# Seasonal Monitoring of Volatiles and Antioxidant Activity of Brown Alga _Cladosteplus spongiosus_
## 3 Materials and Methods
### Gas Chromatography-Mass Spectrometry Analysis (GC-MS)  
GC-MS analysis was performed using an Agilent Technologies (Palo Alto, CA, USA) model 8890A gas chromatograph, coupled to a model 5977E mass selective detector. The compounds were separated on a HP -5 MS column (Agilent Technologies, Santa Clara, CA, USA) 30 m \(\times\) 0.25 mm with a nonpolar stationary phase (5% diphenyl/95% dimethylpolysiloxane) and a film thickness of 0.25 \(\upmu\)m. The following operating conditions were used for the gas chromatograph: 250 \({}^{\circ}\)C injector temperature; 300 \({}^{\circ}\)C detector temperature; column temperature program: 2 min isothermal at 70 \({}^{\circ}\)C, followed by a temperature gradient from 70 \({}^{\circ}\)C to 200 \({}^{\circ}\)C at 3 \({}^{\circ}\)C/min and a further retention of 15 min. The split ratio was 1:50; the carrier gas was helium with a flow rate of 1.0 mL/min; the MSD (EI mode) was operated at 70 eV; the mass range was set to 30 to 300 amu. The compounds were identified by comparing their retention indices (RI), which were based on the retention times of \(n\)-alkanes (C\({}_{9}\)-C\({}_{25}\)), with those reported in the literature (National Institute of Standards and Technology) and their mass spectra with those from the Wiley 9 (Wiley, New York, NY, USA) and NIST 17 (D-Gaithersburg) mass spectral libraries. Percent composition was calculated using the normalisation method (without correction factors). Analyses were performed in duplicate and expressed as the average percentage of peak area.  
Ultra-High Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-ESI-HRMS) of Ethanol Extract  
The UHPLC-ESI-HRMS analyses were performed using the ExionLC AD UHPLC system (AB Sciex, Concord, ON, Canada) equipped with the following ExionLC modules: AD Controller, solvent delivery system (AD Pump and AD Degasser), AD Autosampler and AD Column oven, connected to quadrupole time-of-flight (Q-TOF) mass spectrometer TripleTOF 6600+ (AB Sciex, Concord, ON, Canada) with duospray ion source. Chromatographic separations of the compounds were performed using the Acquity UPLC BEH Phenyl-Hexyl analytical column (Waters, Milford, MA, USA) 2.1 mm \(\times\) 100 mm with a particle size of 1.7 \(\upmu\)m. A continuous flow rate of 0.4 mL/min was set to pump the mobile phases, water (A) and acetonitrile (B), both containing 0.1% formic acid. The oven temperature was set at 30 \({}^{\circ}\)C. Elution started isocartically with 2% B (0.6 min), followed by a gradient program: 0.6-18.5 min (linear B gradient to 100%), 18.5-25 min (100% B). Electrospray ionisation was set in positive mode (ESI\({}^{+}\)) with collision-induced dissociation (CID) in information-dependent acquisition mode (IDA) for MS/MS mass spectra acquisition. In our previous work, description of the parameters in details can be found [40]. ACD/Spectrus Processor 2021.1.0 software (ACD/Labs, Toronto, ON, Canada) was usedto process the mass spectrometer data. Based on the mass spectra and the given elemental compositions of the compounds, and in conjunction with the results of the search in the ChEBI, Lipid Maps, and MassBank databases, the identification of the compounds was proposed.