Thiol Derivatization for LC-MS Identification of Microcystins in Complex Matrices

Microcystins are a group of cyclic heptapeptides originating
from
cyanobacteria. Cyanobacteria also produce a range of peptides and
other compounds that can result in complex chromatograms when samples
are analyzed by LC-MS. Derivatization with appropriate thiols (e.g.,
mercaptoethanol) of the olefin in the α,β-unsaturated
amide present in most microcystins was shown to simplify analysis
of LC-MS chromatograms of sample extracts, making it much easier to
identify peaks corresponding to candidate microcystins. Furthermore,
interpretation of MS2 spectra was facilitated by addition
of the mass associated with the thiol to the α,β-unsaturated
amide of microcystins. Cyanotoxins containing Mdha or Dha reacted
readily with thiols, whereas Mser, Ser, Mdhb, and thiol-derivatives
of Mdha or Dha did not react under the conditions used. This approach
therefore provides a convenient LC-MS method to obtain evidence for
the presence of Mdha or Dha and can likely be used to differentiate
between the isobaric amino acids Mdha and Dhb in candidate cyanotoxin
peaks. When O-(2-mercaptoethyl)-O′-methyl-hexa­(ethylene glycol) (MEMHEG) (Mwt. 356) was used as the thiol, the resulting derivatives
eluted in an LC-MS mass window that was largely free of interferences.
This approach simplifies detection of candidate microcystin analogues
even in the presence of complex mixtures of coeluting components.
The method was used for qualitative analysis of a Microcystis
aeruginosa culture from Lake Naivasha, Kenya, and the results
were verified using precursor-ion scanning and high-resolution mass
spectrometry.