EXTRACTION EFFICIENCIES AND DETERMINATION OF ERGOSTEROL IN A VARIETY OF ENVIRONMENTAL MATRICES

Fungi may be the main decomposers of plant material in an aquatic system and their presence and biomass can be measured indirectly by the detection of ergosterol, a compound generally specific to the cell membranes of fungi. Ergosterol is conveniently used as a measurement of living fungal biomass and is most often detected using high pressure liquid chromatography with a UV detector (HPLC–UV). Described in this manuscript is the general stability of ergosterol in a laboratory setting, the testing of a variety of techniques for the extraction of ergosterol and a general survey of different matrices in an aquatic environment. More importantly, data acquired from the HPLC–UV system was further confirmed with liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI/MS/MS) for the presence of ergosterol. Ergosterol was found to be quite stable over 5 days in a lab setting. Though other procedures gave overall better recoveries of ergosterol extracted, the optimum extraction method proved to be a previously established one of saponification followed by toluene extraction. This method resulted in a relatively clean sample with ergosterol recoveries of above 90%. As expected, ergosterol was widely distributed at ppm levels (2.4 to 302.9 μg ash free dry mass (AFDM)) in matrices of decaying willow leaves, Scirpus stems (living and dead) and sediment collected at the water sediment interface. In the case of water samples, the HPLC–UV system falsely indicated the presence of ergosterol thus pointing to the need for the more selective mass spectral confirmation on samples where chromatographic co-elution is a problem. Care is required when assuming the presence and levels of ergosterol are correct based only on HPLC–UV detection.