Application of compressed fluid-based extraction and purification procedures to obtain astaxanthin-enriched extracts from Haematococcus pluvialis and characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry

The green microalga Haematococcus pluvialis has been widely studied due to its capacity to accumulate great amounts of astaxanthin, a high-value carotenoid with biological activities. In the present work, two green compressed fluid-based processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF), are integrated to obtain an astaxanthin-enriched extract from this microalga. PLE was carried out using pressurized ethanol as solvent, for 20 min, at 10 MPa, and 50 °C as extraction temperature. Subsequently, the obtained extract was processed by SAF to further purify the carotenoid fraction. The SAF process was optimized using a 3-level factorial experimental design and considering three experimental variables: (i) CO₂ pressure (10-30 MPa), (ii) percentage of water in the PLE extract (20-50%), and (iii) PLE extract/supercritical-CO₂ flow rate ratio (0.0125-0.05). Total carotenoid content was evaluated in both extracts and raffinates. Best results were obtained at 30 MPa, 0.05 feed/SC-CO₂ mass flow rate, and 20% (v/v) of water in the feed solution, achieving values of 120.3 mg g^-1 carotenoids in extract (in the SAF extract fraction), which were significantly higher than those obtained in the original PLE extract. In parallel, a new fast two-dimensional comprehensive liquid chromatography (LC×LC) method was optimized to get the full carotenoid profile of these extracts in less than 25 min. This is the first time that the use of a C30 column is reported in an on-line LC×LC system. Graphical abstract.