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 0.43 mg/L and 1.45 mg/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 0.15 mg/L and 0.49 mg/L. Alfalfa was analyzed using these methods, and the method for total ergosterol is more suitable for this application.