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Article Excerpt
ABSTRACT--The analysis of pollutants and their metabolites resulting from biotransformation is important to studies of bioremediation or toxicology of environmental toxicants. Gas chromatography-mass spectrometry (GC-MS) with solid-phase micro-extraction (SPME) has been used to determine the 9-hydroxyphenanthrene levels resulting from phenanthrene biotransformation by yeast. Candida tropicalis CP1-1, derived from a parent strain of Candida tropicalis (ATCC strain 96745), was grown in minimal media containing phenanthrene. Direct extraction of liquid media by SPME served to selectively preconcentrate the pollutants prior to GC-MS analysis. Various derivatizing reagents, including dimethyldichlorosilane, trimethylchlorosilane, acetic anhydride, and trifluoroacetic anhydride, were evaluated for their ability to enhance the volatility of metabolites containing hydroxyl groups, and improve chromatographic separation. Both dimethyldichlorosilane and trifluoroacetic anhydride gave distinctive mass spectra with both molecular and fragment ions that were suitable for selected ion monitoring of 9-hydroxyphenanthrene at trace levels. In addition, trifluoroacetic anhydride on-fiber derivatization of 9-hydroxyphenanthrene after SPME preconcentration yielded a GC-MS method capable of analysis at the parts-per-billion level. The extraction efficiencies of different SPME fiber coatings, including Carbowax[TM]/divinylbenzene, polydimethylsiloxane, and polyacrylate, were compared for their potential application in yeast culture media metabolite analysis. The 100 [micro]m polydimethylsiloxane provided the highest analytical sensitivity for the trifluoroacetyl derivative of 9-hydroxyphenanthrene, while the 70 [micro]m Carbowax[TM]/divinylbenzene provided the next best analytical sensitivity and a simpler chromatogram with less derivatization byproducts. This study demonstrates that SPME with derivatization provides a sensitive method for analyzing hydroxyl group containing metabolites in yeast cultures. The superior analytical sensitivity can be attributed to the highly efficient sample extraction process and the intense signal of the trifluoroacetyl derivative of the molecular ion in GC-MS analysis. Hence, this analytical method is desirable for studying the biochemical transformation of many environmental toxicants via hydroxylation.
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Yeasts isolated from coastal sediments, including Candida lypolytica, Candida maltosa, Candida tropicalis, Candida utilis, Debaromyces hansenii, and Saccharomyces cerevisiae, have been reported to be capable of transforming polycyclic aromatic hydrocarbons (Cerniglia and Crow, 1981; Hofmann, 1986: McGillivray and Sharis, 1993). Yeasts also have been shown to exist in large numbers in petroleum contaminated soils in Alaska that were devoid of bacteria (Atlas et al., 1976). The different yeast species involved in the metabolism of aromatic compounds in a polluted estuary in Rio de Janeiro, Brazil, have been characterized (Hagler et al., 1979; Pinto et al., 1979). Since yeasts are widespread and tolerant of changes in environmental conditions, they could play an important role in the degradation of organic compounds in the environment.
Polycyclic aromatic hydrocarbons (PAHs) are common environmental contaminants that have been listed as priority pollutants by the United States Environmental Protection Agency. Because PAHs may be carcinogenic, mutagenic, and/or toxic to many organisms (Perez et al., 2001; Chiang et al., 1997; Bispo et al., 1999), there is interest in developing remedial approaches for degrading PAHs in polluted environments through PAH biotransformation by microorganisms. It also is important to develop sensitive and efficient techniques for probing the metabolites resulting from PAH biotransformation. Phenanthrene, one of the most abundant PAHs in the environment, is used as a model substrate for studying PAH biotransformation by yeasts because it shares similar molecular features with potent carcinogenic PAHs (Pothuluri and Cerniglia, 1994; Sack et al., 1997). Most microbial pathways for metabolizing phenanthrene rely on the use of dioxygenase among bacteria or cytochrome P-450 mono-oxygenase among yeasts and fungi to form a variety of PAH dihydrodiols and hydroxy PAHs, respectively, with different isomeric configurations. Typically, the bacteria will form the cisisomers of PAH dihydrodiols before being transformed to catechols, whereas yeasts and fungi will produce the intermediate arene oxides (epoxides of PAHs) before undergoing non-enzymatic rearrangement into hydroxy PAHs or conversion into the trans-isomers of PAH dihydrodiols by epoxide hydrolase (Pothuluri and Cerniglia, 1994). The objective of this study was to develop an analytical method capable of characterizing phenanthrene metabolites resulting from biotransformation by Candida tropicalis CP1-1.
Earlier studies of microbial biotransformation were carried out using liquid/liquid extraction of culture media followed by analysis with either GC or high-performance liquid chromatography (HPLC). Selected examples of the analysis of PAHs and their metabolites in both laboratory and field studies using the liquid/liquid extraction approach have been published (Cerniglia and Crow, 1981; Mohammed et al., 1998; Yuan et al., 2000). However, these methods involved laborious sample preparation and the use of large amounts of organic solvents for extraction due to the presence of constituents in the yeast culture media that may not be suitable for GC or HPLC analysis. In addition, the yeast metabolites may be present at very low concentrations that require an extra step of analyte preconcentration during sample preparation. To circumvent these problems, this study describes the use of solid-phase microextraction (SPME) for direct extraction of metabolites from aqueous solution containing phenanthrene or other PAHs followed by GC-MS analysis.
Solid phase microextraction techniques have been used successfully for the analysis of PAHs in a variety of sample matrices including...
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