Analysis of total microcystins and nodularins by oxidative cleavage of their ADMAdda, DMAdda, and Adda moieties.

Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda5]MCs and [DMAdda5]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda5]- and [ADMAdda5]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were <0.25% when analyzed with an Adda enzyme-linked immunosorbent assay (ELISA). Oxidative cleavage experiments identified compounds that could be used in the analysis of total MCs/NODs in a similar fashion to the 2R-methyl-3S-methoxy-4-phenylbutanoic acid (MMPB) technique. Products from oxidative cleavage of both the 4,5- and 6,7-ene of Adda, DMAdda and ADMAdda were observed, and three oxidation products, one from each Adda variant, were chosen for analysis and applied to three field samples and a Nostoc culture. Results from the oxidative cleavage method for total Adda, DMAdda, and ADMAdda were similar to those from the Adda-ELISA, PP2A inhibition, and LC-MS/MS analyses, except for the Nostoc culture where the Adda-ELISA greatly underestimated microcystin levels. This oxidative cleavage method can be used for routine analysis of field samples and to assess the presence of the rarely reported, but toxic, DMAdda/ADMAdda-containing MCs and NODs.

Diagnosing Microcystin Intoxication of Canines: Clinicopathological Indications, Pathological Characteristics, and Analytical Detection in Postmortem and Antemortem Samples.

In the summer of 2018, six dogs exposed to a harmful algal bloom (HAB) of Microcystis in Martin County Florida (USA) developed clinicopathological signs of microcystin (MC) intoxication (i.e., acute vomiting, diarrhea, severe thrombocytopenia, elevated alanine aminotransferase, hemorrhage). Successful supportive veterinary care was provided and led to survival of all but one patient. Confirmation of MC intoxication was made through interpretation of clinicopathological abnormalities, pathological examination of tissues, microscopy (vomitus), and analytical MC testing of antemortem/postmortem samples (vomitus, blood, urine, bile, liver, kidney, hair). Gross and microscopic examination of the deceased patient confirmed massive hepatic necrosis, mild multifocal renal tubular necrosis, and hemorrhage within multiple organ systems. Microscopy of a vomitus sample confirmed the presence of Microcystis. Three analytical MC testing approaches were used, including the MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) technique, targeted congener analysis (e.g., liquid chromatography tandem-mass spectrometry of MC-LR), and enzyme-linked immunosorbent assay (ELISA). Total Adda MCs (as MMPB) were confirmed in the liver, bile, kidney, urine, and blood of the deceased dog. Urinalysis (MMPB) of one surviving dog showed a high level of MCs (32,000 ng mL-1) 1-day post exposure, with MCs detectable >2 months post exposure. Furthermore, hair from a surviving dog was positive for MMPB, illustrating another testable route of MC elimination in canines. The described cases represent the first use of urine as an antemortem, non-invasive specimen to diagnose microcystin toxicosis. Antemortem diagnostic testing to confirm MC intoxication cases, whether acute or chronic, is crucial for providing optimal supportive care and mitigating MC exposure.

The analysis of underivatized ß-Methylamino-L-alanine (BMAA), BAMA, AEG & 2,4-DAB in Pteropus mariannus mariannus specimens using HILIC-LC-MS/MS.

ß-Methylamino-L-alanine (BMAA) has been identified as the potential cause of the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) observed in the Chamorro people of Guam. The principal hypothesis for BMAA exposure and intoxication relies on the biomagnification of BMAA in flying fox specimens ingested by the Chamorro people. Although high levels of BMAA were quantitated in flying fox specimens utilizing liquid chromatography-fluorescence (LC-FL), there have not been any confirmatory analyses conducted to date. Therefore, a method for the tissue homogenization, extraction and direct analysis of BMAA (including BAMA, 2,4-DAB and AEG) was utilized. The approach was applied to mammalian dried skin and hair from various rodent species (negative controls) and archived flying fox (Pteropus mariannus mariannus) specimens. A positive control sample of homogenized mussel (Mytelius edulis) with native BMAA was used to verify the method. It was determined that the direct analysis using HILIC MS/MS required additional quality control in order to allow for the confident identification of BMAA due to the near co-elution of BAMA. BMAA was not present above 0.2 µg g-1 (free fraction) or 2.8 µg g-1 (total fraction) in the flying fox specimens. While analysis did not result in BMAA detection in flying fox or negative control samples, the positive control sample and spiked samples were successfully detected.

Benthic periphyton from Pennsylvania, USA is a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxin-a).

In 2016, the Pennsylvania Department of Environmental Protection conducted a limited survey of streams in the Susquehanna River basin in Pennsylvania, USA, to screen for microcystins/nodularins, anatoxin-a (ATX) and homoanatoxin-a (HTX). Testing revealed the presence of HTX in samples collected from the Pine Creek basin, with ATX present at lower levels. Microcystins/nodularins (MCs/NODs) were also tested and found to be concomitant, with NOD-R confirmed present by LC-MS/MS.

Analysis of free and metabolized microcystins in samples following a bird mortality event.

In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL-1 of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener (m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu1]MC-LR using NMR spectroscopy, LC-HRMS and LC-MS2, which slowly converted to [D-Leu1,Glu(OMe)6]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC-MS2 in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard (Anas platyrhynchos) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC-MS2 were used to measure free MCs, while 'total' MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63-112 ng g-1) accounted for 33-41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu1]MC-LR was quantitated in tissues at 25-67 ng g-1 (LC-MS2). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.

Nodularin from benthic freshwater periphyton and implications for trophic transfer.

In 2013 and 2015, the Pennsylvania Department of Environmental Protection conducted a survey of lotic habitats within the Susquehanna, Delaware, and Ohio River basins in Pennsylvania, USA, to screen for microcystins/nodularins (MCs/NODs) in algae communities and smallmouth bass (Micropterus dolomieu). Periphyton (68 from 41 sites), juvenile whole fish (153 from 19 sites) and adult fish liver (115 from 16 sites) samples were collected and screened using an Adda enzyme-linked immunosorbent assay (ELISA). Samples that were positive for MCs/NODs were further analyzed using LC-MS/MS, including 14 variants of microcystin and NOD-R and the MMPB technique. The ELISA was positive for 47% of the periphyton collections, with NOD-R confirmed (0.7-82.2 ng g-1 d.w.) in 20 samples. NOD-R was confirmed in 10 of 15 positive juvenile whole fish samples (0.8-16.7 ng g-1 w.w.) and in 2 of 8 liver samples (1.7 & 2.8 ng g-1 w.w.). The MMPB method resulted in total MCs/NODs measured in periphyton (2.2-1269 ng g-1 d.w.), juvenile whole fish (5.0-210 ng g-1 d.w.) and adult livers (8.5-29.5 ng g-1 d.w.). This work illustrates that NOD-R is present in freshwater benthic algae in the USA, which has broader implications for monitoring and trophic transfer.

Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS).

Microcystins have been detected in raw and finished drinking water using a variety of techniques, including assays (immunoassay, phosphatase inhibition) and HPLC (UV, MS/(MS)). The principal challenge to microcystin analysis is accounting for the over 150 variants that have been described. A confirmatory individual variant HPLC analysis is prone to under-reporting total microcystins due to method specificity. One method that allows for total microcystin quantitation is the MMPB technique. In this study, water samples with native microcystins were oxidized to cleave the Adda moiety, common to all microcystin variants. LC-MS/MS analysis was conducted on the subsequent MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) molecule and calibrated using a certified reference standard (microcystin-LR) and 4-phenylbutyric acid. Total microcystin concentrations from MMPB were compared to Adda ELISA and individual variant analyses (LC-UV, LC-MS/(MS)). Variants of microcystin, including [DAsp(3)]MC-RR, [Dha(7)]MC-RR, MC-RR, MC-YR, MC-LR, [DAsp(3)]MC-LR, [Dha(7)]MC-LR, MC-WR, MC-LA, and MC-LY were detected and quantified in samples. The individual variant analyses did not account for total microcystins present in samples, as indicated by ELISA and MMPB data. Results demonstrated the MMPB technique is a simple and valuable approach to confirm ELISA data when analyzing microcystins, with method detection limits of 0.05 µg L(-1) for total microcystins.

The extraction and analysis of cylindrospermopsin from human serum and urine.

The naturally derived cyanotoxin, cylindrospermopsin (CYN), has been detected in freshwater systems worldwide and poses a threat to human health. The methods for the extraction and detection of this toxin in source water are well documented, but methods for CYN determination in exposed individuals have not been investigated. In this study, the extraction and detection of CYN from two different matrices, serum and urine, was explored. Both serum and urine matrices inherently produce interference with analytical analyses and require extensive clean-up. Methods for extraction of CYN from both matrices were developed and validated using fortified samples. Serum extraction included homogenization followed by protein precipitation and solid phase extraction (SPE). Urine samples were processed using filtration, pH manipulation, and SPE. Analyses using a commercially available enzyme-linked immunosorbent assay (ELISA) and liquid chromatography coupled with mass spectrometry (LC/MS/MS) were assessed. Matrix effects inhibited ELISA's use as a quantitative tool for both matrices. LC/MS/MS was determined to be the most effective and reproducible means to detect and quantify CYN. The method detection limits determined in this study using LC/MS/MS were 0.25 and 0.50 ng mL⁻¹ for serum and urine, respectively. This method can be used to test individuals exposed to blooms of cyanobacteria producing CYN.

Investigation of extraction and analysis techniques for Lyngbya wollei derived Paralytic Shellfish Toxins.

Paralytic Shellfish Toxins (PSTs) are highly toxic metabolic by-products of cyanobacteria and dinoflagellates. The filamentous cyanobacterium Lyngbya wollei produces a unique set of PSTs, including L. wollei toxins (LWT) 1-6. The accurate identification and quantification of PSTs from Lyngbya filaments is challenging, but critical for understanding toxin production and associated risk, as well as for providing baseline information regarding the potential for trophic transfer. This study evaluated several approaches for the extraction and analysis of PSTs from field-collected L. wollei dominated algal mats. Extraction of PSTs from lyophilized Lyngbya biomass was assessed utilizing hydrochloric acid and acetic acid at concentrations of 0.001-0.1 M. Toxin profiles were then compared utilizing two analysis techniques: pre-column oxidation (peroxide and periodate) High Performance Liquid Chromatography (HPLC) with Fluorescence (FL) detection and LC coupled with Mass Spectrometry (MS). While both acid approaches efficiently extracted PSTs, hydrochloric acid was found to convert the less toxic LWT into the more toxic decarbamoylgonyautoxins 2&3 (dcGTX2&3) and decarbamoylsaxitoxin (dcSTX). In comparison, extraction with 0.1 M acetic acid preserved the original toxin profile and limited the presence of interfering co-extractants. Although pre-chromatographic oxidation with HPLC/FL was relatively easy to setup and utilize, the method did not resolve the individual constituents of the L. wollei derived PST profile. The LC/MS method allowed characterization of the PSTs derived from L. wollei, but without commercially available LWT 1-6 standards, quantitation was not possible for the LWT. In future work, evaluation of the risk associated with L. wollei derived PSTs will require commercially available standards of LWT 1-6 for accurate determinations of total PST content and potency.

Investigation of a Microcystis aeruginosa cyanobacterial freshwater harmful algal bloom associated with acute microcystin toxicosis in a dog.

Microcystin poisoning was diagnosed in a dog exposed to a Microcystis aeruginosa-dominated, freshwater, harmful algal bloom at Milford Lake, Kansas, which occurred during the summer of 2011. Lake water microcystin concentrations were determined at intervals during the summer, using competitive enzyme-linked immunosorbent assays, and indicated extremely high, localized microcystin concentrations of up to 126,000 ng/ml. Multiple extraction and analysis techniques were used in the determination of free and total microcystins in vomitus and liver samples from the poisoned dog. Vomitus and liver contained microcystins, as determined by enzyme-linked immunosorbent assays, and the presence of microcystin-LR was confirmed in vomitus and liver samples using liquid chromatography coupled with tandem mass spectrometry. Major toxic effects in a dog presented for treatment on the day following exposure included fulminant liver failure and coagulopathy. The patient deteriorated rapidly despite aggressive treatment and was euthanized. Postmortem lesions included diffuse, acute, massive hepatic necrosis and hemorrhage, as well as acute necrosis of the renal tubular epithelium. A diagnosis of microcystin poisoning was based on the demonstration of M. aeruginosa and microcystin-LR in the lake water, as well as in vomitus produced early in the course of the poisoning; the presence of microcystin-LR in liver tissue; and a typical clinical course including gastroenteritis and fulminant liver failure.