Integration of membrane processes for the recovery and separation of polyphenols from winery and olive mill wastes using green solvent-based processing.

Winery and olive mill industries generate large amounts of wastes causing important environmental problems. The main aim of this work is the evaluation of different membrane separation processes like microfiltration, ultrafiltration, nanofiltration, and reverse osmosis for the recovery of polyphenols from winery and olive mill wastes in aqueous solutions. Membrane processes were tested separately in a closed-loop system, and by an integration in a concentration mode sequential design (open-loop). Feed flow rate was varied from 1 to 10 mL min-1, and permeate samples were taken in order to measure the polyphenols concentration. The separation and concentration efficiency were evaluated in terms of total polyphenol content, and by polyphenols families (hydroxybenzoic acids (HB), hydroxycinnamic acids (HC), and flavonoids (F)), using high performance liquid chromatography. Results showed that MF and UF membranes removed suspended solids and colloids from the extracts. NF was useful for polyphenols separation (HB rejections were lower than for HC and F: HB rejections of 50 and 63% for lees filters and olive pomace extracts, respectively), and RO membranes were able to concentrate polyphenols streams (86 and 95% rejection from lees filters and olive pomace, respectively). Membranes sequential designs for lees filters and olive pomace extracts, using a selective membrane train composed by UF, NF and RO membranes, were able to obtain polyphenol rich streams and high-quality water streams for reuse purposes.

Metabolic profile modifications in milk after enrofloxacin administration studied by liquid chromatography coupled with high resolution mass spectrometry.

High resolution accurate mass spectrometry (HRMS) operating in full scan MS mode was used in the search and identification of metabolites in raw milk from cows medicated with enrofloxacin. Data consisting of m/z features were taken throughout the entire chromatogram of milk samples from medicated animals and were compared with blank samples. Twenty six different compounds were identified. Some of them were attributed to structures related to enrofloxacin while others were dipeptides or tripeptides. Additionally, enrofloxacin was administered in a controlled treatment for three days. Milk was collected daily from the first day of treatment and until four days after in the search for the identified compounds. The obtained data were chemometrically treated by Principal Component Analysis. Samples were classified by this method into three different groups corresponding to days 1-2, day 3 and days 4-7 considering the different concentration profile evolution of metabolites during the days studied. Tentative metabolic pathways were designed to rationalize the presence of the newly identified compounds.

High-resolution mass spectrometry applied to the study of metabolome modifications in various chicken tissues after amoxicillin administration.

The performance of high resolution accurate mass spectrometry (HRMS) operating in full scan MS mode was investigated for the quantitative determination of amoxicillin (AMX) as well as qualitative analysis of metabolomic profiles in tissues of medicated chickens. The metabolomic approach was exploited to compile analytical information on changes in the metabolome of muscle, kidney and liver from chickens subjected to a pharmacological program with AMX. Data consisting of m/z features taken throughout the entire chromatogram were extracted and filtered to be treated by Principal Component Analysis. As a result, it was found that medicated and non-treated animals were clearly clustered in distinct groups. Besides, the multivariate analysis revealed some relevant mass features contributing to this separation. In this context, recognizing those potential markers of each chicken class was a priority research for both metabolite identification and, obviously, evaluation of food quality and health effects associated to food consumption.

Quechers methodologies as an alternative to solid phase extraction (SPE) for the determination and characterization of residues of cephalosporins in beef muscle using LC-MS/MS.

This work was focused on the comparison of two clean-up methods to be used for the simultaneous determination of seven cephalosporins in cow muscle. In particular, the performance of novel dispersive solid phase extraction (d-SPE) procedures based on QuEChERS methodologies was assessed and compared with conventional SPE. The separation and detection of the analytes using both methods was carried out by LC-MS/MS to reach enough sensitivity to be compatible with the detection of the maximum residue limits (MRL) of cephalosporins as regulated by EU directives. The optimization of the clean-up step relied on experimental design in order to find the most suitable conditions with a reduced number of assays. Besides, multi-objective responses were used to reach an overall compromise in the recovery of all analytes simultaneously. The validation of the two methods was done according to the Directive 2002/657/EC. Linearity, decision limit, detection capability, detection and quantification limits (4-50 µg kg⁻¹), precision (RSD less than 15% except for PIR) and recoveries were determined and adequate results with comparable values using QuEChERS and SPE methodologies. LOQ were better for SPE method (0.1-10 µg kg⁻¹) but both methods show LOQ below MRL values. Precision was slightly better for the QuEChERS method, that also presents better recoveries, higher than 85% except for cephalexin.

Transmission dynamics of highly pathogenic avian influenza at Lake Constance (Europe) during the outbreak of winter 2005-2006.

Highly pathogenic avian influenza virus (HPAI) H5N1 poses a serious threat to domestic animals. Despite the large number of studies on influenza A virus in waterbirds, little is still known about the transmission dynamics, including prevalence, behavior, and spread of these viruses in the wild waterbird population. From January to April 2006, the HPAI H5N1 virus was confirmed in 82 dead wild waterbirds at the shores of Lake Constance. In this study, we present simple mathematical models to examine this outbreak and to investigate the transmission dynamics of HPAI in wild waterbirds. The population dynamics model of wintering birds was best represented by a sinusoidal function. This model was considered the most adequate to represent the susceptible compartment of the SIR model. The three transmission models predict a basic reproduction ratio (R (0)) with value of approximately 1.6, indicating a small epidemic, which ended with the migration of susceptible wild waterbirds at the end of the winter. With this study, we quantify for the first time the transmission of HPAI H5N1 virus at Lake Constance during the outbreak of winter 2005-2006. It is a step toward the improvement of the knowledge of transmission of the virus among wild waterbirds.

Disease awareness of the poultry keepers in Switzerland and their access to information concerning highly pathogenic avian influenza.

The passive surveillance of highly pathogenic avian influenza (HPAI) in domestic poultry is based essentially on the reporting of suspicious clinical cases by the poultry keepers to the veterinary services. As little was known about HPAI disease awareness among Swiss poultry keepers, a cross-sectional study was conducted among poultry keepers in Switzerland in 2007. For data triangulation and complementary information, interviews have been conducted with experts of poultry marketing organizations. The main information source used by the poultry keepers was mass media. Having a non-commercial poultry husbandry was significantly associated with lower knowledge scores. Non-commercial poultry keepers felt neglected by the veterinary authorities. Risks perceived by the poultry keepers reflected well the officially communicated risks for HPAI introduction. By highlighting the needs and the knowledge level of the poultry keepers, we make recommendations with regard to more efficient information exchange between poultry keepers and veterinary authorities. The main challenge will be to consistently integrate non-commercial poultry keepers in the formal information channels.

Production of hybrid lipid-based particles loaded with inorganic nanoparticles and active compounds for prolonged topical release.

The production of particulate hybrid carriers containing a glyceryl monostearate (Lumulse GMS-K), a waxy triglyceride (Cutina HR), silanized TiO(2) and caffeine were investigated with the aim of producing sunscreens with UV-radiation protection properties. Particles were obtained using the supercritical PGSS (Particles from Gas Saturated Solutions) technique. This method takes advantages of the lower melting temperatures of the lipids obtained from the dissolution of CO(2) in the bulk mixture. Experiments were performed at 13 MPa and 345 K, according to previous melting point measurements. Blends containing Lumulse GMS-K and Cutina HR lipids (50 wt%) were loaded with silanized TiO(2) and caffeine in percentile proportions of 6 and 4 wt%, respectively. The particles produced were characterized using several analytical techniques as follows: system crystallinity was checked by X-ray diffraction and differential scanning calorimetry, thermal stability by thermogravimetric analysis, and morphology by scanning and transmission electron microscopy. Further, the UV-shielding ability of TiO(2) after its dispersion in the lipidic matrix was assessed by solid UV-vis spectroscopy. Preliminary results indicated that caffeine-loaded solid lipid particles presented a two-step dissolution profile, with an initial burst of 60 wt% of the loaded active agent. Lipid blends loaded with TiO(2) and caffeine encompassed the UV-filter behavior of TiO(2) and the photoaging prevention properties of caffeine.

Application of principal component analysis to the thermal characterization of silanized nanoparticles obtained at supercritical carbon dioxide conditions.

Samples resulting from reaction of TiO(2) with octyltriethoxysilane, developed using a supercritical carbon dioxide procedure, have been studied by infrared and Raman spectroscopies and thermogravimetric analysis. Different reaction conditions have been applied to the preparation of samples in order to study the influence of experimental factors on the sample properties. Vibrational techniques have first been used to verify the presence of silanized structures on the surface of TiO(2) through the detection of specific bands characteristic of the Si-O-Si cross-linking. Thermogravimetric profiles consisting of weight loss values as a function of temperature have been analyzed by principal component analysis to extract information about the characteristics of the linkage between silane and TiO(2) as well as the thermal stability of the prepared materials. The mathematical treatment of data has provided conclusions on the properties of the samples and analogies and differences with respect to the commercial material.

Time-resolved step-scan FT-IR spectroscopy: focus on multivariate curve resolution.

The present paper describes the application of step-scan FT-IR spectroscopy in combination with chemometric analysis of the spectral data for the study of the photocycle of bacteriorhodopsin. The focus is on the performance of this instrumentation for time-resolved experiments. Three-dimensional data-spectra recorded over time-are studied using various factor analysis techniques, e.g., singular values decomposition, evolving factor analysis, and multivariate curve resolution based on alternating least squares. Transient intermediates formed in the time domain ranging from 1 micros to 6.6 ms are clearly detected through reliable pure time evolving profiles. At the same time, pure difference absorbance spectra are provided. As a result, valuable information about transitions and dynamics of the protein can be extracted. We conclude first that step-scan FT-IR spectroscopy is a useful technique for the direct study of difficult photochemical systems. Second, and this is the essential motivation of this paper, chemometrics provide a step forward in the description of the photointermediates.

Quantitation in multianalyte overlapping peaks from capillary electrophoresis runs using artificial neural networks.

The potentiality of artificial neural networks for multicomponent analysis in unresolved peaks from capillary electrophoresis (CE) is evaluated. The system chosen consists of mixtures of three ebrotidine metabolites, which cannot be successfully separated by CE. Data selected for analysis consist of UV spectra taken at the maximum of the CE peak. The most dissimilar analyte, in terms of spectral differences, is accurately quantitated in any type of mixture with an overall prediction error of 5%. Because of the strong interference of the two most overlapped compounds, a preliminary procedure for spectral data filtering based on principal component analysis is performed to improve their quantitation.

Multivariate calibration methods for quantification in strongly overlapping capillary electrophoretic peaks.

Capillary zone electrophoresis with diode-array detection was applied to the separation of ebrotidine and its metabolites. However, three of these, which are neutral in the conditions studied, co-migrated with the electroosmotic flow signal. Therefore, strongly overlapping peaks were observed. The main aim of this study was to show the potentiality of capillary electrophoresis in combination with chemometrics. Multivariate calibration methods were applied to quantify these analytes in synthetic mixtures. The results obtained using partial least squares (PLS) were in agreement with actual values, with an overall prediction error of 9.7%.

Determination of ebrotidine metabolites in overlapping peaks from capillary zone electrophoresis using chemometric methods.

This paper illustrates the possibilities of chemometric methods in the resolution and quantification of various compounds in overlapping peaks from capillary electrophoresis. Ebrotidine and most of its metabolites were efficiently separated by capillary zone electrophoresis (CZE) in a fused-silica capillary. However, the procedure was not suitable for the physical separation of the three less ionizable metabolites, which comigrated and overlapped with the electroosmotic flow signal. Multivariate curve resolution based on an alternating least squares procedure was used for their mathematical resolution. For such a purpose, data obtained in the CZE system with a diode array detector, which consisted of UV spectra registered over time, were analyzed. The ebrotidine metabolites were successfully resolved and quantified in synthetic mixtures and urine samples.

Potentiometric sensor array for the determination of lysine in feed samples using multivariate calibration methods.

A potentiometric sensor array has been developed for the determination of lysine in feed samples. The sensor array consists of a lysine biosensor and seven ion-selective electrodes for NH4+, K+, Na+, Ca2+, Mg2+, Li+, and H+, all based on all-solid-state technology. The potentiometric lysine biosensor comprises a lysine oxidase membrane assembled on an NH4+ electrode. Because the selectivity of the lysine biosensor towards other cation species is not sufficient, there is severe interference with the potentiometric response. This poor selectivity can be circumvented mathematically by analysis of the richer information contained in the multi-sensor data. The sensor array takes advantage of the cross-selectivity of lysine for each electrode, which differs from the other species and quantification of lysine in complex feed sample extracts is accomplished with multivariate calibration methods, such as partial least-squares regression. The results obtained are in a reasonable agreement with those given by the standard method for amino acid analysis.

Strategies for in-capillary derivatization of amino acids in capillary electrophoresis using 1,2-naphthoquinone-4-sulfonate as a labeling reagent.

This paper examines the potentiality of in-capillary derivatization for improving the sensitivity of the spectrophotometric detection of amino acids in capillary zone electrophoresis. 1,2-Naphthoquinone-4-sulfonate was selected as the labeling agent of amino acids. The underivatized sample and the reagent solution segments are injected by pressure into the capillary prior to applying the running voltage. The corresponding derivatization reaction occurs inside the capillary once the potential is applied, as it induces mixing of the sample with the reagent. Several introduction modes consisting of tandem or sandwich configuration have been evaluated. These techniques result in a straightforward and automated way of carrying out a derivatization. Furthermore, in-capillary procedures may become much more attractive than conventional pre-capillary derivatization in terms of sensitivity and reproducibility. The optimum operation mode found consists of a sandwich system where the sample is injected in between two reagent segments. The method was applied to the determination of amino acids in feed samples. Results show a good concordance with those given by a standard amino acid analyzer.

Sensitivity enhancement by on-line preconcentration and in-capillary derivatization for the electrophoretic determination of amino acids.

This study describes an application of on-line preconcentration by large-volume stacking in combination with in-capillary derivatization for enhancing spectrophotometric detection sensitivity in capillary electrophoresis. The method is illustrated by an example dealing with the determination of amino acids with 1,2-naphthoquinone-4-sulfonate as a labelling agent. Samples are dissolved in water in order to create a stacking process based on differences in the conductivity between this medium and a concentrated running buffer. The in-capillary derivatization is accomplished following a sandwich procedure in which the sample is inserted between two segments of reagent. Amino acid derivatives are obtained and separated in a fused-silica capillary with a sodium borate electrolyte buffer using 2-propanol as an organic modifier. The method is applied to the analysis of amino acids in pharmaceutical and feed samples. A good concordance between the predicted values and those obtained with the standard method is observed, with overall quantification error below 5%. The proposed procedure allows the detection limits sensitivity to be enhanced in 1000-fold with respect to conventional precapillary derivatization.

Potentiality of proton nuclear magnetic resonance and multivariate calibration methods for the determination of dermatan sulfate contamination in heparin samples.

A 1H NMR method for the quantification of dermatan sulfate impurities in heparin industrial samples is proposed. The method is based on the analysis of 1H NMR spectral data by multivariate calibration. The 1H NMR spectra of heparin and dermatan sulfate standards showed characteristic profiles. Thus, differences in the methyl peaks of acetamido groups of heparin and dermatan sulfate were greatly advantageous for the analysis. Other hydrogens of the sugar ring were also relevant in this study. Thus, the determination of dermatan sulfate by multivariate calibration depended on all these differences. Partial least squares regression (PLS) was chosen as the calibration method. In addition, a data standardization procedure was developed in order that 1H NMR spectra registered with different instruments operating under different measurement conditions were comparable. The quantification of dermatan sulfate in the samples was satisfactory, with an overall prediction error of 6%.

Determination of amino acids in overlapped capillary electrophoresis peaks by means of partial least-squares regression.

Amino acid derivatives of 1,2-naphthoquinone-4-sulfonate (NQS) can be separated by capillary electrophoresis at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate-isopropanol (3:1, v/v) solution as background electrolyte. This procedure was suitable for the most common amino acids. However, the peaks of three amino acids (phenylalanine, isoleucine and tyrosine) were only partially resolved and peaks of histidine and leucine derivatives overlapped completely. Partial least-squares regression (PLS) may overcome the lack of selectivity for these amino acids. Spectroelectropherograms of the corresponding amino acid derivative peaks were monitored with a diode-array spectrophotometer in the range 225 to 540 nm. Both spectra and electropherograms can be used as multivariate data for further analysis. In general, the best predictions were obtained using the time domain.

Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares.

The application of chemometric techniques to the resolution of overlapped peaks in capillary electrophoresis (CE) is described. When a physical separation can not be completely accomplished, chemometrics might still resolve the determination of the analytes mathematically. CE with diode array detection can provide a large amount of data consisting of spectra registered over time. In this study, the capillary electrophoretic separation of 1,2-naphthoquinone-4-sulfonate derivatives of amino acids is studied. Most of the common amino acid derivatives can be separated at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate + isopropanol (3:1 v/v) solution as background electrolyte. However, peaks of certain derivatives (Phe, His, Leu and Ile) still overlap. A multivariate curve resolution method based on an alternating least squares optimization procedure is used for the resolution of the overlapped electrophoretic peaks. The method takes advantage of spectral and electrophoretic differences of analytes to recover their pure electrophoretic and spectral profiles. In addition, each analyte in the mixture can be quantified using the corresponding standards.

Principal component analysis and cluster analysis for the characterization of dental composites.

Various experimental dental materials were characterized using chemometric methods. The main aim of the study was to ascertain which composite materials present the best properties for use in restorative dentistry. Bisphenol-alpha-glycidyl methacrylate-based composites containing hydroxyapatite as a filler and a coupling agent were prepared using a photocuring polymerization procedure. Several chemical and mechanical properties of experimental composites were measured and the corresponding data were further studied using principal component analysis and cluster analysis. Results from the characterization allowed the most appropriate materials to be selected. Various composites presented acceptable general properties suggesting their suitability as substitutes for commercial materials in dentistry.

Liquid chromatographic determination of aniline in table-top sweeteners based on pre-column derivatization with 1,2-naphthoquinone-4-sulfonate.

A liquid chromatographic method for the determination of aniline in cyclamate sweeteners based on a pre-column derivatization with 1,2-naphthoquinone-4-sulfonate (NQS) is proposed. Aniline traces were extracted from the cyclamate samples using dichloromethane. After solvent evaporation, the dry residue was derivatized with NQS at pH 9.5 and 85 degrees C for 1 min. The aniline derivative, which was extracted from the reacting mixture, was redissolved in the eluent solution and injected into the chromatographic system. The separation of aniline derivative from other amine impurities was carried out in a C18 column using a 2% acetic acid-methanol (40:60, v/v) mobile phase. Results from the analysis of aniline in the sweetener samples with the proposed method were compared with those from the standard method. A good concordance between the two methods was observed.