Characterization of phase I and phase II metabolites of hop (Humulus lupulus L.) bitter acids: In vitro and in vivo metabolic profiling by UHPLC-Q-Orbitrap

Bitter acids are a class of prenylated phloroglucinol derivatives present in Humulus lupulus L., known for their multiple healthy properties, nevertheless, research regarding their metabolism and stability is lacking. This study was aimed to elucidate the metabolic stability of hop α- and β-acids and characterize I and II phase metabolites in vitro and in vivo. For this purpose, an ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method was developed and validated. Mice liver microsomes were used to assess metabolic stability; in vitro t1/2 and clearance values were calculated, showing a moderate metabolism for α-acids (avgt1/2: 120.01 min, avgCLint 11.96 μL/min/mg), while β-acids were metabolized faster (avgt1/2: 103.01 min, avgCLint: 13.83 μL/min/mg). I and II phase metabolites were characterized both in in vitro, and in vivo, in mouse plasma and urine after oral administration. A combined full scan/data dependent/precursor ion list-triggered neutral loss (FS/dd-MS2/PIL-tNL) strategy was used to detect unknown and expected metabolites. As a result, 33 compounds were detected, including novel metabolites, such as 9 potential oxidized metabolites of humulones (M6-M14), and 10 glucuronide conjugates of α-acids, comprising 7 glucuronide derivatives of oxidized phase I metabolites (M26-M32). The proposed method extends the current knowledge regarding metabolization of hop α- and β-acids and could be applied for the comprehension of the metabolic fate of this class of compounds in different species, as well as for in vivo pharmacokinetic studies.