4.4.3. HR-MS/MS

For UHPLC–qTOF-MS analysis, a UHPLC system (Agilent 1290) with a quadrupole Time-Of-Flight (TOF) mass spectrometer (Agilent 6540 UHD Accurate-Mass, Agilent, Waldbronn, Germany), using an ESI interface, operating in positive ionization mode with a 4 kV capillary voltage, was used. The source operated at 325 ◦C and nitrogen was used as both the drying (40 psi) and nebulizing gas (10 L min-1). The injection volume was 5 μL. The TOF-MS detector was set to acquire MS data over an *m*/*z* range of 100–1600. All-Ions MS/MS experiments were performed to screen and quantify constituents of the sample in a single analysis. Under the aforementioned conditions, fragmentation collision energies ranged from 5–60 eV.

The analytical column was a Waters BEH C18 100 × 2.1 mm, 1.7 μm particle size (Waters, Milford, MA, USA), temperature-controlled at 40 ◦C. The mobile phase consisted of water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) at a flow rate of 200 μL min<sup>−</sup>1. The gradient program changed linearly from 50% to 95% (B) in 25 min, followed by an isocratic elution for 4 min. An equilibration time of 1 min was set for the mobile phase to reach initial conditions again.

For Direct infusion Orbitrap-MS, and in correlation with the UHPLC–qTOF-MS analysis, additional measurements were performed using an Orbitrap MS system, to confirm the molar mass (M) and the mass fragmentation patterns of the suspected NPS. This analysis was performed in positive-ion mode with Electro Spray Ionization (ESI) using a Thermo LTQ Orbitrap MS (Thermo Scientific, Bremen, Germany), and operated with mass resolution of 140,000 at *m*/*z* 200. The sample was infused at a flow rate of 5 μL/min on the system.

### *4.5. Subsection Chemoinformatics Tools*

The ACD/Labs platform (ACD/Labs, Toronto, Canada) was used in combination with Agilent's MassHunter (Agilent Technologies) and XCalibur (Thermo Scientific) for the assessment and evaluation of the obtained data, including all the obtained chromatographic and MS data. This platform allows the confirmation and checking of the consistency of any suggested chemical structures with NMR experimental data, as well as assigning experimental spectra to structures, enabling the creation of a central, fully searchable repository of the assigned NMR spectra. The software was also used to project fragmentation paths, by comparing experimental MS and MS/MS data with theoretical data.
