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Rapid and quantitative analysis of recombinant protein expression using pyrolysis mass spectrometry and artificial neural networks: application to mammalian cytochrome b5 in Escherichia coli.

Recombinant Escherichia coli clones encoding between 0 and 6 copies of the mammalian cytochrome b5 gene were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra so as to obtain quantitative information on the amount of cytochrome b5 produced fully-interconnected feedforward artificial neural networks (ANNs) were studied. It was found that the combination of PyMS and ANNs could be used to predict the amount of cytochrome b5 expressed in E. coli. PyMS is a novel, convenient and rapid method for the screening and analysis of microbial and other cultures producing recombinant proteins.

Animals↗

Rapid inter-strain comparison by pyrolysis mass spectrometry in nosocomial infection with Xanthomonas maltophilia.

Seventeen strains of Xanthomonas maltophilia and one strain of Pseudomonas cepacia were examined by pyrolysis mass spectrometry (PYMS). The Xanthomonas strains comprised 11 clinical and environmental isolates from a suspected outbreak of colonization and infection on a heart-lung transplant intensive care unit, two strains from patients elsewhere in the same hospital and four strains from a national reference collection. The single isolate of Pseudomonas cepacia was from a sink in the same affected intensive care unit. A series of discriminant analyses performed on the PYMS-derived data showed that, whereas six strains of Xanthomonas from the respiratory tract, blood and ventilatory equipment of one of the affected patients were indistinguishable, all the other isolates were distinct. The results of PYMS rapid inter-strain comparison were in accord with those of an epidemiological investigation which suggested that the episode was due to unauthorized reuse of disposable nebulizers and not to cross-infection between patients. Pyrolysis mass spectrometry with rapid data analysis is a potentially useful technique for the investigation of nosocomial infections due to organisms such as X. maltophilia.

Bacteriological Techniques↗

Nosocomial infection due to enterococci attributed to a fluidized microsphere bed. The value of pyrolysis mass spectrometry.

The potential of fluidised microsphere beds as sources of nosocomial Enterococcal infection was investigated with the help of pyrolysis mass spectrometry. Isolates from clinical specimens collected from two patients who were nursed sequentially on a fluidized microsphere bed were compared with similar isolates cultured from the microspheres before and after decontamination. Pyrolysis mass spectrometry confirmed that nosocomial spread had indeed occurred and that the existing decontamination process was inadequate. Recommendations for improvements to this decontamination process appear to have prevented further cases.

Adult↗

Carcinogenic tryptophan pyrolysis products in cigarette smoke condensate and cigarette smoke-polluted indoor air.

The carcinogenic tryptophan pyrolysis products, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido-[4,3-b]indole (Trp-P-2), have been measured in condensate of cigarette mainstream smoke by high-performance liquid chromatography. These carcinogens have been detected in indoor air as well as in the air of the outdoor environment. Levels of these carcinogens in indoor air were much higher than those in outdoor air. The source of these carcinogens in indoor air was determined to be cigarette smoke by the application of smoking machine studies. Concentrations of these carcinogens in indoor air increased markedly with an increase in cigarettes smoked. The results in this investigation suggest that cigarette smoking is a source of carcinogenic tryptophan pyrolysis products in the indoor environment. Our data also suggest that smokers are persistently exposed to the carcinogenic heterocyclic amines together with potent carcinogens such as polynuclear aromatic hydrocarbons and N-nitroso compounds.

Journal Article↗

The interaction of ionizing radiation and food pyrolysis products in producing oncogenic transformation in vitro.

Short term cultures of golden hamster embryo cells were exposed to X-irradiation and/or to 3-amino-1-methyl-5H-pyrido[4,3,-b]indole (Trp-P-2), a pyrolysis product of GL-tryptophan. Oncogenic transformation was scored following treatment with radiation and the pyrolysate, alone or in combination. Pre-treatment of the cells with 50 rad or 150 rad and subsequent exposure to 0.5 microgram/ml Trp-P-2, resulted in a higher transformation frequency as compared to that observed following exposure to the single agents. The enhanced frequency was related to the absorbed dose of radiation. The data suggest a synergistic interaction between X-rays and the pyrolysis product in their oncogenic action.

Animals↗

Pyrolysis mass spectrometry as a method for the classification, identification and selection of actinomycetes.

Pilot experiments were designed to determine the potential of Curie-point pyrolysis mass spectrometry (PyMS) in the classification, identification and typing of industrially significant actinomycetes, and for the detection of target and novel actinomycetes needed for pharmacological screening programmes. The results indicate that the method is of value for the separation of actinomycetes at and below the species level, in the detection and circumscription of novel actinomycetes, and for the detection of identical and duplicated strains. There is also evidence that the pyrolysis system will permit the identification of target actinomycetes directly from selective isolation plates. PyMS is one of the methods that should be used to generate polyphasic taxonomies of actinomycete genera.

Actinomycetales↗

Gas chromatographic-mass spectrometric analysis of tar compounds formed during pyrolysis of rice husks.

Pyrolysis of agricultural waste to produce fuel gas involves formation of tars as noxious by-products. In this paper the qualitative analysis of tars formed during pyrolysis of rice husks is presented, based on identification by gas chromatography-mass spectrometry and interpolation of retention times on a polyaromatic hydrocarbon index scale. The influence of some reaction parameters on product formation is briefly discussed.

Gas Chromatography-Mass Spectrometry↗

Analysis of methylbenactyzium bromide in human urine by thin-layer chromatography and pyrolysis gas chromatography.

A rapid and simple method of utilizing thin-layer chromatography (TLC) and pyrolysis gas chromatography (PyGC) for the identification and determination of methylbenactyzium bromide in human urine was studied in this report. Methylbenactyzium bromide was extracted from urine with ODS-cartridge (Sep-Pak C18), then spotted onto a silica gel 60 F254 TLC plate. After development, the separated spot of methylbenactyzium bromide was scraped and wrapped with a ferromagnetic foil without extraction by any organic solvents. The sample was applied into PyGC analysis. The optimum temperature for pyrolysis was 590 degrees C. The main degradation product of methylbenactyzium bromide was identified as diphenylmethane in this procedure by gas chromatography/mass spectrometry (GC/MS). A calibration graph prepared by absolute calibration method showed a good linearity over the concentration range of 1-75 micrograms/spot for methylbenactyzium bromide. The coefficient of variation obtained for eleven replicate analyses of the 3 micrograms/spot of standard methylbenactyzium bromide was 3.8%. The detection limit of this compound by this procedure was 0.1 micrograms/spot.

Calibration↗

Sonochemistry of nitrone spin traps in aqueous solutions. Evidence for pyrolysis radicals from spin traps.

When argon-saturated aqueous solutions of alpha-phenyl-N-tert-butylnitrone (PBN) were sonicated, the spin adducts PBN-Phenyl (Ph), PBN-X, and PBN-H were observed. It can be inferred that PBN-Ph and -X arise from spin adducts of thermal decomposition products of PBN induced by the high temperature due to ultrasonic cavitation. The ESR signal of PBN-H was observed at a lower PBN concentration than those of PBN-Ph and PBN-X. The ratios of ESR intensity of PBN-H to those of PBN-Ph and PBN-X increased with the final temperatures of the cavitation bubbles created by different rare gases. The spin adducts of methyl and tert-butyl radicals from the pyrolysis of PBN, induced by the high temperatures due to cavitation, were found from spin trapping experiments in which 3,5-dibromo-2,6-dideuterio-4-nitrosobenzene sulfonate was used as a spin trap. Similar spin adducts induced by pyrolysis were also observed in sonicated aqueous solutions of other nitrone spin traps, such as alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, and alpha-(4-nitrophenol) N-tert-butylnitrone. The greater the hydrophobicity of the spin traps, as measured by the 2-octanol/water partition coefficients, the lower the concentration of spin trap at which methyl radicals generated by thermal decomposition of the spin trap can be observed. The present results indicate that the nonvolatile, highly hydrophobic spin traps accumulate preferentially in the interfacial region of cavitation bubbles where they undergo thermal decomposition during cavitation to produce the radicals.

Chemical Phenomena↗

Upgrading of liquid fuel from the pyrolysis of biomass.

Pyrolysis of biomass was carried out in a fluidized bed unit (5 kg/h) with the objective of maximizing liquid yield. Liquid product formed in pyrolysis was separated into two phases: water phase and oil phase. The oil phase was upgraded by sulfided Co-Mo-P catalyst in an autoclave. Effects of reaction conditions on the product distribution were investigated, and optimal conditions were therefore concluded. Comparison was made by analysis between the raw oil phase and the upgraded liquid fuel. The significant difference between the raw pyrolytic oil and the upgraded oil was that the former was methanol-soluble while the latter was oil-soluble.

Biomass↗

Fast pyrolysis of soybean cake: product yields and compositions.

This study was an investigation of the role of important parameters influencing pyrolysis yields from soybean cake. Experiments were carried out at temperatures ranging from 400 to 700 degrees C, for various nitrogen flow rates, heating rates and particle sizes. The maximum liquid yield was 42.83% at a pyrolysis temperature of 550 degrees C with a sweeping gas rate of 200 cm3 min(-1) and heating rate of 700 degrees C min(-1) for a soybean cake sample having 0.425 < D(p) < 0.85 mm particle size. The various characteristics of liquid product were identified. Thus, the aliphatic sub-fraction of the bio-oil was analysed by GC-MS and further structural analyses of bio-oil and aromatic and polar sub-fractions were conducted using FT-IR and 1H-NMR. The H/C ratios and the structural analysis of the fractions obtained from the biocrudes showed that the fractions were quite similar to currently utilised transport fuels.

Carbon↗

Superheated steam pyrolysis of biomass elemental components and Sugi (Japanese cedar) for fuels and chemicals.

To develop a novel noncatalytic biomass refinery process that can be used as a portable process, superheated steam pyrolysis was investigated to produce both carbonized solid fuels and chemicals using a large-scale reactor. Individual biomass components and native biomass (Sugi, Japanese cedar) were pyrolyzed. Between 150 and 400 degrees C, the vaporizing fractions of cellulose, xylan, and kraft lignin were summarized using a numerical model. Cellulose was converted to glycolaldehyde, furfural, 5-hydroxymethyl furfural and levoglucosan, whereas xylan was converted to glycolaldehyde, furfural, and acetic acid. Kraft lignin produced a slight yield of phenol and guaiacol. The total vaporization fraction of Sugi and its vaporizing rate were explained sufficiently using a numerical model based on the weighted average of the vaporizing properties of the individual components. However, the yields of phenol, guaiacol, and acetic acid were underestimated, while the yields of furfurals and levoglucosan were overestimated. Possible synergetic effects among chemicals in the superheated steam pyrolysis of native biomass were also discussed.

Biomass↗

PAH Growth from the pyrolysis of CPD, indene and naphthalene mixture.

In this study, the addition of cyclopentadienyl (CPDyl) moieties to aromatic rings has been investigated experimenNaphthalene was selected as the representative aromatic compound and its pyrolytic reactivity was studied first to obtain background information for the pyrolysis of cyclopentadiene-indene-naphthalene mixture. The experiments were conducted in a 5 s laminar flow reactor over the temperature range of 700-850 degrees C with 50 degrees C increments. PAH growth from naphthalene pyrolysis is mainly attributed to aryl-aryl addition of naphthyl radicals and naphthalene fragmentation, with lower product formation rates comparing with hydrocarbons with CPDyl moieties. The results indicate that naphthalene is less reactive than the CPDyl containing hydrocarbon radical-molecule addition of CPDyl radicals to naphthalene results in phenanthrene, which can also be formed substantially from other growth pathways. This addition occurs mainly at low temperatures and is less favored due to the competition from more reactive indenyl and CPDyl radicals. CPDyl radical addition to naphthalene exhibits limited aromatic growth due to the aromaticity restrictions of naphthalene. The studies of hydrocarbons with and without CPDyl moieties suggest that the reaction pathways of CPDyl bearing hydrocarbons are different from those without these moieties and cannot be adequately accounted for by the existing acetylene addition and aryl-aryl addition mechanisms.

Cyclopentanes↗

Effects of oxygen, catalyst and PVC on the formation of PCDDs, PCDFs and dioxin-like PCBs in pyrolysis products of automobile residues.

End-of-life vehicles are dismantled to recover reusable parts and then sent to a shredding facility for steel material recovery. The residue generated from the shredding process, so-called automobile shredder residue, is now being mostly disposed in landfill, however landfill will be prohibited in the near future. More recycling and energy recovery must be made by the regulation. The most appropriate method is the thermal treatment that will produce various forms of fuels and less volume of vitrified residue. In this research, an automobile shredder residue was simulated with considering the residue from a shredding factory based on the new vehicle and thermally treated by pyrolysis or gasification to observe the yields of gas, oil and char. In the temperature range of 400-800 degrees C, the yields of gas, oil, and char were 38.7-45.8%, 12.3-18.2%, and 36.0-49.0%, respectively. The optimum pyrolyzing temperature for more oil production was around 600 degrees C. Emission characteristics of the byproducts, PCDDs, PCDFs and dioxin-like PCBs were studied at the temperature of 600 degrees C. The analysis on the concentration and distribution of the isomers in various forms of the products were completed with and without oxygen, PVC, and catalytic metals, in order to observe how the existence of oxygen, PVC, and catalytic metals affect the formation of PCDDs, PCDFs, and dioxin-like PCBs from pyrolysis or gasification process. Without PVC, no PCDDs, PCDFs and dioxin-like PCBs were found in any products. PCDFs were predominant in any conditions. Catalyst and oxygen were found to affect to form more PCDDs, PCDFs, and dioxin-like PCBs. Effects of catalyst and oxygen on the byproduct formation seemed to be similar in oil and gas while that was different in char.

Automobiles↗

Gas chromatographic-mass spectrometric determination of polycyclic aromatic hydrocarbons formed during the pyrolysis of phenylalanine.

The formation of polycyclic aromatic hydrocarbons (PAHs) during pyrolysis process of phenylalanine had been studied. Ten PAHs, including fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[k]fluoranthene, benzo[e]pyrene, and benzo[a]pyrene were analyzed by gas chromatography-mass spectrometry using selective ion monitoring mode. This technique offers the capability to analyze trace amounts of PAHs in phenylalanine pyrolyzates. The pyrolysis was carried out in a micro-furnace with quartz furnace liner. The injection was conducted with glass pelletizer syringe to avoid metal contamination. Qualitative results were obtained at 900 degrees C and quantitative analysis of 10 PAHs was done for 700 and 900 degrees C.

Gas Chromatography-Mass Spectrometry↗

Analysis of polycyclic aromatic hydrocarbons in contaminated soil by Curie point pyrolysis coupled to gas chromatography-mass spectrometry, an alternative to conventional methods.

Curie point pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) has been compared with classical extraction procedures (Soxhlet, sonication, KOH digestion, microwave-assisted) followed by GC-MS analysis for the determination of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil. In each case, the efficiency of the technique was examined for 16 PAHs included in the US Environmental Protection Agency Priority Pollutant List. The results indicate that the recovery of PAHs is dependent on the extraction technique. The highest recoveries of PAHs were obtained with Curie point pyrolysis and KOH digestion. Py-GC-MS appeared to be interesting alternative method for the determination of PAHs in contaminated soil. The results were validated by certified soil (CRM 104) analysis.

Gas Chromatography-Mass Spectrometry↗

Analytical characterization of diterpenoid resins present in pictorial varnishes using pyrolysis-gas chromatography-mass spectrometry with on line trimethylsilylation.

A procedure based on the technique of the pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) has been applied, in this work, in order to determine the composition of diterpenoid resin employed in art works. The method is based on the on line derivatization of these resins using hexamethyldisilazane (HMDS). Results obtained were compared with those previously reported in literature from Venice turpentine, Strasbourg turpentine, colophony, sandarac and Manila copal using this same method and with those others from in situ thermally assisted hydrolysis and methylation with tetramethylammonium hydroxide (TMAH). Canada balsam, copper resinate and Copaiba balsam have been also analyzed extending the scope of this method in the field of the analysis of artwork materials. Several non-reported trimethylsilylated derivatives of compounds present in the diterpenoid resins have been identified. An improvement in sensitivity has been obtained by using HMDS as derivatizing reagent, together with a better resolution of the most representative peaks. Additionally, this method reduces the number of pyrolytic fragmentation, recombination, dehydration and isomerization products formed during the pyrolysis process and, in consequence, more simplified chromatograms are obtained. Finally, the reported procedure has been successfully used for characterizing two diterpenoid resin-based varnishes present in the canvas painting "The Betrothal of the Virgin" (Anonymous, 17th century) which is included in the pictorial collection of Saint Joseph Church in Taormina (Italy) and the Magdalena Tryptich (Master of Alzira, 16th century, Valencia, Spain).

Diterpenes↗

First identification of benzo[ghi]naphtho[8,1,2-bcd]perylene as a product of fuel pyrolysis, using high-performance liquid chromatography with diode-array ultraviolet-visible absorbance detection and mass spectrometry.

We present HPLC/UV/MS evidence to support the identification of benzo[ghi]naphtho[8,1,2-bcd]perylene as a product of supercritical toluene pyrolysis. Mass spectral data confirm that compound I-eluting in between co-eluting benzo[a]coronene/phenanthro[5,4,3,2-efghi]perylene and benzo[pqr]naphtho[8,1,2-bcd]perylene, all three of which have been unequivocally identified as C(28)H(14) products of toluene pyrolysis-is also a C(28)H(14) product component. The UV spectrum of compound I is presented, and indicates that it is a benzenoid polycyclic aromatic hydrocarbon (PAH). Five of the eight benzenoid C(28)H(14) PAH isomers have published UV spectra, and characteristics of the remaining three are deduced from annelation theory. Only one of these compounds, benzo[ghi]naphtho[8,1,2-bcd]perylene, is predicted to have a UV spectrum with characteristics that we find in the spectrum of compound I. In addition, benzo[ghi]naphtho[8,1,2-bcd]perylene is the only benzenoid C(28)H(14) isomer whose length-to-breadth ratio is consistent with the HPLC retention time of compound I. The reaction mechanism through which benzo[ghi]naphtho[8,1,2-bcd]perylene is formed in this environment is shown, and is consistent with reaction pathways of other large PAH found in this product mixture.

Aza Compounds↗