Vortex mixer

Sample preparation followed a published protocol 29. Briefly, ground Verbenae herba plant material (VO‐1 to VO‐3) was frozen in liquid nitrogen and homogenized with an analytical ball mill (Mikro‐Dismembrator, Sartorius, Göttingen, Germany). 100.0 ± 0.1 mg plant material was weighed into 1.5 mL polyethylene microcentrifuge tubes (Eppendorf, Hamburg, Germany), mixed with 1.0 mL ethanol/water (1:1, v/v) on a Vortex mixer (VWR, Vienna, Austria) and extracted by sonication for 10 min. After centrifugation (10621 x g for 5 min) supernatants were placed in a 5 mL volumetric flask. This procedure was repeated four more times, and the flask filled up to the final volume with the extraction solvent. All sample solutions were prepared in triplicated and filter prior analysis.

Sodium sulfate (7 g) was added to soil samples (5 g) to remove any moisture and followed by addition of 15 mL of 2:1 hexane:acetone mixture, 5 mL of 1:4 triethylamine:acetone mixture, and p-terphenyl-d14 in a microwave extraction tube (Chigbo et al. 2013 (link)). The content of the tube was mixed using a vortex mixer (VWR, UK) and shaken by inversion to dislodge soil material from the base. Extraction was carried out with a microwave extraction unit (CEM MARS) with the following conditions: temperature ramp to 100 °C at 800 W for 12 min, hold at 100 °C at 800 W for 10 min then cool for 5 min in accordance with the USEPA method 3546 (USEPA 2007 ). Following the extraction, the clear extracts were transferred into glass tubes (20 mL). For the solid phase extraction, SPE HF Mega BE-SI 2 g 12 mL cartridges (Agilent, UK) were conditioned with 5 mL of hexane then, 1 mL of sample extract was added and eluted with 10 mL of 1:1 hexane:dichloromethane mixture. The eluant was collected in a clean 20 mL glass tube and concentrated to a final volume of 1 mL under a gentle stream of nitrogen gas. Samples were prepared in 2 mL vials (Agilent, UK) by adding a semi-volatile internal standard mix to the concentrated sample extracts from soil samples for GC-MS analysis.

Samples (500 mg) in the form of edible films (EFs) were weighed with the addition of methanol (15 mL). EFs solutions were initially mixed vigorously by using a vortex mixer (VWR int., Germany) followed by centrifugation at 10,000 RPM for 5 min at 5 °C. The supernatant obtained was utilized for assessing antioxidant activities.

1M chloride salt solutions were prepared using CaCl₂, MgCl₂, KCl, NaCl, MnCl₂, iron(III)chloride hexahydrate (FeCl₃ + 6 H₂O), CuCl₂, and ZnCl₂ crystals (purity ≥ 98.0%; Sigma-Aldrich, 3050 Spruce St., St. Louis, MO, USA) dissolved in deionized water. These chloride salt solutions were separately kept in polypropylene containers. Before Raman measurements, each solution was well shaken using a vortex mixer (VWR International Ltd., Blanchardstown, Dublin, Ireland) for 1 min to ensure complete dissolution. During the experiment, 10 mL of each solution was transferred into a 50 mL quartz beaker with aluminum foil smoothly covering the outer surface and then placed on an elevating frame under the fiber optic probe stand of the Raman spectrometer (ThermoFisher Scientific UK Ltd., Loughborough, UK). The sample was raised to ensure that its surface was 2 mm away from the quartz screen of the probe head. Detailed measurement procedures and instrumental settings are described in Section 2.2. Each sample was scanned twice at two different surface locations; the mean of these duplicate spectra was used for chemometric analysis.

For the preparation of each sample, 100 mg of B. scorpioides plant material was frozen in liquid nitrogen and homogenized using a Mikro-Dismembrator (Sartorius, Göttingen, Germany). The homogenization procedure was done separately for each of the different experiments (quantitation, precision, and accuracy). For extraction, 7.0 mL of water was added to the plant material, the sample was mixed on a Vortex mixer (VWR, Vienna, Austria) and afterwards extracted in an ultrasonic bath (15 min at room temperature). After centrifugation at 2000× g for 2 min the supernatant was placed in a 25 mL volumetric flask. This procedure was repeated twice and subsequently the flask was filled up to the final volume with the extraction solvent. Finally, the sample solution was filtered through 0.45 µm Phenex-RC 4 mm syringe filters (Phenomenex, Torrance, CA, USA).