HPTLC determination of ergosterol in wheat and structure elucidation by NMR: Toward confirming method selectivity

Abstract

Ergosterol (ERG) is considered as an excellent fungal biomarker, therefore it was utilized for the determination of the degree of fungi infection in wheat grains. A novel, fast, cost-effective and selective high performance thin layer chromatography (HPTLC) with reflectance/absorbance densitometry method was developed and validated for ERG determination. Separation was attained on pre-coated silica gel HPTLC plate with ethyl acetate: petroleum ether (6: 4 v/v) as a mobile phase. This system gave compact bands at retardation factor (Rf) of 0.45 ± 0.002 and quantification was performed at 282 nm over concentration range of 40–600 ng/band. The lower detectable limit was 10.79 ng per band which is three times more sensitive than the previously reported TLC method. The developed method could be utilized efficiently for the detection of ERG in pure powders and in fungi infected wheat samples. Resolution of ERG from other compounds in wheat grain extract allowed utilizing preparative TLC for separating and purifying ERG in a sufficient quantity for structure elucidation by nuclear magnetic resonance spectroscopy (NMR) for the first time. H1 and C13 NMR spectra indicated the purity of ERG extracted from the scrapped silica of preparative TLC plate at the Rf of the standard ERG verifying the method selectivity.