Effw

Anal Bioanal Chem (2009) 395:809–817
DOI 10.1007/s00216-009-3016-1

ORIGINAL PAPER

Identification and quantification of glutathione
and phytochelatins from Chlorella vulgaris by RP-HPLC
ESI-MS/MS and oxygen-free extraction
Denina B. D. Simmons & Allison R. Hayward &
Thomas C. Hutchinson & R. J. Neil Emery

Received: 5 June 2009 / Revised: 20 July 2009 / Accepted: 27 July 2009 /Published online: 18 August 2009
# Springer-Verlag 2009

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 forquantification. Method enhancements include the use of
an argon environment during extraction to reduce autooxidation, 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 forphytochelatin 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
studiesinvolving phytochelatin analyses have likely been
subject to higher variability caused by this propensity for
D. B. D. Simmons : A. R. Hayward
Environmental & Life Sciences Graduate Program,
Environmental Science Building, Trent University,
Room A201, 1600 West Bank Drive,
Peterborough, ON K9J 7B8, Canada
R. J. N. Emery (*)
Department of Biology, Trent University,
Peterborough, ON K9J 7B8,Canada
e-mail: nemery@trentu.ca
T. C. Hutchinson
Environmental and Resource Studies Program, Trent University,
Peterborough, ON K9J 7B8, Canada

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.
Keywords Phytochelatins . Chlorella vulgaris .Auto-oxidation . HPLC-MS . Internal standard . ESI-MS/MS

Background
Phytochelatins (PCs) are short peptides that can bind heavy
metals and are induced in the presence of heavy metals [1–4].
A wide range of higher plants, as well as algae and some
fungi produce PCs [1, 5]. There has been substantial
evidence that demonstrates Cd, Cu, Ag, and As induce the
production of PCs in vivo [2, 6, 7].Numerous studies
indicate that Cd is able to induce and bind to PCs in plants
[6, 8, 9], and some of these studies showed that Cdtolerant ecotypes have a greater concentration of PCs than
non-tolerant ecotypes [1, 10, 11]. Furthermore, PC
deficient mutants were hypersensitive to Cd exposures
[12]. Therefore, PCs are believed to play a role in both
metal homeostasis and metal detoxification [2,13].
Phytochelatins are peptides that have the general
structure (γ-Glu-Cys)n-Gly where n =2–11 [13]. Phytochelatins are rich in the amino acid cysteine (Cys) and, thus,
contain electron-rich thiol groups with which they can
chelate a variety of heavy metal ions [2]. The same
electron-rich thiol groups make PCs sensitive to spontaneous
oxidation, which can cause the formation of peptideaggregates or the formation of organic radicals [14, 15].
PCs chelate metals in amorphous complexes because they
are synthesized in collections of various γ-Glu-Cys chain
lengths [16]. This is important because the longer chains

810

have more stable metal-ion binding [17]. The chelation of
metals always have consistent sulfur atom to metal stoichiometry and, in the case where a plant is...