3.3.2. HPLC-MS/MS and qTOF-MS Analysis of Urine Samples

Figure 4 shows HPLC-MS/MS chromatograms of an exemplary urine sample from one randomly selected participant before (a) and after beta-glucuronidase treatment (b), recorded in MRM-mode. The subsequently mentioned metabolism was observed in the urine of all participants with marginal differences in individual metabolite concentrations and excretion rates. The two peaks (5.39 and 5.69 min) represent the *erythro*- and *threo*asarone diols, respectively, whereas the peak with a retention time of 5.80 min showing the same MRM transition could not be identified with the available standards (Figure 4a). No signal corresponding to 3- OH or asarone ketone was detected in all analyzed urine samples. Furthermore, no hints for a 3- OH glucuronide were found. However, after betaglucuronidase treatment, the signal at 5.80 min disappeared, while the *erythro*-asarone diols peak (5.39 min) slightly and the *threo*-asarone diols peak (5.69 min) strongly increased (Figure 4b). These results suggest that the peak eluting at 5.80 min represents glucuronidated metabolites of the consumed asarone derivatives.

**Figure 4.** HPLC-MS/MS chromatogram of a randomly selected urine sample, which was given after consumption of a calamus tea infusion, (**a**) before; (**b**) after treatment with beta-glucuronidase.

To verify these findings and further to identify further new phase II metabolites, an untargeted HPLC-qTOF-MS approach was applied to human urine samples before betaglucuronidase treatment. For the main peak, a mass of *m*/*z* 417.1404 ([C18H26O11–H]−, Δm: 0.2 ppm) supports the suggestion that *erythro*- and *threo*-asarone diol-glucuronides are potential phase II metabolites in humans (Figure 5a). Moreover, an unknown metabolite with an exact mass of *m*/*z* 403.1256 was detected in human urine. Based on a calculated *m*/*z* of 403.1256 for [C17H24O11–H]−, a mass difference of 1 ppm to the calculated mass

suggested that also demethylated *erythro*- and *threo*-asarone diols-derived glucuronides were formed (Figure 5b). The recorded qTOF-MS spectrum supports our suggestions. The detected fragment ions of *m*/*z* 227.0923 are reported to arise due to the loss of the glucuronic acid moiety, and *m*/*z* 212.0685 with a further loss of a methyl group (Figure 5c).

**Figure 5.** Exemplary HPLC-qTOF-MS chromatograms of a randomly selected urine sample before enzyme treatment. Presented are the extracted-ion chromatogram (XICs) with the calculated mass for (**a**) *erythro*- and *threo*-asarone diol glucuronides (diol-glucuronides, *m*/*z* 417.1404 ± 0.02) and (**b**) demethylated *erythro*- and *threo*-asarone diol glucuronides (demethylated diol-glucuronides, *m*/*z* 403.1247 ± 0.01). (**c**) HPLC-qTOF-MS spectrum of the *O*-demethylated *erythro*and *threo*-asarone diol glucuronides (*m*/*z* 403.1247 ± 0.01) with the respective structural formula. The fragment *m*/*z* 227.0923 ± 0.02 corresponds to the *O*-demethylated metabolites after glucuronic acid cleavage. The loss of a further methyl group is shown by the exact mass of *m*/*z* 212.0685 ± 0.01.

As mentioned before, the calamus infusion used for the human study contains bA as well as *erythro*- and *threo*-asarone diols, thus the potential metabolization of bA to the identified phase II metabolites is not possible. Therefore, in a second proof of concept experiment, an infusion of fresh calamus roots was consumed by three participants and urine samples were collected as described above. It is reported that this tea infusion contains only bA (20 mg/kg) [26]. The analysis of these three urine samples showed that all characterized phase II metabolites are also excreted after single bA intake. Corresponding chromatograms are shown in Supporting Information Figure S1.

#### 3.3.3. Kinetic Studies and Excretion Rate Determination

Kinetic data and excretion rates were determined based on the analysis of the *erythro*and *threo*-asarone diol peaks formed after beta-glucuronidase treatment, because the respec-

tive glucuronides were characterized as main human metabolites. The *O*-demethylated *erythro*- and *threo*-asarone diols-derived glucuronides were not included, because no corresponding reference compounds were available. Based on the above-mentioned finding, that an oral intake of bA also results in a renal excretion of *erythro*- and *threo*-asarone diols glucuronides, the overall amount of bA and *erythro*- and *threo*-asarone-diols was used to calculate total excretion rates. In sum, a total excretion of 42 ± 6% of all participants was determined. Kinetic data over a period of 48 h is shown in Figure 6. The respective phase II metabolites were rapidly excreted, with a maximum excretion between one and six hours. After 24 h only marginal amounts of *erythro*- and *threo*-asarone diols were further detected, thus the period between 24 h and 48 h was summarized in one bar.

**Figure 6.** *Erythro*- and *threo*-asarone diols (diols) excretion kinetic [μg] of ten participants over a period of 48 h. Excretion is classified in two-hour blocks, except for the night hours (14–20 h) and the last 24 h, because the concentrations of the metabolites were mostly under the Limit of Quantification (LOQ).
