*5.5. Validation*

The validation study was performed using spiked analyte-free representative sample materials. The following method parameters were evaluated: LOQ, specificity, linearity, recovery, repeatability (RSDr), reproducibility (RSDwR), matrix e ffects, and expanded measurement uncertainty (MU).

LOQ was defined as the minimum analyte concentration in the spiked samples that produced an SRM transition with a minimum S/N of 10. Specificity of the method was assessed through the analysis of blank matrix samples. The absence of a peak with a S/N ≥ 3 at the expected retention time of the target CG indicated good method specificity. Linearity of the method was evaluated by fortifying blank matrix samples with the target analytes at varying concentrations (minimum five levels). A logarithmic transformation of the axes and a linear regression model were applied. (Apparent) recovery was assessed by a spiking of blank matrix with the target analytes at three concentration levels in triplicate. The measured concentrations were determined using a matrix-matched calibration curve and the recovery was calculated as follows (Equation 1):

$$\text{Recovery } (\%) = \text{Measured concentration} / \text{Theoretical concentration} \times 100. \tag{1}$$

For determination of the repeatability (RSDr), spiking experiments were performed at three concentration levels in triplicate on the same day, while for within-laboratory reproducibility (RSDwR) evaluation, the same experiments were carried out on three days. Matrix e ffects were assessed by comparing the slopes of calibration curves prepared in the matrix extract and neat solvent. The *t*-test was used for statistical evaluation of the matrix e ffect data. The expanded measurement uncertainty (MU) (corresponding to a 95% confidence level and a coverage factor of 2) was estimated according to [44].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/12/4/243/s1, Table S1: Comparison of performance characteristics of di fferent LC-MS/MS methods for analysis of cardiac glycosides; Table S2: Overview of culinary herbs and spices collected in Belgian food stores.

**Author Contributions:** Conceptualization, S.V.M. and J.M.; methodology, S.V.M., P.P.J.M., and J.M.; validation, S.V.M. and J.M.; formal analysis, S.V.M. and J.M.; investigation, S.V.M., P.P.J.M., and J.M.; data curation, S.V.M.; writing—original draft preparation, S.V.M., P.P.J.M., and J.M.; writing—review and editing, S.V.M., P.P.J.M., and J.M.; visualization, S.V.M., P.P.J.M., and J.M.; supervision, J.M.; project administration, S.V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Belgian National Reference Laboratory for Mycotoxins, Plant Toxins, and Marine Biotoxins (contract number FAVV-NRL-GGO-TOX-2017, lot 2).

**Acknowledgments:** Benoît Guillaume and Céline Segers are acknowledged for their technical support, and Séverine Goscinny is thanked for providing a part of the herb samples.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
