Lisa Spoof

Screening for cyanobacterial hepatotoxins, microcystins and nodularin in environmental water samples by reversed-phase liquid chromatography–electrospray ionisation mass spectrometry

Water samples taken from 93 freshwater and brackish water locations in Aland (SW Finland) in 2001 were analysed for biomass-bound microcystins and nodularin, cyanobacterial peptide hepatotoxins, by liquid chromatography-mass spectrometry (LC-MS) in selected ion recording (SIR) and multiple reaction monitoring modes, HPLC-UV, and enzyme-linked immunosorbent assay (ELISA). The extracted toxins were separated on a short C18 column with a gradient of acetonitrile and 0.5% formic acid, and quantified on a Micromass Quattro Micro triple-quadrupole mass spectrometer with an electrospray ion source operated in the positive SIR or scan mode. An injection of 50 pg of microcystin-LR, m/z 995.5, on column gave a signal-to-noise ratio of 17 (peak-to-peak) at the chosen SIR conditions. In-source or MS-MS fragmentation to m/z 135.1, a fragment common to most microcystins and nodularin, was used for confirmatory purposes. Microcystins with a total toxin concentration equal to or higher than 0.2 microg l(-1) were confirmed by all three methods in water samples from 14 locations. The highest toxin concentration in a water sample was 42 microg l(-1). The most common toxins found were microcystins RR, LR and YR with different degrees of demethylation (non-, mono- or didemethylated). Parallel results achieved with ELISA and HPLC-UV were generally in good agreement with the LC-MS SIR results.

First report of the cyanobacterial toxin cylindrospermopsin in the shallow, eutrophic lakes of western Poland.

Cyanobacterial dominance in eutrophic lakes causes water quality problems due to the production of toxins harmful to humans and animals, as well as a number of odorous compounds. Cylindrospermopsin (CYN) is a potent cytotoxic cyanobacterial metabolite involved in triggering illness in humans. The occurrence of CYN has been mostly associated with tropical and subtropical cyanobacteria. We analyzed CYN concentration and phytoplankton assemblages of three lakes located in western Poland during the summers of 2006 and 2007. CYN was detected in 46% of our samples using the HPLC and LC-MS/MS methods. CYN concentrations were in the range of 0.16-1.8 microg L(-1) and exceeded the drinking water guideline value of 1 microgL(-1) in two samples. This is the first report of CYN occurrence in this part of Europe and provides further evidence that this toxin is common not only in subtropical and tropical regions. The lakes were dominated by Planktothrix agardhii but the occurrence of the CYN investigated here might be associated with the invasive species Cylindrospermopsis raciborskii and/or native Aphanizomenon gracile.

Rapid separation of microcystins and nodularin using a monolithic silica C18 column

A monolithic C18-bonded silica rod column (Merck Chromolith) was compared to particle-based C18 and amide C16 sorbents in the HPLC separation of eight microcystins and nodularin-R. Two gradient mobile phases of aqueous trifluoroacetic acid modified with acetonitrile or methanol, different flow-rates and different gradient lengths were tested. The performance of the Chromolith column measured as the resolution of some microcystin pairs, the selectivity, efficiency (peak width) and peak asymmetry equalled, or exceeded, the performance of traditional particle-based columns. The Chromolith column allowed a shortening of the total analysis time to 4.3 min with a flow-rate 4 ml min(-1).

High-performance liquid chromatographic separation of microcystins and nodularin, cyanobacterial peptide toxins, on C18 and amide C16 sorbents

Four C18 columns and a novel amide C16 column were assessed in the HPLC separation of eight microcystins and nodularin-R. Gradient mobile phases of acetonitrile combined with trifluoroacetic acid, formic acid or ammonium acetate were compared. Special attention was paid to the resolution of four possible coeluting microcystin pairs. Generally speaking, the acidic mobile phases were superior to the ammonium acetate-based mobile phase in terms of resolution and selectivity. The amide C16 column had the best overall performance and unique selectivity properties.

Rapid LC-MS detection of cyanobacterial hepatotoxins microcystins and nodularins--comparison of columns.

Eight reversed-phase columns intended for rapid HPLC were assessed for the separation of thirteen microcystins and nodularins, cyclic peptidic hepatotoxins. The instrumentation consisted of an Agilent Technologies 1200 Rapid Resolution high performance liquid chromatography system coupled to a mass spectrometer, Bruker Daltonics Ultra Performance High Capacity Ion Trap MS (HCT Ultra) with electrospray ionisation (RRLC-ESI-IT-MS). The columns tested were 2-2.1 mm x 50 mm in diameter and length, and contained small particles (1.8-2.7 microm), or monolithic silica supports for fast performance. The shortest total run time achieved was 3 min 15 s including equilibration and injection. Critical microcystin pairs were still resolved. Several columns showed excellent performance.

Comparative Cellular Toxicity of Hydrophilic and Hydrophobic Microcystins on Caco-2 Cells

Microcystins (MC), cyanobacterial peptide hepatotoxins, comprise more than 100 different variants. They are rather polar molecules but some variants contain hydrophobic amino acid residues in the highly variable parts of the molecule. In MC-LF and MC-LW, the more hydrophobic phenylalanine (F) and tryptophan (W), respectively, have replaced arginine (R) in MC-LR. Depending on the structure, microcystins are expected to have different in vivo toxicity and bioavailability, but only a few studies have considered the toxic properties of the more hydrophobic variants. The present study shows that MC-LF and MC-LW have more pronounced cytotoxic effects on Caco-2 cells as compared to those of MC-LR. Treatment of Caco-2 cells with MC-LW and especially MC-LF showed clear apoptotic features including shrinkage and blebbing, and the cell–cell adhesion was lost. An obvious reduction of cell proliferation and viability, assessed as the activity of mitochondrial dehydrogenases, was observed with MC-LF, followed by MC-LW and MC-LR. Cytotoxicity was quantified by measuring lactate dehydrogenase leakage. The more hydrophobic MC-LW and MC-LF induced markedly enhanced lactate dehydrogenase leakage compared to controls and MC-LR, indicating that the plasma membrane was damaged. All of the three toxins examined inhibited protein phosphatase 1, with MC-LF and MC-LW to a weaker extent compared to MC-LR. The higher toxic potential of the more hydrophobic microcystins could not be explained by the biophysical experiments performed. Taken together, our data show that the more hydrophobic microcystin variants induce higher toxicity in Caco-2 cells.

Fast separation of microcystins and nodularins on narrow-bore reversed-phase columns coupled to a conventional HPLC system.

Six modern narrow-bore (50 mm length x 2-2.1 mm internal diameter) reversed-phase HPLC columns were tested in the rapid separation of ten microcystins and three nodularins, cyanobacterial peptide hepatotoxins. The columns had either a sub-3 microm particle size or were of monolithic silica technology for high efficiency and rapid run times. A standard low-pressure gradient HPLC equipment with diode-array UV detector was used for the chromatography. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, and the gradient rise times were 1-4 min. Chromatographic performance was assessed by visual judgement and by calculating parameters such as capacity factors, resolution, peak width, selectivity and peak form. Several chromatographic conditions produced excellent results. We recommend sub-3 microm particle-based or monolithic reversed-phase columns for routine use in microcystin and nodularin HPLC analyses.

Diversity of Peptides Produced by Nodularia spumigena from Various Geographical Regions

Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.

Microcystin occurrence in lakes in Åland, SW Finland

Samples from a total of 134 lakes, practically all freshwater lakes in Åland, SW Finland, were screened for microcystins, hepatotoxic peptides of cyanobacteria, using high performance liquid chromatography (HPLC), time-resolved fluoroimmunoassay (TR-FIA), and protein phosphatase inhibition (PPIA) during two summers. Microcystins were recorded in 4 of 54 lakes in 1999 and 14 of 134 lakes in 2000, especially in eutrophic lakes but also in some dystrophic lakes and one oligotrophic. There was a satisfactory agreement between results obtained with different methods. The most likely microcystin-producers involved, as judged by microscopical examination, were Planktothrix agardhii, Microcystis aeruginosa, and Anabaena lemmermannii. One drinking-water reservoir and several lakes used for irrigation were microcystin-positive. Microcystins were also detected in late autumn samples from some eutrophic lakes.

Cyanotoxin production in seven Ethiopian Rift Valley lakes

Abstract We hypothesized that unusual deaths and illnesses in wild and domestic animals in lake areas of the Rift Valley south of Addis Ababa were caused by toxic cyanobacteria. In the first cyanotoxic analyses conducted in samples from Ethiopia, we found lakes Chamo, Abaya, Awassa, Chitu, Langano, Ziway, and Koka all had concentrations of microcystins (MC) ranging from trace to hazardous, whereas only traces less than limits of detection (LOD) of cylindrospermopsin (CYN) were found. In the December 2006 dry season we sampled the lakes for analyses of MC, CYN, species structures, and calculations of cyanobacteria biomass. We used the Utermöhl technique to analyse cyanobacterial biomass and monitored MC toxins using HPLC-DAD, LC-ESI-MS-MRM, and ELISA-test and CYN with HPLC-DAD and ELISA. The various toxicity tests coincided well. In 4 of the lakes (Chamo, Langano, Ziway, and Koka), the inter-lake range of total MC concentration was 1.3–48 μg L−1; in 3 (Abaya, Awassa, and Chitu), we found only traces of MC. Microcystis aeruginosawas the dominant species, with Microcystis panniformis, Anabaena spiroides, and Cylindrospermopsisspp. as subdominants. The MC concentration, especially in Lake Koka, exceeded levels for serious health hazards for humans, cattle, and wildlife.