*2.5. HPLC-MS/MS and HPLC-qTOF-MS Settings*

Chromatographic separation for MS/MS analysis was achieved using a 1260 Infinity LC system (Agilent Technologies, Waldbronn, Germany). MS/MS analysis was performed using a QTrap® 5500 mass spectrometer equipped with a Turbo V ion source and operated with Analyst software 1.6.2 (Sciex, Darmstadt, Germany). The obtained values for the MS parameters declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) were individually determined by infusing standard solutions into the MS system. MS parameters are as follows: Q1 (*m*/*z*), 225; Q3 (*m*/*z*) QN (quantifier transition)/QL (qualifier transition), 193/167; declustering potential (DP), 104; collision energy (CE) QN/QL, 18/23; CXP (V), 11. Retention time (RT): 5.39 min for *erythro*-asarone diols and 5.69 min for *threo*-asarone diols. Further HPLC-MS/MS setup details are presented in Supporting Information Table S2.

Chromatographic separation for qTOF-MS analysis was achieved using a Bruker Elute system (Bruker, Bremen, Germany). Mass spectrometric analysis was carried out on a Bruker impact II qTOF system equipped with an ESI Apollo II ion source operated in positive and negative ionization mode, depending on the analyte of interest (Bruker, Bremen, Germany). For identification of phase II metabolites, a full scan mode within a mass range of *m*/*z* 50 to 1300 as well as Auto MS/MS scan modes, were used. Further HPLC-qTOF-MS setup details for the analysis of liver microsome samples, as well as urine samples are given in the Supporting Information Tables S3 and S4.

#### **3. Results**

#### *3.1. Microsome Experiments*

Incubation of the selected phase I metabolites 3- OH and bAE with pig liver microsomes resulted in formation of different glucuronic acid conjugates. For bAE, it is re-ported that this compound is not stable and hydrolyzes in aqueous solution, with a half-life between 2.4 min and 4 min to *erythro*- and *threo*-asarone diols and asarone ketone [13,28].

Consequently, incubation of bAE with microsomes resulted in diol-derived glucuronic acid conjugates. The extracted ion chromatograms (XICs) with *m*/*z* 399.1297 for 3- OH glucuronide (Figure 2a) and *m*/*z* 417.1402 for *erythro*- and threo-asarone diols-derived glucuronic acid conjugates (Figure 2b) allowed the detection of two peaks with mass differences (Δm) of 0.5 ppm and 0.8 ppm to the calculated masses of [M–H]−. Figure 2c shows the qTOF-MS spectrum of the 3- OH-glucuronide. The fragment with *m*/*z* 223.0984 can be assigned to the loss of the glucuronic acid moiety and corresponds to the [M–H]− of 3- OH (Figure 2c). Due to its low concentration, the spectrum of the *erythro*- and threo-asarone diol-glucuronides did not provide significant fragmentation data. Liver microsomes of human and horse were also used to investigate the phase II metabolism of both phase I metabolites (3- OH, bAE). The respective glucuronic acid conjugates were formed by all species but with slightly different turnover rates (data not shown). Detailed information about species-specific phase II-Metabolism has to be considered in subsequent analyses and are not in the scope of the presented investigations. Sulfuric acid conjugation was not observed at all, indicating that glucuronidation can be considered as the main metabolic phase II pathway in microsomes from all species.
