Determination of Ergosterol in Alfalfa by Gas Chromatography-Mass Spectrometry

Extraction procedures for free ergosterol and total ergosterol from alfalfa were optimized for determination by gas chromatography-mass spectrometry. Free ergosterol was isolated by chloroform/methanol (1:1, v/v), and derivatized with a blended reagent of N-trimethylsilylimidazole and trimethylchlorosilane. The recovery was between 94.5% and 98.7% with relative standard deviations of 3.7% to 8.3%. The limits of detection and quantification were 0.43mg/L and 1.45mg/L, respectively. Total ergosterol was saponified with potassium hydroxide, extracted with methanol and hexane, and derivatized with the blended reagent. The recovery was between 87.5% and 98.2% with relative standard deviations of 5.4% to 7.6%. The limits of detection and quantification for the saponification method were 0.15mg/L and 0.49mg/L. Alfalfa was analyzed using these methods, and the method for total ergosterol is more suitable for this application.