Determination of Isoxanthohumol, Xanthohumol, Alpha and Beta Bitter Acids, and trans and cis-iso-alpha acids by HPLC with UV and Electrochemical Detection: Application to Hop and Beer Analysis

Hops are the female flower clusters of the hop species, Humulus lupulus. They are used as a flavoring and stability agent in beer, and are also used for various purposes in other beverages and as an herbal medicine. Hops contain a number of important phytochemicals including: Xanthohumol (a prenylated chalconoid), and alpha- and beta- acids. As part of the beer brewing process hops or hop extracts are added during the boiling of the wort. The virtually insoluble alpha-acids (humulones) are isomerized into the more soluble iso-acids, the main bittering substances in beer. Beta acids (lupulones) do not isomerize and have a negligible effect on beer taste. Instead, they contribute to beer's bitter aroma but unfortunately may oxidize into compounds that can give beer off-flavors.

Many HPLC methods have been applied to the determination of phytochemicals in hops and beer. HPLC techniques using UV detection typically require a concentration step in the analysis to be able to determine low levels of the bitter acids. Presented here is a global HPLC method using serial UV and Electrochemical Detection and its application to the measurement of numerous phytochemicals in hops and beer. The chromatographic separation was performed on a Dionex Acclaim C30 column (3µm, 3.0×150mm) at 35°C with gradient elution and simultaneous UV (270nm) and EC detection (500 mV and 800 mV). Sample preparation involved extraction with acidified acetonitrile and centrifugation. All calibration curves showed good linear regression (r² >0.996). Peak area RSD's over a twenty-hour run were as follows: isoxanthohumol and xantholhumol, 1.2%; alpha and beta bitter acids, 2.5%; and trans and cis-iso-alpha acids, 2.4%. Limits of detection for most analytes were in the picogram (on column) range.