Factors associated with discharge home in older patients admitted to emergency department observation units: Looking for a predictive scale.

BACKGROUND: The selection of patients who are going to be admitted to an emergency department observation unit (EDOU) is essential for the good management of these units, intended fundamentally to avoid unnecessary hospitalization of patients. This is especially important when dealing with older patients. It would be important to know what factors are associated with discharge home and to have a clinical predictive scale that appropriately selects older patients who are going to be admitted to an EDOU. METHODS: A retrospective cross-sectional study was conducted of all patients ≥65 years of age assisted in 48 Spanish Emergency Departments for 7 consecutive days and were admitted to the EDOU. Demographics-functional, vital signs data and initial laboratory results were analyzed to investigate its association with discharge home and develop and validate a prediction model for discharge home from EDOU. Multivariable logistic regression was performed to develop a prediction model, and a scoring system was created. RESULTS: Among 5457 patients admitted to the EDOU from the emergency room, 2508 (46%) patients were discharged home, and 2949 (54%) were admitted to the hospital. Five variables were strongly associated with discharge home: the absence of fever (adjusted OR: 3.61, 95% CI:1.53-8.54), Glasgow Coma Scale score of 15 points (2.80, 1.63-4.82), absence of tachypnea (2.51, 1.74-3.64) or leukocytosis (2.07, 1.70-2.52) and oxygen saturation >94% (2.00, 1.64-2.43). The final model achieved an area under the receiver operating characteristic curve of 0.648 (IC95% = 0.627-0.668) in the development cohort and 0.635 (0.614-0.656) in the validation cohort. CONCLUSIONS: There are factors associated with a greater probability of discharge home of older patients admitted to EDOUs. Prediction at the individual level remains elusive, as the best model obtained in this study did not have sufficient validity to be applied in the clinical setting.

Use of resources in elderly patients consulting the emergency department: analysis of the Emergency Department and Elder Needs Cohort (EDEN-21).

The elderly population frequently consults the emergency department (ED). This population could have greater use of EDs and hospital health resources. The EDEN cohort of patients aged 65 years or older visiting the ED allowed this association to be investigated. To analyse the association between healthcare resource use and the characteristics of patients over 65 years of age who consult hospital EDs. We performed an analysis of the EDEN cohort, a retrospective, analytical, and multipurpose registry that includes patients over 65 years of age who consulted in 52 Spanish EDs. The impact of age, sex, and characteristics of ageing on the following outcomes was studied: need for hospital admission (primary outcome) and need for observation, stay in the ED > 12 h, prolonged hospital stay > 7 days, need for intensive care unit (ICU) and return to the ED at 30 days related to the index visit (secondary outcomes). The association was analysed by calculating the adjusted odds ratios (aOR) and their 95% confidence intervals (CI), using a logistic regression model. A total of 25,557 patients with a mean age of 78.3 years were analysed, 45% were males. Of note was the presence of comorbidity, a Charlson index ≥ 3 (33%), and polypharmacy (66%). Observation in the ED was required by 26%, 25.4% were admitted to the hospital, and 0.9% were admitted to the ICU. The ED stay was > 12 h in 12.5% and hospital stay > 7 days in 13.5% of cases. There was a progressive increase in healthcare resource use based on age, with an aOR for the need for observation of 2.189 (95% CI 2.038-2.352), ED stay > 12 h 2.136 (95% CI 1.942-2.349) and hospital admission 2.579 (95% CI 2.399-2.772) in the group ≥ 85 years old. Most of the characteristics inherent to ageing (cognitive impairment, falls in the previous 6 months, polypharmacy, functional dependence, and comorbidity) were associated with significant increases in the use of healthcare resources, except for ICU admission, which was less in all the variables studied. Age and the characteristics inherent to ageing are associated with greater use of structural healthcare resources.

A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma.

BACKGROUND: Sarcomas represent an extensive group of malignant diseases affecting mesodermal tissues. Among sarcomas, the clinical management of chondrosarcomas remains a complex challenge, as high-grade tumours do not respond to current therapies. Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes are among the most common mutations detected in chondrosarcomas and may represent a therapeutic opportunity. The presence of mutated IDH (mIDH) enzymes results in the accumulation of the oncometabolite 2-HG leading to molecular alterations that contribute to drive tumour growth. METHODS: We developed a personalized medicine strategy based on the targeted NGS/Sanger sequencing of sarcoma samples (n = 6) and the use of matched patient-derived cell lines as a drug-testing platform. The anti-tumour potential of IDH mutations found in two chondrosarcoma cases was analysed in vitro, in vivo and molecularly (transcriptomic and DNA methylation analyses). FINDINGS: We treated several chondrosarcoma models with specific mIDH1/2 inhibitors. Among these treatments, only the mIDH2 inhibitor enasidenib was able to decrease 2-HG levels and efficiently reduce the viability of mIDH2 chondrosarcoma cells. Importantly, oral administration of enasidenib in xenografted mice resulted in a complete abrogation of tumour growth. Enasidenib induced a profound remodelling of the transcriptomic landscape not associated to changes in the 5 mC methylation levels and its anti-tumour effects were associated with the repression of proliferative pathways such as those controlled by E2F factors. INTERPRETATION: Overall, this work provides preclinical evidence for the use of enasidenib to treat mIDH2 chondrosarcomas. FUNDING: Supported by the Spanish Research Agency/FEDER (grants PID2022-142020OB-I00; PID2019-106666RB-I00), the ISC III/FEDER (PI20CIII/00020; DTS18CIII/00005; CB16/12/00390; CB06/07/1009; CB19/07/00057); the GEIS group (GEIS-62); and the PCTI (Asturias)/FEDER (IDI/2021/000027).

Analytical and functional profiles of paralytic shellfish toxins extracted from Semele proficua and Senilia senilis from Angola.

Paralytic shellfish poisoning is a foodborne illness that typically derive from the consumption of shellfish contaminated with saxitoxin-group of toxins produced by dinoflagellates of the genus Gymnodinium, Alexandrium and Pyrodinium. N-sulfocarbamoyl, carbamate and dicarbamoyl are the most abundant. In 2007 and 2008 some episodes of PSP occurred in Angola where there is not monitoring program for shellfish contamination with marine biotoxins. Therefore, ten samples extracted from Semele proficua from Luanda Bay and Senilia senilis from Mussulo Bay, were analyzed by HPLC finding saxitoxin, decarbamoylsaxitoxin and other three compounds that have an unusual profile different to the known hydrophilic PSP toxins were found in different amounts and combinations. These new compounds were not autofluorescent, and they presented much stronger response after peroxide oxidation than after periodate oxidation. The compounds appear as peaks eluted at 2.5 and 5.6 min after periodate oxidation and 8.2 min after peroxide oxidation. Electrophysiological studies revealed that none of the three unknown compounds had effect at cellular level by decreasing the maximum peak inward sodium currents by blocking voltage-gated sodium channels. Thus, not contributing to PSP intoxication. The presence in all samples of saxitoxin-group compounds poses a risk to human health and remarks the need to further explore the presence of new compounds that contaminate seafood, investigating their activity and developing monitoring programs.

Lysosomal Dysfunction: Connecting the Dots in the Landscape of Human Diseases.

Lysosomes are the main organelles responsible for the degradation of macromolecules in eukaryotic cells. Beyond their fundamental role in degradation, lysosomes are involved in different physiological processes such as autophagy, nutrient sensing, and intracellular signaling. In some circumstances, lysosomal abnormalities underlie several human pathologies with different etiologies known as known as lysosomal storage disorders (LSDs). These disorders can result from deficiencies in primary lysosomal enzymes, dysfunction of lysosomal enzyme activators, alterations in modifiers that impact lysosomal function, or changes in membrane-associated proteins, among other factors. The clinical phenotype observed in affected patients hinges on the type and location of the accumulating substrate, influenced by genetic mutations and residual enzyme activity. In this context, the scientific community is dedicated to exploring potential therapeutic approaches, striving not only to extend lifespan but also to enhance the overall quality of life for individuals afflicted with LSDs. This review provides insights into lysosomal dysfunction from a molecular perspective, particularly in the context of human diseases, and highlights recent advancements and breakthroughs in this field.

Cold plasma-treated medium preferentially eliminates doxorubicin-resistant osteosarcoma cells.

Osteosarcoma (OS) is an aggressive bone cancer with poor prognosis, largely due to the limited effectiveness of current treatments such as doxorubicin (DX). Developing ways to overcome DX resistance is a significant clinical challenge. Here, we used two DX-resistant models to study the potential of Cold Plasma Treated Medium (PTM) to prevent DX resistance in OS. During the acquisition of the resistant phenotype upon long-term DX exposure, OS resistant cells became less proliferative, overexpressed the drug resistance-related efflux pump MDR1 and displayed a concomitant loss of SOD2 or GPX1. According to the reduced expression of these antioxidant enzymes, PTM treatment produced higher levels of oxidative express and was more effective in eradicating DX-resistant cells. Moreover, PTM reduced the expression of MDR1, thus sensitizing resistant cells to DX. These findings uncover new vulnerabilities of DX-resistant cells related with their inability to cope with excessive oxidative stress and their dependence on MDR1 that can be exploited using PTM-based treatments to provide new therapeutic approaches for the management of drug resistance in OS.

Abrogation of stemness in osteosarcoma by the mithramycin analog EC-8042 is mediated by its ability to inhibit NOTCH-1 signaling.

Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has demonstrated an efficient ability to eliminate tumor cells, including cancer stem cell subpopulations (CSC), in sarcomas. In transcriptomic and protein expression analyses, we identified NOTCH1 signaling as one of the main pro-stemness pathways repressed by EC-8042 in osteosarcomas. Overexpression of NOTCH-1 resulted in a reduced anti-tumor effect of EC-8042 in CSC-enriched 3D tumorspheres cultures. On the other hand, the depletion of the NOTCH-1 downstream target HES-1 was able to enhance the action of EC-8042 on CSCs. Moreover, HES1 depleted cells failed to recover after treatment withdrawal and showed reduced tumor growth potential in vivo. In contrast, mice xenografted with NOTCH1-overexpressing cells responded worse than parental cells to EC-8042. Finally, we found that active NOTCH1 levels in sarcoma patients was associated to advanced disease and lower survival. Overall, these data highlight the relevant role that NOTCH1 signaling plays in mediating stemness in osteosarcoma. Moreover, we demonstrate that EC-8042 is powerful inhibitor of NOTCH signaling and that the anti-CSC activity of this mithramycin analog highly rely on its ability to repress this pathway.

Cold plasma and inhibition of STAT3 selectively target tumorigenicity in osteosarcoma.

Osteosarcoma (OS) is a malignant type of bone cancer that arises in periods of increased bone formation. Curative strategies for these types of tumors have remained essentially unchanged for decades and the overall survival for most advanced cases is still dismally low. This is in part due to the existence of drug resistant Cancer Stem Cells (CSC) with progenitor properties that are responsible for tumor relapse and metastasis. In the quest for therapeutic alternatives for OS, Cold Atmospheric Plasmas and Plasma-Treated Liquids (PTL) have come to the limelight as a source of Reactive Oxygen and Nitrogen Species displaying selectivity towards a variety of cancer cell lines. However, their effects on CSC subpopulations and in vivo tumor growth have been barely studied to date. By employing bioengineered 3D tumor models and in vivo assays, here we show that low doses of PTL increase the levels of pro-stemness factors and the self-renewal ability of OS cells, coupled to an enhanced in vivo tumor growth potential. This could have critical implications to the field. By proposing a combined treatment, our results demonstrate that the deleterious pro-stemness signals mediated by PTL can be abrogated when this is combined with the STAT3 inhibitor S3I-201, resulting in a strong suppression of in vivo tumor growth. Overall, our study unveils an undesirable stem cell-promoting function of PTL in cancer and supports the use of combinatorial strategies with STAT3 inhibitors as an efficient treatment for OS avoiding critical side effects. We anticipate our work to be a starting point for wider studies using relevant 3D tumor models to evaluate the effects of plasma-based therapies on tumor subpopulations of different cancer types. Furthermore, combination with STAT3 inhibition or other suitable cancer type-specific targets can be relevant to consolidate the development of the field.

Addressing Doxorubicin Resistance in Bone Sarcomas Using Novel Drug-Resistant Models.

Bone sarcomas have not shown a significant improvement in survival for decades, due, in part, to the development of resistance to current systemic treatments, such as doxorubicin. To better understand those mechanisms mediating drug-resistance we generated three osteosarcoma and one chondrosarcoma cell lines with a stable doxorubicin-resistant phenotype, both in vitro and in vivo. These resistant strains include a pioneer model generated from a patient-derived chondrosarcoma line. The resistant phenotype was characterized by a weaker induction of apoptosis and DNA damage after doxorubicin treatment and a lower migratory capability. In addition, all resistant lines expressed higher levels of ABC pumps; meanwhile, no clear trends were found in the expression of anti-apoptotic and stem cell-related factors. Remarkably, upon the induction of resistance, the proliferation potential was reduced in osteosarcoma lines but enhanced in the chondrosarcoma model. The exposure of resistant lines to other anti-tumor drugs revealed an increased response to cisplatin and/or methotrexate in some models. Finally, the ability to retain the resistant phenotype in vivo was confirmed in an osteosarcoma model. Altogether, this work evidenced the co-existence of common and case-dependent phenotypic traits and mechanisms associated with the development of resistance to doxorubicin in bone sarcomas.

Proof of concept for the use of trained sniffer dogs to detect osteosarcoma.

Sarcomas are mesenchymal cancers which often show an aggressive behavior and patient survival largely depends on an early detection. In last years, much attention has been given to the fact that cancer patients release specific odorous volatile organic compounds (VOCs) that can be efficiently detected by properly trained sniffer dogs. Here, we have evaluated for the first time the ability of sniffer dogs (n = 2) to detect osteosarcoma cell cultures and patient samples. One of the two dogs was successfully trained to discriminate osteosarcoma patient-derived primary cells from mesenchymal stem/stromal cells (MSCs) obtained from healthy individuals. After the training phase, the dog was able to detect osteosarcoma specific odor cues in a different panel of 6 osteosarcoma cell lines with sensitivity and specificity rates between 95 and 100%. Moreover, the same VOCs were also detected by the sniffer dog in saliva samples from osteosarcoma patients (n = 2) and discriminated from samples from healthy individuals with a similar efficacy. Altogether, these results indicate that there are common odor profiles shared by cultures of osteosarcoma cells and body fluid samples from patients and provide a first proof of concept about the potential of canine odor detection as a non-invasive screening method to detect osteosarcomas.

Mithramycin delivery systems to develop effective therapies in sarcomas.

BACKGROUND: Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile. RESULTS: In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM. CONCLUSIONS: Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment.

ATG4D is the main ATG8 delipidating enzyme in mammalian cells and protects against cerebellar neurodegeneration.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

Nano-Encapsulation of Mithramycin in Transfersomes and Polymeric Micelles for the Treatment of Sarcomas.

Sarcomas are aggressive tumors which often show a poor response to current treatments. As a promising therapeutic alternative, we focused on mithramycin (MTM), a natural antibiotic with a promising anti-tumor activity but also a relevant systemic toxicity. Therefore, the encapsulation of MTM in nano-delivery systems may represent a way to increase its therapeutic window. Here, we designed novel transfersomes and PLGA polymeric micelles by combining different membrane components (phosphatidylcholine, Span 60, Tween 20 and cholesterol) to optimize the nanoparticle size, polydispersity index (PDI) and encapsulation efficiency (EE). Using both thin film hydration and the ethanol injection methods we obtained MTM-loaded transferosomes displaying an optimal hydrodynamic diameter of 100-130 nm and EE values higher than 50%. Additionally, we used the emulsion/solvent evaporation method to synthesize polymeric micelles with a mean size of 228 nm and a narrow PDI, capable of encapsulating MTM with EE values up to 87%. These MTM nano-delivery systems mimicked the potent anti-tumor activity of free MTM, both in adherent and cancer stem cell-enriched tumorsphere cultures of myxoid liposarcoma and chondrosarcoma models. Similarly to free MTM, nanocarrier-delivered MTM efficiently inhibits the signaling mediated by the pro-oncogenic factor SP1. In summary, we provide new formulations for the efficient encapsulation of MTM which may constitute a safer delivering alternative to be explored in future clinical uses.

SOX2 Expression and Transcriptional Activity Identifies a Subpopulation of Cancer Stem Cells in Sarcoma with Prognostic Implications.

Stemness in sarcomas is coordinated by the expression of pluripotency factors, like SOX2, in cancer stem cells (CSC). The role of SOX2 in tumor initiation and progression has been well characterized in osteosarcoma. However, the pro-tumorigenic features of SOX2 have been scarcely investigated in other sarcoma subtypes. Here, we show that SOX2 depletion dramatically reduced the ability of undifferentiated pleomorphic sarcoma (UPS) cells to form tumorspheres and to initiate tumor growth. Conversely, SOX2 overexpression resulted in increased in vivo tumorigenicity. Moreover, using a reporter system (SORE6) which allows to monitor viable cells expressing SOX2 and/or OCT4, we found that SORE6+ cells were significantly more tumorigenic than the SORE6- subpopulation. In agreement with this findings, SOX2 expression in sarcoma patients was associated to tumor grade, differentiation, invasive potential and lower patient survival. Finally, we studied the effect of a panel of anti-tumor drugs on the SORE6+ cells of the UPS model and patient-derived chondrosarcoma lines. We found that the mithramycin analogue EC-8042 was the most efficient in reducing SORE6+ cells in vitro and in vivo. Overall, this study demonstrates that SOX2 is a pro-tumorigenic factor with prognostic potential in sarcoma. Moreover, SORE6 transcriptional activity is a bona fide CSC marker in sarcoma and constitutes an excellent biomarker for evaluating the efficacy of anti-tumor treatments on CSC subpopulations.

New Chondrosarcoma Cell Lines with Preserved Stem Cell Properties to Study the Genomic Drift During In Vitro/In Vivo Growth.

For the cancer genomics era, there is a need for clinically annotated close-to-patient cell lines suitable to investigate altered pathways and serve as high-throughput drug-screening platforms. This is particularly important for drug-resistant tumors like chondrosarcoma which has few models available. Here we established and characterized new cell lines derived from two secondary (CDS06 and CDS11) and one dedifferentiated (CDS-17) chondrosarcomas as well as another line derived from a CDS-17-generated xenograft (T-CDS17). These lines displayed cancer stem cell-related and invasive features and were able to initiate subcutaneous and/or orthotopic animal models. Different mutations in Isocitrate Dehydrogenase-1 (IDH1), Isocitrate Dehydrogenase-2 (IDH2), and Tumor Supressor P53 (TP53) and deletion of Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) were detected both in cell lines and tumor samples. In addition, other mutations in TP53 and the amplification of Mouse Double Minute 2 homolog (MDM2) arose during cell culture in CDS17 cells. Whole exome sequencing analysis of CDS17, T-CDS17, and matched patient samples confirmed that cell lines kept the most relevant mutations of the tumor, uncovered new mutations and revealed structural variants that emerged during in vitro/in vivo growth. Altogether, this work expanded the panel of clinically and genetically-annotated chondrosarcoma lines amenable for in vivo studies and cancer stem cell (CSC) characterization. Moreover, it provided clues of the genetic drift of chondrosarcoma cells during the adaptation to grow conditions.

The multikinase inhibitor EC-70124 synergistically increased the antitumor activity of doxorubicin in sarcomas.

Cytotoxic drugs like doxorubicin remain as the most utilized agents in sarcoma treatment. However, advanced sarcomas are often resistant, thus stressing the need for new therapies aimed to overcome this resistance. Multikinase inhibitors provide an efficient way to target several pro-tumorigenic pathways using a single agent and may constitute a valuable strategy in the treatment of sarcomas, which frequently show an aberrant activation of pro-tumoral kinases. Therefore, we studied the antitumor activity of EC-70124, an indolocarbazole analog that have demonstrated a robust ability to inhibit a wide range of pro-survival kinases. Evaluation of the phospho-kinase profile in cell-of-origin sarcoma models and/or sarcoma primary cell lines evidenced that PI3K/AKT/mTOR, JAK/STAT or SRC were among the most highly activated pathways. In striking contrast with the structurally related drug midostaurin, EC-70124 efficiently prevented the phosphorylation of these targets and robustly inhibited proliferation through a mechanism associated to the induction of DNA damage, cell cycle arrest and apoptosis. In addition, EC-70124 was able to partially reduce tumor growth in vivo. Importantly, this compound inhibited the expression and activity of ABC efflux pumps involved in drug resistance. In line with this ability, we found that the combined treatment of EC-70124 with doxorubicin resulted in a synergistic cytotoxic effect in vitro and an increased antitumor activity of this cytotoxic drug in vivo. Altogether, these results uncover the capability of the novel multikinase inhibitor EC-70124 to counteract drug resistance in sarcoma and highlight its therapeutic potential when combined with current treatments.

Paralytic Shellfish Toxins Occurrence in Non-Traditional Invertebrate Vectors from North Atlantic Waters (Azores, Madeira, and Morocco).

Paralytic shellfish toxins (PSTs) are potent alkaloids of microalgal and cyanobacterial origin, with worldwide distribution. Over the last 20 years, the number of poisoning incidents has declined as a result of the implementation of legislation and monitoring programs based on bivalves. In the summer of 2012 and 2013, we collected a total of 98 samples from 23 different species belonging to benthic and subtidal organisms, such as echinoderms, crustaceans, bivalves, and gastropods. The sampling locations were Madeira, São Miguel Island (Azores archipelago), and the northwestern coast of Morocco. The samples were analyzed using post-column oxidation liquid chromatography with a fluorescence detection method. Our main goal was to detect new vectors for these biotoxins. After reporting a total of 59 positive results for PSTs with 14 new vectors identified, we verified that some of the amounts exceeded the limit value established in the EU. These results suggest that routine monitoring of saxitoxin and its analogs should be extended to more potential vectors other than bivalves, including other edible organisms, for a better protection of public health.

Rapid analysis of paralytic shellfish toxins and tetrodotoxins by liquid chromatography-tandem mass spectrometry using a porous graphitic carbon column.

Although paralytic shellfish toxins (PSTs) have traditionally been analyzed by liquid chromatography with either pre- or post-column derivatization, and these methods have been validated successfully through inter-laboratory studies, mass spectrometry methods have also been described in literature for use in monitoring programs. However, methods using liquid chromatography coupled with mass spectrometry (LC-MS) need to be improved in terms of sensitivity, analyte recovery and retention time stability because of undesirable matrix effects. Furthermore, tetrodotoxin (TTX) has been found in northern European bivalves, so it is important to analyze TTX compounds alongside PSTs because characteristics of their toxicity are similar. This paper describes, for the first time, a chemical method that allows determination of PSTs, both hydrophilic and hydrophobic, alongside TTX and its analogue 4,9-anhydro tetrodotoxin (4,9-anhTTX) with LC-MS/MS using a Hypercarb® column. The method was validated for 13 hydrophilic PSTs and TTXs and was able to discriminate six hydrophobic PSTs in 20 min. The method was developed for four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Ruditapes decussatus), scallop (Pecten maximus) and oyster (Ostrae edulis). Clean-up procedure used in this work allowed us to obtain good results for validation parameters for both PSTs and TTXs. No standards were available so strains of Gymnodinium catenatum (G. catenatum) were used instead.