Biological studies with comprehensive 2D-GC-HRMS screening: Exploring the human sweat volatilome

A key issue in GCxGC-HRMS data analysis is how to approach large-sample studies in an efficient and comprehensive way. We have developed a semi-automated data-driven workflow from identification to suspect screening, which allows highly selective monitoring of each identified chemical in a large-sample dataset. The example dataset used to illustrate the potential of the approach consisted of human sweat samples from 40 participants, including field blanks (80 samples). These samples have been collected in a Horizon 2020 project to investigate the capacity of body odour to communicate emotion and influence social behaviour. We used dy-namic headspace extraction, which allows comprehensive extraction with high preconcentration capability, and has to date only been used for a few biological applications. We were able to detect a set of 326 compounds from a diverse range of chemical classes (278 identified compounds, 39 class unknowns, and 9 true unknowns). Unlike partitioning-based extraction methods, the developed method detects semi-polar (log P < 2) nitrogen and oxygen-containing compounds. However, it is unable to detect certain acids due to the pH conditions of un-modified sweat samples. We believe that our framework will open up the possibility of efficiently using GCxGC-HRMS for large-sample studies in a wide range of applications such as biological and environmental studies

Influence of sampling on the determination of warfarin and warfarin alcohols in oral fluid

Background and Objective: The determination of warfarin, RS/SR- and RR/SSwarfarin alcohols in oral fluid may offer additional information to the INR assay. This study aimed to establish an optimized sampling technique providing the best correlation between the oral fluid and the unbound plasma concentrations of these compounds. Materials and Methods: Samples of non-stimulated and stimulated oral fluid, and blood were collected from 14 patients undergoing warfarin therapy. After acidification, analytes were extracted with a dichloromethane/hexane mixture and determined by HPLC with fluorescence detection. Plasma samples were also ultrafiltered for the determination of the unbound fraction. The chromatographic separation was carried out in isocratic conditions with a phosphate buffer/methanol mobile phase on a C-18 reversed-phase column. The absence of interfering compounds was verified by HPLC-ESI-Q-TOF. Results: Stimulation generally increased the oral fluid pH to values close to blood pH in about 6 minutes. The concentration of warfarin and RS/SR-warfarin alcohols in oral fluid followed the same trend, whereas the concentration of RR/SS-warfarin alcohols was not affected. Six minute stimulation with chewing gum followed by collection with a polyester swab was the best sampling procedure, with a good repeatability (RSD 〈10%) and relatively low inter-subject variability (RSD =30%) of the oral fluid to plasma ratio. This procedure provided strong correlations between the measured oral fluid and unbound plasma concentration of warfarin (r = 0.92, p 〈0.001) and RS/SR-warfarin alcohols (r =5 0.84, p 〈0.001), as well as between stimulated oral fluid and total plasma concentration of warfarin (r = 0.78, p 〈0.001) and RS/SR-warfarin alcohols (r = 0.81, p 〈0.001). Conclusion: The very good correlation between oral fluid and unbound plasma concentration of warfarin and RS/SR-warfarin alcohols suggests that oral fluid analysis could provide clinically useful information for the monitoring of anticoagulant therapy, complentary to the INR assay

Multi-Analytical Approach to Characterize the Degradation of Different Types of Microplastics: Identification and Quantification of Released Organic Compounds

Microplastics and nanoplastics represent one of the major environmental issues nowadays due to their ubiquitous presence on Earth, and their high potential danger for living systems, ecosystems, and human life. The formation of both microplastics and nanoplastics strongly depends on both the type of pristine materials and the degradation processes related to biological and/or abiotic conditions. The aim of this study is to investigate the effect of two of the most relevant abiotic parameters, namely temperature and light, taken under direct control by using a Solar box, on five types of reference polymers: high density polyethylene (HDPE), low density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PET). A multi-analytical approach was adopted to investigate in detail the first steps of plastics degradation. Samples of plastic materials at different degradation times were analyzed by means of 1H NMR spectroscopy and thermal desorption gas chromatography mass spectrometry (TD-GC-MS) technique. Several minor molecular species released during degradation were consistently identified by both techniques thus providing a comprehensive view of the various degradation products of these five types of microplastics

HiSorb sorptive extraction for determining salivary short chain fatty acids and hydroxy acids in heart failure patients

Variations in salivary short-chain fatty acids and hydroxy acids (e.g., lactic acid, and 3-hydroxybutyric acid) levels have been suggested to reflect the dysbiosis of human gut microbiota, which represents an additional factor involved in the onset of heart failure (HF) disease. The physical-chemical properties of these metabolites combined with the complex composition of biological matrices mean that sample pre-treatment procedures are almost unavoidable. This work describes a reliable, simple, and organic solvent free protocol for determining short-chain fatty acids and hydroxy acids in stimulated saliva samples collected from heart failure, obese, and hypertensive patients. The procedure is based on in-situ pentafluorobenzyl bromide (PFB-Br) derivatization and HiSorb sorptive extraction coupled to thermal desorption and gas chromatography-tandem mass spectrometry. The HiSorb extraction device is completely compatible with aqueous matrices, thus saving on time and materials associated with organic solvent-extraction methods. A Central Composite Face-Centred experimental design was used for the optimization of the molar ratio between PFB-Br and target analytes, the derivatization temperature, and the reaction time which were 100, 60 degrees C, and 180 min, respectively. Detection limits in the range 0.1-100 & mu;M were reached using a small amount of saliva (20 & mu;L). The use of sodium acetate-1-13C as an internal standard improved the intra- and inter-day precision of the method which ranged from 10 to 23%. The optimized protocol was successfully applied for what we believe is the first time to evaluate the salivary levels of short chain fatty acids and hydroxy acids in saliva samples of four groups of patients: i) patients admitted to hospital with acute HF symptoms, ii) patients with chronic HF symptoms, iii) patients without HF symptoms but with obesity, and iv) patients without HF symptoms but with hypertension. The first group of patients showed significantly higher levels of salivary acetic acid and lactic acid at hospital admission as well as the lowest values of hexanoic acid and heptanoic acid. Moreover, the significant high levels of acetic acid, propionic acid, and butyric acid observed in HF respect to the other patients suggest the potential link between oral bacteria and gut dysbiosis

Salivary lipid mediators: Key indexes of inflammation regulation in heart failure disease

Cardiovascular diseases (CVDs) are the leading cause of premature death and disability in humans and their incidence continues to increase. Oxidative stress and inflammation have been recognized as key pathophysio-logical factors in cardiovascular events. The targeted modulation of the endogenous mechanisms of inflamma-tion, rather than its simple suppression, will become key in treating chronic inflammatory diseases. A comprehensive characterization of the signalling molecules involved in inflammation, such as endogenous lipid mediators, is thus needed. Here, we propose a powerful MS-based platform for the simultaneous quantitation of sixty salivary lipid mediators in CVD samples. Saliva, which represents a non-invasive and painless alternative to blood, was collected from patients suffering from acute and chronic heart failure (AHF and CHF, respectively), obesity and hypertension. Of all the patients, those with AHF and hypertension showed higher levels of isoprostanoids, which are key indexes of oxidant insult. Compared to the obese population, AHF patients showed lower levels (p < 0.02) of antioxidant omega-3 fatty acids, in line with the "malnutrition-inflammation complex syndrome" typical of HF patients. At hospital admission, AHF patients showed significantly higher levels (p < 0.001) of omega-3 DPA and lower levels (p < 0.04) of lipoxin B4 than CHF patients, suggesting a lipid rearrangement typical of the failing heart during acute decompensation. If confirmed, our results highlight the potential use of lipid mediators as predictive markers of re-acutisation episodes, thus providing opportunities for preventive inter-vention and a reduction in hospitalizations

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

Quality of life and emotional distress in sarcoma patients diagnosed during COVID-19 pandemic: a supplementary analysis from the SarCorD study

BackgroundThe COVID-19 outbreak had a negative psychological impact on cancer patients. In this study, we analyzed emotional distress and quality of life in patients diagnosed with sarcoma during the first year of the pandemic compared to the previous year.MethodsWe retrospectively enrolled patients with soft tissue, bone sarcoma, and aggressive benign musculoskeletal diseases diagnosed during the pandemic (COVID group) or the year before (control group) at the IRCCS Regina Elena National Cancer Institute in Rome. Patients who had undergone a psychological assessment with the EORTC QLQ-C30 and the Distress Thermometer at diagnosis were included in the final analysis. We analyzed whether there is a difference in the various domains of quality of life between the two groups and whether there are changes over time in each group.ResultsWe enrolled 114 patients (72 control group; 42 COVID group), affected by soft tissue (64%), bone sarcoma (29%), and aggressive benign musculoskeletal diseases (7%). We did not observe significant differences in the health-related quality of life domains in the control and COVID groups, except for the financial domain (p = 0.039), with 9.7% vs. 23.8% of patients with a score > 0 in the control and COVID groups, respectively. We observed emotional distress at diagnosis in 48.6% of patients in the control group vs. 69.0% in the COVID group (p = 0.034). In the control group, we observed an improvement in physical function (p = 0.043) and in QoL (p = 0.022), while in the COVID group, we observed a deterioration in role function (p = 0.044) during follow-up. In the COVID group, 22.2% of patients were concerned about COVID-19, 61.1% by tumor, 91.1% stated that the pandemic worsened their subjective perception of cancer, and 19.4% perceived that their quality of care had worsened.ConclusionWe observed a higher level of distress among patients diagnosed during the pandemic compared to the year before, probably due to the increased concern for both infection and cancer, the worsened perception of health status, and the perception of a poorer quality of health care

Targeted and untargeted lipidomics of oxygenated metabolites of PUFAs: a new frontier in health and disease

Oxylipins are powerful lipid mediators involved in numerous physiological and pathological processes, generated from both omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) via one or more mono- or dioxygen-dependent reactions. The interest in their profiling has considerably grown during recent years, notably in the active field of biomarker discovery. Mass spectrometry-based metabolomics of oxylipins represents a very interesting tool for various applications in clinical research, but the impact of analytical and biological parameters on the variability of oxylipin patterns needs further understanding. The analytical determination of oxylipins still remains challenging, mostly because of their extremely low concentration levels (ppt range). Therefore, particular attention has to be paid on sample pre-treatment to remove matrix interferents and to concentrate analytes, thus increasing the sensitivity as well as improving the detection limits. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, low-cost and low-solvent extraction, and on-line coupling with analytical instruments. Micro-Extraction by Packed Sorbent (MEPS) presents all these desirable characteristics and, thus, it has emerged as a powerful technique, being much more environment-friendly and less expensive than solid-phase extraction (SPE) and liquid-liquid extraction (LLE) approaches. For the first time in this PhD thesis, the very reliable combination of the off-line semi-automated MEPS technique and liquid chromatography-tandem mass spectrometry was applied to the analysis of oxylipins, as isoprostanes, prostaglandins, epoxyeicosatrienoic and hydroxyeicosatetraenoic acids. The MEPS-UHPLC-MS/MS workflow was fully developed and validated for the targeted and non-targeted profiling of oxylipins in a wide array of biological matrices, as Dried Blood Spots (DBSs), oral fluid and plasma. The novel and fast C18-MEPS procedure guaranteed limits of detection much lower than those reported in literature (5-50 ppts), satisfactory recovery (> 85%) and very good intra- and inter-day precision (RSD < 15%) for most of the oxylipins investigated, by substantially reducing the extraction time (10-20 min), the required volume of both solvents (30-500 μL) and samples (50-500 μL). Furthemore, contrary to the SPE, MEPS cartridge was cost-efficient, being reusable up to 100 times without any loss of extraction efficiency (within ± 10%). Our results clearly demonstrated the usefulness of the internal standard addition to improve the analytical performances of the methods, especially for the DBS assay. For the first time, the innovative internal standard addition performed directly on the card before DBS sampling has been demonstrated to be extremely useful for the correction of analyte variation during the critical storage step. This strategy led to a substantial improvement of the entire assay, thus ensuring a broad applicability of the proposed workflow to the clinical field. The validated MEPS-UHPLC-MS/MS platforms were tested within the framework of three exploratory studies aimed at monitoring inflammation status and oxidant injury in preterm newborns suffering from Patent Ductus Arteriosus, in athletes performing an incremental cycle ergometer test and in acute stroke patients. Most of the oxygenated metabolites forming the complex oxylipin network that were detected in our samples (e.g. (F2/E2-isoprostanes, F2-dihomo-isoprostanes and F4-neuroprostanes, epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids) did not match with the standards and remained unknown. Herein, to greatly expand the repertoire of oxylipins assayed, suspect screening profiling by high resolution mass spectrometry was used, trying to extend the spectrum of oxylipins monitored in humans and provide a foundation for new insight into the role of bioactive lipids in mediating health and disease

NEEDLE TRAP MICRO-EXTRACTION: AN INNOVATIVE APPROACH FOR BREATH ANALYSIS. Method development for volatile organic compounds to monitor heart failure

Over the last few years, breath analysis for monitoring metabolic disorders caused by specific diseases has become more and more attractive due to its non-invasiveness. Several approaches have been proposed for sample collection, treatment, and analysis, devoted to the qualitative evaluation of various chemical compounds. This experimental thesis has been devoted to the development and optimization of analytical procedures for the quantitative determination of several chemical compounds in human breath samples. The optimized analytical procedure includes the simultaneous collection of the alveolar breath fraction, analyte pre-concentration in suitable needle trap device, packed with a combination of sorbent materials (i.e. DVB, Carbopack X and Carboxen 1000) followed by GC-MS/MS analysis. The main critical aspect of this approach resulted the influence of the amount of water in the sorbent material on the thermal desorption step, especially for samples characterized by variable humidity levels. In this work, the use of a combination of labelled internal standards, able to represent the different chemical-physical proprieties of the selected 60 volatile organic compounds, was investigated to solve this issue. Finally, the optimized analytical procedure was applied to the analysis of breath samples gathered from patients with heart failure disease. The preliminary results were very promising, and would seem to indicate that this monitoring technique could be used in clinical applications

Biosensing systems for the detection and quantification of methane gas

Abstract: Climate change due to the continuous increase in the release of green-house gasses associated with anthropogenic activity has made a significant impact on the sustainability of life on our planet. Methane (CH4) is a green-house gas whose concentrations in the atmosphere are on the rise. CH4 measurement is important for both the environment and the safety at the industrial and household level. Methanotrophs are distinguished for their unique characteristic of using CH4 as the sole source of carbon and energy, due to the presence of the methane monooxygenases that oxidize CH4 under ambient temperature conditions. This has attracted interest in the use of methanotrophs in biotechnological applications as well as in the development of biosensing systems for CH4 quantification and monitoring. Biosensing systems using methanotrophs rely on the use of whole microbial cells that oxidize CH4 in presence of O2, so that the CH4 concentration is determined in an indirect manner by measuring the decrease of O2 level in the system. Although several biological properties of methanotrophic microorganisms still need to be characterized, different studies have demonstrated the feasibility of the use of methanotrophs in CH4 measurement. This review summarizes the contributions in methane biosensing systems and presents a prospective of the valid use of methanotrophs in this field. Key points: • Methanotroph environmental relevance in methane oxidation • Methanotroph biotechnological application in the field of biosensing • Methane monooxygenase as a feasible biorecognition element in biosensors.</p