Studio ed ottimizzazione di metodologie analitiche per la determinazione di tossine algali in prodotti ittici destinati al consumo umano

Alghe unicellulari del genere Ostreopsis, Dinophysis, Prorocentrum, Alexandrium e ceppi di Gymnodinium, producono sostanze tossiche che possono mettere a rischio la salute pubblica in quanto responsabili di sindromi a decorso acuto e cronico a carico dell'apparato digerente e del sistema nervoso conseguenti l'assunzione di prodotti ittici contaminati. I molluschi bivalvi in particolare, in quanto organismi filtratori, sono maggiormente implicati nella trasmissione di queste tossine. Il problema delle fioriture di alghe tossiche è particolarmente avvertito sulle coste italiane soprattutto nel periodo estivo. Il presente lavoro di tesi tratterà lo studio ed il confronto di alcune procedure analitiche per la determinazione di tossine algali in polpa di mitile per individuare le condizioni sperimentali di estrazione, purificazione ed analisi strumentale che permettano nella singola analisi l'identificazione e la quantificazione del più ampio spettro di tossine. Per le misure analitiche sarà utilizzata la cromatografia liquida fast-HPLC con rivelazione a Spettrometria di massa ad alta risoluzione

A dual mode breath sampler for the collection of the end-tidal and dead space fractions

This work presents a breath sampler prototype automatically collecting end-tidal (single and multiple breaths) or dead space air fractions (multiple breaths). This result is achieved by real time measurements of the CO2 partial pressure and airflow during the expiratory and inspiratory phases. Suitable algorithms, used to control a solenoid valve, guarantee that a Nalophan® bag is filled with the selected breath fraction even if the subject under test hyperventilates. The breath sampler has low pressure drop (< 0.5 kPa) and uses inert or disposable components to avoid bacteriological risk for the patients and contamination of the breath samples. A fully customisable software interface allows a real time control of the hardware and software status. The performances of the breath sampler were evaluated by comparing a) the CO2 partial pressure calculated during the sampling with the CO2 pressure measured off-line within the Nalophan® bag; b) the concentrations of four selected volatile organic compounds in dead space, end-tidal and mixed breath fractions.Results showed negligible deviations between calculated and off-line CO2 pressure values and the distributions of the selected compounds into dead space, end-tidal and mixed breath fractions were in agreement with their chemical-physical properties

Determination of sevoflurane and isopropyl alcohol in exhaled breath by thermal desorption gas chromatography-mass spectrometry for exposure assessment of hospital staff

Volatile anaesthetics and disinfection chemicals pose ubiquitous inhalation and dermal exposure risks in hospital and clinic environments. This work demonstrates specific non-invasive breath biomonitoring methodology for assessing staff exposures to sevoflurane (SEV) anaesthetic, documenting its metabolite hexafluoroisopropanol (HFIP) and measuring exposures to isopropanol (IPA) dermal disinfection fluid. Methods are based on breath sample collection in Nalophan bags, followed by an aliquot transfer to adsorption tube, and subsequent analysis by thermal desorption gas chromatography-mass spectrometry (TD-GC-MS). Ambient levels of IPA were also monitored. These methods could be generalized to other common volatile chemicals found in medical environments. Calibration curves were linear (r2=0.999) in the investigated ranges: 0.01-1000ppbv for SEV, 0.02-1700ppbv for IPA, and 0.001-0.1ppbv for HFIP. The instrumental detection limit was 10pptv for IPA and 5pptv for SEV, both estimated by extracted ion-TIC chromatograms, whereas the HFIP minimum detectable concentration was 0.5pptv as estimated in SIM acquisition mode. The methods were applied to hospital staff working in operating rooms and clinics for blood draws. SEV and HFIP were present in all subjects at concentrations in the range of 0.7-18, and 0.002-0.024ppbv for SEV and HFIP respectively. Correlation between IPA ambient air and breath concentration confirmed the inhalation pathway of exposure (r=0.95, p&lt;0.001) and breath-borne IPA was measured as high as 1500ppbv. The methodology is easy to implement and valuable for screening exposures to common hospital chemicals. Although the overall exposures documented were generally below levels of health concern in this limited study, outliers were observed that indicate potential for acute exposures

Development of an ImmunoFET for Analysis of Tumour Necrosis Factor- (alfa) in Artificial Saliva: Application for Heart Failure Monitoring

Assessing tumour necrosis factor-(alfa) (TNF-(alfa)) levels in the human body has become an essential tool to recognize heart failure (HF). In this work, label-free, rapid, easy to use ImmunoFET based on an ion-sensitive field effect transistor (ISFET) was developed for the detection of TNF-(alfa) protein. Monoclonal anti-TNF-(alfa) antibodies (anti-TNF-(alfa) mAb) were immobilized on an ISFET gate made of silicon nitride (Si3N4) after salinization with 11-(triethoxysilyl) undecanal (TESUD). The obtained ISFET functionalized with the mAbs (ImmunoFET) was used to detect TNF-(alfa) protein in both phosphate buffer saline (PBS) and artificial saliva (AS). The change in the threshold voltage of the gate (DVT) showed approximately linear dependency on the concentration of the antigens in the range 5-20 pg/mL for both matrixes. The cross-selectivity study showed that the developed ImmunoFET demonstrated to be selective towards TNF-(alfa), when compared to other HF biomarkers such as N-terminal pro-brain natriuretic peptide (NT-proBNP), interleukin-10 (IL-10), and cortisol, even if further experiments have to be carried out for decreasing possible unspecific absorption phenomena. To the best of our knowledge, this is the first ImmunoFET that has been developed based on Si3N4 for TNF-(alfa) detection in AS by electrical measurement

Determination of volatile organic compounds in exhaled breath of heart failure patients by needle trap micro-extraction coupled with gas chromatography-tandem mass spectrometry

The analytical performances of needle trap micro-extraction (NTME) coupled with gas chromatography tandem mass spectrometry were evaluated by analyzing a mixture of twenty-two representative breath VOCs belonging to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics and sulfurs). NTME is an emerging technique that guarantees detection limits in pptv range by pre-concentrating low volumes of sample, and it is particularly suitable for breath analysis. For most VOCs, detection limits between 20 and 500 pptv were obtained by pre-concentrating 25 mL of a humidified standard gas mixture at a flow rate of 15 mL/min. For all compounds, inter- and intra-day precisions were always below 15%, confirming the reliability of the method. The procedure was successfully applied to the analysis of exhaled breath samples collected from forty heart failure patients during their stay in the University Hospital of Pisa. The majority of patients (about 80%) showed a significant decrease of breath acetone levels (a factor of 3 or higher) at discharge compared to admission (acute phase) in correspondence to the improved clinical conditions during hospitalization, thus making this compound eligible as a biomarker of heart failure exacerbation

Predicting Heart Failure Patient Events by Exploiting Saliva and Breath Biomarkers Information

The aim of this work is to present a machine learning based method for the prediction of adverse events (mortality and relapses) in patients with heart failure (HF) by exploiting, for the first time, measurements of breath and saliva biomarkers (Tumor Necrosis Factor Alpha, Cortisol and Acetone). Data from 27 patients are used in the study and the prediction of adverse events is achieved with high accuracy (77%) using the Rotation Forest algorithm. As in the near future, biomarkers can be measured at home, together with other physiological data, the accurate prediction of adverse events on the basis of home based measurements can revolutionize HF management

Chemical biomarkers in human breath and oral fluid: Method development and applications in non-invasive clinical analyses

Early detection plays a crucial role for the planning of treatment and the prognosis of diseases. The capability to evaluate the physiological conditions of a patient, trace the progression of a disease and monitor the efficacy of post-treatment therapies is a primary objective in healthcare research. The analysis of blood, tissues or urine is a common approach to obtain clinical information, but this procedure entails drawbacks. In particular, the collection of the first two specimens is invasive and brings a significant stress for the patient, especially if repeated sampling is needed, whereas urine sampling is simple but with less controllable in frequency. Alternative specimens such as exhaled breath and oral fluid have gained importance as potential sources of clinical information because they reflect the biological activity as well as the health status, and they can be easily and unobtrusively collected. The possibility to obtain clinical information in a non-invasive way and without risk for the patients represents one of the most important goal of health monitoring. The analysis of these alternative matrices thus represents an interesting approach, especially for the screening of a large population. The aim of this work was to develop and validate analytical methods for the quantification of specific biomarkers in human breath and oral fluid, and to apply them for monitoring of disease such as heart failure and psoriasis and the environmental exposure to toxic substances. In a first study, an analytical method was optimized for the chemical characterization of exhaled breath based on two-stage thermal desorption capillary gas chromatography coupled to mass spectrometry. The method was used to monitor the exposure of hospital personnel to sevoflurane, a widely used anaesthetic, and isopropanol, a disinfectant. In addition to these molecules, the determination of eighteen volatile compounds belonging to different chemical classes of environmental concern (i.e. hydrocarbons, ketones, aldehydes, aromatics, tio-compounds and esters) was also possible, which suggests the possible use of exhaled breath analysis for the monitoring of subjects exposed to environmental contaminants or patients suffering from specific disease. In fact, in a second study, several analytical methods were developed and validated to quantify salivary biomarkers related to the clinical status of patients suffering from heart failure. Heart failure is a rapidly diffusing chronic cardiovascular disease and the main cause of mortality and poor quality of life in western societies. Aldosterone, cortisol and 8-iso-Prostaglandin F2 (8-isoPGF2) were determined in oral fluid by liquid chromatography coupled with tandem mass spectrometry, tumor necrosis factor- and interleukin-10 by enzyme-linked immunoassays, uric acid by liquid chromatography with ultraviolet detection, lactate by liquid chromatography with fluorescence detection, and salivary -amylase enzymatic activity by a spectrophotometric method. Each biomarker is representative of a pathological condition occurring with the heart failure syndrome, so that abnormal levels of these molecules could be associated with the severity and progression of the disease. The methods were then used in the framework of the H2020 HEARTEN Project and to support the development of specific biosensors able to anticipate critical exacerbation episodes by rising an alarm in hospital information systems remotely connected by an mHealth application. The third research was focused on the development and validation of suitable analytical method for the determination of stress-related biomarkers in oral fluid, to investigate the response of the two primary neuroendocrine systems, namely the autonomic nervous system and the hypothalamic-pituitary-adrenal axis, whose main biomarkers of activation are salivary -amylase and cortisol, respectively. In this case, a spectrometric method was used for the determination of salivary -amylase enzymatic activity and a Reversed-Phase High-Performance Liquid Chromatography coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry method for cortisol. These methods were then applied to investigate the responsiveness of psoriatic patients to stressors. Psoriasis is a chronic and inflammatory skin disease characterised by a significant psychological distress and psychiatric morbidity, experiences of stigmatization, and decreased quality of life. The aetiology of psoriasis has not been fully understood yet, but it appears to be multifactorial, involving both genetic and environmental influences. Among these factors, emotional stress is considered to play an important role in the onset and exacerbation of the disease. The clinical application of the methods, even if in a limited number of psoriatic volunteers, showed potential to clarify the role of cortisol and salivary -amylase enzymatic activity and the mechanisms of activation of the two biological systems in response to a stressor. Moreover, information was achieved on their play in these mechanisms. The results suggested that the quantification of salivary stress-related biomarkers could be used to assess the effectiveness of stress reduction strategies in the management of psoriasis

DETERMINATION OF BIOMARKERS IN BREATH AND SALIVA FOR MONITORING HEART FAILURE PATIENTS

Heart failure (HF) is a complex clinical syndrome caused by a wide range of cardiovascular disorders, such as structural or functional abnormalities of the heart. The clinical status of a HF patient can be evaluated by detecting specific biomarkers of pathogenic biological processes. Beside conventional clinical investigations, breath and saliva analysis have been recognized as one of the most effective, easy, painless and non-invasive ways of identifying physiological and pathophysiological conditions. This study was focused on the development and validation of analytical methods based on GC-MS/MS, HPLC-MS/MS, spectrophotometric and immunochemical techniques to determine specific breath and saliva biomarkers. Brain natriuretic peptides, 8-iso-prostaglandin F2α, uric acid, tumor necrosis factor-α, interleukin-10, aldosterone, α-amylase, lactate and cortisol in saliva samples, and acetone and isoprene in breath samples were determined. Special attention was paid to the optimization of the sampling procedures and sample handling

Advances in breath and saliva analysis

Breath and saliva share a series of features that allow them to provide complementary and clinically valuable information. Their components originate from normal or abnormal physiology, ingestion and metabolization of food and beverage, exposure to pollutants and bacterial activity. The rapid equilibrium with blood provides these biological fluids with a high potential for the diagnosis of diseases as well as the monitoring of disease progression and therapy. The combination of non-invasive sampling and the fact that their composition mirrors almost in real time processes occurring inside the body makes breath and saliva analysis suitable for monitoring drug therapies, for example to detect potential adverse effects or to provide insights on the pharmacokinetics or on the mechanism of action. From the analytical point of view, breath and saliva offer the advantage of reduced background levels (compared to blood) of chemical interferents, but this is usually paid with lower concentration levels of the target compounds. Sampling procedures of both fluids are critical, as they may have remarkable effects on the composition of the collected specimens. Storage of samples is also critical in the case of breath, whereas saliva does not generally require particular attention. In this work, the use of breath and saliva analysis in the pharmacological field is reviewed and practical applications are shown. After a brief introduction concerning the basics of breath and saliva analysis, more attention is devoted to the sampling procedures and their effects on sample composition. A series of illustrative applications in the pharmacological field concludes the work showing the present use of these techniques and trends for the future

Determination of peppermint compounds in breath by needle trap micro-extraction coupled with gas chromatography–tandem mass spectrometry

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