**4. Conclusions**

This study describes the first detailed validated method for quantification of OLE and other CGs in culinary herbs and human urine. The method displays good specificity, linearity, accuracy, and expanded measurement uncertainty, thus enabling the accurate quantification of OLE, DIGO, DIGI, and CON in culinary herbs and OLE, DIGO, DIGI, CON, and OUB in human urine. This new method was applied to the analysis of more than 60 samples of culinary herbs and herb/spice mixtures containing bay leaves present on the Belgian food market, showing that these products are safe for the consumer. The UHPLC-ESI-MS/MS method described here could, therefore, become a useful tool to determine these plant toxins in culinary herbs and also in urine.

#### **5. Materials and Methods**

#### *5.1. Standards, Reagents, and Consumables*

Analytical standards of OLE, DIGO, DIGI, CON, and OUB octahydrate were purchased from Sigma-Aldrich (Buchs, Switzerland). Individual stock solutions were prepared by dissolving the crystalline standards in MeOH at a concentration of 1 mg/mL. A methanolic solution of DIGO-D (1 mg/mL) was obtained from Cayman Chemicals (Ann Arbor, MI, USA). Intermediate solutions were prepared by diluting the stock solutions in MeOH. The stock and the intermediate solutions were stored at −20 ◦C.

The MeOH absolute ULC-MS, ACN ULC-MS, and HCOOH 99% ULC-MS were purchased from Biosolve (Valkenswaard, the Netherlands). The ammonia solution 28–30% was obtained from Merck (Darmstadt, Germany), while NH4HCO3 LC-MS and HCOONH4 LC-MS were supplied by Sigma-Aldrich (Steinheim, Germany). H2O was purified by a Milli-Q purification system (Millipore Corp., Bedford, MA, USA).

Oasis® MAX (3 cc, 60 mg) LP and Oasis® HLB (3 cc, 60 mg) extraction cartridges were provided by Waters (Wexford, Ireland). Discovery® DSC-18 (6 mL, 500 mg) and Supelclean™ ENVI-Carb™ (6 mL, 500 mg) SPE cartridges were obtained from Sigma-Aldrich. VWR (Randor, PA, USA) was the supplier of 15 and 50 mL centrifuge PP tubes and centrifugal filters (modified nylon, 0.2 μm, 500 μL).

## *5.2. UHPLC-MS*/*MS Conditions*

The UHPLC-MS/MS system consisted of an ACQUITY UPLC H-class system coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA).

The mass spectrometer was operated in the ESI(+) mode. The MS parameters were set as follows: Source and desolvation temperatures: 150 and 350 ◦C, respectively; capillary voltage: 1.50 kV; cone and desolvation gas flows: 150 and 1000 L/h, respectively; collision gas flow: 0.15 mL/min; source offset: 30 V. The SRM acquisition mode was used.

Chromatographic separation was achieved on an AQUITY UPLCTM BEH C18 column (2.1 × 100 mm; 1.7 μm) with an ACQUITY UPLCTM BEH C18 VanGuard precolumn (2.1 × 5 mm; 1.7 μm) (both from Waters). The column temperature was maintained at 40 ◦C. The mobile phase was composed of phase A (H2O containing 10 mM NH4HCO3 pH 9) and phase B (ACN). The flow rate used was 0.45 mL/min and the applied gradient elution program was as follows: 0–1 min: 95% A, 1–6 min: 40% A, 6–7 min: 10% A, 7–7.1 min: 95% A, 7.1–10 min: 95% A. The injection volume was 10 μL.
