to both cross-linking and hydrolysis upon ageing, and to a lesser extent, to oxidation of the side chains. Mercury-protein interactions were also revealed using a cold vapour generation atomic fluorescence spectrometer mercury-specific detector coupled to SEC. The study clearly showed that HgS forms stable complexes with proteins and acts as a sensitizer in cross-linking, hydrolysis and oxidation.

A dynamic surface tension detector (DSTD) has been equipped with an additional pressure sensor for simultaneous viscosity measurements, as a detector for flow injection analysis. The viscosity measurement is based on a single capillary viscometer (SCV) placed in parallel configuration with the DSTD. The viscometer in the optimized conditions consists of a PEEK capillary (i.d.=0.25 mm, L=75 cm) kept at constant temperature using a thermostatic bath, which leads on the two sides to the two arms of a differential piezoelectric pressure transducer with a range of 0-35 psi. The DSTD, described previously, measures the changing pressure across the liquid/air interface of 2 μL drops repeatedly forming at the end of a capillary. SCV performance has been evaluated by measuring dynamic viscosity of water/glycerol mixtures analysed in flow injection and comparing the results with the values reported in the literature. The detection limits of SCV and DSTD, calculated as 3σ of the blank, were 0.012 cP and 0.6 dyn cm(-1), respectively. The FI-SCV-DSTD system has been applied to the study of temperature-induced denaturation/aggregation process in bovine serum albumin (BSA). The results have been supported and discussed with respect to BSA conformational analysis performed using Fourier Transform infrared spectroscopy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The simultaneous application of microwave (MW) power and UV light leads to improved results in photochemical processes. This study investigates the oxidative decomposition of atrazine in water using an innovative MW and UV photochemical reactor, which activates a chemical reaction with MW and UV radiation using an immersed source without the need for a MW oven. We investigated the influence of reaction parameters such as initial H(2)O(2) concentrations, reaction temperatures and applied MW power and identified the optimal conditions for the oxidative decomposition of atrazine. Atrazine was completely degraded by MW/UV/H(2)O(2) in a very short time (i.e. t(1/2) = 1.1 min for 20.8 mg/L in optimal conditions). From the kinetic study, the disappearance rate of atrazine can be expressed as dX/dt = k(PH)[M](0)(b-X)(1-X), where b ≡ [H(2)O(2)](0)/[M](0)+k(OH)[·OH]/k(PH)[M](0), and X is the atrazine conversion, which correlates well with the experimental data. The kinetic analysis also showed that an indirect reaction of atrazine with an OH radical is dominant at low concentrations of H(2)O(2) and a direct reaction of atrazine with H(2)O(2) is dominant when the concentration of H(2)O(2) is more than 200 mg/L., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We developed a photochemical method for the online oxidation of p-hydroxymercurybenzoate (PHMB), an organic mercury species widely used for mercaptan and thiolic compound labeling. The method is based on a fully integrated online UV/microwave (MW) photochemical reactor for the digestion of PHMB, followed by cold vapor generation atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the quantitative conversion of PHMB and thiol-PHMB complexes to Hg(II), with a yield between 91% and 98%, without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. This reaction was followed by the reduction of Hg(II) to Hg(0), performed in a knitted reaction coil with NaBH(4) solution, and AFS detection in an Ar/H(2) miniaturized flame. The low MW power applied (18 W) allowed us to keep constant the temperature of the photochemical reactor (21 ± 1 °C), using a flowing water bath. This avoided peak widening due to diffusion processes generally occurring at high temperatures and in the additional cooling coil. This method has been applied to the determination of thiols in human plasma, blood, and wine.

Background: Patients on warfarin therapy undergo invasive and expensive checks for the coagulability of their blood. No information on coagulation levels is currently available between two controls., Methodology: A method was developed to determine warfarin in oral fluid by HPLC and fluorimetric detection. The chromatographic separation was performed at room temperature on a C-18 reversed-phase column, 65% PBS and 35% methanol mobile phase, flow rate 0.7 mL/min, injection volume 25 µL, excitation wavelength 310 nm, emission wavelength 400 nm., Findings: The method was free from interference and matrix effect, linear in the range 0.2-100 ng/mL, with a detection limit of 0.2 ng/mL. Its coefficient of variation was <3% for intra-day measurements and <5% for inter-day measurements. The average concentration of warfarin in the oral fluid of 50 patients was 2.5±1.6 ng/mL (range 0.8-7.6 ng/mL). Dosage was not correlated to INR (r = -0.03, p = 0.85) but positively correlated to warfarin concentration in the oral fluid (r = 0.39, p = 0.006). The correlation between warfarin concentration and pH in the oral fluid (r = 0.37, p = 0.009) confirmed the importance of pH in regulating the drug transfer from blood. A correlation between warfarin concentration in the oral fluid and INR was only found in samples with pH values ≥7.2 (r = 0.84, p = 0.004)., Conclusions: Warfarin diffuses from blood to oral fluid. The method allows to measure its concentration in this matrix and to analyze correlations with INR and other parameters.