A sampler prototype for the simultaneous collection of exhaled air and breath condensate.

Exhaled air and breath condensate contain a large number of health biomarkers, such as volatile and semi-volatile organic compounds, proteins and lipids. Nowadays, the collection of breath samples is carried out by commercial or lab-made sampling systems that collect only one type of sample (e.g. gaseous or condensate phase), thus limiting the diagnostic capability of breath tests. This work presents a portable prototype optimized for the simultaneous collection of gaseous exhaled breath and exhaled breath condensate within five minutes. The system is fully portable and has a total weight of about 1 Kg. An illustrative determination of ethanol, isoprene, acetone, isopropyl alcohol, 1-propanol, 2-butanone, 2-pentanone, toluene and xylenes in breath, and cortisol and 8-iso-prostaglandin F2α in breath condensate is discussed.

Determination of carbonyl compounds in exhaled breath by on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry.

A reliable method for the determination of carbonyl compounds in exhaled breath based on on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry is described. The analytical performances were optimized for a mixture of C2-C9 aldehydes and C3-C9 ketones, particularly interesting for clinical applications, by using an internal standard and applying a 23 full factorial design. A volume of sample (250 ml) was loaded at 50 ml min-1 into a Tenax GR sorbent tube containing 130 nmol of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride. All compounds showed a limit of detection lower than 200 pptv. The yield of the derivatization procedure was normalized by adding to the sample a known amount of 6D-acetone as an internal standard. This allowed halving the relative standard deviation to 10% and 15% for the mono-and di-carbonyl compounds, respectively, thus improving reliability. The optimized method was applied to the determination of carbonyl compounds in 12 breath samples collected from four patients suffering from heart failure during hospitalization.

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 volatile organic compounds (VOCs) belonging to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics and sulfurs). NTME is an emerging technique that guarantees detection limits in the 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-1. 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 (HF) 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 HF exacerbation.

Temperature and pH sensors based on graphenic materials.

Point-of-care applications and patients' real-time monitoring outside a clinical setting would require disposable and durable sensors to provide better therapies and quality of life for patients. This paper describes the fabrication and performances of a temperature and a pH sensor on a biocompatible and wearable board for healthcare applications. The temperature sensor was based on a reduced graphene oxide (rGO) layer that changed its electrical resistivity with the temperature. When tested in a human serum sample between 25 and 43°C, the sensor had a sensitivity of 110±10Ω/°C and an error of 0.4±0.1°C compared with the reference value set in a thermostatic bath. The pH sensor, based on a graphene oxide (GO) sensitive layer, had a sensitivity of 40±4mV/pH in the pH range between 4 and 10. Five sensor prototypes were tested in a human serum sample over one week and the maximum deviation of the average response from reference values obtained by a glass electrode was 0.2pH units. For biological applications, the temperature and pH sensors were successfully tested for in vitro cytotoxicity with human fibroblast cells (MRC-5) over 24h.

A graphene oxide pH sensor for wound monitoring.

This article describes the fabrication and characterization of a pH sensor for monitoring the wound status. The pH sensitive layer consists of a graphene oxide (GO) layer obtained by drop-casting 5 µÎ of GO dispersion onto the working electrode of a screen-printed substrate. Sensitivity was 31.8 mV/pH with an accuracy of 0.3 unit of pH. Open-circuit potentiometry was carried out to measure pH in an exudate sample. The GO pH sensor proved to be reliable as the comparison with results obtained from a standard glass electrode pH-meter showed negligible differences (<; 0.09 pH units in the worst case) for measurements performed over a period of 4 days.

Comparison of sampling bags for the analysis of volatile organic compounds in breath.

Nalophan, Tedlar and Cali-5-Bond polymeric bags were compared to determine the most suitable type for breath sampling and storage when volatile organic compounds are to be determined. Analyses were performed by thermal desorption gas chromatography mass spectrometry. For each bag, the release of contaminants and the chemical stability of a gaseous standard mixture containing eighteen organic compounds, as well as the CO2 partial pressure were assessed. The selected compounds were representative of breath constituents and belonged to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics, sulfurs and esters). In the case of Nalophan, the influence of the surface-to-volume ratio, related to the bag's filling degree, on the chemical stability was also evaluated. Nalophan bags were found to be the most suitable in terms of contaminants released during storage (only 2-methyl-1,3-dioxalane), good sample stability (up to 24 h for both dry and humid samples), and very limited costs (about 1 € for a 20 liter bag). The (film) surface-to-(sample) volume ratio was found to be an important factor affecting the stability of selected compounds, and therefore we recommended to fill the bag completely.

A breath sampling system assessing the influence of respiratory rate on exhaled breath composition.

This work presents a computerized system to monitor mouth pressure, tidal volume, exhaled airflow, respiration rate and end-tidal partial pressure of CO2 during breath collection. The system was used to investigate the effect of different respiratory rates on the volatile organic compounds (VOCs) concentrations in exhaled breath. For this purpose, VOCs with well-defined biochemical pathways and different chemical and physical properties were selected as biomarkers related to metabolism (acetone and isopropyl alcohol), cholesterol synthesis (isoprene) and intestinal microflora activity (ethanol). Mixed breath was collected from a nominally healthy volunteer in resting conditions by filling a Nalophan bag. The subject followed a regimented breathing pattern at different respiratory rates (10, 30 and 50 breaths per minute). Results highlight that ventilation pattern strongly influences the concentration of the selected compounds. The proposed system allows exhaled breath to be collected also in patients showing dyspnea such as in case of chronic heart failure, asthma and pulmonary diseases.

Post-operative elimination of sevoflurane anesthetic and hexafluoroisopropanol metabolite in exhaled breath: pharmacokinetic models for assessing liver function.

Sevoflurane (SEV), a commonly used anesthetic agent for invasive surgery, is directly eliminated via exhaled breath and indirectly by metabolic conversion to inorganic fluoride and hexafluoroisopropanol (HFIP), which is also eliminated in the breath. We studied the post-operative elimination of SEV and HFIP of six patients that had undergone a variety of surgeries lasting between 2.5 to 8.5 h using exhaled breath analysis. A classical three compartments pharmacokinetic model developed for the study of environmental contaminants was fitted to the breath data. We found that SEV kinetic behavior following surgery (for up to six days) is consistent across all subjects whereas the production and elimination of HFIP varies to some extent. We developed subject specific parameters for HFIP metabolism and interpreted the differences in the context of timing and dose of anesthesia, type of surgery, and specific host factors. We propose methods for assessing individual patient liver function using SEV as a probe molecule for assessing efficiency of liver metabolism to HFIP. This work is valuable not only for the clinical study of metabolism recovery, but potentially also for the study of the interaction of other manufactured and environmental compounds with human systems biology in controlled exposure and observational studies.

Monitoring breath during oral glucose tolerance tests.

The evolution of breath composition during oral glucose tolerance tests (OGTTs) was analysed by thermal desorption/gas chromatography/mass spectrometry in 16 subjects and correlated to blood glucose levels. The glucose tolerance tests classified five of the subjects as diabetics, eight as affected by impaired glucose tolerance and three as normoglycaemic. Acetone levels were generally higher in diabetics (average concentration values: diabetics, 300 ± 40 ppbv; impaired glucose tolerance, 350 ± 30 ppbv; normoglycaemic, 230 ± 20 ppbv) but the large inter-individual variability did not allow us to identify the three groups by this parameter alone. The exhalation of 3-hydroxy-butan-2-one and butane-2,3-dione, likely due to the metabolization of glucose by bacteria in the mouth, was also observed. Future work will involve the extension of the analyses to other volatile compounds by attempting to improve the level of discrimination between the various classes of subjects.

Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry.

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC-MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants.

Development of an SdFFF-ETAAS hyphenated technique for dimensional and elemental characterization of colloids.

Direct hyphenation of electrothermal atomic-absorption spectroscopy (ETAAS) to sedimentation field-flow fractionation (SdFFF) has been developed to enable elemental characterization of submicron particles as a function of size. This hyphenation is particularly suitable for characterizing colloidal particles of environmental interest, for example water-borne particles. The interface is an automatic capillary injection device (CID) which enables direct introduction of large and variable volumes of colloidal particle suspensions into a hot graphite furnace, thus preconcentrating the colloidal particles on the furnace walls. The method was validated by determination of Fe in certified submicron Fe2O3. The procedure was set up by first optimizing the SdFFF fractionation under programmed field conditions, thus enabling optimum fractionation of particle size. The ETAAS procedure was then tested to determine whether it could be used for direct analysis of Fe2O3 slurries without the need for a mineralization step. CID coupled to ETAAS was subsequently exploited for its ability to enhance the sensitivity, because of the increased injection volume. Statistical tests and data handling were conducted to prove the suitability of the ETAAS-CID module. Finally, off-line and on-line ETAAS-CID-SdFFF hyphenation were investigated. These experiments emphasized the advantages of the on-line coupling, because it enables synchronized sampling, enrichment, and elemental analysis of the flowing eluate. The benefits of the proposed hyphenation are the high specificity of analytical detection, increased sensitivity, reduction of analysis time, and minimum sample handling and contamination.

Polychlorobiphenyls and polycyclic aromatic hydrocarbons in the sea-surface micro-layer and the water column at Gerlache Inlet, Antarctica.

The enrichment of PCBs (polychlorobiphenyls) and PAHs (polycyclic aromatic hydrocarbons) in the sea-surface micro-layer and depth profile of these pollutants in the water column were investigated at Gerlache Inlet, Terra Nova Bay, Antarctica. Depth profile samplings were repeated three times during the Antarctic summer (from November to February). PCBs and PAHs showed a concentration range in the water column of 30-120 pg l(-1) and 150-400 pg l(-1), respectively, and these values were very much dependent on the suspended matter content. A nearly two-fold decrease in the pollutant concentration was also observed in the depth profile obtained in February, i.e. late summer, which might be correlated both with the high content of suspended matter and the reduction of the pollutant input. Moreover, isomer ratios of PAHs, such as LMW/HMW and PHE/ANT, highlight that the main PAH source might be petrogenic in nature, whereas the pyrolytic source seems to be less important. Sea surface micro-layer (SML) and sub-surface sea water (SSW) samples were simultaneously collected in the same site by a remote controlled rotating drum-based sampling system, a prototype named MUMS (Multi-User Micro-layer Sampler). Sea surface micro-layer samples showed a total content of PCBs and PAHs in the range 400-450 pg l(-1) and 2000-3000 pg l(-1), respectively, whereas the mean content of the sub-surface sea water samples was 48 pg l(-1) and 325 pg l(-1), respectively. The mean enrichment factors of PCBs and PAHs in sea-surface micro-layer were about 10 and 7, respectively. The surface excess concentrations of PCBs and PAHs were about 35 000 and 200 000, respectively. A fairly good correlation was observed between the concentration of pollutants and water solubility. Based on the assumption that POPs are confined in a very thin top layer of the SML about 0.01-0.001 microm thick, namely the sea-surface nano-layer, and also on an estimated thickness of the sampled sea-surface layer of about 100 microm, an enrichment factor of 10(5)-10(6) for the sea-surface nano-layer was calculated. Such a very high concentration increase was related to the two-fold increase of PAH concentration observed in the underlying 20 cm of the water column in late summer.

Photodegradation of Polychlorinated Dibenzo-p-dioxins in Liquid Samples by Near UV Light Irradiation

Photodegradation of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) in hexane solution was studied under controlled near-UV light exposure in the spectral region from 325 to 269 nm. GC-MS was used to detect the amount of unreacted dioxin and to characterize the relevant degradation products. Irradiation experiments carried out at a constant light energy (700 mJ) showed that the percentage of 1,2,3,4-TCDD left in the solution after irradiation changed from about 55 to 75%, with a minimum of 55% at 310 nm. Further irradiation experiments carried out at two wavelengths, namely 310 and 269 nm, and light energy ranging from 0 to 4000 mJ, showed that the photodegradation reaction of the TCDD always followed a pseudo-first-order kinetic, with a rate constant of 8 x 10(-4) and 5 x 10(-4) mJ-1, respectively. These experiments also showed that trichloro- and dichloro-dibenzo-p-dioxins were produced with less than 15% of the initial quantity of TCDD, leading to the conclusion that the dechlorination process is a minor photolysis pathway.

Decomposition Products of s-Triazine Herbicides by Electron-Transfer in Acidic Aqueous Media

The reduction of atrazine and terbutylazine was preceded by protonation equilibrium. Three protonation sites of the s-triazine molecule determined the structure of the final reduction product. Protonation was investigated by the change of UV-Vis spectra. Two slightly different pKs corresponding to protonation on N5 and N1 heteroatoms were evaluated. The principal reduction pathway involved the cleavage of a chlorine atom. A small quantity of desethylatrazine was detected in the most acidic media.

Polychlorobiphenyls in Antarctica

The presence and the distribution of polychlorobiphenyls (PCBs) in Antarctic environmental components and the effect of the seasonal formation/melting process of pack ice on the pollution level of seawater were investigated. Seawater, marine, and lake sediment and soil samples were collected in a large area of the Ross Sea and Victoria Land during the 1988-1989, 1989-1990, 1990-1991, and 1991-1992 Italian expeditions. The results obtained highlighted a low and quite homogeneous PCB contamination of the studied area. Surface seawater samples from Gerlache Inlet and Wood Bay showed a typical PCB concentration of 130 pg/liter, and an increase after pack ice melting of about 30-40%. Marine sediment, lake sediment, and soil samples showed normalized mean PCB contents of 150, 240, and 130 (pg/g)/(m2/cm3), respectively.

Rapid and sensitive determination of urinary 2,5-hexanedione by reversed-phase high-performance liquid chromatography.

A rapid and sensitive method for the determination of 2,5-hexanedione (HD) (the principal metabolite of n-hexane) in urine samples by reversed-phase high-performance liquid chromatography (HPLC) is described. The sample preparation procedure was based on solid-liquid extraction after acid hydrolysis; it was optimized to enable accurate HD determination in less than 30 min. Analysis of spiked real samples showed a recovery of more than 85% at the 0.1-ppm level, with a relative standard deviation of 5% and a detection limit as low as 0.01 ppm. Intra-assay and inter-assay coefficients of variation at the 0.5-ppm level were 4 and 5%, respectively. The chromatographic peak assigned to HD was identified by collecting the HPLC eluate at the retention time of HD and analysing it using Fourier transform infrared spectrometry coupled with high-resolution gas chromatography. Urine samples of unexposed and exposed subjects were analysed following the proposed analytical procedure. HPLC and high-resolution gas chromatographic analyses were also compared on these samples. A correlation factor of 0.992 was obtained, which showed a good agreement between the two sets of data.

Behaviour of some dialkyl- and trialkyl-lead compounds in the hydride-generation procedure using a non-dispersive atomic-fluorescence detector.

The method of generating covalent hydrides by reduction with sodium tetrahydroborate was applied to aqueous solutions containing traces of R(3)Pb(+) and R(2)Pb(2+) compounds (R = methyl, ethyl). For each compound the effects of sample acidity, sodium tetrahydroborate and hydrogen peroxide concentrations, reaction-vessel and transfer-line materials, were measured and the experimental conditions defined for obtaining maximum sensitivity. Experimental evidence indicating the formation of organolead hydrides during the reduction step at room temperature was adduced. The reaction efficiency was found to be 90% for trimethyl-, triethyl- and diethyl-lead and 59% for dimethyl-lead. In the case of inorganic lead the efficiency was only 27%. Without an intermediate preconcentration step, the detection limits obtained for trimethyl-, triethyl-, dimethyl- and diethyl-lead (3 times the standard deviation) were 3-5 ng of lead per litre. A procedure for discriminating between R(3)Pb(+), R(2)Pb(2+) and Pb(2+) compounds is proposed.

Simultaneous determination of arsenic, selenium, tin and mercury by non-dispersive atomic fluorescence spectrometry.

A procedure is described for simultaneous determination of arsenic, selenium, tin and mercury in aqueous solution by non-dispersive atomic-fluorescence spectrometry. Radiofrequency-excited EDLs, 100% modulated in the kHz region, were used for atom excitation. Sodium tetrahydroborate was used as reductant and a hydrogen-argon miniflame as atomizer. In the optimized procedure, which uses 1 ml of sample, the limits of detection (three times the standard deviation of the blank) were 0.04, 0.08, 0.1 and 0.1 ng ml for arsenic, selenium, tin and mercury respectively. The linear dynamic range was greater than three decades for all analytes and the precision was better than 7% (typically 3%) for concentrations 1 ng ml . Results for mutual interference effects are reported. Copper, nickel, lead and cobalt interfered only with selenium (5 ng ml ), when present in at least 200-fold weight ratio to it. Using 5 ml of sample improved the limits of detection for selenium and arsenic (0.01 and 0.02 ng ml respectively), but at the expense of greater interference. Recovery from spiked natural water samples was better than 95% at the ng ml level, except for selenium in sea-water, when the recovery was only 85%. Determination of the four elements, including standard-addition and background measurements, requires about 10 min.

Determination of manganese at ng ml levels in natural waters by differential pulse polarography.

A procedure based on differential pulse polarography is described for the determination of manganese at ng ml levels in fresh, estuarine and sea-water buffered at pH 9.5 with a citrate-borate solution that also serves as supporting electrolyte. The procedure is unaffected by the potentially interfering compounds most likely to occur in natural waters. Furthermore, iron (in ascorbate-borate buffer, pH 9.5) or chromium (in ascorbate-ammonia-ammonium-chloride buffer, pH 9.8) can be determined together with manganese. Some results for the concentration of manganese, iron and chromium in the River Arno are reported.