# Development and validation of an alternative method for ergosterol determination in alder leaves using liquid-liquid extraction and LC-MS/MS after saponification

## TL;DR

This paper introduces a new method to measure ergosterol in alder leaves using a faster and more accurate technique than traditional methods.

## Contribution

A novel analytical method for ergosterol quantification using LLE and LC-MS/MS instead of SPE and HPLC-UV is developed and validated.

## Key findings

- The new method achieved mean recoveries between 95.1 and 100.2% with relative standard deviations below 10%.
- The method was validated for ergosterol determination at concentrations of 10 µg/g and 500 µg/g dw in alder leaves.
- The use of 7-dehydrocholesterol as an internal standard improved accuracy and compensated for signal drift.

## Abstract

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

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

Image, graphical abstract