Quantification of sterols via gas chromatography-mass spectrometry (GC–MS)

Quantification of sterol components was described by Li et al.⁸⁹. The yeast strains (2 × 10⁵ cells/ml in YPD medium), treated or untreated with the Xylaria extract or ECQ. The wild type cells were treated with 1000 or 225 µg/ml of Xylaria extract or ECQ, respectively. While the ∆erg6 cells were treated with 225 or 112 µg/ml of Xylaria extract or ECQ, respectively. Concentration at MIC/4 was used to treat ∆erg6 strain with the Xylaria extract or ECQ and wild type strain with ECQ. For wild-type cells, 1000 µg/ml of Xylaria extract was used for treatment because could not find MIC from susceptibility test. For GC–MS, cells were grown for 24 h at 30 °C with shaking. Cells were then collected and resuspended in 40% alcoholic KOH. The mixture was saponified by heating at 85 °C for 1 h and allowed to cool to room temperature. The sterol was then extracted in petroleum ether and vaporized and analyzed by GC–MS (AGILENT 7890B). The gas chromatograph was equipped with a 5% phenyl and 95% dimethylpolysiloxane column (length, 30 m; inner diameter, 0.25 mm; film thickness, 0.25 µm). The settings were as follows: initial GC temperature of 120 °C for 4 min, 290 °C for 3 min with gradient of 15 °C/min; detector temperature, 340 °C; sample injection temperature, 320 °C; carrier gas, nitrogen gas; flow rate, 1 ml/min. Sterols were identified from their retention times and specific mass spectrometric patterns.