Skip to main content

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

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.

Graphical abstract

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Vílchez C, Forján E, Cuaresma M, Bédmar F, Garbayo I, Vega JM. Marine carotenoids: biological functions and commercial applications. Mar Drugs. 2011;9:319–33.

    Article  Google Scholar 

  2. Rodriguez-Concepcion M, Avalos J, Bonet ML, Boronat A, Gomez-Gomez L, Hornero-Mendez D, et al. A global perspective on carotenoids: metabolism, biotechnology, and benefits for nutrition and health. Prog Lipid Res. 2018;70:62–93.

    CAS  Article  Google Scholar 

  3. Guerin M, Huntley ME, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 2003;21:210–6.

    CAS  Article  Google Scholar 

  4. Sánchez-Camargo AP, Ibáñez E, Cifuentes A, Herrero M. Bioactives obtained from plants, seaweeds, microalgae and food by-products using pressurized liquid extraction and supercritical fluid extraction. In: Ibáñez E, Cifuentes A, editors. Comprehensive analytical chemistry, vol. 76. Amsterdam: Elsevier; 2017. p. 27–51.

    Google Scholar 

  5. Gallego R, Bueno M, Herrero M. Sub- and supercritical fluid extraction of bioactive compounds from plants, food-by-products, seaweeds and microalgae – an update. TrAC-Trends Anal Chem. 2019;116:198–213.

    CAS  Article  Google Scholar 

  6. Saini RK, Keum YS. Carotenoid extraction methods: a review of recent developments. Food Chem. 2018;240:90–103.

    CAS  Article  Google Scholar 

  7. Gallego R, Martínez M, Cifuentes A, Ibáñez E, Herrero M. Development of a green downstream process for the valorization of Porphyridium cruentum biomass. Molecules. 2019;24:1564.

    CAS  Article  Google Scholar 

  8. Reyes FA, Mendiola JA, Ibañez E, Del Valle JM. Astaxanthin extraction from Haematococcus pluvialis using CO 2-expanded ethanol. J Supercrit Fluids. 2014;92:75–83.

    CAS  Article  Google Scholar 

  9. Jaime L, Rodríguez-Meizoso I, Cifuentes A, Santoyo S, Suarez S, Ibáñez E, et al. Pressurized liquids as an alternative process to antioxidant carotenoids’ extraction from Haematococcus pluvialis microalgae. LWT Food Sci Technol. 2010;43:105–12.

    CAS  Article  Google Scholar 

  10. Gallego R, Montero L, Cifuentes A, Ibáñez E, Herrero M. Green extraction of bioactive compounds from microalgae. J Anal Test. 2018;2:109–23.

    Article  Google Scholar 

  11. Visentín A, Cismondi M, Maestri D. Supercritical CO2 fractionation of rosemary ethanolic oleoresins as a method to improve carnosic acid recovery. Innov Food Sci Emerg Technol. 2011;12:142–5.

    Article  Google Scholar 

  12. Gonzalez-Coloma A, Martín L, Mainar AM, Urieta JS, Fraga BM, Rodríguez-Vallejo V, et al. Supercritical extraction and supercritical antisolvent fractionation of natural products from plant material: comparative results on Persea indica. Phytochem Rev. 2012;11:433–46.

    CAS  Article  Google Scholar 

  13. Sánchez-Camargo AP, Mendiola JA, Valdés A, Castro-Puyana M, García-Cañas V, Cifuentes A, et al. Supercritical antisolvent fractionation of rosemary extracts obtained by pressurized liquid extraction to enhance their antiproliferative activity. J Supercrit Fluids. 2016;107:581–9.

    Article  Google Scholar 

  14. Kraujalis P, Venskutonis PR, Ibáñez E, Herrero M. Optimization of rutin isolation from Amaranthus paniculatus leaves by high pressure extraction and fractionation techniques. J Supercrit Fluids. 2015;104:234–42.

    CAS  Article  Google Scholar 

  15. Cacciola F, Donato P, Giuffrida D, Torre G, Dugo P, Mondello L. Ultra high pressure in the second dimension of a comprehensive two-dimensional liquid chromatographic system for carotenoid separation in red chili peppers. J Chromatogr A. 2012;1255:244–51.

    CAS  Article  Google Scholar 

  16. Dugo P, Giuffrida D, Herrero M, Donato P, Mondello L. Epoxycarotenoids esters analysis in intact orange juices using two-dimensional comprehensive liquid chromatography. J Sep Sci. 2009;32:973–80.

    CAS  Article  Google Scholar 

  17. Dugo P, Giuffrida D, Dugo G, Mondello L, Herrero M, Kumm T. Application of comprehensive two-dimensional liquid chromatography to elucidate the native carotenoid composition in red orange essential oil. J Agric Food Chem. 2008;56:3478–85.

    CAS  Article  Google Scholar 

  18. Cacciola F, Giuffrida D, Utczas M, Mangraviti D, Dugo P, Menchaca D, et al. Application of comprehensive two-dimensional liquid chromatography for carotenoid analysis in red mamey (Pouteria sapote) fruit. Food Anal Methods. 2016;9:2335–41.

    Article  Google Scholar 

  19. Giuffrida D, Donato P, Dugo P, Mondello L. Recent analytical techniques advances in the carotenoids and their derivatives determination in various matrixes. J Agric Food Chem. 2018;66:3302–7.

    CAS  Article  Google Scholar 

  20. Gilbert-López B, Mendiola JA, van den Broek LAM, Houweling-Tan B, Sijtsma L, Cifuentes A, et al. Green compressed fluid technologies for downstream processing of Scenedesmus obliquus in a biorefinery approach. Algal Res. 2017;24:111–21.

    Article  Google Scholar 

  21. Li X, Stoll DR, Carr PW. Equation for peak capacity estimation in two-dimensional liquid chromatography. Anal Chem. 2009;81:845–50.

    CAS  Article  Google Scholar 

  22. Camenzuli M, Schoenmakers PJ. A new measure of orthogonality for multi-dimensional chromatography. Anal Chim Acta. 2014;838:93–101.

    CAS  Article  Google Scholar 

  23. Holtin K, Kuehnle M, Rehbein J, Schuler P, Nicholson G, Albert K. Determination of astaxanthin and astaxanthin esters in the microalgae Haematococcus pluvialis by LC-(APCI)MS and characterization of predominant carotenoid isomers by NMR spectroscopy. Anal Bioanal Chem. 2009;395:1613–22.

    CAS  Article  Google Scholar 

  24. Catchpole OJ, Grey JB, Mitchell KA, Lan JS. Supercritical antisolvent fractionation of propolis tincture. J Supercrit Fluids. 2004;29:97–106.

    CAS  Article  Google Scholar 

  25. Dugo P, Herrero M, Kumm T, Giuffrida D, Dugo G, Mondello L. Comprehensive normal-phase × reversed-phase liquid chromatography coupled to photodiode array and mass spectrometry detection for the analysis of free carotenoids and carotenoid esters from mandarin. J Chromatogr A. 2008;1189:196–206.

    CAS  Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Miguel Herrero.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

ABC Highlights: authored by Rising Stars and Top Experts.

Electronic supplementary material

ESM 1

(PDF 690 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

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

Keywords

  • Supercritical antisolvent fractionation
  • SAF
  • Haematococcus pluvialis
  • PLE
  • Astaxanthin
  • LC×LC