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

IF 1.9 Q2 MULTIDISCIPLINARY SCIENCES

MethodsX Pub Date : 2025-07-07 DOI:10.1016/j.mex.2025.103495

Björn Kusebauch, Stefan Bader, Steffen Carl

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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.

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Selective and flexible analytical method for ergosterol quantification in alder leaves using LLE and LC-MS/MS instead of SPE and HPLC-UV
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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
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Method validation for three mass transitions according to guidance document SANTE/2020/12830 including matrix effect evaluation and stability investigations

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皂化后液-液萃取- LC-MS/MS测定桤木叶中麦角甾醇的方法的建立与验证

麦角甾醇被广泛用于估算凋落叶相关真菌生物量。根据常用的教科书方法，麦角甾醇可以用甲醇氢氧化钾从植物组织中提取，然后用固相萃取（SPE）纯化，用高效液相色谱-紫外（HPLC-UV）检测。作为替代，我们开发了一种环己烷液-液萃取法（LLE）来代替耗时且容易出错的固相萃取法。麦角甾醇检测用灵敏度更高、选择性更强的液相色谱-串联质谱法（LC-MS/MS）代替HPLC-UV。该方法在10µg/g dw和500µg/g dw的桤木叶中进行了验证，平均回收率为95.1% ~ 100.2%(相对标准偏差<；10%)，并已成功应用于杀真菌剂对凋落叶相关水生真菌的效果测定。•可选使用7-脱氢胆固醇作为内标，以补偿样品制备过程中的体积变化和LC-MS/MS中的信号漂移•根据指导文件SANTE/2020/12830进行三次质量转移的方法验证，包括基质效应评估和稳定性调查

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