Rapid, accurate and routine HPLC method for large-scale screening of pro-vitamin A carotenoids in oilseeds

Abstract

Plant carotenoids, the precursors of vitamin A display several important biological functions as antioxidants and anti-carcinogens. The oilseed crops, owing to their high oil content, form a good matrix for the bioavailability of β-carotene, thereby providing potential targets for biofortification to combat vitamin A deficiency (VAD). However, the screening and characterization of these crops, that otherwise contain very low levels of pro-vitamin A carotenoids has been difficult owing to their poor recovery and strong binding to the oil matrix. Here, we report a rapid method for high volume HPLC analysis involving the extraction and determination of β-carotene in four oilseed crops (peanut, soybean, sunflower and mustard). This included a comprehensive study of the factors that potentially influence the qualitative and quantitative yields of β-carotene in these crops. This is the first crop-independent HPLC method for the quantification of pro-vitamin A carotenoids that shows excellent recovery and reproducibility (>90 percentage recovery in oil) using small tissue sample and is capable of processing up to 30 samples per day. The protocol is sensitive, and enables better detection and separation of individual carotenoids by reducing artefacts during extraction, purification and chromatography that can be used for routine screening of oilseeds.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

BHT:

Butylated hydroxyltoluene

DE:

Diethyl ether

HPLC:

High-performance liquid chromatography

PE:

Petrolium ether

PDA:

Photodiode array detector

PTFE:

Polytetrafluoroethylene

THF:

Tetrahydrofuran

VAD:

Vitamin A deficiency

References

  1. Barua AB, Olson JA (1998) Reversed-phase gradient high-performance liquid chromatographic procedure for simultaneous analysis of very polar to non polar retinoids, carotenoids and tocopherols in animal and plant samples. J Chromatogr B 707:69–79

    CAS  Article  Google Scholar 

  2. Bouis HE (2003) Micronutrient fortification of plants through plant breeding: can it improve nutrition in man at low cost? Proc Nutr Soc 62:403–411

    PubMed  Article  Google Scholar 

  3. Broszeit G, Diepenbrock F, Graf O, Hecht D, Heinze J, Martin HD, Mayer D, Schaper K, Smie A, Strehblow HH (1997) Generation, storage, and delocalization of charge in carotenoids. Liebigs Ann-Recueil 11:2205–2213

    Article  Google Scholar 

  4. Cooper DA (2004) Carotenoids in health and disease: recent scientific evaluations, research recommendations and the consumer. J Nutr 134:221S–224S

    CAS  PubMed  Google Scholar 

  5. Davey MW, Keulemans J, Swennen R (2006) Methods for the efficient quantification of fruit provitamin A contents. J Chromatogr A 1136:176–184

    CAS  PubMed  Article  Google Scholar 

  6. Graham PH, Vance CP (2003) Legumes: importance and constraints to greater utilization. Plant Physiol 131:872–877

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  7. Granado F, Olmedilla B, Gil-Martinez E, Blanco I (2001) A fast, reliable and low-cost saponification protocol for analysis of carotenoids in vegetables. J Food Compos Anal 14:479–489

    CAS  Article  Google Scholar 

  8. Hart DJ, Scott KJ (1995) Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem 54:101–111

    CAS  Article  Google Scholar 

  9. Humphrey JH, West KP, Sommer A (1992) Vitamin A deficiency and attributable mortality among under 5 year olds. Bull World Org 70:225–232

    CAS  Google Scholar 

  10. Kimura M, Rodriguez-Amaya DB, Godoy HT (1990) Assessment of the saponification step in the quantitative determination of carotenoids and provitamins A. Food Chem 35:187–195

    CAS  Article  Google Scholar 

  11. Kurilich AC, Juvik JA (1999) Simultaneous quantification of carotenoids and tocopherols in corn kernel extracts by HPLC. J Liquid Chromatogr relat Technol 22:2925–2934

    CAS  Article  Google Scholar 

  12. Patte HE, Purcell AE, Johns EB (1967) Carotenoid pigments in peanut oil. J Am Oil Chem Soc 44:328–330

    Article  Google Scholar 

  13. Patte HE, Purcell AE, Johns EB (1969) Changes in carotenoid and oil content during maturation of peanut seeds. J Am Oil Chem Soc 46:629–631

    Article  Google Scholar 

  14. Ping BTY, Gwendoline ECL (2006) Identification of lutein in crude palm oil and evaluation of carotenoids at various ripening stages of the oil palm fruit. J Oil Palm Res 18:189–197

    CAS  Google Scholar 

  15. Schierle J, Pietsch B, Ceresa A, Fizet C, Waysek EH (2004) Method for the determination of beta-carotene in supplements and raw materials by reversed-phase liquid chromatography: single laboratory validation. J Am Oil Chem Soc 87:1070–1082

    CAS  Google Scholar 

  16. Shewmaker CK, Sheehy JA, Daley M, Colburn S, Ke DYD (1999) Seed specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. Plant J 20:410–412

    Article  Google Scholar 

  17. Siong TE, Heng GA, Choo KS (1995) Carotenoid composition and content of legumes, tubers and starchy roots by HPLC. J Nutr 1:63–74

    Google Scholar 

  18. Van Lieshout M, West CE, Muhilal PD, Wang Y, Xu X, Van Breemen RB, Creemers AFL, Verhoeven MA, Lugtenburg J (2001) Bioefficacy of β-carotene dissolved in oil studied in children in Indonesia. Am J Clin Nutr 73:949–958

    PubMed  Google Scholar 

  19. Weber EJ (1987) Carotenoids and tocols of corn grain determined by HPLC. J Am Oil Chem Soc 64:1129–1134

    CAS  Article  Google Scholar 

  20. Yu B, Lydiate DJ, Young LW, Schäfer UA, Hannoufa A (2008) Enhancing the carotenoid content of Brassica napus seeds by down regulating lycopene epsilon cyclase. Transgenic Res 17:573–585

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgments

This research was supported by grants from HarvestPlus Challenge Program and the Department of Biotechnology, Government of India. We thank the Directorate of Oilseed Research, Hyderabad, India and the Peanut Breeding unit of ICRISAT for providing the seeds for this research. The technical support received from Mohd. Yousuf, D. Pandary and C. Lakshminarayana is duly acknowledged.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Kiran K. Sharma.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bhatnagar-Panwar, M., Bhatnagar-Mathur, P., Bhaaskarla, V.V. et al. Rapid, accurate and routine HPLC method for large-scale screening of pro-vitamin A carotenoids in oilseeds. J. Plant Biochem. Biotechnol. 24, 84–92 (2015). https://doi.org/10.1007/s13562-013-0239-1

Download citation

Keywords

  • β-carotene
  • Biofortification
  • High Performance Liquid Chromatography
  • Mustard
  • Oilseeds
  • Peanut
  • Pro-vitamin A carotenoids
  • Soybean
  • Sunflower