*2.4. Method Validation*

The developed HPLC-MS/MS method for quantitation of *erythro*- and *threo*-asarone diols was evaluated with regard to the following parameters: linearity, limit of detection (LOD), LOQ, recovery as well as intraday and interday repeatability [27].

For quantitation of *erythro*- and *threo*-asarone diols in the urine samples, a matrixmatched calibration in blank urine of different volunteers was prepared. To that end, urine was processed as described in Section 2.3.4. and fortified with the analytes at concentrations of 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 ng/mL, respectively. Linearity across the whole working range was verified by the Mandel's fitting test and a coefficient of determination (R2) ≥ 0.995.

LOD and LOQ were determined using a matrix-matched approach in blank urine of different volunteers. Analytes were spiked in the following concentrations 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 and 10 ng/mL. Procedure was performed in triplicate. LOD and LOQ were determined using a Signal to Noise (S/N) approach receiving a S/N ratio of three for LOD and ten for LOQ.

For determination of recovery rates, blank urine was spiked with distinct analyte concentrations (0.25, 0.5, 1, 2.5, 5, 10, 25, 50 ng/mL) prior to sample preparation (Section 2.3.4). For calculation, the matrix-matched calibration was measured along with the matrix calibration and the slope of both calibration curves was compared. Each calibration point was prepared in triplicate.

The precision of the method is described by interday and intraday repeatability. Intraday repeatability was evaluated by preparing and analyzing one randomly chosen urine sample of one test person ten times. For interday repeatability one sample was prepared three times and repeatedly injected throughout the measurement of all urine samples of the study.
