*2.6. Chemical Analysis of Ergosterol*

Ergosterol (ERG) in 60 grain samples was determined by HPLC as described by Young [29] with modifications [30,31]. A detailed evaluation of the method was given by Perkowski et al. [31]. Samples containing 100 mg of ground grains were placed into 17-mL culture tubes, suspended in 2 mL of methanol, treated with 0.5 mL of 2 M aqueous sodium hydroxide and tightly sealed. The culture tubes were then placed within 250-mL plastic bottles, tightly sealed and placed inside a microwave oven (Model AVM 401/1WH, Whirlpool, Sweden) operating at 2450 MHz and 900 W maximum output. Samples were irradiated (370 W) for 20 s and after about 5 min for an additional 20 s. After 15 min the contents of culture tubes were neutralized with 1 M aqueous hydrochloric acid, 2 mL MeOH were added and extraction with pentane (3 × 4 mL) was carried out within the culture tubes. The combined pentane extracts were evaporated to dryness in a nitrogen stream. Before analysis samples were dissolved in 4 mL of MeOH, filtered through 13-mm syringe filters with a 0.5 mm pore diameter (Fluoropore Membrane Filters, Millipore, Ireland) and evaporated to dryness in a N2 stream. The sample extract was dissolved in 1ml of MeOH and 50 μL were analyzed by HPLC. Separation was performed on a 150 × 3.9 mm Nova Pak C-18, 4 mm column and eluted with methanol/acetonitrile (90:10) at a flow rate of 0.6 mL min−1. Ergosterol was detected with a Waters 486 Tunable Absorbance Detector (Milford, MA, USA) set at 282 nm. The presence of ERG was confirmed by a comparison of retention times and by co-injection of every tenth sample with an ergosterol standard.
