Validation of a rapid and sensitive reversed-phase liquid chromatographic method for the quantification of prenylated chalcones and flavanones in plasma and urine

Abstract

Prenylated chalcones and flavanones from hops (Humulus lupulus), such as 6-prenylnaringenin (6PN), 8-prenylnaringenin (8PN), xanthohumol (XN) and isoxanthohumol (IXN), are increasingly investigated for their potential health benefits, but validated and easy-to-implement methods for their sensitive quantification in biological samples are scarce. We therefore developed and validated a reversed-phase HPLC method with UV and fluorescence detection for the sensitive analysis of 6PN, 8PN, XN and IXN in human plasma and urine. Separation of all analytes and naringenin (a potential internal standard) was achieved within 20min using gradient elution and a pentafluorophenyl column. The method was validated according to the guidelines for bioanalytical method validation of the Center for Drug Evaluation and Research of the U.S. Food and Drug Administration. Recovery, precision and accuracy deviated <20% from the known values, with the exception of XN, which isomerized to IXN during sample preparation. Using the sum of XN+IXN as a measure of XN in the samples improved recovery, precision and accuracy to <25% deviation from the known values. Sensitivity (lower limits of detection and quantification) was comparable to published LC methods with UV-detection or better. The lower limit of quantification for IXN was 1.8ng/mL, which is near the reported sensitivity of tandem MS detection of 1.0ng/mL. In conclusion, we report a validated and sensitive LC method for the quantification of prenylated chalcones and flavanones in human plasma and urine with potential for adaptation to other sample matrices.