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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.

Background and Objective: The determination of warfarin, RS/SR- and RR/SS-warfarin 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  =  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, complementary to the INR assay.

Background: Ultraviolet (UV) absorbance of spent dialysate has been proposed as a method for monitoring hemodialysis efficiency. The contribution of the various uremic toxins to UV absorption, however, needs clarifying., Methods: Urea, creatinine and uric acid were measured in blood and dialysate before and during dialysis in 22 maintenance hemodialysis patients. Absorbance was measured in dialysate., Results: High-performance liquid chromatography (HPLC) analyses of dialysate revealed uric acid as the predominant peak. Spent dialysate absorbance decreased, during dialysis, similarly to serum and dialysate urea, creatinine and uric acid. Dialysate urea correlated closely with absorbance, though urea did not contribute to absorbance, which was determined mostly by uric acid. Uric acid and urea removals were very similar. The spectrophotometric Kt/V correlated with spKt/V urea, with slight but significant differences between the two measurements., Conclusions: UV absorbance is determined mostly by uric acid. Absorbance measurements seem suitable as a method for monitoring dialysis efficiency.

Two analytical procedures are presented for the determination of the total content and unbound fraction of both warfarin and warfarin alcohols in human plasma. Chromatographic separation was carried out in isocratic conditions at 25°C on a C-18 reversed-phase column with a mobile phase consisting of a 70% buffer phosphate 25mM at pH=7, 25% methanol and 5% acetonitrile at a flow rate of 1.2mL/min. Fluorescence detection was performed at 390nm (excitation wavelength 310nm). Neither method showed any detectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcohols at a concentration level of 1000ng/mL was 89±3% and 73±3%, respectively, whereas for their unbound fraction (at a concentration level of 10ng/mL) was 66±8% and 90±7%, respectively. The intra- and inter-day precision (assessed as relative standard deviation) was <10% for both methods. The limits of detection were 0.4 and 0.2ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfully applied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

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.

Background: Patients on warfarin therapy undergo invasive and expensive checks for the coagulability of their blood. No information on coagulation levels is currently available between two controls., Methodology: A method was developed to determine warfarin in oral fluid by HPLC and fluorimetric detection. The chromatographic separation was performed at room temperature on a C-18 reversed-phase column, 65% PBS and 35% methanol mobile phase, flow rate 0.7 mL/min, injection volume 25 µL, excitation wavelength 310 nm, emission wavelength 400 nm., Findings: The method was free from interference and matrix effect, linear in the range 0.2-100 ng/mL, with a detection limit of 0.2 ng/mL. Its coefficient of variation was <3% for intra-day measurements and <5% for inter-day measurements. The average concentration of warfarin in the oral fluid of 50 patients was 2.5±1.6 ng/mL (range 0.8-7.6 ng/mL). Dosage was not correlated to INR (r = -0.03, p = 0.85) but positively correlated to warfarin concentration in the oral fluid (r = 0.39, p = 0.006). The correlation between warfarin concentration and pH in the oral fluid (r = 0.37, p = 0.009) confirmed the importance of pH in regulating the drug transfer from blood. A correlation between warfarin concentration in the oral fluid and INR was only found in samples with pH values ≥7.2 (r = 0.84, p = 0.004)., Conclusions: Warfarin diffuses from blood to oral fluid. The method allows to measure its concentration in this matrix and to analyze correlations with INR and other parameters.

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

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.

This study investigates the potential of pervaporation (a selective membrane separation technique) combined with an electronic nose based on metal oxide sensors for analyzing wine model solutions. Choosing a suitable membrane polymer, it is shown that subtle variations in aroma composition can be detected by the metal oxide sensors, even when 12% (v/v) ethanol is present in the original sample. Simulations of the composition of the permeate demonstrate that, despite the low molecular density of the permeate, the respective solute concentrations are sufficiently high to generate a reproducible and significant response from the metal oxide sensors.

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 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.

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.

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.

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.

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.

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.

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.