Development and validation of an alternative method for ergosterol determination in alder leaves using liquid-liquid...

Ergosterol is widely used as proxy for the estimation of leaf litter associated fungal biomass. According to a common textbook method, ergosterol can be extracted from plant tissue with methanolic potassium hydroxide followed by purification with solid-phase extraction (SPE) and detection via high-performance liquid chromatography-ultraviolet (HPLC-UV). As an alternative, we developed a method using liquid-liquid extraction (LLE) of the methanolic extract with cyclohexane instead of time consuming and error prone SPE. For ergosterol detection, HPLC-UV was replaced by the more sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated for alder leaves at 10 µg/g dw (LOQ) and 500 µg/g dw with mean recoveries between 95.1 and 100.2 % (relative standard deviations < 10 %) and has been successfully applied for the measurement of fungicide effects on leaf litter associated aquatic fungi.•Selective and flexible analytical method for ergosterol quantification in alder leaves using LLE and LC-MS/MS instead of SPE and HPLC-UV•Optional use of 7-dehydrocholesterol as internal standard to allow for compensation of volumetric variations during sample preparation and signal drift in LC-MS/MS•Method validation for three mass transitions according to guidance document SANTE/2020/12830 including matrix effect evaluation and stability investigations.