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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

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Abstract

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.

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Acknowledgements

The authors thank Microphyt (Baillargues, France) for kindly providing H. pluvialis biomass.

Funding

The authors would like to thank projects ABACUS (Algae for a Biomass Applied to the production of added value compounds—funded by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 745668) and AGL2017-89417-R (MINECO, Spain) for funding.

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Authors and Affiliations

  1. Laboratory of Foodomics, Institute of Food Science Research (CIAL, CSIC-UAM), Calle Nicolás Cabrera 9, 28049, Madrid, Spain

    Rocío Gallego, Elena Ibáñez & Miguel Herrero

  2. Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Annunziata, 98168, Messina, Italy

    Katia Arena, Paola Dugo & Luigi Mondello

  3. Unit of Food Science and Nutrition, Department of Medicine, University Campus Bio-Medico, Via Álvaro del Portillo 21, 00128, Rome, Italy

    Paola Dugo & Luigi Mondello

  4. Chromaleont s.r.l., c/o Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Annunziata, 98168, Messina, Italy

    Paola Dugo & Luigi Mondello

  5. BeSep s.r.l., c/o Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Annunziata, 98168, Messina, Italy

    Luigi Mondello

Authors
  1. Rocío Gallego
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  2. Katia Arena
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  3. Paola Dugo
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  4. Luigi Mondello
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  5. Elena Ibáñez
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  6. Miguel Herrero
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Correspondence to Miguel Herrero.

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Gallego, R., Arena, K., Dugo, P. et al. 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. Anal Bioanal Chem 412, 589–599 (2020). https://doi.org/10.1007/s00216-019-02287-y

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  • DOI: https://doi.org/10.1007/s00216-019-02287-y

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