Potentially Toxic Planktic and Benthic Cyanobacteria in Slovenian Freshwater Bodies: Detection by Quantitative PCR

Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE, cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.     

Cyanobacterial Toxins and Peptides in Lake Vegoritis,Greece

Cyanotoxins (CTs) produced by cyanobacteria in surface freshwater are a major threat for public health and aquatic ecosystems. Cyanobacteria can also produce a wide variety of other understudied bioactive metabolites such as oligopeptides microginins (MGs), aeruginosins (AERs), aeruginosamides (AEGs) and anabaenopeptins (APs). This study reports on the co-occurrence of CTs and cyanopeptides (CPs) in Lake Vegoritis, Greece and presents their variant-specific profiles obtained during 3-years of monitoring (2018–2020). Fifteen CTs (cylindrospermopsin (CYN), anatoxin (ATX), nodularin (NOD), and 12 microcystins (MCs)) and ten CPs (3 APs, 4 MGs, 2 AERs and aeruginosamide (AEG A)) were targeted using an extended and validated LC-MS/MS protocol for the simultaneous determination of multi-class CTs and CPs. Results showed the presence of MCs (MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, and MC-HilR) and CYN at concentrations of <1 μg/L, with MC-LR (79%) and CYN (71%) being the most frequently occurring. Anabaenopeptins B (AP B) and F (AP F) were detected in almost all samples and microginin T1 (MG T1) was the most abundant CP, reaching 47.0 μg/L. This is the first report of the co-occurrence of CTs and CPs in Lake Vegoritis, which is used for irrigation, fishing and recreational activities. The findings support the need for further investigations of the occurrence of CTs and the less studied cyanobacterial metabolites in lakes, to promote risk assessment with relevance to human exposure.     

Optimization of the QuEChERS-Based Analytical Method for Investigation of 11 Mycotoxin Residues in Feed Ingredients and Compound Feeds

Mycotoxins are toxic substances naturally produced by various fungi, and these compounds not only inflict economic damage, but also pose risks to human and animal health. The goal of the present study was to optimize the QuEChERS-based extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the analysis of 11 mycotoxins, such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), T-2 toxin, HT-2 toxin, zearalenone (ZEN), and deoxynivalenol (DON), commonly found in feed. The QuEChERS method, characterized by being “quick, easy, cheap, effective, rugged, and safe”, has become one of the most common extractions and clean-up procedures for mycotoxin analyses in food. Therefore, in this experiment, an optimal method for the analysis of 11 mycotoxins in feed was established by modifying the general QuEChERS method. In this process, it was confirmed that even if feed samples of different weights were extracted, the quantitative value of mycotoxins in the feed was not affected. To reduce matrix effects, 13C-labeled compounds and deuterium were used as internal standards. This optimized method was then applied in the determination of 11 mycotoxins in 736 feed ingredients and compound feeds obtained from South Korea. The results showed that the occurrence rates of FBs, ZEN, and DON were 59.4%, 38.0%, and 32.1%, respectively, and OTA, AFs, and T-2 toxin and HT-2 toxin were found in fewer than 1% of the 736 feeds. The mean concentration ranges of FBs, ZEN, and DON were 757–2387, 44–4552, and 248–9680 μg/kg, respectively. Among the samples in which DON and ZEN were detected, 10 and 12 samples exceeded the management recommendation standards presented by the Ministry of Agriculture, Food and Rural Affairs (MAFRA). However, when the detected concentrations of DON and ZEN were compared with guideline levels in foreign countries, such as the US, Japan, China, and the EU, the number of positive samples changed. In addition, the co-occurrence of mycotoxins in the feed was analyzed, and the results showed that 43.8% of the samples were contaminated with two or three mycotoxins, among which the co-occurrence rate of FBs, ZEN, and DON was the highest. In conclusion, these results suggest the need for stricter management standards for FBs, DON, and ZEN in South Korea, and emphasize the importance of the continuous monitoring of feeds.     

Residue Analysis and Assessment of the Risk of Dietary Exposure to Domoic Acid in Shellfish from the Coastal Areas of China

Harmful algal blooms in Chinese waters have caused serious domoic acid (DA) contamination in shellfish. Although shellfish are at particular risk of dietary exposure to DA, there have been no systematic DA risk assessments in Chinese coastal waters. A total of 451 shellfish samples were collected from March to November 2020. The presence of DA and four of its isomers were detected using liquid chromatography–tandem mass spectrometry. The spatial-temporal distribution of DA occurrence and its potential health risks were examined. DA was detected in 198 shellfish samples (43.90%), with a maximum level of 942.86 μg/kg. DA was recorded in all 14 shellfish species tested and Pacific oysters (Crassostrea gigas) showed the highest average DA concentration (82.36 μg/kg). The DA concentrations in shellfish showed distinct spatial-temporal variations, with significantly higher levels of occurrence in autumn than in summer and spring (p < 0.01), and particularly high occurrence in Guangdong and Fujian Provinces. The detection rates and maximum concentrations of the four DA isomers were low. While C. gigas from Guangdong Province in September showed the highest levels of DA contamination, the risk to human consumers was low. This study improves our understanding of the potential risk of shellfish exposure to DA-residues.     

Paralytic Shellfish Poisoning (PSP) in Mussels from the Eastern Cantabrian Sea: Toxicity,Toxin Profile,and Co-Occurrence with Cyclic Imines

In the late autumn of 2018 and 2019, some samples taken by the official monitoring systems of Cantabria and the Basque Country were found to be paralytic shellfish poisoning (PSP)-positive using a mouse bioassay. To confirm the presence of PSP toxins and to obtain their profile, these samples were analyzed using an optimized version of the Official Method AOAC 2005.06 and using LC–MS/MS (HILIC). The presence of some PSP toxins (PSTs) in that geographical area (~600 km of coast) was confirmed for the first time. The estimated toxicities ranged from 170 to 983 µg STXdiHCl eq.·kg⁻¹ for the AOAC 2005.06 method and from 150 to 1094 µg STXdiHCl eq.·kg⁻¹ for the LC–MS/MS method, with a good correlation between both methods (r² = 0.94). Most samples contained STX, GTX2,3, and GTX1,4, and some also had NEO and dcGTX2. All of the PSP-positive samples also contained gymnodimine A, with the concentrations of the two groups of toxins being significantly correlated. The PSP toxin profiles suggest that a species of the genus Alexandrium was likely the causative agent. The presence of gymnodimine A suggests that A. ostenfeldii could be involved, but the contribution of a mixture of Alexandrium species cannot be ruled out.     

Occurrence of Alternaria and Other Toxins in Cereal Grains Intended for Animal Feeding Collected in Slovenia: A Three-Year Study

In recent years, the less-studied Alternaria mycotoxins have attracted increasing interest due to the lack of survey data and their ability to cause toxic effects in animals and humans. To fill the gap, the aim of this three-year survey was to investigate the presence and co-occurrence of Alternaria and other mycotoxins in a total of 433 cereal grain samples from Slovenian farms and agricultural cooperatives from 2014 to 2016. Using the multi-mycotoxin method, 14 mycotoxins were determined. In 53% of 433 analysed samples, contamination with at least one mycotoxin was found. Deoxynivalenol (DON) and tenuazonic acid (TeA) were present in 32% and 26% of cereal grain samples, respectively, whereas alternariol (AOH), tentoxin (TEN), alternariol monomethyl ether (AME), 3- and 15-acetyldeoxynivalenol (3- and 15-AcDON), and zearalenone (ZEN) were present in fewer than 15% of the samples. Ochratoxin A (OTA) was found in one rye sample, while diacetoxyscirpenol (DAS), HT-2 and T-2 toxin, and fumonisins B₁ and B₂ (FB1 and FB2) were not detected. The highest maximum and median concentrations of Alternaria toxins were determined in spelt in 2016 (TeA, 2277 µg/kg and 203 µg/kg, respectively), and those of Fusarium toxins in wheat in 2015 (DON, 4082 µg/kg and 387 µg/kg, respectively). The co-occurrence of two or more mycotoxins was found in 43% of the positive samples. The correlations between Alternaria toxins were very weak but statistically significant (r: 0.15–0.17, p: 0.0042–0.0165). A well-known correlation between Fusarium toxins DON and ZEN was weak and highly significant (r = 0.28, p < 0.0001).     

Development of a LC–MS/MS Method for the Simultaneous Determination of the Mycotoxins Deoxynivalenol (DON) and Zearalenone (ZEA) in Soil Matrix

Mycotoxins, toxins of fungal origin, can directly or indirectly contaminate food and feed and are poisonous to livestock and humans. While a large amount is known about their occurrence in crops, food, and feeds, little is known about mycotoxin amounts in soil. However, soil is known as a major fungal habitat and a potential sink for mycotoxins in the environment. Furthermore, there is neither a reliable detection nor an extraction method for mycotoxins testing in different soil textures or for potential deficits due to aging processes. Therefore, the aim of the present study was to present a reliable extraction and detection method for the simultaneous quantification of the most common mycotoxins, deoxynivalenol (DON) and zearalenone (ZEA), via liquid chromatography-tandem mass spectrometry (LC–MS/MS). This method was validated with six different samples with different textures and different soil organic matter (SOM). Deuterated standards were used to overcome possible matrix effects. This extraction method could eliminate potential aging processes. The recovery rate was always >80% for DON and >82% for ZEA. The quantification limits were 1 ng per g soil for DON and 0.5 ng per g soil for ZEA.     

Rapid,Sensitive and Reliable Ricin Identification in Serum Samples Using LC–MS/MS

Ricin, a protein derived from the seeds of the castor bean plant (Ricinus communis), is a highly lethal toxin that inhibits protein synthesis, resulting in cell death. The widespread availability of ricin, its ease of extraction and its extreme toxicity make it an ideal agent for bioterrorism and self-poisoning. Thus, a rapid, sensitive and reliable method for ricin identification in clinical samples is required for applying appropriate and timely medical intervention. However, this goal is challenging due to the low predicted toxin concentrations in bio-fluids, accompanied by significantly high matrix interferences. Here we report the applicability of a sensitive, selective, rapid, simple and antibody-independent assay for the identification of ricin in body fluids using mass spectrometry (MS). The assay involves lectin affinity capturing of ricin by easy-to-use commercial lactose–agarose (LA) beads, following by tryptic digestion and selected marker identification using targeted LC–MS/MS (Multiple Reaction Monitoring) analysis. This enables ricin identification down to 5 ng/mL in serum samples in 2.5 h. To validate the assay, twenty-four diverse naive- or ricin-spiked serum samples were evaluated, and both precision and accuracy were determined. A real-life test of the assay was successfully executed in a challenging clinical scenario, where the toxin was identified in an abdominal fluid sample taken 72 h post self-injection of castor beans extraction in an eventual suicide case. This demonstrates both the high sensitivity of this assay and the extended identification time window, compared to similar events that were previously documented. This method developed for ricin identification in clinical samples has the potential to be applied to the identification of other lectin toxins.     

Determination of hydroxy metabolites of cocaine in hair samples for proof of consumption

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC‐positive samples for these compounds was performed by liquid chromatography–quadrupole time of flight–mass spectrometry (LC–QTOF–MS). Once identified, mass transitions for o‐, p‐ and m‐isomers of hydroxy COC as well as p‐ and m‐isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography–tandem mass spectrometry (LC–MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC‐positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p‐OH‐BE, m‐OH‐BE as well as p‐OH‐NCOC and m‐OH‐NCOC have been identified for the first time in COC‐positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC‐positive hair samples.     

Stability of mephedrone and five of its phase I metabolites in human whole blood

Mephedrone is a new psychoactive substance known to be unstable in biological matrices stored at room temperature or refrigerated. While the instability of mephedrone has been investigated before, there is currently no data regarding the stability of mephedrone metabolites. In this study, a liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of mephedrone and five of its phase I metabolites (dihydro‐mephedrone, nor‐mephedrone, hydroxytolyl‐mephedrone, 4‐carboxy‐mephedrone and dihydro‐nor‐mephedrone) in human whole blood has been developed and validated. Samples were extracted by a mixed mode solid‐phase extraction and analyzed on a pentafluorophenylpropyl column. The method was successfully validated for selectivity, linearity (0.2–2 to 10–100 ng/mL), limits of detection (50–500 pg/mL) and quantification (200–2000 pg/mL), precision (0.924–8.27%), accuracy (86.6–115%), carryover, recovery (32.5–88.3%), and matrix effects (71.0–108%). Analyte stability in human whole blood preserved with sodium fluoride/potassium oxalate was assessed at +4°C and ?20°C after 24 hours, 48 hours, 4 days, and 10 days of storage. Instability was observed in samples stored at +4°C: nor‐mephedrone and 4‐carboxy‐mephedrone lost 40.2 ± 6.7% and 48.1 ± 4.8%, respectively, of their initial concentration at low concentration level and 33.8 ± 4.2% and 44.6 ± 6.5%, respectively, at high concentration level after 10 days. All analytes were more stable at ?20°C where the highest loss of 22.6 ± 6.9% was observed for 4‐carboxy‐mephedrone after 10 days. This is the first time stability of mephedrone metabolites in human whole blood has been assessed, indicating ?20°C to be the recommended storage condition for all analytes in clinical settings.     

"In-source" fragmentation of an isobaric impurity of lamotrigine for its measurement by liquid chromatography tandem mass spectrometry after pre-concentration using solid phase extraction

An analytical method has been developed for trace analysis (i.e. sub-ppm levels) of a key synthetic impurity, 14W80 ((Z)-2-(2,3-dichlorophenyl)-2-(guanidinylimino)acetonitrile) in lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine). 14W80 is isobaric with lamotrigine, which gives an extra layer of complexity to its determination and the various problems associated with development of an appropriate methodology are discussed in this work. Ultimately, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method using in-source fragmentation with Atmospheric Pressure Chemical Ionisation (APCI) followed by multiple reaction monitoring (MRM) has been found to provide adequate sensitivity and specificity. A detection limit of 25 ppb mass fraction relative to lamotrigine was achieved for 14W80. The use of solid phase extraction (SPE) enhanced the detection limit to 2 ppb mass fraction relative to lamotrigine.     

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) – a group of new psychoactive substances (NPS) – is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography—tandem mass spectrometry (LC?MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography?high resolution tandem mass spectrometry (LC?HRMS/MS) and finally included to the MS/MS spectra in‐house library. Sample preparation was performed using a cheap‐and‐easy salting‐out liquid–liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05 ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.     

First Report on Microcystin-LR Occurrence in Water Reservoirs of Eastern Cuba,and Environmental Trigger Factors

The factors related to cyanotoxin occurrence and its social impact, with comprehension and risk perception being the most important issues, are not yet completely understood in the Cuban context. The objectives of this research were to determine the risk extension and microcystin-LR levels, and to identify the environmental factors that trigger the toxic cyanobacteria growth and microcystin-LR occurrence in 24 water reservoirs in eastern Cuba. Samplings were performed in the early morning hours, with in situ determination and physicochemical analysis carried out in the laboratory. Microcystin-LR were determined in water and within the cells (intracellular toxins) using UPLC–MS analysis after solid phase extraction. The reservoirs studied were found to be affected by eutrophication, with high levels of TN:TP ratio and phytoplankton cell concentrations, high water temperatures and low transparency, which cause collateral effect such as cyanobacterial bloom and microcystin-LR occurrence. In Hatillo, Chalóns, Parada, Mícara, Baraguá, Cautillo, La Yaya, Guisa and Jaibo reservoirs, concentrations of MC-LR higher than the WHO limits for drinking water (1 µg·L⁻¹), were detected.     

Fenton's oxidation: a tool for the investigation of potential drug metabolites

Within the scope of searching for new lead structures in the field of anti-infectives, we ascertained Fenton's reagent to be an easy-to-handle and non-expensive tool for screening the metabolic profile of new bioactive compounds. The underlying chemistry of the Fenton's one-electron oxidation is comparable to that of cytochrome P450, which is the main metabolism enzyme. To study the metabolic screening capability, we subjected different antibiotics and the antiplasmodial naphthylisoquinoline alkaloid dioncophylline A to Fenton's reagent and examined the obtained compound libraries by liquid chromatography/tandem mass spectrometry (LC-MS/MS). For ciprofloxacin and linezolid about half of literature-known metabolites were identified as products of Fenton's oxidation. For dioncophylline A six new possible metabolites were discovered.     

Liquid chromatography-tandem mass spectrometry assay for the quantitation of plagiochin E and its main metabolite plagiochin E glucuronides in rat plasma

Plagiochin E, a macrocyclic bisbibenzyl isolated from liverwort Marchantia polymorpha, was found to have antifungal activity. To evaluate the pharmacokinetics of plagiochin E in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantitation of plagiochin E and its total conjugated metabolites in rat plasma. For detection, a Sciex API 4000 LC-MS/MS with a TurboIonSpray ionization (ESI) inlet in the negative ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pretreated by a simple liquid-liquid extraction with ethyl acetate. The concentration of plagiochin E parent form was determined directly and the concentration of plagiochin E conjugated metabolites was assayed in the form of plagiochin E after treatment with beta-glucuronidase/sulfatase. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.5-1000.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.5 ng/mL for plagiochin E in 50 microL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of plagiochin E in rats after an oral and an intravenous administration.     

Reductive Amination for LC–MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) or high-resolution MS (LC–HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC–MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard’s reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC–MS/MS and LC–HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1–GRT derivatization strategy mitigates many of the shortcomings of current LC–MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes.     

Doping control analysis of four JWH‐250 metabolites in equine urine by liquid chromatography–tandem mass spectrometry

JWH‐250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH‐250 metabolites (JWH‐250 4‐OH‐pentyl, JWH‐250 5‐OH‐pentyl, JWH‐250 5‐OH‐indole, and JWH‐250 N‐pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The chromatographic separation was carried out using a Phenomenex Lux^® 3 μm AMP column (150 x 3.0 mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50 pg/mL, respectively. The linear dynamic range of quantification was 50–10000 pg/mL. Enzymatic hydrolysis indicated that JWH‐250 4‐OH‐pentyl, JWH‐250 5‐OH‐pentyl, and JWH‐250 5‐OH indole are highly conjugated whereas JWH‐250 N‐pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post‐race urine samples collected from horses suspected of being exposed to JWH‐250. All four JWH‐250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.     

Biomonitoring of Multiple Mycotoxins in Urine by GC–MS/MS: A Pilot Study on Patients with Esophageal Cancer in Golestan Province,Northeastern Iran

A pilot study to investigate the occurrence of 10 mycotoxins (deoxynivalenol, DON; 3-acetyldeoxynivalenol, 3-ADON; 15-acetyldeoxynivalenol, 15-ADON; fusarenon-X, FUS-X; diacetoxyscirpenol, DAS; nivalenol, NIV; neosolaniol, NEO; zearalenone, ZON; zearalanone, ZAN; T-2 toxin, T-2; and HT-2 toxin, HT-2) in esophageal cancer patients was performed with the urinary biomarkers approach in Golestan, Iran. Urine multimycotoxin analysis was performed by dispersive liquid–liquid microextraction and gas chromatography–tandem mass spectrometry (GC–MS/MS) analysis, and values were normalized with urinary creatinine (μg/g). Four mycotoxins, namely NEO (40%), HT-2 (17.6%), DON (10%), and HT-2 (5.8%), were detected in the analyzed urine samples. DON was only detected in the control group (5.09 μg/g creatinine), while T-2 (44.70 μg/g creatinine) was only present in the esophageal cancer group. NEO and HT-2 were quantified in both control and case groups, showing average of positive samples of 9.09 and 10.45 μg/g creatinine for NEO and 16.81 and 29.09 μg/g creatinine for HT-2, respectively. Mycotoxin co-occurrence was observed in three samples as binary (NEO/HT-2 and T-2/HT-2) and ternary (DON/NEO/HT-2) combinations, reaching total concentrations of 44.58, 79.13, and 30.04 µg/g creatinine, respectively. Further investigations are needed to explore a causal association between mycotoxin contamination and esophageal cancer. For this pilot study in Golestan, the low sample size was a very limiting factor.     

Tetrodotoxin and Its Analogues in Cephalothrix cf. simula (Nemertea: Palaeonemertea) from the Sea of Japan (Peter the Great Gulf): Intrabody Distribution and Secretions

Some nemertean species from the genus Cephalothrix accumulate tetrodotoxin (TTX) in extremely high concentrations. The current study is the first to provide high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) data on tetrodotoxin and its analogues (TTXs) profile and concentration in different regions and organs of Cephalothrix cf. simula, and its secretions produced in response to stimulation. Different specimens of C. cf. simula possessed 7–11 analogues, including nine previously found in this species and two new for nemerteans—4,9-anhydro-8-epi-5,6,11-trideoxyTTX and 1-hydroxy-8-epi-5,6,11-trideoxyTTX. The study of the toxins’ distribution in different regions and organs of nemerteans revealed the same qualitative composition of TTXs throughout the body but differences in the total concentration of the toxins. The total concentration of TTXs was highest in the anterior region of the body and decreased towards the posterior; the ratio of the analogues also differed between regions. The data obtained suggest a pathway of TTXs uptake in C. cf. simula and the role of toxins in the life activity of nemerteans.     

First Report of Cylindrospermopsin Production by Two Cyanobacteria (Dolichospermum mendotae and Chrysosporum ovalisporum) in Lake Iznik,Turkey

Cylindrospermopsin (CYN) is a cytotoxic alkaloid produced by cyanobacteria. The distribution of this toxin is expanding around the world and the number of cyanobacteria species producing this toxin is also increasing. CYN was detected for the first time in Turkey during the summer months of 2013. The responsible species were identified as Dolichospermum (Anabaena) mendotae and Chrysosporum (Aphanizomenon) ovalisporum. The D. mendotae increased in May, however, C. ovalisporum formed a prolonged bloom in August. CYN concentrations were measured by LC-MS/MS and ranged from 0.12 µg·mg⁻¹to 4.92 µg·mg⁻¹ as dry weight, respectively. Both species were the only cyanobacteria actively growing and CYN production was attributed solely to these species. Despite CYN production by C. ovalisporum being a well-known phenomenon, to our knowledge, this is the first report of CYN found in D. mendotae bloom.