High-Performance Separation of Unmodified and Reduced Hop and Beer Bitter Compounds by a Single High-Performance Liquid Chromatographic Method

Abstract

An improved high-performance liquid chromatographic method for the separation of the isoxanthohumol iso-α-acids, rho-iso-α-acids, tetrahydro-iso-α-acids, α-acids, and β-acids in beer is presented. This is the first time a complete separation of all six isomers of iso-α-acids has been described. It is done by using an isocratic elution system in beer. A gradient elution method is used for measuring all the main hop-bittering substances such as α-acids, β-acids, iso-α-acids, and all reduced compounds in light-stable beer, hop extracts, and wort. A chemically cross-linked “base deactivated” C18 column with a citrate-buffered (pH 7) acetonitrile-methanol gradient mobile phase enables the separation of the beer bitter components within 50 min. The analysis of wort samples was achieved with a tris-buffered (tris(hydroxymethyl)-aminomethane) (pH 7.5) acetonitrile-methanol gradient mobile phase within 60 min. Sample preparation was by preconcentration on SPE C8 cartridges, which gave good reproducibility and separation of bitter components from the beer matrix. Due to the complex matrix of beer and wort, an optimized sample preparation procedure is used to increase the life of the column.