Determination of Endogenous Gibberellins in Germinating Barley by Combined Gas Chromatography-Mass Spectrometry

Abstract

This study was undertaken to elucidate the dominant endogenous gibberellins (GA) and the changes in their levels in germinating barley during malting. Also, a method for determining gibberellins in barley and malt by combined gas chromatography-mass spectrometry (GC-MS) was developed. Endogenous gibberellins in germinating Fuji Nijo II and Betzes barley were sequentially extracted with ethanol and ethyl acetate. Fractions corresponding to GA1, GA3, GA4, and GA7 were separated and collected from the ethyl acetate extract by Sephadex LH-20 column chromatography, silicic acid partition column chromatography, silica gel thin-layer chromatography, and kieselguhr thin-layer chromatography. Biological activity of each fraction was examined by bioassay with dwarf rice seedlings (var. Tan-ginbozu) and deembryonated barley endosperm. Identification of each gibberellin was performed by analysis of the methyl ester trimethylsilyl ether derivative of gibberellin in each fraction by GC-MS. Determination of gibberellin content was performed by mass fragmentography with GC-MS. The data indicated that the dominant gibberellin in germinating barley is GA1 and that GA3 is a minor gibberellin. GA4 and GA7 were not identified. GA1 and GA3 contents in germinating barley under malting conditions reached their maximum concentrations on the second day; levels decreased on the fourth and sixth days of germination. The behavior of authentic GA3 added to steeped barley during germination and the contents of residual GA3 in commercial malts were also examined.