Elisa Robotti

Determination of perfluorochemicals in biological, environmental and food samples by an automated on-line solid phase extraction ultra high performance liquid chromatography tandem mass spectrometry method

A rapid on-line solid phase extraction ultra high performance liquid chromatography tandem mass spectrometry method was developed for the identification and quantitation of nine perfluorinated compounds in matrices of environmental, biological and food interest. Pre-treatment, solid phase extraction, chromatographic and mass detection conditions were optimised, in order to apply the whole methodology to the analysis of different matrices. Particular attention was devoted to the evaluation of matrix effect and the correlated phenomena of ion enhancement or suppression in mass spectrometry detection. LOD and LOQ range from 3 to 15ngL(-1) and from 10 to 50ngL(-1), respectively. Method detection limits (MDLs) were also calculated for each kind of matrix. The recovery, evaluated for each analyte, does not depend on analyte concentration in the explored concentration range: average R¯% values are always greater than 82.9%. In the whole, the results obtained for samples of river waters, blood serum, blood plasma, and fish confirm the ubiquitous presence of perfluorinated compounds, as recently denounced by many sources.

Automated Online Solid Phase Extraction Ultra High Performance Liquid Chromatography Method Coupled with Tandem Mass Spectrometry for Determination of Forty-Two Therapeutic Drugs and Drugs of Abuse in Human Urine

The study deals with a fast (analysis times of around 11 min) simultaneous identification and quantification in human urine of 42 drugs (21 therapeutic and 21 of abuse), through an automated online solid phase extraction ultra high performance liquid chromatography method coupled with tandem mass spectrometry (SPE UHPLC-MS/MS). In the method validation, particular attention was devoted to the matrix effect, through matrix-matched calibration in blank urine, suitably diluted. For all the abuse drugs investigated, the limit of quantitation (LOQ) values are lower than the legal threshold concentration levels, making the method suitable for routine control. The whole procedure was applied in the analysis of urine of patients positive to the I level screening test.

High-Performance Liquid Chromatography−Ultraviolet Detection Method for the Simultaneous Determination of Typical Biogenic Amines and Precursor Amino Acids. Applications in Food Chemistry

A reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination in food of biogenic amines and their precursor amino acids after a precolumn derivatization with dansyl chloride. The chromatographic conditions, selected to be suitable for mass spectrometry detection, were optimized through experimental design and artificial neural networks. The HPLC-UV method was validated by comparing the separation results with those obtained through a HPLC method, working under the same chromatographic conditions but employing mass spectrometry detection. The HPLC-UV method was then applied to the analysis of different food samples, namely, cheese, clams, salami, and beer. For all of the matrices, recoveries (relative standard deviation always <5%) always >92% were obtained. The results are discussed as a function of the total biogenic amine content and of the concentration ratio between amines and precursor amino acids.

Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged ageing

To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bulls muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Students t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes.

Simultaneous determination of thirteen polycyclic aromatic hydrocarbons and twelve aldehydes in cooked food by an automated on-line solid phase extraction ultra high performance liquid chromatography tandem mass spectrometry.

An on-line solid phase extraction (SPE) ultra high performance liquid chromatography tandem mass spectrometry method has been developed for the simultaneous identification and determination of thirteen polycyclic aromatic hydrocarbons (PAHs) and twelve aldehydes (derivatized with 2,4-dinitrophenylhydrazine). The chromatographic conditions have been optimized to obtain the maximum of sensitivity and resolution taking into account the different retention interactions and the different ionization conditions of PAHs and derivatized aldehydes. LOD values ranging from 0.028 to 0.768 μg L(-1) for PAHs and from 0.002 to 0.125 μg L(-1) for aldehydes were obtained. The resolution permitted the separation of four couples of PAH isomers. Sample pre-treatment and SPE were optimized in order to apply the whole methodology to the analysis of different food matrices as salmon, frankfurter, steak, and pork chop, subjected to different cooking modes (smoked, grilled, cooked in oil or in butter). Particular attention was devoted to the evaluation of matrix effect that was significantly reduced through the on-line SPE treatment. For each food matrix the method detection limits, the method quantitation limits, and the recovery R were evaluated. R was shown not to depend on analyte concentration in the explored concentration range (LOQ - 50.000 μg L(-1)): the average R percent ranges from 70.6% to 120.0%.

Monitoring of pigmented and wooden surfaces in accelerated ageing processes by FT-Raman spectroscopy and multivariate control charts.

Two of the most suitable analytical techniques used in the field of cultural heritage are NIR (near-infrared) and Raman spectroscopy. FT-Raman spectroscopy coupled to multivariate control charts is applied here for the development of a new method for monitoring the conservation state of pigmented and wooden surfaces. These materials were exposed to different accelerated ageing processes in order to evaluate the effect of the applied treatments on the goods surfaces. In this work, a new approach based on the principles of statistical process control (SPC) to the monitoring of cultural heritage, has been developed: the conservation state of samples simulating works-of-art has been treated like an industrial process, monitored with multivariate control charts, owing to the complexity of the spectroscopic data collected. The Raman spectra were analysed by principal component analysis (PCA) and the relevant principal components (PCs) were used for constructing multivariate Shewhart and cumulative sum (CUSUM) control charts. These tools were successfully applied for the identification of the presence of relevant modifications occurring on the surfaces. CUSUM charts however proved to be more effective in the identification of the exact beginning of the applied treatment. In the case of wooden boards, where a sufficient number of PCs were available, simultaneous scores monitoring and residuals tracking (SMART) charts were also investigated. The exposure to a basic attack and to high temperatures produced deep changes on the wooden samples, clearly identified by the multivariate Shewhart, CUSUM and SMART charts. A change on the pigment surface was detected after exposure to an acidic solution and to the UV light, while no effect was identified on the painted surface after the exposure to natural atmospheric events.

Application of partial least squares discriminant analysis and variable selection procedures: a 2D-PAGE proteomic study

Abstract2D gel electrophoresis is a tool for measuring protein regulation, involving image analysis by dedicated software (PDQuest, Melanie, etc.). Here, partial least squares discriminant analysis was applied to improve the results obtained by classic image analysis and to identify the significant spots responsible for the differences between two datasets. A human colon cancer HCT116 cell line was analyzed, treated and not treated with a new histone deacetylase inhibitor, RC307. The proteins regulated by RC307 were detected by analyzing the total lysates and nuclear proteome profiles. Some of the regulated spots were identified by tandem mass spectrometry. The preliminary data are encouraging and the protein modulation reported is consistent with the antitumoral effect of RC307 on the HCT116 cell line. Partial least squares discriminant analysis coupled with backward elimination variable selection allowed the identification of a larger number of spots than classic PDQuest analysis. Moreover, it allows the achievement of the best performances of the model in terms of prediction and provides therefore more robust and reliable results. From this point of view, the multivariate procedure applied can be considered a good alternative to standard differential analysis, also taking into account the interdependencies existing among the variables.

Proteins involved in biotic and abiotic stress responses as the most significant biomarkers in the ripening of Pinot Noir skins

We propose an integrated approach, obtained by the combination of multivariate statistics and proteomics, useful to isolate candidate biomarkers for the evaluation of grape ripening. We carried out a comparative 2-DE analysis of grape skins collected in three moments of ripening and analyzed the spot volume dataset through the application of principal component analysis followed by forward stepwise-linear discriminant analysis. This technique allowed to discriminate véraison, quite mature and mature samples, and to sort the matched spots according to their significance. We identified 36 spots showing high discriminating coefficients through liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). Most of them were involved in biotic and abiotic stress responses indicating these enzymes as good candidate markers of berry ripening. These evidences hint at a likely developmental role of these proteins, in addition to their reported activity in stress events. Restricting the same statistical analysis to the samples belonging to the two last stages, it was indicated that this approach can clearly distinguish these close and similar phases of berry development. Taken all together, these results bear out that the employment of the combination of 2-DE and multivariate statistics is a reliable tool in the identification of new protein markers for describing the ripening phases and to assess the overall quality of the fruit.

Development of a technique based on multi-spectral imaging for monitoring the conservation of cultural heritage objects

A new approach for monitoring the state of conservation of cultural heritage objects surfaces is being developed. The technique utilizes multi-spectral imaging, multivariate analysis and statistical process control theory for the automatic detection of a possible deterioration process, its localization and identification, and the wavelengths most sensitive to detecting this before the human eye can detect the damage or potential degradation changes occur. A series of virtual degradation analyses were performed on images of parchment in order to test the proposed algorithm in controlled conditions. The spectral image of a Dead Sea Scroll (DSS) parchment, IAA (Israel Antiquities Authority) inventory plate # 279, 4Q501 Apocryphal Lamentations B, taken during the 2008 Pilot of the DSS Digitization Project, was chosen for the simulation.

New approach based on fuzzy logic and principal component analysis for the classification of two-dimensional maps in health and disease: Application to lymphomas

Two-dimensional (2D) electrophoresis is the most wide spread technique for the separation of proteins in biological systems. This technique produces 2D maps of high complexity, which creates difficulties in the comparison of different samples. The method proposed in this paper for the comparison of different 2D maps can be summarised in four steps: (a) digitalisation of the image; (b) fuzzyfication of the digitalised map in order to consider the variability of the two-dimensional electrophoretic separation; (c) decoding by principal component analysis of the previously obtained fuzzy maps, in order to reduce the system dimensionality; (d) classification analysis (linear discriminant analysis), in order to separate the samples contained in the dataset according to the classes present in said dataset. This method was applied to a dataset constituted by eight samples: four belonging to healthy human lymph-nodes and four deriving from non-Hodgkin lymphomas. The amount of fuzzyfication of the original map is governed by the sigma parameter. The larger the value, the more fuzzy theresulting transformed map. The effect of the fuzzyfication parameter was investigated, the optimal results being obtained for sigma = 1.75 and 2.25. Principal component analysis and linear discriminant analysis allowed the separation of the two classes of samples without any misclassification.