2.3.1. Study Conditions and Subjects

Ten healthy enrolled participants (five females and five males, age 25.8 ± 4.0, BMI 23.8 ± 1.9) were informed about the aim and scope of the study and gave their written consent to the study conditions prior to their commencement. Samples and food diaries were equipped with a six-digit number and assigned to each participant randomly. The study was approved by the research ethical committee of the University Hospital Münster, Germany (File reference: 2020-002-f-S).

#### 2.3.2. Study Design

Study participants were not allowed to consume *A. calamus*-derived preparations or herbal products three days before intake of the tea infusion (wash-out phase), and wrote a food diary during the complete study progress (Figure 1).

**Figure 1.** Study design including wash-out phase, diary keeping and urine collection after intake of 300 mL calamus tea infusion. Urine samples were collected for 48 h and total urine volume was determined. The participants collected a spot urine sample every time they urinated.

On day one, participants passed a morning urine sample as blank before calamus tea consumption. Tea preparation is described in (Section 2.3.3 "Calamus Tea selection"). All participants consumed at the same time 300 mL of the prepared tea infusion to ensure a consistent intake of asarone isomers. The tea infusion was consumed within half an hour and urine was collected for 48 h. The total urine volume over 48 h was determined by each participant by summarizing the volumes of each spot urine sample. Thereafter, an aliquot of 2 mL of each urine sample was collected for every time point (Figure 1). Urine samples were stored at 20 ◦C prior to sample preparation.

The total urine volume and the concentrations of *erythro*- and *threo*-asarone diols after beta-glucuronidase treatment was used to determine the excretion rate. Excretion values over 48 h were totalized and were compared with the amounts of *erythro*- and *threo*-asarone diols and bA in 300 mL of the consumed tea infusion. The average of all values for each participant was used as overall excretion rate (%).

The kinetic curve was determined using point in time of urination (aliquot of each urine delivery of each participant), given urine volume and concentrations of *erythro*- and *threo*-asarone diols in the urine samples over the period of 48 h. Time points were classified in two-hour blocks, except for the night hours (14–20 h) and the last 24 h, because in this period only a small number of samples was above the limit of quantitation (LOQ). The results were represented in a box-plot-whisker diagram. The average 50% of the data is located within the box, whose dimensions are defined by the lower and upper quartiles. The lowest and highest data points are shown by the whiskers if the values fall within the 1.5 interquartile range otherwise they are outliers and demonstrated as dots below and above the whiskers.

#### 2.3.3. Calamus Tea Selection

Calamus tea consisted of organically grown calamus roots, which have been dried and chopped prior to disposal. The used calamus infusion classified as food was analyzed within a previous product screening study [26]. Tea was prepared by weighing 48.5 g of dried calamus roots, infusing it with 3300 mL of boiling water and steeping for 15 min. For analysis of the asarone amount, an aliquot was filtrated using a 0.45 μM regenerated cellulose (RC) membrane (Phenomenex, Aschaffenburg, Germany), diluted with acetonitrile/0.1% formic acid in water (12/88, *v*/*v*) and analyzed by HPLC-MS/MS as reported previously [26]. 300 mL of the infusion contained 0.76 mg bA, 0.65 mg *erythro*-asarone diols and 1.38 mg *threo*-asarone diols. The amounts are in the mean of commercially available calamus infusions [26]. The fresh calamus tea was prepared by a separation of the rhizome from the *A. calamus* plant, which was bought in a local garden center (Vechta, Germany) The outer root layer was cleaned, and 3 g of calamus roots were crushed. Thereafter the roots were infused with 200 mL boiling water (100 ◦C). Steeping time, filtration, dilution and analysis were carried out as described above.

#### 2.3.4. Urine Sample Preparation

To 100 μL of each urine sample of each collection point, 100 μL of ammonium hydrogen carbonate (NH4HCO3) buffer (pH 6.6) were added which contained 6000 U/mL of betaglucuronidase. The samples were incubated for 16 h at 37 ◦C by gently shaking. A volume of 200 μL acetonitrile were added to stop the enzyme reaction and after homogenization samples were centrifugated at 14,000× *g* for 5 min at 5 ◦C. Afterwards, the supernatant was diluted 1:10 with acetonitrile/0.1% formic acid in water (12/88, *v*/*v*) prior to HPLC-MS/MS or qTOF-MS analysis. Workability of beta-glucuronidase was verified by the 4-methylumbelliferyl-β-D-glucuronide converted to the highly fluorescent 4-methylumbelliferon and glucuronic acid. Fluorescence was measured at 365/440 nm.
