Synap g2

An Ultra Performance Liquid Chromatography (UPLC) (Acquity H Class, Waters Corporation, Manchester, UK) coupled by quadrupole-time-of-flight mass spectrometry (Synap G2, Waters Corporation, Manchester, UK) was employed for all high-resolution mass spectrometry analysis. Prior to mass spectrometry analysis, all powdered samples (100 mg) were extracted with 10 mL of acidified water (pH 2), and filtered through 0.22 μm nylon disk filters (Millipore, Billerica, MA, USA). 10 µL of the final solution were injected into the chromatograph. Analytical separation of peptides and proteins was performed on an Acquity BEH 300 C4 analytical column (100 mm × 2.1 mm internal diameter, 1.7 μm; Waters Corporation, Manchester, UK). A mobile phase consisting in a gradient program combining deionized water with 0.1% of acid formic as solvent A and acetonitrile with 0.1% of acid formic as solvent B was used. The initial conditions were 95% A and 5% B. A linear gradient was then established to reach 95% (v/v) of B at 15 min. Total run time was 25 min and post-delay time 5 min. Mobile phase flow rate was 0.4 mL/min.
After chromatographic separation, a high-resolution mass spectrometry analysis was carried out in positive electro spray ionization (ESI+ve). The gas used for desolvation (800 L/h) and Cone (25 L/h) was high-purity nitrogen. Spectra were recorded over the mass/charge (m/z) range 50–1200.

Ethanolic extracts of untreated or treated N. gaditana were analyzed by ultra performance liquid chromatography (UPLC) (Acquity H Class, Waters, Milford, MA, USA) coupled with quadrupole time-of-flight mass spectrometry (Synap G2, Waters). Ten-microliter aliquots of the extracts were injected into the chromatograph. Separation was carried out on an Acquity HSST33 analytical column (100 mm × 2.1 mm internal diameter, 1.8 μm). A gradient program that combined deionized water with 0.5% of acetic acid as solvent A and acetonitrile with 0.5% of acetic acid as solvent B was used as the mobile phase. The initial conditions were 95% A and 5% B. A linear gradient was then established to reach 95% (v/v) of B at 18 min (total run time 18 min, post-delay time 5 min, mobile phase flow rate 0.4 mL/min). Afterward, a high-resolution mass spectrometry analysis was carried out in negative electrospray ionization (ESI-ve). Spectra were recorded over the mass/charge (m/z) range of 50–1500. The retention times (RTs) and mass (MS) fragments were used to identify all the compounds. Chromatogram analysis was performed using MassLynx V4.1 software (Waters Corporation, Milford, MA, USA). The Chemspider database was used to validate the identified compounds by matching at least three of their subfragments.