Early detection of ureteropelvic junction obstruction in neonates with prenatal diagnosis of renal pelvis dilatation using 1H NMR urinary metabolomics.

Renal pelvis dilatation (RPD) is diagnosed in utero on prenatal ultrasonography (US) and can resolve spontaneously. However, isolated RPD can also reflect ureteropelvic junction obstruction (UPJO), which requires surgical treatment to prevent progressive renal deterioration. The diagnosis of UPJO can only be confirmed after birth with repeat US and renal isotope studies. 1H Nuclear Magnetic Resonance spectroscopy (NMR) was performed on urine of newborns with prenatally diagnosed unilateral RPD and healthy controls to identify specific urinary biomarkers for UPJO. The original combination of EigenMS normalization and sparse partial-least-squares discriminant analysis improved selectivity and sensitivity. In total, 140 urine samples from newborns were processed and 100 metabolites were identified. Correlation network identified discriminant metabolites in lower concentrations in UPJO patients. Two main metabolic pathways appeared to be impaired in patients with UPJO i.e. amino acid and betaine metabolism. In this prospective study, metabolic profiling of urine samples by NMR clearly distinguishes patients who required surgery for UPJO from patients with transient dilatations and controls. This study will pave the way for the use of metabolomics for the diagnosis of prenatal hydronephrosis in clinical routine.

Determination of enantiomeric and stable isotope ratio fingerprints of active secondary metabolites in neroli (Citrus aurantium L.) essential oils for authentication by multidimensional gas chromatography and GC-C/P-IRMS.

Neroli essential oil (EO), extracted from bitter orange blossoms, is one of the most expensive natural products on the market due to its poor yield and its use in fragrance compositions, such as cologne. Multiple adulterations of neroli EO are found on the market, and several authentication strategies, such as enantioselective gas chromatography (GC) and isotope ratio mass spectrometry (IRMS), have been developed in the last few years. However, neroli EO adulteration is becoming increasingly sophisticated, and analytical improvements are needed to increase precision. Enantiomeric and compound-specific isotopic profiling of numerous metabolites using multidimensional GC and GC-C/P-IRMS was carried out. These analyses proved to be efficient for geographical tracing, especially to distinguish neroli EO of Egyptian origin. In addition, δ2H values and enantioselective ratios can identify an addition of 10% of petitgrain EO. These results demonstrate that enantioselective and stable isotopic metabolite fingerprint determination is currently a necessity to control EOs.

Synthesis, molecular properties, anti-inflammatory and anticancer activities of novel 3-hydroxyflavone derivatives.

A new series of 3-hydroxyflavones (1-46) were synthesized according to the Claisen-Schmidt followed by Algar-Flynn-Oyamada reactions (AFO) in one step. The synthesized flavonoids were characterized by 1H NMR, 13C NMR and DCI-HRMS. All the synthesized compounds were tested in vitro for their 15-lipoxygenase inhibitory and cytotoxic activity against the human cell lines HCT-116 (Human colon carcinoma), IGROV-1 and OVCAR-3 (human ovarian carcinoma). It has been found that the derivatives 25, 37 and 45 were the most actives against HCT-116 (IC50 = 8.0, 9.0 and 9.0 µM, respectively) and against IGROV-1 (IC50 = 2.4, 5.0 and 6.0 µM, respectively). The derivatives 14 and 21 exhibited the higher anti-inflammatory activity at 100 µM with PI values of 76.50 and 72.70%, respectively. Molecule description was performed with DFT calculations, the drug likeness and bioactivity scores. The results exhibted that some compounds are in linear correlation with Lipinski's rule of five showing good drug likeness and bioactivity score for drug targets.

Antibacterial Properties of Polyphenols: Characterization and QSAR (Quantitative Structure-Activity Relationship) Models.

Besides their established antioxidant activity, many phenolic compounds may exhibit significant antibacterial activity. Here, the effect of a large dataset of 35 polyphenols on the growth of 6 foodborne pathogenic or food-spoiling bacterial strains, three Gram-positive ones (Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes) and three Gram-negative ones (Escherichia coli, Pseudomonas aeruginosa, and Salmonella Enteritidis), have been characterized. As expected, the effects of phenolic compounds were highly heterogeneous ranging from bacterial growth stimulation to antibacterial activity and depended on bacterial strains. The effect on bacterial growth of each of the polyphenols was expressed as relative Bacterial Load Difference (BLD) between a culture with and without (control) polyphenols at a 1 g L-1 concentration after 24 h incubation at 37°C. Reliable Quantitative Structure-Activity Relationship (QSAR) models were developed (regardless of polyphenol class or the mechanism of action involved) to predict BLD for E. coli, S. Enteritidis, S. aureus, and B. subtilis, unlike for L. monocytogenes and P. aeruginosa. L. monocytogenes was generally sensitive to polyphenols whereas P. aeruginosa was not. No satisfactory models predicting the BLD of P. aeruginosa and L. monocytogenes were obtained due to their specific and quite constant behavior toward polyphenols. The main descriptors involved in reliable QSAR models were the lipophilicity and the electronic and charge properties of the polyphenols. The models developed for the two Gram-negative bacteria (E. coli, S. Enteritidis) were comparable suggesting similar mechanisms of toxic action. This was not clearly observed for the two Gram-positive bacteria (S. aureus and B. subtilis). Interestingly, a preliminary evaluation by Microbial Adhesion To Solvents (MATS) measurements of surface properties of the two Gram-negative bacteria for which QSAR models were based on similar physico-chemical descriptors, revealed that MATS results were also quite similar. Moreover, the MATS results of the two Gram-positive bacterial strains S. aureus and B. subtilis for which QSARs were not based on similar physico-chemical descriptors also strongly differed. These observations suggest that the antibacterial activity of most of polyphenols likely depends on interactions between polyphenols and bacterial cells surface, although the surface properties of the bacterial strains should be further investigated with other techniques than MATS.

Raman spectroscopy combined with advanced chemometric methods: A new approach for detergent deformulation.

Deformulation of a commercial surfactant mixture using Raman spectroscopy and advanced chemometric tools have been investigated. Since the use of surfactants is drastically expanding, their fine identification and quantification are required for quality control and regulation. Dilution of the detergent mixtures combined with Raman spectroscopy for signal extraction tools allowed the extraction of the first information concerning the composition of the mixture. The raw materials identified were thus used in an experimental design to obtain a robust model for the determination of detergent composition. The combination of chemometric tools (independent component analysis and Partial Least Square) and spectroscopic methods provided pertinent information for detergent composition. This methodology can easily be transposed to the industrial world.

Heavy Metal Pollution of Chari River Water during the Crossing of N'Djamena (Chad).

This study was carried out to identify and assess the water quality of the Chari River. The Chari, 1200 km long, is Chad's major water source. Municipal sewage, industrial wastewater discharge, and seasonal run-off from agriculture are regularly fed into the river. Several trace metals such as Cu, Zn, Fe, Ni, Cr, Mn, and Cd, were measured in different sampling stations located along the Chari River at N'Djamena in different campaigns from 2008 to 2010. Overall, manganese, zinc, chromium, and copper concentration levels were mainly in the range of the permissible limits prescribed by WHO guidelines (WHO 2011). Nickel, iron, and cadmium concentrations were still high. This preliminary study allowed us to identify the magnitude of toxic pollutants, which are responsible for Chari River water contamination in the study area. This study revealed that urgent measures must be taken to protect the local people from health problems resulting from high concentrations of heavy metals.

Pectin gelation with chlorhexidine: Physico-chemical studies in dilute solutions.

Low methoxyl pectin is known to gel with divalent cations (e.g. Ca(2+), Zn(2+)). In this study, a new way of pectin gelation in the presence of an active pharmaceutical ingredient, chlorhexidine (CX), was highlighted. Thus chlorhexidine interactions with pectin were investigated and compared with the well-known pectin/Ca(2+) binding model. Gelation mechanisms were studied by several physico-chemical methods such as zeta potential, viscosity, size measurements and binding isotherm was determined by Proton Nuclear Magnetic Resonance Spectroscopy ((1)H NMR). The binding process exhibited similar first two steps for both divalent ions: a stoichiometric monocomplexation of the polymer followed by a dimerization step. However, stronger interactions were observed between pectin and chlorhexidine. Moreover, the dimerization step occurred under stoichiometric conditions with chlorhexidine whereas non-stoichiometric conditions were involved with calcium ions. In the case of chlorhexidine, an additional intermolecular binding occurred in a third step.

Determination of surfactant bio-sourced origin by isotope-ratio mass spectrometry.

RATIONALE: To develop more eco-friendly laundry detergents, renewable surfactants synthesized from vegetal sources are increasingly being used. In a more stringent regulation context, the determination of bio-sourced surfactant origin thus appears essential to assess the claims of detergent manufacturers. Radiocarbon determination, the standard method for the analysis of bio-sourced materials, is an expensive technique, so there is a need for a cheaper method. METHODS: Here, the use of an elemental analyzer linked to isotope-ratio mass spectrometry (EA/IRMS) is evaluated as an alternative approach to the official method. The δ(18) O, δ(13) C and δ(2) H isotope-ratio values were determined to investigate the bio-sourced origin of surfactant raw materials and mixtures. RESULTS: A sample library of 26 commercial surfactants representative of detergent raw materials was first analyzed by EA/IRMS. The δ(18) O, δ(13) C and δ(2) H values allowed discrimination of synthetic and bio-sourced surfactants. Moreover, in this latter group, C4 plant-derived surfactants were distinguished by their δ(13) C values. Binary and ternary mixtures made of synthetic and bio-sourced surfactants were also analyzed and indicated a linear relationship between mixture isotope-ratio values and surfactant proportions. CONCLUSIONS: IRMS represents a viable alternative to radiocarbon determination for the evaluation of surfactant bio-sourced origin. It is a faster and cheaper technique, allowing discrimination of petroleum- and biomass-derived surfactants and identification of their carbon sources (C4 or C3 plants).

Characterization of surfactant complex mixtures using Raman spectroscopy and signal extraction methods: Application to laundry detergent deformulation.

This paper presents the analysis of surfactants in complex mixtures using Raman spectroscopy combined with signal extraction (SE) methods. Surfactants are the most important component in laundry detergents. Both their identification and quantification are required for quality control and regulation purposes. Several synthetic mixtures of four surfactants contained in an Ecolabel laundry detergent were prepared and analyzed by Raman spectroscopy. SE methods, Independent Component Analysis and Multivariate Curve Resolution, were then applied to spectral data for surfactant identification and quantification. The influence of several pre-processing treatments (normalization, baseline correction, scatter correction and smoothing) on SE performances were evaluated by experimental design. By using optimal pre-processing strategy, SE methods allowed satisfactorily both identifying and quantifying the four surfactants. When applied to the pre-processed Raman spectrum of the Ecolabel laundry detergent sample, SE models remained robust enough to predict the surfactant concentrations with sufficient precision for deformulation purpose. Comparatively, a supervised modeling technique (PLS regression) was very efficient to quantify the four surfactants in synthetic mixtures but appeared less effective than SE methods when applied to the Raman spectrum of the detergent sample. PLS seemed too sensitive to the other components contained in the laundry detergent while SE methods were more robust. The results obtained demonstrated the interest of SE methods in the context of deformulation.

Use of designed experiments for the improvement of pre-analytical workflow for the quantification of intracellular nucleotides in cultured cell lines.

The present study is focused on the development of a pre-analytical strategy for the quantification of intracellular nucleotides from cultured cell lines. Different protocols, including cell recovery, nucleotide extraction and purification, were compared on a panel of nucleoside mono-, di- and triphosphates from four cell lines (adherent and suspension cells). The quantification of nucleotides was performed using a validated technique with on-line solid-phase extraction coupled with liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS). Designed experiments were implemented to investigate, in a rigorous and limited-testing experimental approach, the influence of several operating parameters. Results showed that the technique used to harvest adherent cells drastically affected the amounts of intracellular nucleotides. Scraping cells was deleterious because of a major leakage (more than 70%) of intracellular nucleotides during scraping. Moreover, some other tested conditions should be avoided, such as using pure methanol as extraction solvent (decrease over 50% of intracellular nucleotides extracted from NCI-H292 cells) or adding a purification step with chloroform. Designed experiments allowed identifying an interaction between the percentage of methanol and the presence of chloroform. The mixture methanol/water (70/30, v/v) was considered as the best compromise according to the nucleoside mono-, di-, or triphosphates and the four cell lines studied. This work highlights the importance of pre-analytical step combined with the cell lines studied associated to sensitive and validated assay for the quantification of nucleotides in biological matrices.

Data-handling strategies for metabonomic studies: example of the UHPLC-ESI/ToF urinary signature of tetrahydrocannabinol in humans.

Metabonomics has become a very valuable tool and many research fields rely on results coming out from this combination of analytical techniques, chemometric strategies, and biological interpretation. Moreover, the matrices are more and more complex and the implications of the results are often of major importance. In this context, the need for pertinent validation strategies comes naturally. The choice of the appropriate chemometric method remains nevertheless a difficult task due to particularities such as: the number of measured variables, the complexity of the matrix and the purposes of the study. Consequently, this paper presents a detailed metabonomic study on human urine with a special emphasis on the importance of assessing the data's quality. It also describes, step by step, the statistical tools currently used and offers a critical view on some of their limits. In this work, 29 urine samples among which 15 samples obtained from tetrahydrocannabinol (delta-9-tetrahydrocannabinol)-consuming athletes, 5 samples provided by volunteers, and 9 samples obtained from athletes were submitted to untargeted analysis by means of ultra high-pressure liquid chromatography-electrospray ionization-time-of-flight mass spectrometry. Next, the quality of the obtained data was assessed and the results were compared to those found in databases. Then, unsupervised (principal component analysis (PCA)) and supervised (ANOVA/PCA, partial least-square-discriminant analysis (PLS-DA), orthogonal PLS-DA) univariate and multivariate statistical methods were applied.

Total ApoE and ApoE4 isoform assays in an Alzheimer's disease case-control study by targeted mass spectrometry (n=669): a pilot assay for methionine-containing proteotypic peptides.

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE ε2, ApoE ε3 and ApoE ε4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE ε4 allele is a risk factor for developing Alzheimer's disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the ε4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n=68) of heterozygous ε3ε4 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large case-control study (n=669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoring-based assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance.

A design of experiment approach to the sol­gel synthesis of titania monoliths for chromatographic applications.

A design of experiement approach is described for the optimization of the microscopic morphology of macro-mesoporous titania monoliths that were elaborated for the chromatographic enrichment of phosphorylated compounds. The monolithic titania gels were formed via an alkoxy-derived sol-gel route in association with a phase separation mechanism. The synthesis was performed at mild temperatures of gelation using starting mixtures of titanium n-propoxide, hydrochloric acid, N-methylformamide, water, and poly (ethylene oxide). The gelation temperature and the chemical compositions of N-methylformamide, water, and poly (ethylene oxide) were chosen as the most relevant experimental factors of the sol­gel process. Using the sizes of the skeletons and macropores as morphological descriptors of the dried porous monoliths, the statistical analyses simultaneously revealed the effects and interactions between the different factors. Crack-free TiO2 monolithic rods of 8 to 10 cm long with well-defined co-continuous macropores and micro-structured skeletons were obtained after selection of the sol-gel parameters and optimization of the drying and heat-treatment steps of the gels. The bimodal texture of the rods exhibited macropores of 1.5 µm and mesopores centered at 5.2 nm with a total surface area of 140 m2 g(-1). The ability of the macro-mesoporous titania rods to selectively bind phosphorylated compounds was demonstrated for O-phosphoamino acids (P-Ser, P-Thr, P-Tyr).

A steroidomic approach for biomarkers discovery in doping control.

Anti-doping authorities have high expectations of the athlete steroidal passport (ASP) for anabolic-androgenic steroids misuse detection. However, it is still limited to the monitoring of known well-established compounds and might greatly benefit from the discovery of new relevant biomarkers candidates. In this context, steroidomics opens the way to the untargeted simultaneous evaluation of a high number of compounds. Analytical platforms associating the performance of ultra-high pressure liquid chromatography (UHPLC) and the high mass-resolving power of quadrupole time-of-flight (QTOF) mass spectrometers are particularly adapted for such purpose. An untargeted steroidomic approach was proposed to analyse urine samples from a clinical trial for the discovery of relevant biomarkers of testosterone undecanoate oral intake. Automatic peak detection was performed and a filter of reference steroid metabolites mass-to-charge ratio (m/z) values was applied to the raw data to ensure the selection of a subset of steroid-related features. Chemometric tools were applied for the filtering and the analysis of UHPLC-QTOF-MS(E) data. Time kinetics could be assessed with N-way projections to latent structures discriminant analysis (N-PLS-DA) and a detection window was confirmed. Orthogonal projections to latent structures discriminant analysis (O-PLS-DA) classification models were evaluated in a second step to assess the predictive power of both known metabolites and unknown compounds. A shared and unique structure plot (SUS-plot) analysis was performed to select the most promising unknown candidates and receiver operating characteristic (ROC) curves were computed to assess specificity criteria applied in routine doping control. This approach underlined the pertinence to monitor both glucuronide and sulphate steroid conjugates and include them in the athletes passport, while promising biomarkers were also highlighted.

Urinary signature of anabolic steroids and glucocorticoids in humans by LC-MS.

A metabonomic strategy based on LC-MS was employed to investigate the metabolic profile of urine samples from 20 athletes who had been tested positive for corticoids and anabolic steroids and 29 controls. In this aim, different sample preparations and chromatographic conditions were compared. The acquired LC-MS data of doped athletes and controls were subjected to analysis of variance (ANOVA) and principal component analysis (PCA). Using this approach, molecular signature of human urine was obtained showing that metabonomics could be a complementary tool to discriminate different urinary profiles and to track down metabolic changes in humans.

Optimisation of pressurised liquid extraction by experimental design for quantification of pesticides and alkyl phenols in sludge, suspended materials and atmospheric fallout by liquid chromatography-tandem mass spectrometry.

The study proposes an analytical method to quantify 10 alkylphenols and 12 pesticides at ultra-trace levels by liquid chromatography in reverse mode coupled to positive electrospray ionisation-tandem mass spectrometry. The extraction procedure from environmental solids was optimised by pressurised liquid extraction using acetonitrile:isopropanol (1:1, v/v). The influence of several extraction experimental factors, temperature, pressure, duration and number of cycles, related to the PLE was investigated by an original and efficient chemometric approach. The optimised extraction method (80 degrees C, 40 bar, 10 min, 1 cycle) exhibited recoveries between 67 and 127% with RSD mostly under 13%. The whole method was applied to real samples: sludge, suspended materials, atmospheric fallouts and roof deposit. Pollutant levels were between 1 microg kg(-1) and 5.9 mg kg(-1).

Development and optimisation of a single extraction procedure for the LC/MS/MS analysis of two pharmaceutical classes residues in sewage treatment plant.

A unique extraction procedure leading to the separation of 2 different pharmaceutical classes molecules has been developed and optimised by chemometric tools. From only one sampling, this analytical method allows the determination of 21 pharmaceuticals from corticosteroids and beta-blockers classes. Performing the SPE on Oasis MCX (mixed-mode cation exchange), the sequential elution of each pharmaceutical class is achievable, allowing a high purity level of extracts as well as high recovery rates. Performing a unique sample preparation results in an important save of time. The extracts were then analysed by LC/MS/MS, using a Hibar Purospher Star column for beta-blockers and an X-Bridge column for corticosteroids with formate buffer (pH 3.8)/AcN and water/AcN mobile phases, respectively. This work also includes a study of the chromatographic and mass spectrometric parameters in order to increase the analyte signal. The optimised SPE-LC/MS/MS method was then applied to environmental samples from sewage treatment plant (STP). beta-Blockers and corticosteroids were detected, respectively, in concentrations up to 318 ng L(-1) (sotalol) and 174 ng L(-1) (cortisone), in STP influents. Moreover, both pharmaceutical classes have also been detected in STP effluents. As far as we know, this is the first paper reporting the detection of corticosteroids in environmental waters. The developed analytical method can be used in further studies to investigate the environmental contamination by these drugs.

Speciation of selenium in a commercial dietary supplement by liquid chromatography coupled with inductively coupled plasma-mass spectrometry (ICP-MS).

Size exclusion and anion-exchange chromatographies coupled with inductively coupled plasma-mass spectrometry (ICP-MS) were used for the speciation of selenium (Se) in a dietary supplement. A sequential extraction method resulted in 85% recovery of Se and 78% of the Se extracted could be identified. The results obtained show that selenomethionine and its oxide are the predominant compounds, while selenite and selenomethylcysteine are present at low concentrations. Methane seleninic acid, probably arising from the oxidation of selenomethylcysteine, accounted for 22% of total Se. High-molecular-weight compounds, probably proteins, were detected in sodium dodecyl sulfate (SDS) and driselase extracts by size exclusion chromatography.

Spray-drying nanocapsules in presence of colloidal silica as drying auxiliary agent: formulation and process variables optimization using experimental designs.

PURPOSE: Spray-drying process was used for the development of dried polymeric nanocapsules. The purpose of this research was to investigate the effects of formulation and process variables on the resulting powder characteristics in order to optimize them. MATERIALS AND METHODS: Experimental designs were used in order to estimate the influence of formulation parameters (nanocapsules and silica concentrations) and process variables (inlet temperature, spray-flow air, feed flow rate and drying air flow rate) on spray-dried nanocapsules when using silica as drying auxiliary agent. The interactions among the formulation parameters and process variables were also studied. Responses analyzed for computing these effects and interactions were outlet temperature, moisture content, operation yield, particles size, and particulate density. Additional qualitative responses (particles morphology, powder behavior) were also considered. RESULTS: Nanocapsules and silica concentrations were the main factors influencing the yield, particulate density and particle size. In addition, they were concerned for the only significant interactions occurring among two different variables. None of the studied variables had major effect on the moisture content while the interaction between nanocapsules and silica in the feed was of first interest and determinant for both the qualitative and quantitative responses. The particles morphology depended on the feed formulation but was unaffected by the process conditions. CONCLUSION: This study demonstrated that drying nanocapsules using silica as auxiliary agent by spray drying process enables the obtaining of dried micronic particle size. The optimization of the process and the formulation variables resulted in a considerable improvement of product yield while minimizing the moisture content.