Analysis of fatty acids via gas chromatography and mass spectrometry

Dried microalgal extracts were prepared for fatty acid analysis, as described in the analysis of antimicrobial activity of microalgal extracts above. Total lipid extraction was performed using 20 mg dried microalgal extracts, as described previously [80]. The microalgal extracts were suspended in 2-mL saponification reagent (7.5 M NaOH:CH₃OH, 1:1 v/v), shaken manually for 30 s, and incubated at 100 °C for 30 min. To produce fatty acid methyl esters (FAMEs), the mixture was cooled, 4-mL methylation reagent (6 N HCl:CH₃OH, 1:1 v/v) added, and thereafter incubated at 80 °C for 10 min. To extract FAMEs, 2.5-mL extraction solvent (hexane:methyl tert-butyl ether, 1:1 v/v) was added, and the mixture incubated with shaking for 10 min, followed by centrifugation at 1500g for 10 min. The upper hexane phase containing FAMEs was collected and washed with 6-mL washing solution (0.5 M NaOH).

The FAMEs were validated and quantified using a GC–MS system (7890A-5957C; Agilent Technologies, Santa Clara, CA, USA) equipped with a capillary GC column (HP-5MS Ultra Inert; Agilent Technologies), as described previously, with some modifications [81]. For GC analysis of the FAMEs, the oven temperature was initially maintained at 180 °C for 5 min, raised to 220 °C at a rate of 2 °C min⁻¹, held for 5 min, raised to 300 °C at a rate of 80 °C min⁻¹, and then held for 5 min. The helium carrier gas was maintained at a flow rate of 1 mL min⁻¹. Mass spectrometry was carried out with a transfer line temperature of 280 °C, source temperature of 230 °C, quadrupole temperature of 150 °C, ionization potential of 70 eV, and mass range of m/z = 50–350 amu. Peaks in the GC chromatograms were identified using the mass spectral library of the National Institute of Standards and Technology (Gaithersburg, MD, USA).