Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.     

Complex Toxin Profile of French Mediterranean Ostreopsis cf. ovata Strains,Seafood Accumulation and Ovatoxins Prepurification

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg⁻¹ of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg⁻¹ in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell⁻¹. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.     

Occurrence of Lipophilic Marine Toxins in Shellfish from Galicia (NW of Spain) and Synergies among Them

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.     

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.     

Hunt for Palytoxins in a Wide Variety of Marine Organisms Harvested in 2010 on the French Mediterranean Coast

During the summer of 2010, 31 species including fish, echinoderms, gastropods, crustaceans, cephalopods and sponges were sampled in the Bay of Villefranche on the French Mediterranean coast and screened for the presence of PLTX-group toxins using the haemolytic assay. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for confirmatory purposes and to determine the toxin profile. The mean toxin concentration in the whole flesh of all sampled marine organisms, determined using the lower- (LB) and upper-bound (UB) approach was 4.3 and 5.1 µg·kg⁻¹, respectively, with less than 1% of the results exceeding the European Food Safety Authority (EFSA) threshold of 30 µg·kg⁻¹ and the highest values being reported for sea urchins (107.6 and 108.0 µg·kg⁻¹). Toxins accumulated almost exclusively in the digestive tube of the tested species, with the exception of octopus, in which there were detectable toxin amounts in the remaining tissues (RT). The mean toxin concentration in the RT of the sampled organisms (fishes, echinoderms and cephalopods) was 0.7 and 1.7 µg·kg⁻¹ (LB and UB, respectively), with a maximum value of 19.9 µg·kg⁻¹ for octopus RT. The herbivorous and omnivorous organisms were the most contaminated species, indicating that diet influences the contamination process, and the LC-MS/MS revealed that ovatoxin-a was the only toxin detected.     

Cyanobacterial Toxic and Bioactive Peptides in Freshwater Bodies of Greece: Concentrations,Occurrence Patterns,and Implications for Human Health

Cyanobacterial harmful algal blooms represent one of the most conspicuous waterborne microbial hazards in aquatic environments mostly due to the production of toxic secondary metabolites, mainly microcystins (MCs). Other bioactive peptides are frequently found in cyanobacterial blooms, yet their concentration and ecological relevance is still unknown. In this paper we studied the presence and concentration of cyanobacterial peptides (microcystins, anabaenopeptins, anabaenopeptilides) in 36 Greek freshwater bodies, using HPLC-DAD, ELISA, and PP1IA. Microcystins were found in more than 90% of the samples investigated, indicating that microcystin-producing strains seem to also occur in lakes without blooms. Microcystins MC-RR, MC-LR, and MC-YR were the main toxin constituents of the bloom samples. Anabaenopeptin A and B were predominant in some samples, whereas anabaenopeptolide 90A was the only peptide found in Lake Mikri Prespa. The intracellular concentrations of anabaenopeptins produced by cyanobacterial bloom populations are determined for the first time in this study; the high (>1000 µg·L⁻¹) anabaenopeptin concentration found indicates there may be some impacts, at least on the ecology and the food web structure of the aquatic ecosystems. The maximum intracellular MC values measured in Lakes Kastoria and Pamvotis, exceeding 10,000 µg·L⁻¹, are among the highest reported.     

Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]⁺ ions.     

First Identification of 5,11-Dideoxytetrodotoxin in Marine Animals,and Characterization of Major Fragment Ions of Tetrodotoxin and Its Analogs by High Resolution ESI-MS/MS

Even though tetrodotoxin (TTX) is a widespread toxin in marine and terrestrial organisms, very little is known about the biosynthetic pathway used to produce it. By describing chemical structures of natural analogs of TTX, we can start to identify some of the precursors that might be important for TTX biosynthesis. In the present study, an analog of TTX, 5,11-dideoxyTTX, was identified for the first time in natural sources, the ovary of the pufferfish and the pharynx of a flatworm (planocerid sp. 1), by comparison with totally synthesized (−)-5,11-dideoxyTTX, using high resolution ESI-LC-MS. Based on the presence of 5,11-dideoxyTTX together with a series of known deoxy analogs, 5,6,11-trideoxyTTX, 6,11-dideoxyTTX, 11-deoxyTTX, and 5-deoxyTTX, in these animals, we predicted two routes of stepwise oxidation pathways in the late stages of biosynthesis of TTX. Furthermore, high resolution masses of the major fragment ions of TTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX were also measured, and their molecular formulas and structures were predicted to compare them with each other. Although both TTX and 5,6,11-trideoxyTTX give major fragment ions that are very close, m/z 162.0660 and 162.1020, respectively, they are distinguishable and predicted to be different molecular formulas. These data will be useful for identification of TTXs using high resolution LC-MS/MS.     

Structural Analysis of a Heteropolysaccharide from Saccharina japonica by Electrospray Mass Spectrometry in Tandem with Collision-Induced Dissociation Tandem Mass Spectrometry (ESI-CID-MS/MS)

A fucoidan extracted from Saccharina japonica was fractionated by anion exchange chromatography. The most complex fraction F0.5 was degraded by dilute sulphuric acid and then separated by use of an activated carbon column. Fraction Y1 was fractionated by anion exchange and gel filtration chromatography while Fraction Y2 was fractionated by gel filtration chromatography. The fractions were determined by ESI-MS and analyzed by ESI-CID-MS/MS. It was concluded that F0.5 had a backbone of alternating 4-linked GlcA and 2-linked Man with the first Man residue from the nonreducing end accidentally sulfated at C6. In addition, F0.5 had a 3-linked glucuronan, in accordance with a previous report by NMR. Some other structural characteristics included GlcA 1→3 Man 1→4 GlcA, Man 1→3 GlcA 1→4 GlcA, Fuc 1→4 GlcA and Fuc 1→3 Fuc. Finally, it was shown that fucose was sulfated at C2 or C4 while galactose was sulfated at C2, C4 or C6.     

Isolation of Araguspongine M,a New Stereoisomer of an Araguspongine/Xestospongin alkaloid,and Dopamine from the Marine Sponge Neopetrosia exigua Collected in Palau

A new stereoisomer of an araguspongine/xestospongin alkaloid, named araguspongine M (1), has been isolated together with 12 known compounds, araguspongines B (2) and D (3), dopamine, three galactosyl diacylglycerols, 24-methyl cholesterol, 5,6-dihydrocholesterol, β-sitosterol, and three 5α,8α-epidioxy sterols (11–13), from the marine sponge Neopetrosia exigua (formerly Xestospongia exigua) collected in Palau. The structure of 1 was assigned on the basis of its spectral data analysis. This is the first report on the isolation of dopamine from a marine sponge. This compound may be produced by an endosymbiotic Synechococcus-like cyanobacterium. Compounds 1–3 and 11–13 showed cytotoxicity against HL-60 at IC₅₀’s of 5.5, 5.5, 5.9, 22.4, 9.5, and 9.6 μM, respectively. The possible biosynthesis origin of the isolated metabolites is discussed.     

Smenamides A and B,Chlorinated Peptide/Polyketide Hybrids Containing a Dolapyrrolidinone Unit from the Caribbean Sponge Smenospongia aurea. Evaluation of Their Role as Leads in Antitumor Drug Research

An in-depth study of the secondary metabolites contained in the Caribbean sponge Smenospongia aurea led to the isolation of smenamide A (1) and B (2), hybrid peptide/polyketide compounds containing a dolapyrrolidinone unit. Their structures were elucidated using high-resolution ESI-MS/MS and homo- and heteronuclear 2D NMR experiments. Structures of smenamides suggested that they are products of the cyanobacterial metabolism, and 16S rRNA metagenomic analysis detected Synechococcus spongiarum as the only cyanobacterium present in S. aurea. Smenamides showed potent cytotoxic activity at nanomolar levels on lung cancer Calu-1 cells, which for compound 1 is exerted through a clear pro-apoptotic mechanism. This makes smenamides promising leads for antitumor drug design.