Quantification of xanthohumol, isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin in hop extracts and derived capsules using secondary standards

Hop (Humulus lupulus) is a frequently used estrogenic herbal medicinal product, containing the prenylflavonoids xanthohumol (XN), isoxanthohumol (IXN), 8- and 6-prenylnaringenin (8-PN and 6-PN). Although many analytical methods have been developed for the quantification of these compounds, there still is a lack of validated methods for routine control. Therefore we have developed an accessible HPLC-DAD method using quercetin and naringenin as secondary standards for the analysis of hop extract and capsules. After optimization of sample preparation and HPLC conditions, the analysis was validated according to the ICH guidelines. The response function of XN, 8-PN, quercetin and naringenin showed a linear relationship. For the determination of XN, a calibration line of at least three concentrations of quercetin was constructed. The correction factors for XN (quercetin) and 8-PN (naringenin) were validated and determined to be 0.583 for XN, and 1.296 for IXN, 8-PN and 6-PN. The intermediate precision was investigated and it could be concluded that the standard deviation of the method was equal considering time and concentration (RSD of 2.5–5%). By means of a recovery experiment, it was proven that the method is accurate (recoveries of 96.1–100.1%). Several hop-containing preparations available on the Belgian market were analysed in order to check their compliance to the guideline concerning the maximal daily intake of selected medicinal plants, establised by the Advisory Committee for Plant Preparations. For hop the maximal daily dose is limited to 400µg of 8-PN. These analyses showed that the reported method was applicable for this conformity testing.