4.5. Instrumentation

The operating configuration used here was based on those described by Dasenaki et al. [48] (method A) and Silva et al. [22] (method B), with minor adaptations as described below.

The separation of the analytes present in the extracts was performed in an Accela 1250 Pump UHPLC liquid chromatography system from Thermo Scientific (Bremen, Germany) equipped with a binary pump, vacuum degasser, oven, and autosampler. In both methods, an Acquity^TM UHPLC BEH C18 reserved-phase column (50 × 2.1 mm i.d., 1.7 μm particle size) was used coupled to a VanGuardAcquity^TM UPLC BEH C18 pre-column (5 × 2.1 mm i.d., 1.7 mm), both from Waters (Wexford, Ireland).

For method A, the column and precolumn were operated at 30 °C and the autosampler was maintained at 15 °C. The mobile phases consisted of 0.01% formic acid in water (A) and methanol (B). The gradient program applied was a linear ramp from 95% A and 5% B to 0% A and 100% B in 7.0 min, and from 7.0 to 10.0 min it was held constant at 100% B; decreasing linearly to 5% B and 95% A in 0.1 min, maintaining the initial condition up to 17.0 min. The flow was 0.1 mL min⁻¹ and the injection volume was 5 µL.

For method B, two conditions for the separation of analytes were applied to each extract of the sample, using a flow rate of 0.4 mL min⁻¹ and an injection volume of 10 µL. For the aqueous extract, the column and autosampler temperatures were 35 and 15 °C, respectively. The mobile phases consisted of 5 mmol L⁻¹ ammonium formate + 0.1% formic acid in water (A) and 0.1% formic acid in water/ACN (5:95, v/v) (B). The elution gradient was as follows: 100% A to 80% A in 8.0 min, 80% A to 5% A from 8.0 to 14.0 min, with hold up to 18.0 min; from 5% A to 100% from 18.0 to 20.0 min. For the organic extract, the column was operated at a temperature of 30 °C, while the automatic sample was at 15 °C. The mobile phases were composed of ammonium formiate 0.2 mol L⁻¹ + formic acid 0.1% in water (A) and formic acid 0.1% in water/ACN (5:95, v/v) (B). Gradient elution was 100% A up to 7.5 min; 100% A up to 2% A in 2.5 min, with maintenance up to 17.5 min, and re-establishment of the initial gradient with 100% A up to 20 min (Table S2 in Supplementary Materials).

The high-resolution mass spectrometer (HRMS) used to identify the substances was the Q-Exactive^TM hybrid quadrupole-Orbitrap^TM (Thermo Scientific, Bremen, Germany), equipped with a heated electrospray ionization source (HESI-II). The analyses were performed by two acquisition methods: the MS full scan acquisition mode which operates by switching between positive and negative ionization modes, for data acquisition at a resolution of 35,000 full width at half maximum (FWHM) (MS¹) in the range from 65 to 975 m/z, and full scan mode with all ion fragmentation (AIF) or MS² experiments, operated at a resolution power of 17,500 FWHM in order to increase the number of detected signals for each product ion, using a three-step scaled normalized collision energy (NCE) for target analytes at values on 10, 35, and 80 eV, in high energy collisional dissociation (HCD) to ensure complete fragmentation of precursor ions. In both cases, the automatic gain control (AGC) representing the C-trap was 3 × 10⁶ ions for the maximum injection time (IT) of 100 ms and 1.0 microscans for the final mass spectra acquisition. The following source conditions were employed to assist the ionization process: electrospray voltage of the HESI interface was adjusted to 3.9 and 2.9 kV in positive and negative ionization modes, respectively; there was a capillary temperature of 350 °C; lens voltage S was set to 50 (arbitrary unit—arb); and a sheath and auxiliary gas flow rate (N₂ 0.545 mbar) of 40 arb and 15 arb, respectively (Table S3 in Supplementary Materials).

A study of the adequacy of procedures was performed, as well as the optimization of method conditions for detection by HESI-Orbitrap^TM mass spectrometry (MS). A database of substances, which contained information on the molecular formula, the exact mass of precursor ions, and the corresponding potential adducts (including a selection of protonated ions [M]⁺, [M+H]⁺, [M+Na]⁺, [M+H₂O+H]⁺, [M-H₂O+H]⁺, [M+H+CH₃OH]⁺, [(M+H₂CO)+H]⁺, [(M+H₂-CO)+H]⁺, [M+NH₄]⁺, [M+Ca]⁺, [M+H₃O]⁺, deprotonated ions [M-H]⁻, double charged ions [M+2H]²⁺, [M+H₂O+2H]²⁺, or tri-charged ions [M+3H]³⁺ in the positive operation mode, with up to five MS/MS of the most abundant fragment ions being defined considering the theoretical m/z of the substance, in addition to the respective chromatographic retention times. Furthermore, the structures and details of the physicochemical properties of each analyte were presented, such as partition coefficient (log p), acid/base dissociation constants (pKa), and intrinsic solubility (data in Supplementary Materials, Table S4) obtained after direct infusion of individual standard solutions into the MS system with ionization by electrospray in the positive and negative modes (HESI⁺ e HESI⁻) to select the most intense ions by means of fragmentation energy scans, in a solvent at a concentration of 500 μg L⁻¹ in MeOH:H₂O 0.1% (v/v) of formic acid. Retention times were evaluated by injecting a fortified extract into the matrix in the UHPLC-Q-Orbitrap^TM at the same concentration as the other parameters.

The acquisition and processing of raw data were performed using the software Xcalibur Analysis 3.0 (Thermo Fisher Scientific Inc., Waltham, MA, USA) for the creation of methods and for the execution of the samples, with the exact mass of the substances calculated using Qualbrowser.