Varriale, Fabio (2021) Emerging toxins of European concern: identification, development of reference material and methods. [Tesi di dottorato]

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Phytoplankton is the autotrophic component of marine and freshwater ecosystems whose activity is crucial for the well-being of all the living organisms, including terrestrial and aquatic ones. However, a certain number of species belonging to the wide group of dinoflagellates and cyanobacteria may pose a serious threat for the safety of humans and wild animals due to the production of toxic secondary metabolites known as biotoxins. These noxious microorganisms, under specific and not fully clarified environmental conditions can massively enhance their proliferation rate through the so-called harmful algal bloom (HAB). This scenario raises even more concerns as the effects of the anthropogenic pressure on the whole ecosystem are leading to tremendous environmental changes, which are promoting the incidence and the spread of HABs all over the world. The impact of HABs is drastic since the increased density of harmful algae and consequently, the presence of high toxin levels in the aquatic systems, strongly affect the economy of coastal areas and, more importantly, represents an actual risk for both environment and human health. The biotoxins can indeed accumulate in the edible tissues of a wide number of organisms within the marine trophic chain, thus ending up on the table of unaware consumers. As a consequence, the consumption of contaminated seafood can give rise to characteristic food-borne illnesses. Beside the oral route, phycotoxins can result in human poisoning following inhalation of toxic aerosols and/or direct skin contact. In order to safeguard the public health and limiting the adverse effects of HABs, governments in collaboration with food safety agencies released specific legislations to regulate the maximum permitted level of toxins in seafood. These regulations require a strict surveillance of toxins in food chain and in the environment through the implementation of routine monitoring programs which are conducted by national and local competent health protection authorities. Unfortunately, a wide range of factors are steadily increasing the proliferation of the microalgal community, with the displacement of known and unknown alien toxin-producing species in regions where they were not historically confined like the Mediterranean basin. This has determined the appearance in temperature regions of new structurally-related compounds designated as ''Emerging Toxins'', which are currently non-regulated in EU, thus not regularly monitored through surveillance activities. Their presence in waters and seafood is a matter of concern for competent authorities, which required efforts from the scientific community before establishing a meaningful regulation. The development of sensitive and effective analytical methods for the detection of toxins and their metabolites in seafood, as well as the production of reference material, which is fundamental for the optimization of analytical techniques and for conducting toxicological studies, are high priority tasks for facing the emerging toxins related issues. Among the variety of instrumental techniques developed so far, the hyphenated techniques mainly based on the combination of liquid chromatography coupled to mass spectrometry (LC-MS) have proven to be effective and robust enough for monitoring phycotoxins in environmental and food samples. In addition, the employment of high-resolution multiple stage mass spectrometry (HRMSn) demonstrated suitable for the identification and characterization of new structural analogues contained at trace levels in complex matrices. At this regard, the aim of my PhD project was the study of the main classes of emerging toxins of European concern using LC-HRMS as method of choice. Different LC-HRMS methods were developed and optimized for each group of analytes to achieve the best analytical performances in terms of sensitivity, reproducibility and specificity. Such methods were subsequently applied to the analysis of complex matrices for determination of known compounds, as well as for identification and tentative structural characterization of new toxins and their biotransformation products. Notably: Chapter 1 is a general introduction which reports on: i) the importance of phytoplankton in the aquatic ecosystems, ii) the real threat of harmful algal blooms for living species, iii) toxins currently regulated in EU, and iv) a detailed description of the emerging toxins. Chapter 2 describes the development of a LC-HRMS method for the analysis of assorted cyclic imines (CIs) and its application to shellfish samples from the Mediterranean basin (Italy and Tunisia) and the Galician coastline (Spain). A mixture of CI standards containing pinnatoxin G and A (PnTX-G and -A), gymnodimine A (GYM-A) and 13desmethyl spirolide C (13desMeSPX-C) was used to optimize the chromatography, the MS parameters, and to evaluate the analytical performances and the matrix interference. The optimized HRMS2 conditions provided for each toxin highly informative fragmentation spectra, whose complete interpretation allow to discover previously unreported fragment ions, and a new fragmentation pathway co-occurring with the main retro-Diels-Alder ring opening. The application of the implemented LC-HRMS method to the analysis of Tunisian shellfish revealed high levels of gymnodimine A (376.5 µg/Kg) together with lower levels of five isobaric analogues of GYM-B/C and a new structural congener, which was named GYM-F, whose structure was proposed based on its fragmentation patterns. The high level of GYMs in the Tunisian sample prompted to deeply investigate the metabolic profile of the contaminated shellfish. So, a LC-HRMS data-dependent acquisition (DDA) based-approach was implemented and successfully applied, in combination with targeted HRMS2 experiments, to the analysis of GYM fatty acid ester metabolites. The optimized methodology revealed the presence of a wide number of esters of GYM-A and -B/C, including new metabolites esterified with atypical hydroxylated, polyhydroxylated and odd-chain fatty acids. The study of the fragmentation pattern of GYM esters, in association with the finding of several isobaric ester metabolites, led to set up a new MS-based strategy, labeled as backward analysis, whose application successfully revealed the presence of new GYMs starting from the identification of their ester metabolites; the new congeners were named GYM-G, -H, -I and J. A careful interpretation of their HRMS2 spectra allowed to propose the chemical structure of GYM-G and partially that of GYM-H, whereas only structural hints were obtained for the others due to their low relative abundance. In addition, the application of the optimized LC-HRMS method brought to the light the presence of PnTX-G (6.8 µg/Kg) for the first time in M. galloprovincialis from Sardinia (Thyrrenean Sea, Italy) and in mussels from the Atlantic coast of Spain (Galicia) in the range 3.1-7.7 µg/Kg. The same Spanish mussels were found to be even contaminated by 13desMeSPX-C (11.0-29.0 µg/Kg). Chapter 3 reports on the development, the analytical comparison and the application of 3 HILIC-HRMS methods, labeled as method 1, 2 and 3, for the simultaneous determination of 13 paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) in seafood by using the Orbitrap MS. The main challenge was the implementation of a reliable and highly sensitive multi-analyte method as the reduced scan frequency due to long injection times, which are essential for the acquisition of the accurate masses, strongly affects the instrumental limits of detection and method applicability. Although methods 1 and 2 differed for chromatographic conditions and consequently, for ESI source parameters, they shared the acquisition mode, which was based on the time segmentation technique. The latter allows to select a defined number of analytes to monitor in a specific time segment, or window, thus resulting in an increased instrumental sensitivity due to the decrease of MS2 scans within the entire run. A rigorous comparison between method 1 and 2 revealed that: the former was characterized by higher reproducibility of retention times within and between different batches of analysis, while the latter provided the best chromatographic resolution and peak shape; both methods showed high linearity and analytical sensitivity. However, the analysis of several PST-contaminated shellfish samples revealed a low specificity for method 2, that, associated with poor chromatographic reproducibility, made it not applicable for determination of toxins in seafood. As part of method development, intriguing insight emerged in the use of the LTQ Orbitrap XL FTMS for the analysis of such compounds. A careful investigation revealed a drastic impact of the ion transmission system (LTQ-C-trap-Orbitrap) on the stability of the sulfated PST analogues in the MS analyzer, thus influencing the HRMS2 conditions set in both methods 1 and 2. HILIC-HRMS method 3, which shared the same chromatography of method 2 but different MS conditions, was successfully applied to the analysis of environmental and food samples. Firstly, it was employed to determine the toxin profile of culture strains of A. pacificum originally isolated from plastic debris harvested in the Syracuse Bay (Ionian Sea, Southern Italy) in the frame of a surveillance program between 2016-2017. Secondly, it was used to confirm the presence of TTX in mussels harvested in the Marano Lagoon (Northern Adriatic Sea, Italy) during an official monitoring program between 2017-2018. Chapter 4 reports on the developments of LC-HRMS methods for the analysis of a wide number of cyanotoxins. Notably, an effective and sensitive reverse-phase LC-HRMS method was optimized for the analysis of microcystins (MCs) and nodularins (NODs), and successfully applied to a cyanobacterial biomass sample collected from the Greek lake Kastoria. A large number of MCs were detected, with MC-RR and MC-LR being the most abundant variants. In addition, two new MC analogues were identified and named MC-prHcysR and MC-prHcys(O)R according to structural features emerging from the interpretation of their HRMS2 spectra. The high biodiversity observed in the cyanobacterial biomass led to carefully explore the metabolic profile of the occurring cyanobacterial species. At this purpose, an effective workflow based on the combination of HRMS DDA approach with a new vendor-free published database of cyanometabolites was designed, and successfully applied. The implemented methodology turned out to be a powerful analytical tool for high throughput analysis since a large number of known and new cyanobacterial secondary metabolites belonging to microginin, anabaenopeptin and cyanopeptoline-type peptide classes was revealed. Tentative HRMS2-based structural characterization was conducted for all the new metabolites. In addition, the HILIC-HRMS method 1 reported in chapter 3, which was optimized for the determination of PSTs and TTX in seafood, was exploited to evaluate its suitability for the analysis of small polar cyanotoxins like anatoxin a (ATXa), cylindrospermopsin (CYN) and lynbyawolleytoxin 1 (LWTX1). As a result, a satisfactory sensitivity and linearity was achieved from the analysis of the relevant toxin standards, thus a multi-toxin HILIC-HRMS method based on time segmentation was implemented for the simultaneous analysis of a wide number of cyanotoxins (PSTs, ATXa, CYN and LWTX1). In Chapter 5 the optimization of LC-HRMS and LC-MS2 methods for the analysis of ciguatoxins (CTXs) on different MS instruments is reported. The untargeted approach was optimized for the analysis of Pacific (P) congeners by using a mixture of 5 reference standards. Although the method refinement still requires more efforts, which are currently hampered by the lack of adequate CRM, the optimized ESI source conditions turned out to be a valuable tool for confirmation of toxin identity. Under the implemented conditions, P-CTXs ionized through a complex pattern of in-source ions – [M+H]+, [M+H-nH2O]+, [M+Na]+, [M+K]+, [M+NH4]+ – whose presence and relative ion abundance ratio, which was toxin-dependent, represented a characteristic fingerprint that can be exploited to detect known congeners in complex matrices, as well as for identification of new putative analogues. The targeted approach was optimized for the analysis of Caribbean (C) CTXs on a triple quadrupole (QqQ) MS during a 6 month-period that I spent as visiting PhD student at the Centre for Environment, Fisheries and Aquaculture Science (CEFAS, Weymouth, United Kingdom) under the supervision of Dr Andrew Turner. The LC-MS2 method was optimized by using fish extracts contaminated by C-CTX1 and -2 (lab RM) and employed to confirm and study the presence of CTXs in frozen Red Snapper fillets imported from India which were suspected to be cause of a food poisoning occurred in 2017 in Stoke-on-Trent (UK). The LC-MS2 analysis of a wide number of fish fillets revealed the presence of peaks attributable to Caribbean (C-) or Indian (I-) CTX1 and -2 in the multiple reaction monitoring (MRM) chromatograms, whereas a noteworthy ciguatoxicity was measured by the cell-based assay (CBA-N2a). In light of these findings, part of the contaminated production batch was used as starting material for a preparative work aimed at isolating the toxic compounds for the production of RM. At this purpose, several experiments were designed and performed to optimize a large-scale procedure to extract toxins from fish tissue with high yield and low variability. As a result, 89 Kg of homogenized fish tissue were processed on small-scale and analyzed by LC-MS2. An aliquot of 10 Kg, identified among the most contaminated ones, was subjected to the large-scale extraction, and 74.5 g of liposoluble residue were obtained. Currently, further purification and isolation steps are ongoing using a combination of LC-MS2 and N2a experiments for toxin/toxicity monitoring. Chapter 6 describes the optimization and the successful application of a preparative procedure aimed at extracting, purifying and isolating ovatoxin-a (OVTX-a) with high grade of purity from 219 liters of a cultured strain of O. ovata. The final goal was to isolate enough material with a grade of purity greater than 90% to measure, in collaboration with national and foreign partners, in vivo acute toxicity by different routes of administration, and to support preliminary stability studies for the production of OVTX-a CRM which is not commercially available yet. LC-HRMS analysis of the toxin profile revealed the suitability of O. ovata cell culture for isolation of OVTX-a as it represented the main component (78%), with other analogues (OVTX-d/e) representing only 22% of the total toxin content. Starting from a previously optimized procedure, the isolation of OVTX-a was achieved through several steps including: extraction of toxin with solvents, ii) clean-up of the extracts through medium-pressure liquid chromatography (MPLC, flash chromatography), iii) a semi-preparative HPLC, iv) a final preparative HPLC, and v) multiple evaporation steps within the whole protocol. However, the procedure was strictly influenced by critical aspects which are related to the chemical-physical properties of OVTXs and palytoxin. Notably, the evaporation of solvents, which represents the most critical but unavoidable step, the irreversible adsorption of toxins to different materials and the usage of acids in the chromatographic purifications, drastically reduce recovery yields. On balance, the optimized procedure allowed to successfully isolate 3.4 mg of OVTX-a with a grade of purity of 93.3% (calculated on the total OVTXs content). The extraction procedure of toxins from cell pellets, the clean-up of the extracts by flash chromatography and the semi-preparative HPLC provided the highest yields of recovery, whilst the concentration steps, the storage of the crude extracts and the final preparative HPLC still need to be improved. Chapter 7 is a summary reporting two collaborative studies outside the PhD project. The first one reports on the development of an ESI- HRMS direct injection method for the analysis of bisphenol (BP) AF and BPM, and its application to the analysis of beverage samples. BPs are a group of small organic molecules massively used to manufacture a wide range of commercial products. However, they are endocrine disruptors whose toxicity on living organisms is well-known. Their occurrence in foodstuff is frequent and mainly due to a migration from the packaging materials. Therefore, the HRMS approach was used to confirm the presence of BPAF and BPM in 2 processed beer samples, with the aim to support and validate the identification of different BPs in 52 beverage samples, which was conducted through a previously validated LC-fluorescence detection (FD) method. The second collaborative study describes the optimization of a HILIC-HRMS method for the analysis of sapropterin and its structurally related compounds. Sapropterin is the active ingredient of the Kuvan®, a drug approved for the treatment of phenylalaninemia, a rare illness due to a reduced activity of the phenylalanine hydroxylase. Sapropterin-containing drugs have to guarantee high quality standards since dangerous impurities originating from the synthetic process of the active ingredient or degradation reactions may be found. In this context, the HILIC-HRMS approach was employed to corroborate the results of a LC-UV method, which was applied to identify and quantify sapropterin and its impurities in Kuvan® and Diterin ®, the branded and the generic drug, respectively.

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