Kent J. Voorhees

Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was investigated as a method for the rapid identification of whole bacteria, either by comparison with archived reference spectra or by co-analysis with cultures of known bacteria. Bacteria were sampled from colonies on an agar plate, mixed with the matrix, air-dried, and introduced in batches into the mass spectrometer for analysis. In the first experiment, both bacterial strains that had been previously analyzed to obtain reference spectra and other strains that had not been analyzed were blind-numbered and their spectra were obtained. Those strains that matched reference spectra were found to be correctly identified. A second experiment involved co-analysis of reference strains and bind-numbered strains under identical conditions; species-specific identification was demonstrated by comparison of spectra of the blind-numbered strains with those of the standards. In all of the spectra obtained in these experiments, each bacterial strain showed a few characteristic high-mass ions which are thought to be derived from bacterial proteins. This work represents the first reported instance of successful bacterial chemotaxonomy by MALDI-TOFMS analysis of whole cells. For the strains tested, the method is rapid and simple.

Hydrolysis of Polylactic Acid (PLA) and Polycaprolactone (PCL) in Aqueous Acetonitrile Solutions: Autocatalysis

Polylactic acid (PLA) is a hydrolytically degradable aliphatic polyester. The rate of polymer hydrolysis increases with time, and that has been attributed to the high reactivity of the terminal ester and the kinetics of autocatalysis. Hydrolysis is carried out in an acetonitrile/water solution to eliminate any solid-state contributions such as diffusion and crystallinity to the degradation process. A kinetic equation is derived to describe the autocatalytic effect of the increasing carboxylic acid end-group concentration. The results of solution hydrolysis are examined and found to fit the derived equation. Hydrolysis was also carried out with polycaprolactone (PCL) in acetonitrile, where reaction kinetics were found to differ from those of PLA. The PCL polymer required external acid catalysis by the addition of HCl, whereas hydrolysis of PLA was “selfcatalyzed” by the carboxylic acid end-groups.

Water transport in polylactic acid (PLA), PLA/ polycaprolactone copolymers, and PLA/polyethylene glycol blends

Polylactic acid (PLA) is a hydrolytically degradable aliphatic polyester, and water vapor permeability may have a significant influence on the rate of degradation. A method is devised to use bags prepared from PLA films and filled with molecular sieves to determine the water vapor permeability in the polymer, its copolymers with caprolactone, and blends with polyethylene glycol. The “solution-diffusion” model is used to determine the permeability parameters. These include the solubility coefficient,S, a measure of the equilibrium water concentration available for hydrolysis and the diffusion coefficient,D, which characterizes the rate of water vapor diffusion into the film under specific conditions. Values ofS andD at 50‡C and 90% relative humidity ranged from 400 × 10-6 to 1000 × 10-6 cm3 (STP)/(cm3 Pa) and 0.20 × 10-6 to 1.0 × 10-6 cm2/s, respectively. TheS andD coefficients were also measured at 20 and 40‡C and compared to those of other polymers. The degree of crystallinity was found to have little influence on the measured permeability parameters. The heat of sorption, δHS, and the activation energy of diffusion, ED, were used to show that the permeability process is best described by the “water cluster” model for hydrophobic polymers. Finally, the diffusion coefficient is used to compare the rate of water diffusion to the rate of water consumption by ester hydrolysis. Results indicate that hydrolytic degradation of PLA is reaction-controlled.

Pathogenic bacteria: Their detection and differentiation by rapid lipid profiling with pyrolysis mass spectrometry

Abstract Pyrolysis mass spectrometry (Py–MS) can be used to profile methylated fatty acids from bacterial pathogens without a chromatographic step. An in situ thermal hydrolysis and methylation (THM) step incorporated into the Py–MS analysis of whole bacteria reduces the sample preparation time from 60 min to less than 1 min. Detection of four bacterial pathogens with a field-portable aerosol-sampling Py–ion trap MS using FAME profiles from whole bacteria is demonstrated with a total analysis time of less than 10 min/lipid profile.

A rapid approach for the detection of dipicolinic acid in bacterial spores using pyrolysis/mass spectrometry.

Curie-point pyrolysis/triple quadrupole mass spectrometry and micro-tube furnace pyrolysis/quadrupole ion trap mass spectrometry have been used to detect dipicolinic acid (DPA) in sporulated whole bacteria. DPA in whole cells of sporulated Bacillus anthracis reacted in situ during pyrolysis with tetramethylammonium hydroxide to form the dimethyl ester derivative of DPA, dimethyl-2,6-dipicolinate (mDPA). The mDPA was identified by its positive-ion electron ionization fragmentation pattern and confirmed with tandem mass spectrometry. In an oxidative pyrolysis/quadrupole ion trap instrument, the mDPA mass spectrum showed characteristic positive-ion electron ionization fragmentation along with a significant [M+1]+ ion due to self-chemical ionization. The characteristic collision-induced dissociation fragments of mDPA were used to establish the presence of sporulation in B. anthracis whole cells at a concentration of 2.2 x 10(7) CFU (colony-forming units)/mL using the triple quadrupole instrument. The total time for analysis, including sample preparation, was less than 10 minutes for both instruments.

An investigation of the thermal degradation of poly(ethylene glycol)

Abstract The products from the thermal degradation of poly(ethylene glycol) (PEG) in a nitrogen atmosphere have been studied by GC/MS. The results of these studies show that PEG degrades in nitrogen by a series of competitive intermolecular and intramolecular processes. Following the homolytic cleavage of the CC or CO bonds, disproportionation and hydrogen abstraction reactions can be used to explain the observed thermal decomposition products. This proposed mechanism is compared with the existing PEG thermal decomposition literature.

Diesel particle filter and fuel effects on heavy-duty diesel engine emissions.

The impacts of biodiesel and a continuously regenerated (catalyzed) diesel particle filter (DPF) on the emissions of volatile unburned hydrocarbons, carbonyls, and particle associated polycyclic aromatic hydrocarbons (PAH) and nitro-PAH, were investigated. Experiments were conducted on a 5.9 L Cummins ISB, heavy-duty diesel engine using certification ultra-low-sulfur diesel (ULSD, S ≤ 15 ppm), soy biodiesel (B100), and a 20% blend thereof (B20). Against the ULSD baseline, B20 and B100 reduced engine-out emissions of measured unburned volatile hydrocarbons and PM associated PAH and nitro-PAH by significant percentages (40% or more for B20 and higher percentage for B100). However, emissions of benzene were unaffected by the presence of biodiesel and emissions of naphthalene actually increased for B100. This suggests that the unsaturated FAME in soy-biodiesel can react to form aromatic rings in the diesel combustion environment. Methyl acrylate and methyl 3-butanoate were observed as significant species in the exhaust for B20 and B100 and may serve as markers of the presence of biodiesel in the fuel. The DPF was highly effective at converting gaseous hydrocarbons and PM associated PAH and total nitro-PAH. However, conversion of 1-nitropyrene by the DPF was less than 50% for all fuels. Blending of biodiesel caused a slight reduction in engine-out emissions of acrolein, but otherwise had little effect on carbonyl emissions. The DPF was highly effective for conversion of carbonyls, with the exception of formaldehyde. Formaldehyde emissions were increased by the DPF for ULSD and B20.

Analysis of insoluble carbonaceous materials from airborne particles collected in pristine regions of colorado

Abstract Atmospheric particulate samples collected from remote mountainous locations in Colorado were extracted in sequence with methanol, methylene chloride, acetone and hexane. The resulting insoluble material was investigated using pyrolysis-mass spectrometry (Py-MS) and accelerator mass spectrometric 14 C analysis. The pyrolysis produced a complex mixture of aromatic and aliphatic hydrocarbons. The aliphatic hydrocarbons were postulated as being derived from vegetation. The 14 C analysis and the GC/MS results suggested that the aromatics occurred from combustion of living materials and also from long-range transport of industrial pollution. Appropriate samples from power plants and controlled forest fires were used to substantiate the conclusions.

Amino acid and oligopeptide analysis using Curie-point pyrolysis mass spectrometry with in-situ thermal hydrolysis and methylation: mechanistic considerations

Abstract In-situ thermal hydrolysis and methylation-mass spectrometry has been used to study amino acids and oligopeptides containing glycine and alanine. Using tetramethylammonium hydroxide (TMAH) during Curie-point pyrolysis, methylation occurred on groups containing acidic hydrogen moeities (O–H and N–H groups). Multiple methylation reactions were found to occur on compounds containing more than one such functionality. Thermal dehydration reactions to form cyclic dipeptides were observed for oligopeptides as well as amino acids during pyrolysis with TMAH. The methylation and thermal dehydration mechanisms are discussed. In addition, the decomposition mechanisms of amino acids and oligopeptides are presented.

The use of biomarker compounds for the identification of bacteria by pyrolysis-mass spectrometry

Abstract Several recent advances in using biomarkers for bacteria characterization are discussed. A study using Gram-positive and Gram-negative bacteria has been conducted to compare both gas chromatographic and mass spectrometric analyses of the separately isolated and methylated fatty acids to an in situ reaction in which the saponification and methylation of the fatty acids is achieved using tetramethylammoninium hydroxide (TMAH). Principal components analysis of the three data sets showed the same three cluster patterns. In situ methylation using TMAH has also been conducted on free nucleotides, oligonucleotides, DNA, various amino acids, and oligopeptides. An increase in the volatility of important biomarkers was observed in all analyses which leads to an increase in the information content of the data. High resolution mass spectrometry has also been applied to the peaks corresponding to a series of biomarker compounds. The data show that most nominal masses are composed of several contributing species.