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

Abstract

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 MS(2) 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) (M(w)t. 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.

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

“…For cyanotoxin analysis, 10 L of lake water from the surface was sampled in a plastic container, frozen, thawed and then shaken with 30 g of activated HP-20 resin (DIAION, Mitsubishi Chemical Corporation, Tokyo, Japan) overnight to extract microcystins (Miles et al 2012). The sample was filtered through nylon netting (200 mm mesh) and the resin recovered and stored at 48C until transportation to Norway.…”

“…For analysis of MCs, the filtrate (100 mL) was mixed with MeOH (100 mL) (Miles et al 2012), and for analysis of CYN and ATX the filtrate was mixed with acetonitrile (1: 4). Ballot, unpubl.…”

“…Aliquots of the extract were treated with pH 9.7 carbonate buffer (Miles et al 2012) (to produce [DMAdda 5 ]-microcystins by hydrolysis) at 308C, and progress of the reaction monitored by LC-MS 2 for 2.5 days. Treatment of hydrolysed and unhydrolysed aliquots (in carbonate buffer) with mercaptoethanol (to derivatise the Mdha 7 -group), followed by LC-MS 2 analysis (Miles et al 2012), was used to confirm the identity of the major hydrolysis products ([DMAdda 5 ]MC-LR, [DMAdda 5 ]MC-LHar and [Asp 3 , DMAdda 5 ]MC-LR) and the hydrolysed extract was then used as a qualitative standard for these microcystins.…”

“…LC-MS 2 analysis with and without mercaptoethanol derivatisation was performed as described by Miles et al (2012). Briefly liquid chromatography was performed on a Symmetry C18 column (3.5 mm, 100 Â 2.1 mm; Waters, Milford, MA, USA), using a Surveyor MS Pump Plus and a Surveyor Auto sampler Plus (Finnigan, Thermo Electron Corp., San Jose, CA, USA) eluted (0.3 mL min À1 ) with a linear gradient (300 mL min À1 ) of acetonitrile (A) and water (B) each containing 0.1% formic acid.…”

“…Microcystins were analysed by LC-MS 2 , and quantitated from their [MþH] (Miles et al 2012). Oxidised MC-WR analogues in the samples were identified by comparison with MS 2 spectra of related compounds (Puddick 2012).…”