Development and implementation of an analytical procedure for the quantification of natural and synthetic steroid hormones in whole surface waters.

Natural and synthetic steroid hormones are chronically released into aquatic spheres. Whereas knowledge on their combined mode of action and the cocktail effect are needed, only few multi-class methods address the challenge of their trace quantification in surface waters. The current study describes a sensitive multi-residue analytical strategy aiming to quantify 23 steroid hormones belonging to androgens, estrogens, glucocorticoids and progestogens in whole surface waters. The procedure relies on a two-step solid-phase extraction followed by an ultra-performance liquid chromatography separation coupled to tandem mass spectrometry detection (UPLC-MS/MS). Isotope dilution was implemented when possible in order to ensure the reliability of the measurement. The procedure was optimized toward the reliable quantification of the 23 target compounds at the predicted no-effect concentrations when existing or below the ng L-1 level. Satisfactory absolute global recoveries ≥ 77% were obtained for almost all compounds (21 out of 23) in intermediate precision conditions. Measurement errors were comprised between -27% and +17% for the great majority of compounds (21 out of 23) with standard deviations < 20% in intermediate precision conditions. Despite signal suppression was observed in water samples, satisfactory limits of quantification were achieved, ranging from 0.035 ng L-1 for 17alpha-ethinylestradiol to 1 ng L-1 for 6beta-hydroxycortisol and 6beta-hydroxydexamethasone. Abiotic stability was demonstrated for the great majority of target compounds (22 out of 23) in reference water samples stored at 4 ± 3 °C during 48 h, driving our sampling strategy. To demonstrate its fitness for purpose, the procedure was implemented in a preliminary monitoring survey of Belgian surface waters. As a result, 6 out of 23 target compounds were detected or quantified, showing a contamination by some estrogens and glucocorticoids at levels ranging from 0.1 to 0.9 ng L-1.

Is quantitative sensory testing helpful in the management of oxaliplatin neuropathy? a two-year clinical study.

PURPOSE: To better understand how quantitative sensory testing could help the clinician in the management of oxaliplatin-induced peripheral neuropathy in terms of earlier and more reliable detection, we conducted a two-year prospective study. METHODS: Thermal sensory assessment, tactile sensory assessment, neuropathic pain assessment and adverse events gradation (NCI-CTC) were performed during treatment and 6 months after treatment completion. RESULTS: 35 patients were enrolled and followed-up during one year. Cold and Warm Detection Thresholds were higher 6 months after treatment completion than at enrollment. Mechanical detection thresholds didn't change significantly. Neurotoxicity was mostly grade-1, only 18% grade-2 and no grade-3. Grade-2 patients received lower oxaliplatin cumulative dose than grade-1, which reveals effective dose adaptation and grade-2 patients were more likely to develop painful neuropathy. CONCLUSION: Thermal thresholds impairment emerges too late to help the clinician in the prophylaxis of neuropathy. Management of OXA-treatment based on NCI-CTC, as currently recommended, remains the best way to detect neuropathy and ensure treatment adaptation.

IFPA meeting 2014 workshop report: Animal models to study pregnancy pathologies; new approaches to study human placental exposure to xenobiotics; biomarkers of pregnancy pathologies; placental genetics and epigenetics; the placenta and stillbirth and fetal growth restriction.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction.

Ultra high performance liquid chromatography-quadrupole-time of flight analysis for the identification and the determination of resveratrol and its metabolites in mouse plasma.

Resveratrol is a polyphenol that has numerous interesting biological properties, but, per os, it is quickly metabolized. Some of its metabolites are more concentrated than resveratrol, may have greater biological activities, and may act as a kind of store for resveratrol. Thus, to understand the biological impact of resveratrol on a physiological system, it is crucial to simultaneously analyze resveratrol and its metabolites in plasma. This study presents an analytical method based on UHPLC-Q-TOF mass spectrometry for the quantification of resveratrol and of its most common hydrophilic metabolites. The use of (13)C- and D-labeled standards specific to each molecule led to a linear calibration curve on a larger concentration range than described previously. The use of high resolution mass spectrometry in the full scan mode enabled simultaneous identification and quantification of some hydrophilic metabolites not previously described in mice. In addition, UHPLC separation, allowing run times lower than 10 min, can be used in studies that requiring analysis of many samples.

Simultaneous quantitation of histamine and its major metabolite 1-methylhistamine in brain dialysates by using precolumn derivatization prior to HILIC-MS/MS analysis.

In nonclinical drug development targeting the central nervous system (CNS), the quantitative determination of extracellular brain concentrations of neurotransmitters is a key challenge. In some CNS disorders, the monitoring of the modified profile of neurotransmitter release such as that of histamine may explain the mechanism of action of the drug candidate. Microdialysis is a commonly used method for sampling extracellular levels of neurotransmitters/drug candidates in small laboratory animals. Detection and quantification of extracellular levels of neurotransmitters remain an analytical and technical challenge owing to the low concentrations of neurotransmitters collected, the small microdialysis sample size, and the high amount of inorganic salts. A precolumn derivatization strategy prior to hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry analysis is proposed to quantify histamine release after administration of a CNS research compound. Derivatization using propionic anhydride dissolved in organic solvent combined with the HILIC approach effectively eliminated three time-consuming steps, organic layer transfer, dry down, and reconstitution, all of which are required by traditional reversed-phase liquid chromatography. The formation of propionylated amides, performed under mild conditions, required no further sample cleanup. After a dual microdialysis probe implantation into the prefrontal cortex (neurotransmitters) and in the inferior vena cava of rat (drug candidate), microdialysate fractions were collected every 15 min for 8 h and stored frozen at -20 °C until analysis. The method was validated using 10 µL microdialysate, achieving low limits of quantitation of 83.4 and 84.5 pg.mL(-1) for histamine and 1-methylhistamine, respectively. These limits were suitable to assess kinetic release of neurotransmitters and are compatible with those obtained by microdialysis sampling. This method provided the required selectivity, sensitivity, accuracy, and precision to assess release kinetics of histamine and 1-methylhistamine in several hundred rat brain microdialysates after intravenous infusion of CNS drug candidates.

Hyperphosphataemia sensitizes renally impaired rats to the profibrotic effects of gadodiamide.

BACKGROUND AND PURPOSE: Hyperphosphataemia is common in patients with nephrogenic systemic fibrosis (NSF). NSF has been linked to administration of gadolinium (Gd) chelates (GCs) and elevated serum phosphate levels accelerate the release of Gd from linear, non-ionic GCs but not macrocyclic GCs. Hence, we determined whether hyperphosphataemia is a cofactor or risk factor for NSF by investigating the role of hyperphosphataemia in renally impaired rats. EXPERIMENTAL APPROACH: Firstly, the clinical, pathological and bioanalytical consequences of hyperphosphataemia were investigated in subtotal nephrectomized (SNx) Wistar rats following i.v. administration of the non-ionic, linear GC gadodiamide (5 × 2.5 mmol·kg(-1) ·day(-1) ). Secondly, the effects of several GCs were compared in these high-phosphate diet fed rats. Total Gd concentration in skin, femur and plasma was measured by inductively coupled plasma mass spectrometry (ICP-MS) and free Gd(3+) in plasma by liquid chromatography coupled to ICP-MS. Relaxometry was used to measure dissociated Gd in skin and bone. KEY RESULTS: Four out of seven SNx rats fed a high-phosphate diet administered gadodiamide developed macroscopic and microscopic (fibrotic and inflammatory) skin lesions, whereas no skin lesions were observed in SNx rats treated with saline, the other GCs and free ligands or in the normal diet, gadodiamide-treated group. Unlike the other molecules, gadodiamide gradually increased the r(1) relaxivity value, consistent with its in vivo dissociation and release of soluble Gd. CONCLUSIONS AND IMPLICATIONS: Hyperphosphataemia sensitizes renally impaired rats to the profibrotic effects of gadodiamide. Unlike the other GCs investigated, gadodiamide gradually dissociates in vivo. Our data confirm that hyperphosphataemia is a risk factor for NSF.

Kaurenoic acid from pulp of Annona cherimolia in regard to Annonaceae-induced Parkinsonism.

Guadeloupean Parkinsonism has been linked epidemiologically to the consumption of Annonaceae fruits. These were proposed to be etiological agents for sporadic atypical Parkinsonism worldwide, because of their content of neurotoxins such as isoquinolinic alkaloids and Annonaceous acetogenins. The pulp of Annona cherimolia Mill. from Spain was screened for these toxic molecules using Matrix-Assisted Laser Desorption Ionisation - Time of Flight mass spectrometry (MALDI-TOF MS) and it was found not to be a source of exposure. However, kaurenoic acid, a diterpene considered to be cytotoxic, was detected in high amounts (66 mg/fresh fruit). Treatment of rat embryonic striatal primary cultures, up to a high concentration (50 µM), did not cause neuronal death nor astrogliosis, suggesting that this molecule is not at risk of implication in human neurodegenerative diseases.

A layered structure in the organic envelopes of the prismatic layer of the shell of the pearl oyster Pinctada margaritifera (Mollusca, Bivalvia).

The organic interprismatic layers of the mollusc Pinctada margaritifera are studied using a variety of highly spatially-resolved techniques to establish their composition and structure. Our results show that both the interlamellar sheets of the nacre and interprismatic envelopes form layered structures. Additionally, these organic layers are neither homogeneous in composition, nor continuous in their structure. Both structures play a major role in the biomineralization process and act as a boundary between mineral units.

Chemical characterization of Titan's tholins: solubility, morphology and molecular structure revisited.

In this work Titan's atmospheric chemistry is simulated using a capacitively coupled plasma radio frequency discharge in a N(2)-CH(4) stationnary flux. Samples of Titan's tholins are produced in gaseous mixtures containing either 2 or 10% methane before the plasma discharge, covering the methane concentration range measured in Titan's atmosphere. We study their solubility and associated morphology, their infrared spectroscopy signature and the mass distribution of the soluble fraction by mass spectrometry. An important result is to highlight that the previous Titan's tholin solubility studies are inappropriate to fully characterize such a heterogeneous organic matter and we develop a new protocol to evaluate quantitatively tholins solubility. We find that tholins contain up to 35% in mass of molecules soluble in methanol, attached to a hardly insoluble fraction. Methanol is then chosen as a discriminating solvent to characterize the differences between soluble and insoluble species constituting the bulk tholins. No significant morphological change of shape or surface feature is derived from scanning electron microscopy after the extraction of the soluble fraction. This observation suggests a solid structure despite an important porosity of the grains. Infrared spectroscopy is recorded for both fractions. The IR spectra of the bulk, soluble, and insoluble tholins fractions are found to be very similar and reveal identical chemical signatures of nitrogen bearing functions and aliphatic groups. This result confirms that the chemical information collected when analyzing only the soluble fraction provides a valuable insight representative of the bulk material. The soluble fraction is ionized with an atmospheric pressure photoionization source and analyzed by a hybrid mass spectrometer. The congested mass spectra with one peak at every mass unit between 50 and 800 u confirm that the soluble fraction contains a complex mixture of organic molecules. The broad distribution, however, exhibits a regular pattern of mass clusters. Tandem collision induced dissociation analysis is performed in the negative ion mode to retrieve structural information. It reveals that (i) the molecules are ended by methyl, amine and cyanide groups, (ii) a 27 u neutral moiety (most probably HCN) is often released in the fragmentation of tholin anions, and (iii) an ubiquitous ionic fragment at m/z 66 is found in all tandem spectra. A tentative structure is proposed for this negative ion.

Atmospheric pressure photoionization mass spectrometry of oligodeoxyribonucleotides.

Small oligonucleotides (di- and trimers) were investigated by atmospheric pressure photoionization (APPI) with focus on the fragmentation mechanisms. The fragmentation patterns of these biomolecular ions have been monitored under dopant-assisted photoionization (DA-APPI) conditions. Our results reveal new aspects of the gas- phase chemistry of ions formed from such biomolecules. They illustrate that the reaction between low-energy electrons released from photoionization processes and di- and trinucleotides lies in dissociative electron attachment processes leading to phosphodiester bond cleavages and to the formation of numerous fragments in the ion source. The conditions of DA-APPI, which involve protic solvents and atmospheric pressure conditions, seem to be relevant for the study of radiation damages to biological molecules.

[Imaging mass spectrometry: a new tool for the analysis of skin biopsy. Application in Fabry's disease]. / Imagerie par spectrométrie de masse: un nouvel outil pour l'analyse de biopsies cutanées. Application à la maladie de Fabry.

The advent of innovative techniques in mass spectrometry, especially in the area of imaging, prompted us to evaluate two promising techniques: secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. For this purpose, sections of cutaneous biopsies from patients affected by Fabry's disease and control patients were analyzed. In the course of this disease, two physiological glycosphingolipids [globotriasylceramide (Gb3) and the galabiosylceramide (Ga2)] accumulate in certain tissues owing to a catabolism failure. The ability of these techniques to localize sites of accumulation in body tissues and their capacity to identify the accumulated lipid structures by mass spectra were evaluated. Results demonstrated that these two techniques provide complementary information:-secondary ion mass spectrometry enabled precise localization of areas of accumulation with lateral resolution in the micrometer range;-the signal obtained with matrix-assisted laser desorption/ionization mass spectrometry was high enough to identify these structures according to their molecular weight.

Fragmentation induced in atmospheric pressure photoionization of peptides.

In this work, the fragmentation of peptides under atmospheric pressure photoionization conditions is investigated. Intensive fragmentations into b/y- and c-sequence ions are reported. Abundance of these c-ions appeared to be related to the quantity of dopant infused and to the disappearance of the doubly protonated peptide ion. A careful analysis of the role of the dopant indicates that the fragmentations are not dependent on the nature of the dopant but on their ionization efficiencies. This result shows that the fragmentation arises from the reaction of the protonated peptide with photoelectrons released upon ionization of the dopant in an electron capture dissociation/electron transfer dissociation (ECD/ETD) type mechanism. Experiments with peptides bearing a single proton indicate that additional mechanisms are involved. H-atom transfer reactions are suggested to be responsible for the fragmentations as well. Those atoms could arise either from the dopant ions or from negatively charged solvent nanodroplets. This is the first report of an ECD/ETD mechanism in a dense medium and at atmospheric pressure.

Study of a bisquaternary ammonium salt by atmospheric pressure photoionization mass spectrometry.

A comprehensive atmospheric pressure photoionization (APPI) mass spectrometry investigation of hexamethonium bromide is reported. This bisquaternary ammonium salt is a model system for the investigation of multiply charged species and elucidation of ion formation processes. It has been used to elucidate the physicochemical phenomenon occurring when photoionization is carried out at atmospheric pressure. First, the in-source fragmentations were studied for aqueous solutions of the salt with the photoionization lamp switched off, i.e. under thermospray conditions. It is shown that, in this mode of operation, fragmentations are minor and may be classified into two classes, namely dequaternization and charge separation, arising from the two precursors, M2+ and [M+Br]+. Second, the fragmentation patterns have been monitored in dopant- assisted APPI for different dopants (toluene, toluene-d8, anisole and hexafluorobenzene) at various amounts. At low dopant flow rates, the [M+Br]+ and M2+ ions are still observed. As the flow rate is increased, these precursor ions lose intensity and are finally suppressed for all three dopants. Comparison of toluene and toluene-d8 reveals that H atoms may be transferred from the dopant to the molecular ions, very likely mediated by the solvent. The role of the solvent (water) was also investigated by using heavy water. Apart from the thermospray fragmentations, which are also observed in APPI, several fragmentation pathways appear to be specific to the photoionization process. Photoionization efficiencies are measured by determination of the relative photoionization cross sections with respect to toluene. It is found that, when the ionization efficiencies are taken into account, the depletion of the precursors as a function of the dopant flow rates is the same for all three dopant molecules. This result shows that the precursor ions are depleted by reactions with the photoelectrons released from the dopant. Three additional mechanisms are proposed to account for this effect: electron transfer or H atom transfer from negatively charged water nanodroplets and H atom transfer from the dopant.

Liquid chromatography on porous graphitic carbon with atmospheric pressure photoionization mass spectrometry and tandem mass spectrometry for the analysis of glycosphingolipids.

The study of several structural variations (the length, the degree of unsaturation and hydroxylation of the alkyl chains, the number and nature of osidic residues) helped understand the behaviour of neutral glycosphingolipids (GSLs) on porous graphitic carbon stationary phase (PGC). Atmospheric pressure photoionization mass spectrometry (APPI) and tandem mass spectrometry were used to perform the detection and the identification of molecular species in positive mode where [M+H](+) and [M-H(2)O+H](+) ions provided structural information on the fatty acid and the sphingoid base. The retention of GSLs increased with the hydrocarboneous volume of their alkyl chains and with the number of osidic residues in agreement with hydrophobic properties and polar retention effect of graphite, respectively. The presence of polar groups, such as OH-group or double bond within alkyl chains, decreased their retention. The coupling of chromatography on PGC with APPI tandem mass spectrometry detection appeared a powerful technique to discriminate isobaric molecules. Isobaric solutes differing by the position of two double bonds or by the repartition of hydrocarboneous skeleton were discriminated according to their chromatographic comportment or their mass spectrum, respectively. Among isobaric molecules, only few structures differing by the nature of osidic residue were not discriminated (i.e. glucosylceramide and galactosylceramide with similar ceramide skeleton were co-eluted and no difference in mass spectra was observed).

N-Acyl derivatives of Asn, new bacterial N-acyl D-amino acids with surfactant activity.

New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.

Sesquiterpenes and alkaloids from Scorodocarpus borneensis.

A new sesquiterpene, scodopin, and a mixture of three tryptamine-type alkaloids, scorodocarpines A-C, were isolated from the fruits of Scorodocarpus borneensis, together with a known hemisynthetic sesquiterpene, cadalene-beta-carboxylic acid, which was isolated from the bark. The structures of the new compounds were elucidated by interpretation of spectral data, especially tandem mass spectrometry for the alkaloid mixture.

Trypanocidal withanolides and withanolide glycosides from Dunalia brachyacantha.

Two new withanolide glycosides, (20R,22R)-O-(3)-alpha-L-rhamnopyranosyl(1-->4)-beta-D-glucopyranosyl-1 alpha,12 beta-diacetoxy-20-hydroxywitha-5,24-dienolide (3) and (20R,22R)-O-(3)-beta-D-xylopyranosyl(1-->3)-[beta-D-xylopyranosyl(1-->4)]-beta-D-glucopyranosyl-1 alpha-acetoxy-12 beta,20-dihydroxywitha-5,24-dienolide (4), were isolated from the leaves and root of Dunalia brachyacantha. Their aglycones, (20R,22R)-1 alpha,12 beta-diacetoxy-3 beta,20-dihydroxywitha-5,24-dienolide (or 1 alpha,12 beta-diacetyldunawithagenine) and (20R,22R)-1 alpha-acetoxy-3 beta,12 beta,20-trihydroxywitha-5,24-dienolide (or 1 alpha-acetyl-12 beta-hydroxydunawithagenine), are novel. The known 18-acetoxywithanolide D (1) and 18-acetoxy-5,6-deoxy-5-withenolide D (2) were also isolated from the leaves. These last two compounds were shown to be responsible for the trypanocidal, leishmanicidal, and bactericidal activities manifested by the crude ethanolic extract. The structures were deduced from spectroscopic data and on the basis of chemical evidence.

Stabilization of gas-phase noncovalent macromolecular complexes in electrospray mass spectrometry using aqueous triethylammonium bicarbonate buffer.

The use of triethylammonium bicarbonate (TEAB) solution in electrospray mass spectrometry proved to be a very efficient way for studying proteins or noncovalent protein complexes under "nondenaturing" conditions. The low charge states observed in the mass spectra improve the separation of ions arising from macromolecular species of close masses. Moreover, the multiply charged ions generated in a TEAB solution are significantly more stable than those formed under more conventional conditions (for example, with ammonium bicarbonate or acetate solution). The analytical interest of TEAB for the analysis of macromolecular species that can easily dissociate in the gas phase, such as hemoglobin or other macromolecular noncovalent complexes, is demonstrated.

Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid.

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

Structural investigation of cyclic peptidolipids from Bacillus subtilis by high-energy tandem mass spectrometry.

The natural products belonging to the surfactin family are cycloheptapeptides bearing a long beta-hydroxy-fatty acyl chain at the N-terminal position. The structure of these compounds, often isolated as complex mixtures, can be elucidated by high-energy tandem mass spectrometry (MS/MS). The protonated molecules generated by cesium ion bombardment (LSIMS) undergo charge-proximate fragmentations leading to the b- and y-type ion series useful for the sequence determination. The sodium-cationised molecules show a radically different behaviour towards high-energy collisional activation. Besides the well-known charge-remote dissociation products of the alkyl side chain, complete series of d- and w-type fragments allow easy distinction between leucine and isoleucine. The complementary MS/MS data obtained from the protonated and cationised molecules prove to be of great interest for the structural characterisation of this type of compounds.