Determination of nitrate in blood by gas chromatography and gas chromatography–mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu²⁺ and Zn²⁺ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography–mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Analysis of procarbazine and metabolites by gas chromatography—mass spectrometry

Twelve compounds representing procarbazine, seven metabolites, and an internal standard were analyzed by gas chromatography—mass spectrometry on a 3% OV-1 column. Procarbazine and four metabolites were derivatized with acetic anhydride.A sensitive, specific and quantitative assay was established by selected ion monitoring using a synthetic analogue of the drug as an internal standard. The limits of detection were approximately 1 ng/ml of plasma while the limits of quantitation were 10 ng/ml of plasma.Studies on the degradation of procarbazine - HCl in 0.05 M phosphate buffer (pH 7.4) were compared to in vivo studies. At 1 h after incubation of procarbazine - HCl in buffer, the azo and aldehyde metabolites were detected in the highest concentrations representing 27.2% and 20.3% of total drug and metabolites. In the in vivo studies, analyses of rat plasmas indicated that 1 h after an oral dose of procarbazine - HCl, the aldehyde metabolite represented 72% of the total drug and metabolites, and that relatively little of the azo metabolite was present.     

Mutagenic/carcinogenic agents in indoor pollutants; the dinitropyrenes generated by kerosene heaters and fuel gas and liquefied petroleum gas burners

Incomplete combustion of kerosene heater, and fuel gas and liquefied petroleum gas-burner emissions produces indoor pollutants that may be carcinogenic. The incomplete-combustion products from each type of appliance were therefore collected by adsorption on about 3 g of XAD-2 resin, and were extracted with benzene-methanol as a solvent for determination and identification of mutagens in the Salmonella-microsome test system. Benzene-methanol extracts of the particulates generated by a heater and two burners showed extreme mutagenicity for strains TA97 and TA98 without S9 mix. Based on the results of analysis, a combination of high performance liquid chromatography (h.p.l.c.) and gas chromatography (GC), about 40-80% of the direct-acting mutagenicity in each crude extract showed the same h.p.l.c. and GC retention times as dinitropyrenes (1,3-, 1,6- and 1,8-isomers), and 1-nitropyrene. Moreover, other nitroarenes, 2-nitrofluorene, 1,5- and 1,8-dinitronaphthalene, and 4,4'-dinitrobiphenyl, were detectable in almost all samples, but their contribution to the mutagenicity of each extract was very low. Kerosene heaters were found to generate small amounts (0.2 ng/h) of dinitropyrenes, which are potential mutagens/carcinogens, only after 1 h of operation.     

Novel approaches for regulating gas supply to plant systems <Emphasis Type="Italic">in vitro</Emphasis>: Application and benefits of artificial gas carriers

Summary The composition of the gaseous environment within tissue culture vessels is a critical factor in determining in vitro plant growth and morphogenic responsiveness. Consequently, the provision of an adequate and sustainable oxygen supply for cultured plant cells (including isolated protoplasts), tissues and organs is a crucial prerequisite for optimization and regulation of such cultural responses. During the past decade, research has focused on improving growth and development using artificial gas carriers based on inert perfluorocarbon (PFC) liquids and hemoglobin (Hb) solution. Supplementation of culture media with such artificial oxygen carriers has demonstrated beneficial effects of increased and sustainable cellular mitotic division and subsequent biomass production in a diverse range of plant species, during both short- and longer-term culture. Studies have targeted systems where oxygen availability is actually or potentially a major growth-limiting factor. Undoubtedly, gas carrier-facilitated improvements in regulating the supply of respiratory gases to cultured cells, tissues and organs will have increasingly important biotechnological and practical implications in the context of plant micropropagation, somatic hybridization, transgenic plant production, and secondary product biosynthesis.     

H<Subscript>2</Subscript> enrichment from synthesis gas by <Emphasis Type="Italic">Desulfotomaculum</Emphasis><Emphasis Type="Italic">carboxydivorans</Emphasis> for potential applications in synthesis gas purification and biodesulfurization

Desulfotomaculum carboxydivorans, recently isolated from a full-scale anaerobic wastewater treatment facility, is a sulfate reducer capable of hydrogenogenic growth on carbon monoxide (CO). In the presence of sulfate, the hydrogen formed is used for sulfate reduction. The organism grows rapidly at 200 kPa CO, pH 7.0, and 55°C, with a generation time of 100 min, producing nearly equimolar amounts of H₂ and CO₂ from CO and H₂O. The high specific CO conversion rates, exceeding 0.8 mol CO (g protein)⁻¹ h⁻¹, makes this bacterium an interesting candidate for a biological alternative of the currently employed chemical catalytic water–gas shift reaction to purify synthesis gas (contains mainly H₂, CO, and CO₂). Furthermore, as D. carboxydivorans is capable of hydrogenotrophic sulfate reduction at partial CO pressures exceeding 100 kPa, it is also a good candidate for biodesulfurization processes using synthesis gas as electron donor at elevated temperatures, e.g., in biological flue gas desulfurization. Although high maximal specific sulfate reduction rates (32 mmol (g protein)⁻¹ h⁻¹) can be obtained, its sulfide tolerance is rather low and pH dependent, i.e., maximally 9 and 5 mM sulfide at pH 7.2 and pH 6.5, respectively.     

Reduction of molecular gas diffusion through gaskets in leaf gas exchange cuvettes by leaf‐mediated pores

There is an ongoing debate on how to correct leaf gas exchange measurements for the unavoidable diffusion leakage that occurs when measurements are done in non‐ambient CO₂ concentrations. In this study, we present a theory on how the CO₂ diffusion gradient over the gasket is affected by leaf‐mediated pores (LMP) and how LMP reduce diffusive exchange across the gaskets. Recent discussions have so far neglected the processes in the quasi‐laminar boundary layer around the gasket. Counter intuitively, LMP reduce the leakage through gaskets, which can be explained by assuming that the boundary layer at the exterior of the cuvette is enriched with air from the inside of the cuvette. The effect can thus be reduced by reducing the boundary layer thickness. The theory clarifies conflicting results from earlier studies. We developed leaf adaptor frames that eliminate LMP during measurements on delicate plant material such as grass leaves with circular cross section, and the effectiveness is shown with respiration measurements on a harp of Deschampsia flexuosa leaves. We conclude that the best solution for measurements with portable photosynthesis systems is to avoid LMP rather than trying to correct for the effects.     

The use of formic acid in carrier gas : Rapid method for identification and determination of phytanic acid by gas chromatography—mass spectrometry—chemical ionization

A rapid gas chromatographic method to determine phytanic acid in plasma from Refsum's disease is described. After a brief alkaline hydrolysis of lipids, the biological sample is directly injected into a glass pre-column; an acid carrier gas (formic acid in nitrogen) is used to displace the long-chain fatty acids from their sodium salts and from their binding to proteins. Formic acid introduced through the column may also be used as a reagent gas for chemical ionization in combined gas chromatography—mass spectrometry; fatty acids (C₁ to C_16:2 and phytanic acid) are easily identified by their M + 1 (base peak) and M − 17 peaks. The described procedure is also suitable for studying normal fatty acids from plasma lipids.     

Comparative investigation of fine bubble and macrobubble aeration on gas utility and biotransformation productivity

The sufficient provision of oxygen is mandatory for enzymatic oxidations in aqueous solution, however, in process optimization this still is a bottleneck that cannot be overcome with the established methods of macrobubble aeration. Providing higher mass transfer performance through microbubble aerators, inefficient aeration can be overcome or improved. Investigating the mass transport performance in a model protein solution, the microbubble aeration results in higher k_La values related to the applied airstream in comparison with macrobubble aeration. Comparing the aerators at identical k_La of 160 and 60 1/h, the microbubble aeration is resulting in 25 and 44 times enhanced gas utility compared with aeration with macrobubbles. To prove the feasibility of microbubbles in biocatalysis, the productivity of a glucose oxidase catalyzed biotransformation is compared with macrobubble aeration as well as the gas‐saving potential. In contrast to the expectation that the same productivities are achieved at identically applied k_La, microbubble aeration increased the gluconic acid productivity by 32% and resulted in 41.6 times higher oxygen utilization. The observed advantages of microbubble aeration are based on the large volume‐specific interfacial area combined with a prolonged residence time, which results in a high mass transfer performance, less enzyme deactivation by foam formation, and reduced gas consumption. This makes microbubble aerators favorable for application in biocatalysis.     

Metabolism,gas exchange,and acid‐base balance of giant salamanders

The giant salamanders are aquatic and paedomorphic urodeles including the genera Andrias and Cryptobranchus (Cryptobranchidae), Amphiuma (Amphiumidae), Siren (Sirenidae), and Necturus (Proteidae, of which only N. maculosus is considered ‘a giant'). Species in the genera Cryptobranchus and Necturus are considered aquatic salamanders well adapted for breathing water, poorly adapted for breathing air, and with limited abilities to compensate acid‐base disturbances. As such, they are water‐breathing animals with a somewhat fish‐like respiratory and acid‐base physiology, whose habitat selection is limited to waters that do not typically become hypoxic or hypercarbic (although this assertion has been questioned for N. maculosus). Siren and Amphiuma species, by contrast, are dependent upon air‐breathing, have excellent lungs, inefficient (Siren) or no (Amphiuma) gills, and are obligate air‐breathers with an acid‐base status more similar to that of terrestrial tetrapods. As such, they can be considered to be air‐breathing animals that live in water. Their response to the aquatic hypercarbia that they often encounter is to maintain intracellular pH (pH_i) and abandon extracellular pH regulation, a process that has been referred to as preferential pH_i regulation. The acid‐base status of some present‐day tropical air‐breathing fishes, and of Siren and Amphiuma, suggests that the acid‐base transition from a low PCO₂‐low [] system typical of water‐breathing fishes to the high PCO₂‐high [] systems of terrestrial tetrapods may have been completed before emergence onto land, and likely occurred in habitats that were typically both hypoxic and hypercarbic.     

Measurement of testosterone and pregnenolone in nails using gas chromatography–mass spectrometry

An efficient method for the determination of testosterone and pregnenolone in human nails using gas chromatography–mass spectrometry (GC–MS) with d₃-testosterone as an internal standard is described. The method involves alkaline digestion and liquid–liquid extraction, with subsequent conversion to mixed pentafluoropenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected-ion monitoring (SIM) mode. The limit of detection (LOD) and limit of quantification (LOQ) were lowered to 0.1 and 0.2 pg/g, respectively, when 100 mg of nail-clippings were used. The mean recoveries of testosterone and pregnenolone were 89.8 and 86.7%, respectively, while good overall precision (% C.V.; 4.5–9.5) and accuracy (% bias; 3.9–8.4) were demonstrated. Linearity as a correlation coefficient was 0.9913 (testosterone) and 0.9965 (pregnenolone). When applied to fingernail and toenail samples from seven healthy men and nine healthy women, testosterone and pregnenolone were positively detected in the concentration range of 0.24–5.80 ng/g. The levels of two steroids studied in the nails were found to be higher in the male subjects than in the female subjects, and except for the toenails of the females, the levels of testosterone were higher than those of pregnenolone.     

Subnational greenhouse gas and land‐based biodiversity footprints in the European Union

Insights into subnational environmental impacts and the underlying drivers are scarce, especially from a consumption‐based perspective. Here, we quantified greenhouse gas (GHG) emissions and land‐based biodiversity losses associated with final consumption in 162 regions in the European Union in 2010. For this purpose, we developed an environmentally extended multi‐regional input–output (MRIO) model with subnational European information on demand, production, and trade structures subdivided into 18 major economic sectors, while accounting for trade outside Europe. We employed subnational data on land use and national data on GHG emissions. Our results revealed within‐country differences in per capita GHG and land‐based biodiversity footprints up to factors of 3.0 and 3.5, respectively, indicating that national footprints may mask considerable subnational variability. The per capita GHG footprint increased with per capita income and income equality, whereas we did not find such responses for the per capita land‐based biodiversity footprint, reflecting that extra income is primarily spent on energy‐intensive activities. Yet, we found a shift from the domestic to the foreign part of the biodiversity footprints with rising population density and income. Because our analysis showed that most regions are already net importers of GHG emissions and biodiversity losses, we conclude that it is increasingly important to address the role of trade in national and regional policies on mitigating GHG emissions and averting further biodiversity losses, both within and outside the region itself. To further increase the policy relevance of subnational footprint analyses, we also recommend the compilation of more detailed subnational MRIO databases including harmonized environmental data.     

Land management change greatly impacts biofuels’ greenhouse gas emissions

Harvesting corn stover for biofuel production may decrease soil organic carbon (SOC) and increase greenhouse gas (GHG) emissions. Adding additional organic matter into soil or reducing tillage intensity, however, could potentially offset this SOC loss. Here, using SOC and life cycle analysis (LCA) models, we evaluated the impacts of land management change (LMC), that is, stover removal, organic matter addition, and tillage on spatially explicit SOC level and biofuels’ overall life cycle GHG emissions in US corn–soybean production systems. Results indicate that under conventional tillage (CT), 30% stover removal (dry weight) may reduce baseline SOC by 0.04 t C ha^?1 yr^?1 over a 30‐year simulation period. Growing a cover crop during the fallow season or applying manure, on the other hand, could add to SOC and further reduce biofuels’ life cycle GHG emissions. With 30% stover removal in a CT system, cover crop and manure application can increase SOC at the national level by about 0.06 and 0.02 t C ha^?1 yr^?1, respectively, compared to baseline cases without such measures. With contributions from this SOC increase, the life cycle GHG emissions for stover ethanol are more than 80% lower than those of gasoline, exceeding the US Renewable Fuel Standard mandate of 60% emissions reduction in cellulosic biofuels. Reducing tillage intensity while removing stover could also limit SOC loss or lead to SOC gain, which would lower stover ethanol life cycle GHG emissions to near or under the mandated 60% reduction. Without these organic matter inputs or reduced tillage intensity, however, the emissions will not meet this mandate. More efforts are still required to further identify key practical LMCs, improve SOC modeling, and accounting for LMCs in biofuel LCAs that incorporate stover removal.