*3.4. Extraction Procedures*

Extraction optimization was carried out using *G. verum* and after, under optimized conditions, the microextraction procedure was applied for the other four *Galium* species. The following microextraction procedure were investigated: DLLME, ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME), Salting-out liquid-liquid extraction (SA-LLE), and Sugaring-out liquid-liquid extraction (SULLE). The general procedure for the extractions reported in the following paragraphs was described in Figure 3.

**Figure 3.** General extraction procedure.

#### 3.4.1. DLLME and UA-DLLME

10 mg of the dry plant material of *G. verum* were accurately weighted and placed into a 2 mL Eppendorf tube. Subsequently, 700 μL of solvent medium (water, 10% NaCl, NADES, IL or 1% β-cyclodextrin (β-CD)), 400 μL of ethyl acetate, and 300 μL of ethanol were added to the Eppendorf tube by automatic pipette. The solution was vortexed during 30 s until a cloudy solution was formed. In the case of UA-DLLME, after those steps, the test tube was placed into the ultrasound bath for 5 min. Then, the solution was kept at rest for 1 min, for the analytes to distribute into the extraction solvent. For the phase separation, the solution was centrifuged at 12000× *g* for 5 min. The extraction solvent was found on the top of the Eppendorf tube, and its whole volume was collected using a microsyringe and transferred to the new Eppendorf tube, and then dried under a gentle stream of nitrogen. The dried residue was redissolved in 50 μL of mobile phase under ultrasonication for 5 min and 20 μL of the obtained solution were injected into the HPLC system.
