*3.2. Method Validation*

Method validation of the used HPLC-MS/MS method was performed prior to analysis of the urine samples from the human study. As *erythro*- and *threo*-asarone diols were found to be the dominant metabolites in urine after beta-glucuronidase treatment, quantitation of these compounds with a matrix-matched calibration in blank urine was performed. *Erythro*- and *threo*-asarone diols are diastereomers, which represent a pair of enantiomers, respectively (Figure 3a).

Accordingly, with the used HPLC-MS/MS method, for the diastereomers *erythro*and *threo*-asarone diols could be chromatographically separated, while the enantiomers coeluted. Figure 3b shows the analysis of one selected urine sample spiked with erythro and threo-asarone diols at a concentration of 5 ng/mL

From matrix-matched calibration, the validation parameters LOD and LOQ as well as linearity were determined. Linearity across the applied concentration range was confirmed by means of the Mandel's fitting test as well as a R2 ≥ 0.995 for the analytes. The analytical precision via interday and intraday repeatability reached values of between 3% and 12% and recovery rates of 83% or 103% were determined. All values fall into an acceptable range considering the respective US Food and Drug Administration regulations [27]. The validation parameters are illustrated in Table 1.

**Figure 2.** HPLC-qTOF-MS chromatograms after incubation of (**a**) 3- OH and (**b**) bAE in pig liver microsomes. Presented are the extracted-ion chromatogram (XICs) with the calculated mass of (**a**) *m*/*z* 399.1305 ± 0.01 for the 3- OH glucuronide and (**b**) *m*/*z* 417.1397 ± 0.01 for *erythro*- and *threo*-asarone diols-derived glucuronic acid conjugates. (**c**) HPLC-qTOF-MS spectrum of 3- OH glucuronide (*m*/*z* 399.1305 ± 0.01) with the respective structural formula and the suggested cleavage of the glucuronic acid majority to *m*/*z* 223.0984.

**Figure 3.** (**a**) Structural illustration of *erythro*- and *threo*-asarone diols and their stereochemistry. (**b**) HPLC-MS/MS chromatogram of a 1:10 diluted urine sample spiked with 5 ng/mL of erythro- and threo-asarone diols. Presented are the quantifier (*m*/*z* 225→193) and qualifier (*m*/*z* 225→167) SRM transition.


**Table 1.** Method performance characteristics of the LC-MS/MS method used for quantitation of *erythro*- and *threo*-asarone diols in urine samples.

#### *3.3. Human Study*

3.3.1. Analysis of the Consumed Tea Infusion

The amounts of bA (0.76 mg) as well as *erythro*- (0.65 mg) and *threo*-diols (1.38 mg) in 300 mL of the consumed tea were used in total (2.79 mg) for calculation of the excretion rates.
