Microcystin profiles in European noble crayfish Astacus astacus and water in Lake Steinsfjorden, Norway.

Lake Steinsfjorden, an important noble crayfish (Astacus astacus) habitat, is often affected by blooms of Planktothrix spp. that produce microcystins (MCs). A poor correlation between MCs by ELISA in the water and in crayfish tissue in a study in 2015 prompted further investigation by LC-HRMS. LC-HRMS analyses of filters from water samples and on selected crayfish tissue extracts from the 2015 study revealed the presence of known and previously unreported MCs. Crayfish samples from May and June 2015 were dominated by MCs from the Planktothrix bloom, whereas in September novel MCs that appeared to be metabolites of MC-LR were dominant, even though neither these nor MC-LR were detected in the water in 2015. A water sample from October 2016 also showed MCs typical of Planktothrix (i.e., [d-Asp3]- and [d-Asp3,Dhb7]MC-RR and -LR), but low levels of MC-RR and MC-LR were detected in the lake water for the first time. In late summer and autumn, the MC profiles of crayfish were dominated by the homonorvaline (Hnv) variant MC-LHnv, a putative metabolite of MC-LR. Taken together, ELISA, LC-HRMS and previous PCR analyses showed that although Planktothrix was part of the crayfish diet, it was not the sole source of MCs in the crayfish. Possibly, crayfish in Lake Steinsfjorden may be ingesting MCs from benthic cyanobacteria or from contaminated prey. Therefore, information on the cyanobacterial or MC content in the water column cannot safely be used to make predictions about MC concentrations in the crayfish in Lake Steinsfjorden. Interestingly, the results also show that targeted LC-MS analysis of the crayfish would at times have underestimated their MC content by nearly an order of magnitude, even if all previously reported MC variants had been included in the analysis.

Isolation of Caribbean Ciguatoxin-5 (C-CTX5) and confirmation of its structure by NMR spectroscopy.

Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south St. Thomas, US Virgin Islands. The highest CTX-producing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 µg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions.

Preparation of 18O-labelled azaspiracids for accurate quantitation using liquid chromatography-mass spectrometry.

Azaspiracids (AZAs) are a group of polyether marine algal toxins known to accumulate in shellfish, posing a risk to human health and the seafood industry. Analysis of AZAs is typically performed using LC-MS, which can suffer from matrix effects that significantly impact the accuracy of measurement results. While the use of isotopic internal standards is an effective approach to correct for these effects, isotopically labelled standards for AZAs are not currently available. In this study, 18O-labelled AZA1, AZA2, and AZA3 were prepared by reaction with H218O under acidic conditions, and the reaction kinetics and sites of incorporation were studied using LC-HRMS/MS aided by mathematical analysis of their isotope patterns. Analysis of the isotopic incorporation in AZA1 and AZA3 indicated the presence of four exchangeable oxygen atoms. Excessive isomerization occurred during preparation of 18O-labelled AZA2, suggesting a role for the 8-methyl group in the thermodynamic stability of AZAs. Neutralized mixtures of 18O-labelled AZA1 and AZA3 were found to maintain their isotopic and isomeric integrities when stored at -20 °C and were used to develop an isotope-dilution LC-MS method which was applied to reference materials of shellfish matrices containing AZAs, demonstrating high accuracy and excellent reproducibility. Preparation of isotopically labelled compounds using the isotopic exchange method, combined with the kinetic analysis, offers a feasible way to obtain isotopically labelled internal standards for a wide variety of biomolecules to support reliable quantitation.

Liquid chromatography-high-resolution tandem mass spectrometry of anatoxins, including new conjugates and reduction products.

Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins for which only a handful of structural analogues have been well characterized. Here, we report the development of an LC-HRMS/MS method for the comprehensive detection of ATXs. Application of this method to samples of benthic cyanobacterial mats and laboratory cultures showed detection of several new ATXs. Many of these result from nucleophilic addition to the olefinic bond of the α,ß-unsaturated ketone functional group of anatoxin-a (ATX) and homoanatoxin-a (hATX), analogous to the conjugation chemistry of microcystins, which contain similar α,ß-unsaturated amide functionality. Conjugates with glutathione, γ-glutamylcysteine, methanethiol, ammonia, methanol and water were detected, as well as putative C-10 alcohol derivatives. Structural confirmation was obtained by simple and selective analytical-scale semisynthetic reactions starting from available ATX standards. Methanol, water and ammonia conjugates were found to result primarily from sample preparation. Reduction products were found to result from enzymatic reactions occurring primarily after cell lysis in laboratory cultures of Kamptonema formosum and Cuspidothrix issatschenkoi. The relative contributions of the identified analogues to the anatoxin profiles in a set of 22 benthic-cyanobacterial-mat field samples were estimated, showing conjugates to account for up to 15% of total ATX peak area and 10-hydroxyanatoxins up to 38%. The developed methodology, new analogues and insight into the chemical and enzymatic reactivity of ATXs will enable a more comprehensive study of the class than possible previously.

A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States.

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.

Thermal stability of cannabinoids in dried cannabis: a kinetic study.

This study was undertaken to quantitatively explore the effect of temperature on the degradation of cannabinoids in dried cannabis flower. A total of 14 cannabinoids were monitored using liquid chromatography and tandem mass spectrometry in temperature environments from - 20 to + 40 ∘C lasting up to 1 year. We find that a network of first-order degradation reactions is well-suited to model the observed changes for all cannabinoids. While most studies focus on high-temperature effects on the cannabinoids, this study provides high-precision quantitative assessment of room temperature kinetics with applications to shelf-life predictions and age estimates of cannabis products.

Oxidative Release of Thiol-Conjugated Forms of the Mycotoxin 4-Deoxynivalenol.

Deoxynivalenol (DON) is a trichothecene mycotoxin that is produced by several species of Fusarium, which may infect grain crops. DON, as well as other type-B trichothecenes, contain an α,ß-unsaturated carbonyl group that may react with sulfhydryl groups in, for example, amino acids and peptides. Such conjugates have been shown to occur in plants. Nucleophilic addition of thiols to the conjugated double bond in DON afforded several isomeric reaction products, and the thermodynamically favored isomers of DON-10-cysteine and DON-10-glutathione have been prepared and characterized previously. This study reports the preparation and characterization of the kinetically favored DON-10-cysteine isomer. We subsequently studied and compared the rate of the deconjugation reaction of the two DON-10-cysteine isomers and the thermodynamically favored DON-10-glutathione adduct. The deconjugation rate of the thermodynamically favored thiol conjugates was slow with half-lives of weeks even at pH 10.7, while the kinetically favored DON-10-cysteine isomer deconjugated within a few hours, affording free DON. We adapted a simple and rapid oxidation protocol in which the sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with the base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate to afford free DON. The deconjugation reactions of the sulfoxides and sulfones of thermodynamically favored DON-10-thiols were complete within hours or minutes at pH 10.7, respectively. The increase in deconjugation rates for the kinetically favored DON-10-cysteine were less dramatic. Oxidation of sulfides to sulfoxides is known to occur in vivo, and thus, our data show that thiol-conjugated DON might become bioavailable via sulfide oxidation followed by elimination to regenerate DON. The oxidation-elimination approach could also be useful for the indirect quantification of DON-10-thiol conjugates in plant and animal tissues.

LC-HRMS and Chemical Derivatization Strategies for the Structure Elucidation of Caribbean Ciguatoxins: Identification of C-CTX-3 and -4.

Ciguatera poisoning is linked to the ingestion of seafood that is contaminated with ciguatoxins (CTXs). The structural variability of these polyether toxins in nature remains poorly understood due to the low concentrations present even in highly toxic fish, which makes isolation and chemical characterization difficult. We studied the mass spectrometric fragmentation of Caribbean CTXs, i.e., the epimers C-CTX-1 and -2 (1 and 2), using a sensitive UHPLC-HRMS/MS approach in order to identify product ions of diagnostic value. We found that the fragmentation of the ladder-frame backbone follows a characteristic pattern and propose a generalized nomenclature for the ions formed. These data were applied to the structural characterization of a pair of so far poorly characterized isomers, C-CTX-3 and -4 (3 and 4), which we found to be reduced at C-56 relative to 1 and 2. Furthermore, we tested and applied reduction and oxidation reactions, monitored by LC-HRMS, in order to confirm the structures of 3 and 4. Reduction of 1 and 2 with NaBH4 afforded 3 and 4, thereby unambiguously confirming the identities of 3 and 4. In summary, this work provides a foundation for mass spectrometry-based characterization of new C-CTXs, including a suite of simple chemical reactions to assist the examination of structural modifications.

Improved Isolation Procedures for Okadaic Acid Group Toxins from Shellfish (Mytilus edulis) and Microalgae (Prorocentrum lima).

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin-2 (DTX2) from shellfish (M. edulis), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima, that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL-1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L-1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.

Novel Microcystins from Planktothrix prolifica NIVA-CYA 544 Identified by LC-MS/MS, Functional Group Derivatization and 15N-labeling.

Microcystins are cyclic heptapeptides from cyanobacteria that are potent inhibitors of protein phosphatases and are toxic to animals and humans. At present, more than 250 microcystin variants are known, with variants reported for all seven peptide moieties. While d-glutamic acid (d-Glu) is highly-conserved at position-6 of microcystins, there has been only one report of a cyanobacterium (Anabaena) producing microcystins containing l-Glu at the variable 2- and 4-positions. Liquid chromatography-mass spectrometry analyses of extracts from Planktothrix prolifica NIVA-CYA 544 led to the tentative identification of two new Glu-containing microcystins, [d-Asp3]MC-ER (12) and [d-Asp3]MC-EE (13). Structure determination was aided by thiol derivatization of the Mdha7-moiety and esterification of the carboxylic acid groups, while 15N-labeling of the culture and isotopic profile analysis assisted the determination of the number of nitrogen atoms present and the elemental composition of molecular and product-ions. The major microcystin analog in the extracts was [d-Asp3]MC-RR (1). A microcystin with an unprecedented high-molecular-mass (2116 Da) was also detected and tentatively identified as a sulfide-linked conjugate of [d-Asp3]MC-RR (15) by LC-HRMS/MS and sulfide oxidation, together with its sulfoxide (16) produced via autoxidation. Low levels of [d-Asp3]MC-RW (14), [d-Asp3]MC-LR (4), [d-Asp3,Mser7]MC-RR (11), [d-Asp3]MC-RY (17), [d-Asp3]MC-RF (18), [d-Asp3]MC-RR-glutathione conjugate (19), and [d-Asp3]MC-RCit (20), the first reported microcystin containing citrulline, were also identified in the extract, and an oxidized derivative of [d-Asp3]MC-RR and the cysteine conjugate of 1 were partially characterized.

Identification of 21,22-Dehydroazaspiracids in Mussels ( Mytilus edulis) and in Vitro Toxicity of Azaspiracid-26.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 µg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

Occurrence of cyclic imines in European commercial seafood and consumers risk assessment.

Cyclic imines constitute a quite recently discovered group of marine biotoxins that act on neural receptors and that bioaccumulate in seafood. They are grouped together due to the imino group functioning as their common pharmacore, responsible for acute neurotoxicity in mice. Cyclic imines (CIs) have not been linked yet to human poisoning and are not regulated in the European Union (EU), although the European Food Safety Authority (EFSA) requires more data to perform conclusive risk assessment for consumers. Several commercial samples of bivalves including raw and processed samples from eight countries (Italy, Portugal, Slovenia, Spain, Ireland, Norway, The Netherlands and Denmark) were obtained over 2 years. Emerging cyclic imine concentrations in all the samples were analysed on a LC-3200QTRAP and LC-HRMS QExactive mass spectrometer. In shellfish, two CIs, pinnatoxin G (PnTX-G) and 13-desmethylspirolide C (SPX-1) were found at low concentrations (0.1-12µg/kg PnTX-G and 26-66µg/kg SPX-1), while gymnodimines and pteriatoxins were not detected in commercial (raw and processed) samples. In summary, SPX-1 (n: 47) and PnTX-G (n: 96) were detected in 9.4% and 4.2% of the samples, respectively, at concentrations higher than the limit of quantification (LOQ), and in 7.3% and 31.2% of the samples at concentrations lower than the LOQ (25µg/kg for SPX-1 and 3µg/kg for PnTX-G), respectively. For the detected cyclic imines, the average exposure and the 95th percentile were calculated. The results obtained indicate that it is unlikely that a potential health risk exists through the seafood diet for CIs in the EU. However, further information about CIs is necessary in order to perform a conclusive risk assessment.

Stereochemical Definition of the Natural Product (6R,10R,13R, 14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 by Total Synthesis and Comparative Analyses.

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R, 33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned 20R epimer did not.

Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S, 25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals Non-Identity with the Natural Product.

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved by a late-stage Nozaki-Hiyama-Kishi coupling to form the C21-C22 bond with the C20 configuration unambiguously established from l-(+)-tartaric acid. Postcoupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the resulting C1 primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

Rapid and Convenient Oxidative Release of Thiol-Conjugated Forms of Microcystins for Chemical Analysis.

Microcystins are potent cyclic heptapeptide toxins found in some cyanobacteria, and usually contain an α,ß-unsaturated carbonyl group that is readily conjugated to thiol-containing amino acids, peptides, and proteins in vivo and in vitro. Methods for deconjugating these types of adducts have recently been reported, but the reactions are slow or result in derivatized microcystins. Mercaptoethanol derivatives of a range of microcystins were therefore used as model compounds to develop deconjugation procedures in which the dialkyl sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate via ß-elimination to afford free microcystins with the α,ß-unsaturated carbonyl group intact. These free microcystins can be analyzed by LC/MS to determine the toxin profile of bound microcystins. The method was tested on Cys- and GSH-derivatives of [Dha7]MC-LR. In solution, the deconjugation reactions were complete within minutes at pH 10.7 and within a few hours at pH 9.2. Oxidation of sulfides to sulfoxides is easier and more rapid than oxidation to sulfones, allowing the use of milder oxidants and shorter reaction times. Oxidation of any methionine residues present in the microcystins occurs inevitably during these procedures, and interpretation of the microcystin profile obtained by LC/MS analysis needs to take this into account. Oxidation of tryptophan residues and degradation of microcystins by excess oxidant were circumvented by the addition of Me2SO as a sacrificial reducing agent. These methods may be useful for other compounds that undergo conjugation via thia-Michael addition, such as acrylamide and deoxynivalenol. Oxidation of sulfides to sulfoxides can occur in vivo and could affect the bioavailability of toxins and drugs conjugated via thia-Michael addition, potentially exacerbating oxidative stress by catalytically converting GSH to its sulfenate via conjugation, oxidation, and elimination to regenerate the free toxin.

Conjugation of Microcystins with Thiols Is Reversible: Base-Catalyzed Deconjugation for Chemical Analysis.

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,ß-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method's utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,ß-unsaturated carbonyl groups and can be expected to behave in a similar manner.

Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.

Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure-activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2-epi-DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 >> 2-epi-DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs' activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors.

Epimers of azaspiracids: Isolation, structural elucidation, relative LC-MS response, and in vitro toxicity of 37-epi-azaspiracid-1.

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.

Indole-diterpenoid profiles of Claviceps paspali and Claviceps purpurea from high-resolution Fourier transform Orbitrap mass spectrometry.

RATIONALE: The biological activities most commonly associated with indole-diterpenoids are tremorgenicity in mammals and toxicity in insects through modulation of ion channels. The neurotoxic effects of some analogues are the cause of syndromes such as 'ryegrass staggers' and 'Paspalum staggers' in cattle and sheep. Our purpose was to obtain and interpret mass spectra of some pure Claviceps-related indole-diterpenoids (paspaline, paspalinine, paxilline, paspalitrems A and B) to facilitate identification of related compounds for which standards were not available. METHODS: C. paspali-infected Paspalum dilatatum as well as C. purpurea sclerotia obtained from infected Phalaris arundinacea were extracted and the extracts separated via liquid chromatography. Low- and high-resolution mass spectra were then obtained of known and potentially unknown indole-diterpenoids. RESULTS: At least 20 different indole-diterpenoids were detected in the C. paspali extract with molecular masses ranging from 405 Da (C28H40NO) to 517 Da (C32H40NO5). The C. purpurea sclerotia were shown to contain several indole-diterpenoids with molecular masses ranging from 405 Da (C28H40NO) to 419 Da (C28H38NO2). CONCLUSIONS: This study demonstrates for the first time that C. purpurea may also produce indole-diterpenoids. This might explain why grazing of Phalaris spp. is occasionally connected with a tremorgenic syndrome in cattle, called 'phalaris staggers'.

Sulfide oxidations for LC-MS analysis of methionine-containing microcystins in Dolichospermum flos-aquae NIVA-CYA 656.

Microcystins are cyclic heptapeptides produced by a range of cyanobacteria. More than 150 microcystin analogues have been reported from cultures, algal blooms, or other contaminated samples. Relatively few analytical standards are available, making identification and quantitation of these toxins a challenge, even with LC-MS technology. We developed a two-step oxidative procedure that allows LC-MS identification of microcystins containing methionine and methionine sulfoxide, and reveals the oxidation state of the methionyl sulfur atom. The procedure was used in parallel with mercaptoethanol derivatization and LC-MS(2) analysis to demonstrate the presence of [Asp(3)]MC-MR (12) and MC-MR (17) in a culture of Dolichospermum flos-aquae, together with low levels of [Asp(3)]MC-M(O)R (5) and MC-M(O)R (7), as well as 20 other microcystins. Fresh culture contained only traces of sulfoxides 5 and 7, but these increased during storage or sample extraction and preparation. This suggests that microcystins containing methionine sulfoxide are primarily postextraction oxidation artifacts, rather than being produced by biosynthesis in cyanobacteria. A simple, rapid extraction under inert gas followed promptly by LC-MS analysis minimized oxidation artifacts for D. flos-aquae.