Determining the selectivity of a tetra-phosphorylated biomimetic peptide towards uranium in the presence of competing cations through the simultaneous coupling of HILIC to ESI-MS and ICP-MS.

A cyclic tetra-phosphorylated biomimetic peptide (pS1368) has been proposed as a promising starting structure to design a decorporating agent of uranyl (UO22+) due to its affinity being similar to that of osteopontin (OPN), a target UO22+ protein in vivo. The determination of this peptide's selectivity towards UO22+ in the presence of competing endogenous elements is also crucial to validate this hypothesis. In this context, the selectivity of pS1368 towards UO22+ in the presence of Ca2+, Cu2+ and Zn2+ was determined by applying the simultaneous coupling of hydrophilic interaction chromatography (HILIC) to electrospray ionization (ESI-MS) and inductively coupled plasma (ICP-MS) mass spectrometry. Sr2+ was used as Ca2+ simulant, providing less challenging ICP-MS measurements. The separation of the complexes by HILIC was first set up. The selectivity of pS1368 towards UO22+ was determined in the presence of Sr2+, by adding several proportions of the latter to UO2(pS1368). UO22+ was not displaced from UO2(pS1368) even in the presence of a ten-fold excess of Sr2+. The same approach has been undertaken to demonstrate the selectivity of pS1368 towards UO22+ in the presence of Cu2+, Zn2+ and Sr2+ as competing endogenous cations. Hence, we showed that pS1368 was selective towards UO22+ in the presence of Sr2+, but also in the presence of Cu2+ and Zn2+. This study highlights the performance of HILIC-ESI-MS/ICP-MS simultaneous coupling to assess the potential of molecules as decorporating agents of UO22+.

Determination of the affinity of biomimetic peptides for uranium through the simultaneous coupling of HILIC to ESI-MS and ICP-MS.

Several proteins have been identified in the past decades as targets of uranyl (UO22+) in vivo. However, the molecular interactions responsible for this affinity are still poorly known which requires the identification of the UO22+ coordination sites in these proteins. Biomimetic peptides are efficient chemical tools to characterize these sites. In this work, we developed a dedicated analytical method to determine the affinity of biomimetic, synthetic, multi-phosphorylated peptides for UO22+ and evaluate the effect of several structural parameters of these peptides on this affinity at physiological pH. The analytical strategy was based on the implementation of the simultaneous coupling of hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). An essential step had been devoted to the definition of the best separation conditions of UO22+ complexes formed with di-phosphorylated peptide isomers and also with peptides of different structure and degrees of phosphorylation. We performed the first separations of several sets of UO22+ complexes by HILIC ever reported in the literature. A dedicated method had then been developed for identifying the separated peptide complexes online by ESI-MS and simultaneously quantifying them by ICP-MS, based on uranium quantification using external calibration. Thus, the affinity of the peptides for UO22+ was determined and made it possible to demonstrate that (i) the increasing number of phosphorylated residues (pSer) promotes the affinity of the peptides for UO22+, (ii) the position of the pSer in the peptide backbone has very low impact on this affinity (iii) and finally the cyclic structure of the peptide favors the UO22+ complexation in comparison with the linear structure. These results are in agreement with those previously obtained by spectroscopic techniques, which allowed to validate the method. Through this approach, we obtained essential information to better understand the mechanisms of toxicity of UO22+ at the molecular level and to further develop selective decorporating agents by chelation.

Separation of multiphosphorylated cyclopeptides and their positional isomers by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS).

Peptides are efficient models used in different fields such as toxicology to study the interactions of several contaminants at the molecular scale, requiring the development of bio-analytical strategies. In this context, Hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) was used to separate synthetic multiphosphorylated cyclopeptides and their positional isomers at physiological pH. We assessed (i) the selectivity of eleven HILIC columns, from different manufacturers and packed with diverse polar sorbents, and (ii) the effect of mobile phase composition on the separation selectivity. The best selectivity and baseline resolution were achieved with the columns grafted by neutral sorbents amide and diol. Furthermore, we investigated the HILIC retention mechanism of these peptides by examining the effect of the number of phosphorylated residues in the peptide scaffold on their retention. The peptide behavior followed the classical hydrophilic partitioning mechanism exclusively on amide and diol columns. This trend was not fully respected on bare and hybrid silica due to the attractive/repulsive interactions of the deprotonated surface silanol groups with the Arginine or Glutamate residues in the peptide scaffold according to the peptide sequence. The position of the phosphorylated amino acid in the peptide backbone also showed to have an impact on the retention, making possible the separation of positional isomers of these multiphosphorylated cyclic peptides using HILIC.

Hollow fibre supported liquid membrane extraction for BTEX metabolites analysis in human teeth as biomarkers.

The use of human teeth as biomarkers has been previously applied to characterize environmental exposure mainly to metal contamination. Difficulties arise when the contaminants are volatile or its concentration level is very low. This study presents the development of a methodology based on the transport through hollow fibre membrane liquid-phase microextraction (HF-LPME), followed by HPLC-UV measurement, to determine three different metabolites of BTEX contaminants, mandelic acid (MA), hyppuric acid (HA), and methylhippuric acid (4mHA). The driving force for the liquid membrane has been studied by using both non-facilitated (pH gradient 2-12) and facilitated transport (ionic and non-ionic carriers). Enrichment factors of several hundreds were accomplished. Different ionic and non-ionic water insoluble compounds were used as metabolite carriers for the facilitated transport at HF-LPME. Three organic solvents were used to constitute the liquid membrane, dodecane, dihexyl ether and n-decanol. Other parameters affecting the extraction process, such as extraction time, stirring speed, acceptor buffer and salt content were optimised in spiked solutions and selected those that presented the best enrichment factors for all analytes. Final conditions were established for donor solution as 20mL, pH2 of 0.5M NaCl, the OLM (Organic Liquid Membrane) as n-decanol and the acceptor solution as 40µL of 1M NaOH. The selected extraction time was 20h with stirring speed of 500rpm. Validation of the optimised method included the determination of individual linearity range (MA: 0.002-5.7µg; HA: 0.01-7.9µg; 4mHA 0.002-5.3µg), limits of detection (MA: 1.6ng; HA: 0.2ng; 4mHA 0.2ng), repeatability (RSD 7-10%) and reproducibility (5-8%). The developed method was applied to the analysis of MA, HA and 4mHA in teeth samples of 8 workers exposed to BTEX.

Simultaneous determination of BTEX and their metabolites using solid-phase microextraction followed by HPLC or GC/MS: An application in teeth as environmental biomarkers.

Applications of benzene, toluene, ethylbenzene, and o-, m-, and p-xylenes (BTEX) release them into the environment exposing living organism. These endocrine disruptors are toxic, highly volatile and easily absorbed by the lungs and can cause adverse consequences for the human health as neurological diseases and cancer. A method for the analysis of BTEX and its metabolites (phenols and aromatic acids) in teeth is presented. The method consists in a one-step simple extraction procedure from spiked tooth using NaOH solution followed by SPME-HPLC or HS-SPME-GC/MS determination. Optimization of both, spiking procedure and extraction step of these analytes from tooth, was carried out. Two fibers CAR/PDMS for BTEX and PA for BTEX metabolites were used for the SPME and variables were optimized for analytes at 30°C using spiked solutions. The optimized adsorption times were 30, 75 and 30min and desorption times were 10, 40 and 30min for BTEX, phenols and aromatic acids, respectively. Linearity for SPME-HPLC method was established using spiked solutions with both, BTEX and metabolites, at 2.5, 5.0, 10.0, 25.0µg/mL. The obtained results indicated a good linearity (r2 above 0.994) for all analytes. Triplicate analyses were performed with RSD lower than 15%. LODs were in the range 0.2-33.3ng/mL for SPME-HPLC and 0.06-0.09pg/mL for HS-SPME-GC/MS methods in spiking solutions. Once the method was optimized, bovine teeth were used as biological matrix model for the tuning of spiking and extraction steps. Optimal adsorption and desorption times were 4h for both procedures. Micrograms per tooth gram of BTEX and phenols were quantified in ten human teeth and aromatic acids were not identified. The developed method for BTEX and metabolites analyses using SPME-HPLC or HS-SPME-GC/MS shows good precision, linearity and sensitivity. The method was successfully applied in human teeth as environmental biomarker of BTEX and metabolites.

LC-ICP-MS analysis of arsenic compounds in dominant seaweeds from the Thermaikos Gulf (Northern Aegean Sea, Greece).

The content of total arsenic and arsenic compounds in the dominant seaweed species in the Thermaikos Gulf, Northern Aegean Sea was determined in samples collected in different seasons. Total arsenic was determined by acid digestion followed by ICP-MS. Arsenic speciation was analyzed by water extraction followed by LC-ICP-MS. Total arsenic concentrations in the seaweeds ranged from 1.39 to 55.0 mg kg(-1). Cystoseira species and Codium fragile showed the highest total As contents, while Ulva species (U. intestinalis, U. rigida,U. fasciata) had the lowest Arsenosugars, the most common arsenic species in seaweeds, were found in all samples, and glycerol-arsenosugar was the most common form; however, phosphate-arsenosugar and sulfate-arsenosugar were also present. Inorganic arsenic was measured in seven algae species and detected in another. Arsenate was the most abundant species in Cystoseira barbata (27.0 mg kg(-1)). Arsenobetaine was measured in only one sample. Methylated arsenic species were measured at very low concentrations. The information should contribute to further understanding the presence of arsenic compounds in dominant seaweeds from the Thermaikos Gulf.

Occurrence of arsenic species in algae and freshwater plants of an extreme arid region in northern Chile, the Loa River Basin.

This study reports data on arsenic speciation in two green algae species (Cladophora sp. and Chara sp.) and in five aquatic plants (Azolla sp., Myriophyllum aquaticum, Phylloscirpus cf. desserticola, Potamogeton pectinatus, Ruppia filifolia and Zannichellia palustris) from the Loa River Basin in the Atacama Desert (northern Chile). Arsenic content was measured by Mass spectrometry coupled with Inductively Coupled Plasma (ICP-MS), after acidic digestion. Liquid chromatography coupled to ICP-MS was used for arsenic speciation, using both anionic and cationic chromatographic exchange systems. Inorganic arsenic compounds were the main arsenic species measured in all samples. The main arsenic species in the extracts of freshwater algae and plants were arsenite and arsenate, whereas glycerol-arsenosugar (gly-sug), dimethylarsinic acid (DMA) and methylarsonic acid (MA) were present only as minor constituents. Of the samples studied, algae species accumulated more arsenic than aquatic plants. Total arsenic content ranged from 182 to 11100 and from 20 to 248 mg As kg(-1) (d.w.) in algae and freshwater plants, respectively. In comparison with As concentration in water samples, there was hyper-accumulation (>0.1% d.w.) in Cladophora sp.