Gunilla Westerdahl

Determination of environmental levels of peptidoglycan and lipopolysaccharide using gas chromatography with negative-ion chemical-ionization mass spectrometry utilizing bacterial amino acids and hydroxy fatty acids as biomarkers.

D-Alanine and diaminopimelic acid originating from bacterial peptidoglycans and hydroxy fatty acids from lipopolysaccharides (endotoxins) were analysed by gas chromatography using a chiral column (Chirasil-Val as stationary phase) and selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry. The amino acids were analysed as N-heptafluorobutyryl isobutyl esters after rapid hydrolysis of peptidoglycan followed by isolation of the amino acids with disposable ion-exchange columns. Racemization of amino acid enantiomers was controlled by using deuterium chloride in the hydrolysis. The hydroxy acids were analysed as O-pentafluorobenzoyl methyl esters. Most of the bacteria present in airborne dust from a poultry confinement building were found to be Gram-positive according to the analytical chemical method whereas the Limulus amoebocyte lysate test suggested the presence of appreciable amounts of lipopolysaccharides of Gram-negative bacteria. Further studies are required to compare the utility of these two methods for determining endotoxins in complex environments.

Determination of microbial fatty acid profiles at femtomolar levels in human urine and the initial marine microfouling community by capillary gas chromatography-chemical ionization mass spectrometry with negative ion detection

Room temperature esterification with the electron capturing pentafluorobenzyl bromide in glass capillaries, with analysis by capillary gas-liquid chromatography coupled with chemical ionization mass spectrometry and negative ion detection in the selected ion mode, allowed detection and identification of fatty acids from microbial biofilms at the femtomolar level. This sensitivity was achieved without loss of specificity of the mass spectrometric analysis. The detection of all the fatty acids commonly associated with bacteria was quantitative and linearly related to their concentration at a sensitivity permitting detection of about 600 bacteria the size of Escherichia coli. With this technique it was possible to detect the characteristic 3-hydroxy fatty acid of the lipopolysaccharide lipid A of E. coli infecting human urine at concentrations representing 10 4 bacteria and define the community structure of the initial marine microfouling community attached to a teflon surface at concentrations below the detectability by gas chromatography with flame ionization detection.

Glass capillary gas-chromatographic analysis of free amino acids in biological microenvironments using electron capture or selected ion-monitoring detection

Abstract Studies on analysis of free animo acids using a support-coated, open-tube capillary column, and electron-capture detection or selective ion monitoring have been performed on samples from biological microenvironments. For most amino acids the detection limit was found to be less than 1 pg. The preparation of the support-coated open-tube capillary column is described as well as the gas-chromatographic conditions for direct injection and temperature-programmed separation of the N -heptafluorobutyryl iso butyl ester derivatives. Electron-capture detection and selected ion monitoring are compared with respect to linearity and sensitivity and the bases for the greater sensitivity of electron-capture detection compared with flame-ionization detection using halogenated derivatives is discussed. Applications of the gas-chromatographic method for analysis of free amino acids in environments deliberately chosen very small are demonstrated.

Determination Of Fatty Acids And Carbohydrate Monomers In Micro-Organisms By Means Of Glass Capillary Gas Chromatography: Analysis Of Mycobacterium gordonae and Mycobacterium scrofulaceum

Trifluoroacetylated whole-cell methanolysates of four strains each of Mycobacterium gordonae and Mycobacterium scrofulaceum were analysed by gas chromatography, using a glass capillary column. The major chromatographic peaks were identified by mass spectrometry as derivatives of fatty acids and carbohydrates. In addition, two predominant peaks, present in chromatograms representing M. scrofulaceum, were identified as 2-octadecanol and 2-eicosanol. These secondary alcohols were not found in any of the strains of M. gordonae studied. The amount of tuberculostearic acid in the latter species was less than 1% of that in M. scrofulaceum.

Determination of endotoxins by gas chromatography: evaluation of electron-capture and negative-ion chemical-ionization mass spectrometric detection of halogenated derivatives of β-hydroxymyristic acid

The sensitivity and selectivity of gas chromatography for analysing several halogenated ester derivatives of beta-hydroxymyristic acid were studied using both selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry and electron-capture detection. Six different derivatization methods were compared with respect to yield, chemical stability and formation of by-products. Procedures for removal of excess reagents using disposable silica columns and thin-layer chromatography were elaborated. The 3-O-pentafluorobenzoyl-methyl ester was the preferred derivative since it provided high sensitivity and had the molecular ion as the base peak in the mass spectrum. The detection limit was 0.5 pg with electron-capture detection and 0.3 pg with the mass spectrometric system. Using beta-hydroxymyristic acid as a chemical marker it was possible to detect Escherichia coli endotoxin in aqueous solution at a level of 1 ng/ml.

Determination of 13C-enrichment in bacterial fatty acids using chemical ionization mass spectrometry with negative ion detection

Saturated, monoenoic and β-hydroxysubstituted fatty acids, 13C-labelled at the carboxyl group, were prepared from natural or synthetic unlabelled analogues. The synthetic route involves decarboxylation of the unlabelled fatty acid to the next lower iodide, displacement of iodide for [13C]cyanide and hydrolysis. The fatty acids were converted to their pentafluorobenzyl esters and analysed by selected ion monitorint using chemical ionization and negative ion detection. Measurements of the signal ratios for the negative carboxylate ion (m) and the (m + 1) ion showed that at 95% confidence level and n = 5, mean values differing by 1.0 atom% 13C will be significantly resolved. The calculated standard deviation was the same for the studied bacterial acids including the phospholipid ester-linked palmitoleic acid, β-hydroxymyristic acid in the lipopolysaccharides and β-hydroxybutyric acid in the storage polymer poly-β-hydroxyalkanoate. Sodium [1-13C]acetate or D-[13C6]glucose were pulse administered to a Gram-negative marine bacterium isolate. Phospholipid ester-linked fatty acids and β-hydroxybutyric acid showed extensive 13C-incorporation within 15 min after the pulse. After approximately 60 min a maximum of 10 atom% excess of 13C was reached for palmitoleic acid. The method provides the potential to measure the metabolic activity of bacterial communities by measuring the incorporation of 13C-labelled substrates into specific fatty acids that can be utilized as biomarkers for biomass, community structure and nutritional status. (Less)

Determination of muramic acid by high-performance liquid chromatography-plasma spray mass spectrometry

Abstract A high-performance liquid chromatographic-plasma spray mass spectrometric method was developed for the determination of muramic acid, a unique compound present in bacterial peptidoglycan. The method included hydrolysis of bacterial samples by hydrochloric acid, purification using a disposable extraction column, and separation using cation-exchange chromatography with a mobile phase composed of ammonium acetate-trifluoroacetic acid-water. Muramic acid was detected in cells of Bacillus subtilis ; the presence of yeast cells ( Saccharomyces cerevisiae ) in excess amounts did not interfere with the analyses. In contrast to currently applied liquid and gas chromatographic methods, the described method permits highly selective determination of underivatized muramic acid, and may therefore be useful for detecting bacterial and bacterial cell debris in complex biological matrices.

Selected ion monitoring for rapid differentiation of mycobacteria isolated from domestic animals

Abstract Gas chromatography/mass spectrometry adapted for selected ion monitoring was used to detect C 32 mycocerosic acid in short-term incubated cultures of procineand canine strains of mycobacteria. The method can be employed for rapid differentiation of Mycobacterium tuberculosis from M. avium-intracellulare .