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Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction

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Abstract

Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-13C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 μM for glutathione, 0.440 μM for phytochelatin 2, and 0.120 μM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation.

This figure was created using Adobe Photoshop CS3 (San Jose, CA). It displays an image of a chromatogram from our manuscript (a phytochelatin extraction) overlaid upon an image of Chlorella vulgaris and laboratory equipment and materials used in our methods.

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Acknowledgments

The authors would like to acknowledge the Canadian National Science and Engineering Council (NSERC) for funding DS with a PGS-D3, a NSERC Discovery grant to RJNE, and the Arctic Institute of North America (AINA) for funding ARH with Grants-in-Aid.

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Authors and Affiliations

  1. Environmental & Life Sciences Graduate Program, Environmental Science Building, Trent University, Room A201, 1600 West Bank Drive, Peterborough, ON, K9J 7B8, Canada

    Denina B. D. Simmons & Allison R. Hayward

  2. Department of Biology, Trent University, Peterborough, ON, K9J 7B8, Canada

    R. J. Neil Emery

  3. Environmental and Resource Studies Program, Trent University, Peterborough, ON, K9J 7B8, Canada

    Thomas C. Hutchinson

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  1. Denina B. D. Simmons
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  2. Allison R. Hayward
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Correspondence to R. J. Neil Emery.

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Simmons, D.B.D., Hayward, A.R., Hutchinson, T.C. et al. Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction. Anal Bioanal Chem 395, 809–817 (2009). https://doi.org/10.1007/s00216-009-3016-1

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  • DOI: https://doi.org/10.1007/s00216-009-3016-1

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