Formula predictor software

The analytical characterization of phytochemical compounds contained in the cocoa extract was performed on a Shimadzu Nexera RP-UHPLC-PDA system (Reversed Phase-Ultra High Performance Liquid Chromatography-Photodiode Array, Kyoto, Japan) coupled online with a LC-MS-IT-TOF (Liquid Chromatography-Mass Spectrometry-Ion Trap-Time of Flight, Shimadzu, Milan, Italy) equipped with an electrospray source (ESI). LC-MS data elaboration was performed by the LCMS solution^® software (Version 3.50.346, Shimadzu, Milan, Italy).
Chromatographic separation was accomplished on a Kinetex^® EVO C18 column (150 × 2.1 mm × 2.6 µm, 100 Å, Phenomenex^®, Bologna, Italy) maintained at 45 °C, employing H₂O and ACN plus 0.1% (v/v) CH₃COOH as mobile phases delivered at constant flow rate of 0.5 mL min⁻¹. Analysis was performed in gradient elution as follows: 0–10 min, 5–26% ACN; 10–13 min, 26–95% ACN; isocratic at 95% for 3 min and, finally 5 min for column re-equilibration. Data acquisition was set in the range 190–800 nm and chromatograms were monitored at 280 nm.
MS detection of bioactive compounds was operated in positive and negative mode ionization. Full scan MS data were acquired in the range 150–1500 m/z and MS/MS experiments were conducted in data dependent acquisition. Molecular formulas of identified compounds were determined by “Formula Predictor” software (Shimadzu, Milan, Italy).

The flavonoid fraction of bergamot juice (BJe) has been provided by the company “Agrumaria Corleone” (Palermo, Italy). The fruits of Citrus bergamia were coming from crops located in the province of Reggio Calabria (Italy). The extract was centrifuged at 6000 rpm/min for 15 minutes to remove any impurities and successively transformed into a dry powder by the method of spray drying. Small aliquots of BJe were stored at −20°C. Finally, the drug was defrosted, diluted in culture media, pH adjusted to 7.4 and filtered just prior to use.
Chemical composition of BJe was investigated as previously described [32] (link). Briefly, BJe was solubilized in methanol to a concentration of 1 mg/mL, ultrasonicated and filtered by a 0.2 µm nylon membrane (Millipore, Milan, Italy). Qualitative and quantitative determination of flavonoids in BJe was performed using a UHPLC coupled online to an LCMS–IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Flavonoids were identified on the basis of diode array spectra, MS molecular ions and MS/MS fragmentation patterns. Data obtained were compared with those available in scientific literature. Molecular formula was calculated by the Formula Predictor software (Shimadzu).

Qualitative and quantitative composition of FFBJ and FFOJ were determined using previously described protocol [21 (link), 22 (link)]. Briefly, both FFBJ and FFOJ were solubilized in methanol to a concentration of 1 mg/mL⁻¹, ultrasonicated and filtered through a 0.2 μm nylon membrane (Millipore, Milan, Italy), and then injected into a UHPLC coupled online to an LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Identification of flavonoids was carried out on the basis of diode array spectra, MS molecular ions, and MS/MS fragmentation patterns. Data obtained were compared with those available in scientific literature. Molecular formulae were calculated by the Formula Predictor software (Shimadzu).