USE OF SOLID-PHASE EXTRACTION TO DETERMINE ERGOSTEROL CONCENTRATIONS IN PLANT-TISSUE COLONIZED BY FUNGI

At present, the ergosterol and acetate-to-ergosterol techniques are ge nerally considered to be the methods of choice to quantify fungal biom ass, growth rate, and productivity under natural conditions. Both meth ods rely on the accurate isolation and quantification of ergosterol, a major membrane component of eumycotic fungi. Taking advantage of the solid-phase extraction (SPE) technique, we present a novel method to d etermine the ergosterol concentration in lipid extracts derived from p lant tissues and dead organic matter colonized by a fungi. In this met hod, a primary alkaline extract is acidified and passed through a reve rsed-phase (C-18) SPE column. The column is then washed with an alkali ne methanol-water solution to eliminate interfering substances and inc rease pH and is thoroughly dried in air. Ergosterol is eluted with alk aline isopropanol. This eluting solvent was chosen to produce a strong ly basic pH of the final extract and thus confer stability on the ergo sterol molecule before high-performance liquid chromatography analysis . The recovery of ergosterol from plant tissues and the O-hf horizon o f a woodland soil ranged from 85 to 98%, and the overall extraction ef ficiency was similar to that obtained by a conventional procedure invo lving liquid-liquid extraction. Potential pitfalls of ergosterol analy sis by SPE include (i) insufficient acidification before sample loadin g on the extraction column, resulting in a poor affinity of ergosterol for the sorbent; () incomplete drying of the sorbent bed before II th e elution step; and (iii) chemical breakdown of ergosterol after eluti on, which was found to be related to a low pH of the final extract and a high concentration of matrix compounds. When these pitfalls are avo ided, SPE is an attractive alternative to existing methods of ergoster ol analysis of environmental samples.