Hyphenated Solid Phase Extraction/Supercritical Fluid Extraction Methods for Extraction of Phenolic Compounds from Algae

Hyphenated SPE/SFE method was developed for the extraction of phenolic compounds from plant matrices (6 algae species). 20 ml of liquid solvent was injected to the SPE cartridge and extracted at following conditions: 40 MPa, 80°C, restrictor 10.5 cm x 50 μm i.d. for 60 min into 20 ml of MeOH as a trapping solvent. Two different methods were developed for different SPE sorbents (OASIS MCX 3cc, Waters and ADVANTA Spe-ed, Applied Separations) to prove that the principles work at different conditions. Each sorbent needs different modifier: 400 μL of 2M HAc in situ + 50 % MeOH / 5 % ammonia in ultra-pure water on-line is best for OASIS, ADVANTA needs 50 % MeOH and 5 % ether (v/v) on-line. SPE/SFE method has higher recovery, gives cleaner extracts, enables the use of liquid samples via SPE cartridge and higher precision was achieved (no restrictor plugging). Reversed phase liquid chromatography-electrospray mass spectrometry analysis was used for analyte determination: the chromatograph was HP 1100 with single quadruple massselective HP MSD detector and chromatographic column Zorbax SB – C 18 (75 mm x 4.6 mm, 3.5 μm, Agilent Technologies). LOD varied in the range 0.8 – 3.7 ng/l, combined RSDs for both SFE and HPLC were in range 1.4 – 5.1 %. Newly developed methods were applied on real algae samples to evaluate them as a prospective source of functional food additives. 11 of 13 monitored phenolic compounds were found, some of them in concentrations high enough to use the algae as a source of antioxidants.