Dad 3000

The sample analysis was performed by using a Dionex UltiMate 3000 UHPLC system (Thermo Scientific, San Jose, CA, USA), comprising a LPG-3400SD binary pump, WPS-3000TSL thermostat autosampler, TCC-3000SD thermostat column compartment and DAD-3000 diode array detector. The data were recorded and analyzed by Chromeleon™ 7.2 Chromatography Data System Software (Thermo Scientific, San Jose, CA, USA).
The chromatographic separation of MI, MCI, MP, EP, PP and BP was achieved through an ACCLAIM™ 120 C₈ analytical column with the dimensions 150 mm × 2.1 mm and 5 μm of particles size (Thermo Scientific, San Jose, USA). The optimal separation was obtained by using binary mobile phase: water (0.1% trifluoroacetic acid, pH 2.1, solvent A) and acetonitrile (solvent B) at a flow rate of 0.5 mL/min. The gradient mobile phase elution was 0–2 min (B, 12.5%), 2–4 min (B 20–30%), 4–16 min (B, 30–50%), 16–22 min (B, 50–100%), return to its equilibrium conditions and 22–30 min. The column temperature was kept at 35 °C, and the sample injection volume was 10 µL. The column was also washed with a mixture (50:50, v/v) of methanol and Milli-Q water solution, for five minutes, following the analysis of every ten samples. The optimal detection wavelength was performed in the UV range at 255 nm.

The HAAs were analyzed using HPLC (Thermo Ultimate 3000, Thermo Scientific, Waltham, MA, USA) equipped with an autosampler (WPS-3000), a pump (LPG-3400SD), and a diode array detector (DAD-3000). A reversed-phase analytical column (Acclaim^TM 120 C₁₈, 3 μm, 4.6 × 150 mm) from Tosoh Bioscience GmbH (Stuttgart, Germany) was used for separation, and the column temperature was maintained at 35 °C. A gradient program was applied using a mobile phase of methanol: acetonitrile: deionized water: acetic acid (8:14:76:2, v/v/v/v) as a solvent A (pH = 5.0 adjusted with ammonium hydroxide (25%)); and acetonitrile as a solvent B. HAAs were identified by their retention times and quantified using an external calibration curve.

The cannabinoids were identified and quantified by high pressure liquid chromatography (HPLC) (Ultimate 3000, ThermoFisher) and photodiode (DAD-3000, ThermoFisher) array detection. Certified reference materials purchased from Cayman Chemical Company (Ann-Arbor, MI, USA) and Sigma-Aldrich were used to prepare calibration curves. Samples were injected in 5 μL volumes and eluted through a Raptor ARC-18 analytical column (Restek, Bellefonte, PA, USA) with 77% acetonitrile (buffered with 0.1% v/v formic acid) and 23% water (buffered with 5 m m ammonium formate and 0.1% v/v formic acid). Flow rate was set to 0.95 mL/min, the column compartment was maintained at 25°C and the UV-Vis signals were recorded using a DAD-3000 photodiode array detector. Calibrations and quantifications were based on absorbances measured at 228 nm.