Jørgen Øgaard Madsen

Application of head-space solid-phase microextraction for the analysis of volatile metabolites emitted by Penicillium species

Head-space solid-phase microextraction (HS-SPME) has been used to collect volatile organic compounds (VOCs) emitted from fungi of the genus Penicillium. Gas chromatography combined with mass spectrometry (GC-MS) was employed for the analysis of the profiles of volatile metabolites characteristic for each species. The results obtained by HS-SPME compared favourably with those obtained by Tenax adsorption. The following characteristic metabolites were detected: isopentyl alcohol, 1-octene-3-ol, 3-octanone, 3-octanol, 2-methylisoborneol and geosmin, together with the not previously reported acetates of 1-octene-3-ol and 3-octanol. Further, several mono- and sesquiterpene hydrocarbons and alcohols were identified, many of those for the first time from Penicillium species. The growth conditions were found to be of major importance for the quantitative aspects. Distribution coefficients between the fibre coating and air were determined for some typical fungal metabolites using polyacrylate and polydimethylsiloxane as fibre coatings. No significant differences in the results for the two fibre coatings were observed for non-polar to slightly polar compounds, whereas the more polar compounds were better extracted by the polyacrylate fibre coating. However, the equilibration times are longer with this fibre coating. Increasing responses were observed with decreasing volatility of the compounds for both types of fibre coating. Being a rapid, simple and practically non-interfering technique, HS-SPME has a great potential use in the analysis of biogenic VOC emissions, including chemotaxonomical studies.

Analysis of chloro- and nitroanilines and -benzenes in soils by headspace solid-phase microextraction

Abstract Quantitative analysis by headspace solid-phase microextraction (HS-SPME) of twenty chloro- and nitrobenzenes and -anilines spiked into soil samples was possible when the calibration was performed with the same matrix. The SPME response increased with addition of water to the air dried soil samples, and by optimisation of extraction conditions such as temperature, extraction time and sample agitation. Distribution constants between the fibre coating materials and water were determined. They showed that polyacrylate, rather than polydimethylsiloxane, should be used in the analysis of polar compounds. Matrix effects, mainly depending on the organic carbon content of the soil, were so large that quantitative analyses of real soil samples would not be reliable with model matrix calibration. This problem would be overcome if a nearly exhaustive extraction could be achieved. Recovery studies showed that this was possible only in the case of extraction of the lightest non-polar analytes from a soil with a very low content of organic carbon. Instead quantification could be performed with calibration by standard addition. By this approach the non-linear calibration curves at concentrations near the detection limits would cause inaccurate results at trace level, whereas it could be applied successfully within the linear ranges. Hence, the potential of HS-SPME in soil analysis is primarily as a rapid screening technique.

Derivatisation/solid-phase microextraction followed by gas chromatography–mass spectrometry for the analysis of phenoxy acid herbicides in aqueous samples

Abstract Different combinations of derivatisation and solid-phase microextraction followed by gas chromatography–mass spectrometry were optimised and evaluated for the analysis of phenoxy acid herbicides in water. The most successful derivatisation approach was aqueous-phase derivatisation with benzyl bromide. The benzyl esters were extracted most efficiently by the solid-phase microextraction fibre coated with polydimethylsiloxane–divinylbenzene. No carry-over problems were encountered with this fibre upon desorption at 250°C. Detection limits in the ng/l range were obtained, while the relative standard deviations were between 14 and 42%.

Determination of ergosterol on mouldy building materials using isotope dilution and gas chromatography-tandem mass spectrometry.

Ergosterol content of building materials was quantified using gas chromatography-tandem mass spectrometry (GC-MS-MS) in an ion trap with external ionisation. Hydrolysing the samples by classic extraction at 85 degrees C for 90 min in vials was faster, more precise and safer than microwave assisted extraction. [4-2H2]ergosterol was synthesised and used as internal standard, giving method standard deviation of 5-10% from 10 to 30 ng to 10-15 microg ergosterol in the sample. The use of GC-MS-MS meant that no solid-phase extraction clean-up was needed, so one person could easily prepare 40-80 samples per day.

BAKER'S YEAST : PRODUCTION OF D- AND L-3-HYDROXY ESTERS

Abstract Bakers yeast grown under oxygen limited conditions and used in the reduction of 3-oxo esters results in a shift of the stereoselectivity of the yeast towards d -hydroxy esters as compared with ordinary bakers yeast. The highest degree of stereoselectivity was obtained with growing yeast or yeast harvested while growing. In contrast, the stereoselectivity was shifted towards l -hydroxy esters when the oxo esters were added slowly to ordinary bakers yeast supplied with gluconolactone as co-substrate. The reduction rate with gluconolactone was increased by active aeration. Ethyl l -( S )-3-hydroxybutanoate was afforded in >99% ee. Both enantiomers of ethyl 3-hydroxypentanoate, d -( R ) in 96% ee and l -( S ) in 93% ee, and of ethyl 4-chloro-3-hydroxybutanoate, d -( S ) in 98% ee and l -( R ) in 94% ee, were obtained. The results demonstrate that the stereoselectivity of bakers yeast can be controlled to a large extent without the use of inhibitors, heat treatment, etc.

Baker's yeast: improving the d-stereoselectivity in reduction of 3-oxo esters

Abstract The stereoselectivity of bakers yeast in the reduction of ethyl 3-oxopentanoate was shifted towards the corresponding ( R )-hydroxy ester by sugar, heat treatment and allyl alcohol. The highest enantiomeric excesses obtained with bakers yeast with a good reduction capacity, 92–97%, were achieved by combining allyl alcohol and sugar; heat treatment did not increase the stereoselectivity further. With the use of this technique, ethyl ( R )-3-hydroxyhexanoate, >99% ee , and ethyl ( S )-4-chloro-3-hydroxybutanoate, 82–90% ee , were produced from the corresponding esters, and for the first time an excess of the ( R )-enantiomer of ethyl 3-hydroxybutanoate was obtained with ordinary bakers yeast.

Onion-like off-flavour in beer: Isolation and identification of the culprits

Beer with an onion-like off-flavour is found to contain two unusual sulphur compounds: (1-methylethyl)-thiirane and 2-mercapto-3-methyl-1-butanol. The off-flavour is attributable to the latter, previously unknown compound. The methods used are: extraction of beer with trichlorofluoromethane, GC analyses of extractives and reference compounds, using a sulphur sensitive flame photometric detector, and GC-MS. Both of the new sulphur constituents are synthesized and their structures ascertained by NMR and MS.

Nonan-2-ol esters in sorghum leaf epicuticular wax and their collection by preparative gas chromatography

Epicuticular wax from sorghum seedling leaves was isolated and analyzed by TLC and GC. Its composition was remarkably similar to that of waxes on many other Gramineae leaves with the exception that three alkan-2-ol esters (nonan-2-ol docosanoate, nonan-2-ol tetracosanoate, and nonan-2-ol hexacosanoate) were present. This was unexpected as such esters have hitherto only been reported in waxes of plants with the capability of synthesizing β-diketone lipids. The C31−C35 alkan-2-ol esters were separated from the C38−C56 alkan-1-ol esters by preparative GC. The column effluent was passed through a small Dexsil-300 trap maintained at 150°C, and the esters eluted therefrom with chloroform. This technique gave better than 90% recovery of a [1-14C]−C32 ester.

Diastereospecific, enzymically catalysed transmethylation from S-methyl-L-methionine to L-homocysteine, a naturally occurring process

A known catabolic pathway of S-methyl-L-methionine in higher plants: donation of a methyl group to L-homocysteine resulting in the production of two molecules of L-methionine, is subjected to stereochemical studies. The two, diastereoisomeric (2-2H, methyl-13C)-S-methyl-L-methionines are synthesized and utilised in transmethylation reactions with L-homocysteine as the acceptor and an enzyme preparation from jack beans as a catalyst. The resulting, variously labelled methionine species are converted into butyl esters of the N-trifluoroacetylated derivatives and, as such, subjected to g.l.c. combined with mass spectrometry in two ionisation modes. Experimentally determined parameters such as mass peak intensities, isotopic enrichment factors, diastereoisomeric purities, and protein-derived methionine, are utilised for calculating the stereoselectivity in the enzyme transfer of the diastereoisotopic methyl groups from S-methyl-L-methionine to L-homocysteine. Together, the independent results from the two series of diastereoisomers reveal an enzymic preference of the pro-(R)-methyl group to the extent of 94% or more.

Isolation of dihydromyricetin and dihydroquercetin from bark of pinus contorta

Bark of shore pine has high contents of the dihydroflavonols dihydromyricetin and dihydroquercetin. A method for isolation of the pure, optically active compounds in gram amounts has been developed, involving Soxhlet extraction of finely ground, dry bark and preparative scale reversed-phase HPLC. Among a number of inexpensive solvents tested ethyl acetate is useful, being reasonably effective in the extraction step and giving a crude extractive that is suitable for HPLC without any pre-treatment. The use of bark from dead trees, which have been lying in the open for a couple of years, is advantageous because of the lower content of resinous material. 270 MHz1H- and13C-NMR and electron impact mass spectra are presented.