Smartformula

Samples were analyzed using an UPLC Dionex Ultimate 3000 RS LC coupled to QTOF Bruker Maxis Impact HD (Bruker Daltonics, Bremen, Germany), equipped with an ESI interface operating in negative ion mode, with a mass range of m/z 50-1000, the capillary voltage was 2500 V; dry N₂ gas flow 8.0 l/min (200°C); nebulizer pressure 2.0 bar; end plate offset 500 V; collision energy 25 eV, and acquisition time factor 1 s.
Chromatographic separation was carried out using an Acclaim RSLC 120 C18 column (2.2 µm, 120 Å, 2.1 x 100 mm) (Dionex). The mobile phase consisted of 90% methanol with 5 mM ammonium acetate and 50% methanol with 5 mM ammonium acetate. Injection volume was 1.0 µl. Mass calibration was performed using 1 mM sodium formate/acetate in 50% isopropanol with 0.2% acid; HCOO(NaCOOH)₁ (m/z 112.9856), Ac(NaAc)₁ (m/z 141.0169), and Ac(NaF)₁ (m/z 127.0013).
Data analysis and calculation were performed using the software programs Data Analysis 4.1 (SmartFormula, SmartFormula 3D, and Fragmentation Explorer) and Profile Analysis 2.1 (PCA, HCA, and SmartFormula) from Bruker Daltonics, Bremen, Germany.

The UHPLC-MS system consisted of the UltiMate 3000 RS liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled with a tandem mass spectrometer with quadrupole and time-of-flight analyzers (Micro-TOF-Q II, Bruker, Bremen, Germany). A mobile phase consisted of water with 0.1% (v/v) formic acid (phase A) and acetonitrile with 0.1% formic acid (phase B). In each analysis, 5 μL of sample was injected by an autosampler, and the separation was carried out in isocratic mode (30% A, 70% B) within a Hypersil Gold Phenyl column (50 mm × 2.1 mm I.D., particles 1.9 µm, Dionex, Sunnyvale, CA, USA) for 16 min. The flow rate of the mobile phase was 0.4 mL min⁻¹. The column was thermostated at 25 °C. The electrospray ion source was operated under positive ionization conditions (ESI+). The other MS parameters were as follows: nebulizer pressure: 2.0 bar; dry gas flow: 5.5 L min⁻¹; drying gas temperature: 200 °C; capillary voltage: −500 V. The accurate mass of each ion was calibrated using sodium formate clusters. SmartFormula (Bruker) was used for the prediction of molecular formulas. The mass range was from 50 to 1500 m/z.

Selected VLC-derived marine cyanobacterial fractions were analyzed in the positive-ion-mode electrospray ionization using an MS system (Bruker MAXIS II equipped with a Quadrupole-Time-of-Flight mass analyzer) coupled to an HPLC (Agilent 1290 Infinity equipped with a diode array detector) on a Phenomenex analytical C18 column (2.5 µm, 100 Å, 4.6 × 150 mm). Each fraction was eluted with a starting mobile phase of 5% ACN:95% H₂O (0.1% formic acid), followed by a gradient of up to 100% ACN for 15 min at a flow rate of 1 mL/min. The raw data file was analyzed using compact Data Analyst (Bruker software) to provide accurate and high-resolution mass per charge molecular ions in the fractions and generate molecular formulae using a Bruker SmartFormula manually.