Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography–tandem mass spectrometry

Abstract

A method for quantitation of six prenylflavonoids (xanthohumol, isoxanthohumol, desmethylxanthohumol, 6- and 8-prenylnaringenins and 6-geranylnaringenin) in hops and beer by HPLC–tandem mass spectrometry has been developed. The method allows direct analysis of beer and crude methanolic extracts of hops. After HPLC separation, prenylflavonoids were detected by positive ion multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with a heated nebulizer–atmospheric pressure chemical ionization interface. The accuracy and precision were evaluated by replicate analyses of (spiked) samples. Thirteen commercial beers were analysed with the method. Isoxanthohumol, formed by isomerization of xanthohumol during the brewing process, was the most abundant flavonoid in hopped beers, ranging from 0.04 to 3.44 mg/l.

Introduction

Hops, the female inflorescences of the hop plant (Humulus lupulus L.), are used in the brewing industry to add bitterness and aroma to beer. Most research on beer flavour has therefore been focused on bitter acids and essential oils. It has been suggested that the hop flavonoids moderate beer bitterness and increase stability, but the brewing value of these polyphenolics is not well understood [1]. Xanthohumol and other prenylated flavonoids from hops [2]have recently received attention because of their potential anti-cancer properties 3, 4, 5.

Little is known about the dietary intake (estimates vary from 50 mg to about 1 g per person per day [6]) and pharmacokinetics of flavonoids in general [7]. Food products such as vegetables and fruits contain mainly glycosides of common flavonols, flavones and flavanones [8]. Prenylated flavonoids are found in a limited number of plant families, of which the Moraceae-Cannabinaceae (with Humulus lupulus), Leguminosae and Asteraceae host roughly 80% of the ca. 1100 known prenylflavonoids [9]. Although a number of food plants belong to prenylflavonoid-bearing families, none of these can be identified as a rich nutritional source of these phenolics in the diets of western countries. While the presence of prenylflavonoids in beer has not been reported in the literature previously, beer may well represent the most important source of dietary prenylflavonoids.

Xanthohumol has been quantified in hops by high-performance liquid chromatography (HPLC) with UV detection [10], but this technique offers insufficient sensitivity and selectivity for quantitative analysis of the minor prenylflavonoids in beer. As a detection technique, tandem mass spectrometry (MS–MS) can provide improved sensitivity, and its greater selectivity makes it especially useful for analysis of minor components in complex matrices.

Liquid chromatography (LC) coupled with (tandem) mass spectrometry has been successfully applied to the quantitative analysis of soya isoflavones in plasma [11]and in baby foods and soya flour [12]. We now wish to report on the development of a method for quantitation of xanthohumol and five other prenylflavonoids in hops and beer (Fig. 1) by HPLC–MS–MS. To demonstrate the suitability of the method, a selection of beers and two hop-containing herb teas have also been analysed.