Ltq tune plus 2

For the measurement of metabolites release from the MCF-7 and A375 cells, the samples were separated on a HPLC Accela (Thermo Fischer Scientific, Bremen, Germany) using an ACE Equivalence C18 column (VWR/Avantar, Alfragide, Portugal), 5 µm particle size and dimensions of 3.0 mm ID × 75 mm. The samples were eluted over a gradient of 100% solvent A (CH₃COOBH₄, 10 mM) to reach 100% Solvent B (acetonitrile with HCOOH 0.1%) during 8 min at a flow rate of 0.4 mL/min. Analyses were done on an LTQ OrbitrapTM XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0. The capillary voltage of the electrospray ionization source (ESI) was set to 3.1 kV. The capillary temperature was 275 °C. The sheath gas and auxiliary gas flow rate were 40 and 10 (arbitrary units, as provided by the software settings). The capillary voltage was 32 V, and the tube lens voltage was 55 V. An MS data handling software (Xcalibur QualBrowser software, Thermo Fischer Scientific, Waltham, MA USA) was used to search for metabolites by their m/z value.

The samples were diluted tenfold in 30% aqueous acetonitrile containing 0.1% formic acid and infused at 5 µL/min flowrate directly into a Finnigan Ion Max 2 electrospray (ESI) by using a glass microliter syringe. Sample nebulization and ionization were provided by using a metal capillary 140 mm long and with a 160 µm inner diameter, and applying a spray voltage of 5 kV, 20 units of sheath gas (nitrogen), and a capillary temperature of 275 °C. MS spectra were collected by an LTQ-Orbitrap™ XL-ETD mass spectrometer within a scan range from 60 to 600 m/z. The analyzer was working on 5 × 10⁵ ion per scan, with a resolution of 100,000 (full width at half-maximum at 400 m/z). Lock mass option was enabled to provide real time internal mass calibration. Reference masses were 20 ions identified as background ESI trace contaminants and already listed as common mass spectrometry contaminants [36 (link)]. Instrument control was provided by the software LTQ Tune plus 2.5.5 and data extraction and analysis by Xcalibur 2.1 (Thermo Fisher Scientific, Rodano, MI, Italy).

¹H NMR data were acquired on a 400 MHz Bruker Avance III spectrometer. LC-HRESIMS data for the spent medium extract and the purified portoamide mixture were acquired on an Accela HPLC fitted with a Gemini C18 column (5 μm, 110 A, 4.6 mm ID × 150 mm, Phenomenex) column, coupled to an Accela PDA detector, Accela autosampler, and Accela 600 pump and to an LTQ Orbitrap XL spectrometer, controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1 (Thermo Scientific). Twenty microliters of each sample were injected at a concentration of 0.1 mg mL^-1 (MeOH). The separation was carried out using a gradient from 20% MeCN (aq) to 100% MeCN over 30 min. The LTQ spectrometer was operated in positive ion mode, the capillary voltage of the electrospray ionization source (ESI) was set to 3.0 kV and the capillary temperature was 300°C. For the separation and analysis leading to the isolation of portoamides A and B, an HPLC system composed of an Alliance 2695 HPLC (Waters) coupled to a PDA 2998 detector, fitted with a XB-C18 Aeris PEPTIDE column (150 mm × 4.6 mm, 3.6 μm, Phenomenex, kept at 35°C during the chromatography) was used. Monitored wavelengths during separation were 210 and 280 nm.
Solvents used were MS-grade or HPLC-gradient grade for MS and HPLC procedures, and ACS grade for extraction and column chromatography. NMR solvents were acquired from BDH Prolabo (VWR).