*Article* **Phase II Metabolism of Asarone Isomers In Vitro and in Humans Using HPLC-MS/MS and HPLC-qToF/MS**

**Lena Hermes, Janis Römermann, Benedikt Cramer and Melanie Esselen \***

Institute of Food Chemistry, University of Muenster, Corrensstraße 45, 48149 Muenster, Germany; lena.hermes@uni-muenster.de (L.H.); ja.roemermann@gmail.com (J.R.); cramerb@uni-muenster.de (B.C.) **\*** Correspondence: esselen@uni-muenster.de; Tel.: +49-251-8333874

**Abstract:** (1) Background: Metabolism data of asarone isomers, in particular phase II, in vitro and in humans is limited so far. For the first time, phase II metabolites of asarone isomers were characterized and human kinetic as well as excretion data after oral intake of asarone-containing tea infusion was determined. (2) Methods: A high pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-qTOF-MS) approach was used to identify phase II metabolites using liver microsomes of different species and in human urine samples. For quantitation of the respective glucuronides, a beta-glucuronidase treatment was performed prior to analysis via high pressure liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). (3) Results: Ingested beta-asarone and *erythro* and *threo*-asarone diols were excreted as diols and respective diol glucuronide conjugates within 24 h. An excretion rate about 42% was estimated. *O*-Demethylation of beta-asarone was also indicated as a human metabolic pathway because a corresponding glucuronic acid conjugate was suggested. (4) Conclusions: Already reported *O*-demethylation and epoxide-derived diols formation in phase I metabolism of beta-asarone in vitro was verified in humans and glucuronidation was characterized as main conjugation reaction. The excretion rate of 42% as *erythro* and *threo*-asarone diols and respective asarone diol glucuronides suggests that epoxide formation is a key step in beta-asarone metabolism, but further, as yet unknown metabolites should also be taken into consideration.

**Keywords:** asarone isomers; human study; metabolites; asarone glucuronides
