New azaspiracid analogues detected as bi-charged ions in Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters.

Lipophilic marine biotoxin azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters, and detailed toxin profiles of two strains (mdd421 and HM536) among them were clarified by several detection techniques on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). In our present study, AZA analogues in seven strains of A. poporum from Japanese coastal waters (including two previously reported strains) were determined by these detection techniques. The dominant AZA in the seven strains was AZA2 accompanied by small amounts of several known AZAs and twelve new AZA analogues. Eight of the twelve new AZA analogues discovered in our present study were detected as bi-charged ions on the positive mode LC/MS/MS. This is the first report describing AZA analogues detected as bi-charged ions with hexose and sulfate groups in their structures.

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

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

Co-localisation of Azaspiracid Analogs with the Dinoflagellate Species Azadinium spinosum and Amphidoma languida in the Southwest of Ireland.

Phytoplankton and biotoxin monitoring programmes have been implemented in many countries to protect human health and to mitigate the impacts of harmful algal blooms (HABs) on the aquaculture industry. Several amphidomatacean species have been confirmed in Irish coastal waters, including the azaspiracid-producing species Azadinium spinosum and Amphidoma languida. Biogeographic distribution studies have been hampered by the fact that these small, armoured dinoflagellates share remarkably similar morphologies when observed by light microscopy. The recent releases of species-specific molecular detection assays have, in this context, been welcome developments. A survey of the south west and west coasts of Ireland was carried out in August 2017 to investigate the late summer distribution of toxic amphidomataceans and azaspiracid toxins. Azadinium spinosum and Am. languida were detected in 83% of samples in the southwest along the Crease Line and Bantry Bay transects between 20 and 70 m depth, with maximal cell concentrations of 7000 and 470,000 cells/L, respectively. Azaspiracid concentrations were well aligned with the distributions of Az. spinosum and Am. languida, up to 1.1 ng/L and 4.9 ng/L for combined AZA-1, -2, -33, and combined AZA-38, -39, respectively. Although a snapshot in time, this survey provides new insights in the late summer prominence of AZAs and AZA-producing species in the southwest of Ireland, where major shellfish aquaculture operations are located. Results showed a substantial overlap in the distribution of amphidomatacean species in the area and provide valuable baseline information in the context of ongoing monitoring efforts of toxigenic amphidomataceans in the region.

Spirolides in Bivalve Mollusk of the Galician (NW Spain) Coast: Interspecific, Spatial, Temporal Variation and Presence of an Isomer of 13-Desmethyl Spirolide C.

Spirolides are cyclic imines whose risks to human health have not been sufficiently evaluated. To determine the possible impact of these compounds in Galicia (NW Spain), their presence and concentration in bivalve mollusk were studied from 2014 to 2021. Only 13-desmethyl spirolide C (13desmSPXC) and an isomer have been detected, and always at low concentrations. Mussel, Mytilus galloprovincialis, was the species which accumulated more spirolides, but the presence of its isomer was nearly restricted to cockle, Cerastoderma edule, and two clam species, Venerupis corrugata and Polititapes rhomboides. On average, the highest 13desmSPXC levels were found in autumn-winter, while those of its isomer were recorded in spring-summer. Both compounds showed decreasing trends during the study period. Geographically, the concentration tends to decrease from the southern to the north-eastern locations, but temporal variability predominates over spatial variability.

Characterization of equine liver microsome-generated metabolites of growth hormone secretagogue small molecule ibutamoren.

RATIONALE: Interest in growth hormone secretagogues has intensified during the past several years based on capable, ever-widening investigational applications of recombinant growth hormone in animals and humans. Ibutamoren is a potent, long-acting, selective and orally active non-peptide growth hormone secretagogue, which has a great potential for abuse as a performance-enhancing agent in sports. METHODS: To support drug metabolism and pharmacokinetic studies of chiral pharmaceuticals, it is necessary to combine the resolving power of high-performance liquid chromatography with the sensitivity of mass spectrometric techniques. This paper describes the metabolic conversion of ibutamoren using equine liver microsomes and metabolite characterization using a QExactive high-resolution mass spectrometer. RESULTS: A total of 32 metabolites for ibutamoren (20 phase I and 12 phase II) were detected. The important findings of the current research are as follows: (1) the growth hormone secretagogue ibutamoren was prone to oxidation, resulting in corresponding hydroxylated metabolites; (2) in ibutamoren, the dissociation of the phenyl ring and 2-amino-2-methylpropanamide side chain was also observed; (3) the glucuronic acid conjugates of mono-, di- and trihydroxylated analogues were detected; and (4) no sulfonic acid conjugated metabolites were observed in this study of ibutamoren. CONCLUSIONS: The reported data help in the speedy detection of the growth hormone secretagogue ibutamoren and reveal its illegal use in competitive sports.

Complex profiles of azaspiracid analogues in two culture strains of Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters determined by LC-MS/MS.

Lipophilic marine biotoxins azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters. In our present study, AZA analogues in two strains (mdd421 and HM536) of A. poporum were analyzed by several detection techniques on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). The dominant AZA analogue in the Japanese A. poporum strains was AZA2. Other known AZA analogues were AZA11, AZA35, AZA2 methyl ester and AZA2 phosphate ester. Besides these AZAs, thirteen new AZA analogues were discovered in the two strains. A putative AZA analogue (Compound 1) with the smallest molecular weight ever found in nature was also discovered in the two strains. This is the first report describing detailed AZA profiles in Japanese isolates of A. poporum.

A mussel tissue certified reference material for multiple phycotoxins. Part 5: profiling by liquid chromatography-high-resolution mass spectrometry.

A freeze-dried mussel tissue-certified reference material (CRM-FDMT1) was prepared containing the marine algal toxin classes azaspiracids, okadaic acid and dinophysistoxins, yessotoxins, pectenotoxins, cyclic imines, and domoic acid. Thus far, only a limited number of analogues in CRM-FDMT1 have been assigned certified values; however, the complete toxin profile is significantly more complex. Liquid chromatography-high-resolution mass spectrometry was used to profile CRM-FDMT1. Full-scan data was searched against a list of previously reported toxin analogues, and characteristic product ions extracted from all-ion-fragmentation data were used to guide the extent of toxin profiling. A series of targeted and untargeted acquisition MS/MS experiments were then used to collect spectra for analogues. A number of toxins previously reported in the literature but not readily available as standards were tentatively identified including dihydroxy and carboxyhydroxyyessotoxin, azaspiracids-33 and -39, sulfonated pectenotoxin analogues, spirolide variants, and fatty acid acyl esters of okadaic acid and pectenotoxins. Previously unreported toxins were also observed including compounds from the pectenotoxin, azaspiracid, yessotoxin, and spirolide classes. More than one hundred toxin analogues present in CRM-FDMT1 are summarized along with a demonstration of the major acyl ester conjugates of several toxins. Retention index values were assigned for all confirmed or tentatively identified analogues to help with qualitative identification of the broad range of lipophilic toxins present in the material.

Dissipation kinetics and dietary risk assessment of spiromesifen on major summer vegetables using good agricultural practices.

Dissipation kinetics and dietary risk assessment of spiromesifen is worked out on four summer vegetables, viz. okra, chilli, capsicum and brinjal (eggplant or aubergine) during March-April 2015 at the experimental farm of the Department of Entomology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry Nauni, Solan using good agricultutral practices. Two foliar applications of spiromesifen @ 144.0 g.a.i./ha each were given at 10 days interval with a knapsack sprayer with the first application at the fruit initiation stage. Sample were collected up to 15 days after pesticide application and processed using a modified QuEChERS method, which was validated by doing recovery studies having recovery range and RSD within established guidelines of SANCO. Estimation of spiromesifen residues was conducted on GC-MS. The initial deposits after spraying of spiromesifen on okra, capsicum, chilli and brinjal fruit after 2 h of treatment were 1.327, 0.727, 0.800 and 0.738 mg/kg, respectively. The residues persisted up to 7 days and further dissipated and declined below the limit of quantification of <0.025 mg/kg at 10 days after treatment in all of the crops under investigation. Dissipation of spiromesifen followed first-order kinetics. The spiromesifen residues dissipated to half in 1.6, 1.8, 1.9 and 1.7 days with the suggested safe waiting period of 8.9, 5.2, 6.0 and 7.0 in the respective crops. The hazard quotient was <1 and theoretical maximum dietary intake was less than the maximum permissible intake, which was less than the maximum residue limit in all of the vegetable crops under investigation.

Enzyme and lateral flow monoclonal antibody-based immunoassays to simultaneously determine spirotetramat and spirotetramat-enol in foodstuffs.

Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC50 value for the sum of spirotetramat and spirotetramat-enol of 0.1 µg/L was developed using selected antibodies and a novel heterologous bioconjugate carrying a rationally-designed hapten. Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%.

Determination of lipophilic marine biotoxins by liquid chromatography-tandem mass spectrometry in five shellfish species from Washington State, USA.

Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.

Simultaneous determination of spirodiclofen, spiromesifen, and spirotetramat and their relevant metabolites in edible fungi using ultra-performance liquid chromatography/tandem mass spectrometry.

A fast, sensitive, and reliable analytical method was developed and validated for simultaneous identification and quantification of spirodiclofen, spiromesifen, and spirotetramat and their relevant metabolites in edible fungi by ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). First, sample extraction was done with acetonitrile containing 1% formic acid followed by phase separation with the addition of MgSO4:NaOAc. Then, the supernatant was purified by primary secondary amine (PSA), octadecylsilane (C18), and graphitized carbon black (GCB). The linearities of the calibrations for all analytes were excellent (R2 ≥ 0.9953). Acceptable recoveries (74.5-106.4%) for all analytes were obtained with good intra- and inter- relative standard deviations of less than 14.5%. The limit of quantification (LOQs) for all analytes was 10 µg kg-1. For accurate quantification, matrix-matched calibration curve was applied to normalize the matrix effect. The results indicated that the method was suitable for detecting the three acaricides and their relevant metabolites in edible fungi.

Development of a data dependent acquisition-based approach for the identification of unknown fast-acting toxins and their ester metabolites.

Phycotoxins in the marine food-web represent a serious threat to human health. Consumption of contaminated shellfish and/or finfish poses risk to consumer safety: several cases of toxins-related seafood poisoning have been recorded so far worldwide. Cyclic imines are emerging lipophilic toxins, which have been detected in shellfish from different European countries. Currently, they are not regulated due to the lack of toxicological comprehensive data and hence the European Food Safety Authority has required more scientific efforts before establishing a maximum permitted level in seafood. In this work, a novel data dependent liquid chromatography - high resolution mass spectrometry (LC-HRMS) approach has been successfully applied and combined with targeted studies for an in-depth investigation of the metabolic profile of shellfish samples. The proposed analytical methodology has allowed: i) to discover a plethora of unknown fatty acid esters of gymnodimines and ii) to conceive a brand new MS-based strategy, termed as backward analysis, for discovery and identification of new analogues. In particular, the implemented analytical workflow has broadened the structural diversity of cyclic imine family through the inclusion of five new congeners, namely gymnodimine -F, -G, -H, -I and -J. In addition, gymnodimine A (376.5 µg/kg), 13-desmethyl spirolide C (11.0-29.0 µg/kg) and pinnatoxin G (3.1-7.7 µg/kg) have been detected in shellfish from different sites of the Mediterranean basin (Tunisia and Italy) and the Atlantic coast of Spain, with the confirmation of the first finding of pinnatoxin G in mussels harvested in Sardinia (Tyrrhenian Sea, Italy).

Evaluation of different strategies to minimize the matrix effects on LC-MS/MS analysis of multiple lipophilic shellfish toxins in both acidic and alkaline chromatographic conditions.

Lipophilic shellfish toxins (LSTs) accumulated by shellfish pose a potential threat to consumer health. A mandatory routine monitoring of LSTs has been adopted for seafood products by liquid chromatography-mass spectrometry (LC-MS) in many countries. In this study, two methods developed on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) under acidic and alkaline chromatographic conditions were assessed for the determination of multiple LSTs. Different strategies including matrix solid-phase dispersion (MSPD), solid phase extraction (SPE) and sample dilution were applied and evaluated the matrix effects of mussel, scallop, clam, and oyster samples on the signal response of mass spectrometry. Results showed that the alkaline method achieved a lower limit of detection (LOD) and more robust compared to the acidic method. The obvious signal suppression of OA and DTX1 (55%-76%) and signal enhancement of PTX2 (27%-34%) occurred in the crude extracts of shellfish under acidic chromatography. In the alkaline method, no remarkable matrix effects of crude extracts were found except for the scallop matrix on the signal intensity of DTX1, AZA3 and GYM-A (121%-130%). Clean-up methods MSPD, SPE and sample dilution obviously reduced the inhibition of shellfish matrices on the signal response of OA and DTX1, however, which were still subject to signal inhibition under acidic condition. Sample dilution was more effective than SPE and MSPD in minimizing the matrix interference in both acidic and alkaline methods. Furthermore, sample dilution in combination with the alkaline chromatography was the most effective method. Bivalve mollusks harvested from Beibu Bay, South China Sea, were generally contaminated by GYM-A and SPX1 at low concentrations.

Detection of Cyclic Imine Toxins in Dietary Supplements of Green Lipped Mussels (Perna canaliculus) and in Shellfish Mytilus chilensis.

Seafood represents a significant part of the human staple diet. In the recent years, the identification of emerging lipophilic marine toxins has increased, leading to the potential for consumers to be intoxicated by these toxins. In the present work, we investigate the presence of lipophilic marine toxins (both regulated and emerging) in commercial seafood products from non-European locations, including mussels Mytilus chilensis from Chile, clams Tawerea gayi and Metetrix lyrate from the Southeast Pacific and Vietnam, and food supplements based on mussels formulations of Perna canaliculus from New Zealand. All these products were purchased from European Union markets and they were analyzed by UPLC-MS/MS. Results showed the presence of the emerging pinnatoxin-G in mussels Mytilus chilensis at levels up to 5.2 µg/kg and azaspiracid-2 and pectenotoxin-2 in clams Tawera gayi up to 4.33 µg/kg and 10.88 µg/kg, respectively. This study confirms the presence of pinnatoxins in Chile, one of the major mussel producers worldwide. Chromatograms showed the presence of 13-desmethyl spirolide C in dietary supplements in the range of 33.2-97.9 µg/kg after an extraction with water and methanol from 0.39 g of the green lipped mussels powder. As far as we know, this constitutes the first time that an emerging cyclic imine toxin in dietary supplements is reported. Identifying new matrix, locations, and understanding emerging toxin distribution area are important for preventing the risks of spreading and contamination linked to these compounds.

Discovery of chemical markers for improving the quality and safety control of Sinomenium acutum stem by the simultaneous determination of multiple alkaloids using UHPLC-QQQ-MS/MS.

Sinomenium acutum stem is a popular traditional Chinese medicine used to treat bone and joint diseases. Sinomenine is considered the only chemical marker for the quality control of S. acutum stem in mainstream pharmacopeias. However, higenamine in S. acutum stem is a novel stimulant that was banned by the World Anti-Doping Agency in 2017. Therefore, enhancing the quality and safety control of S. acutum stem to avoid potential safety risks is of utmost importance. In this study, a fast, sensitive, precise, and accurate method for the simultaneous determination of 11 alkaloids in S. acutum stem by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) was established. This method successfully analyzed thirty-five batches of S. acutum stem samples. The average contents of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine were 24.9 mg/g, 6.35 mg/g, 435 µg/g, 435 µg/g, 288 µg/g, 44.4 µg/g, 22.5 µg/g, 21.1 µg/g, 15.8 µg/g, 9.30 µg/g, and 8.75 µg/g, respectively. Multivariate analysis, including principal component analysis (PCA), orthogonal partial least square method-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA), were performed to characterize the importance and differences among these alkaloids in S. acutum stem samples. As a result, sinomenine, magnoflorine, coclaurine, acutumine, and higenamine are proposed as chemical markers for quality control. Higenamine and coclaurine are also recommended as chemical markers for safety control. This report provides five alkaloids that can be used as chemical markers for improving the quality and safety control of S. acutum stem. It also alerts athletes to avoid the risks associated with consuming S. acutum stem.

The occurrence of lipophilic toxins in shellfish from the Middle Adriatic Sea.

The first survey of the phycotoxin profile in mussels (Mytilus galloprovincialis) from the coastal waters of Bosnia and Herzegovina (The Bay of Neum, Middle Adriatic Sea) in correlation to the Makarska City Bay (Croatia, Middle Adriatic Sea) was conducted in 2017. Throughout the monitoring period, occasions of gymnodimine (GYM) and azaspiracid (AZA2) shellfish toxicity were recorded in concentrations that do not endanger human health. The occurrence of yessotoxins (YTXs), the most common toxins found in the Adriatic Sea, was correlated to the presence of the Gonyaulax species, a potential source of YTX. The DSP group of toxins is represented by the ester-OA. Phytoplankton analysis confirmed the presence of dinoflagellates from the Prorocentrum genus, a species associated with DSP toxicity. Occurrence frequency and variability of toxin composition were investigated in conjunction to physico-chemical parameters in the surrounding sea water. In the central Adriatic Sea, the infestation period ranges in general from June to August. However, the depuration phase extended beyond September in the Bay of Neum, increasing the length of the decontamination period.

Distribution and abundance of azaspiracid-producing dinophyte species and their toxins in North Atlantic and North Sea waters in 2018.

Representatives of the marine dinophyte family Amphidomataceae produce lipophilic phycotoxins called azaspiracids (AZA) which may cause azaspiracid shellfish poisoning (AZP) in humans after consumption of contaminated seafood. Three of the four known toxigenic species are observed frequently in the eastern North Atlantic. In 2018, a research survey was performed to strengthen knowledge on the distribution and abundance of toxigenic Amphidomataceae and their respective toxins in Irish coastal waters and in the North Sea. Species-specific quantification of the three toxigenic species (Azadinium spinosum, Azadinium poporum and Amphidoma languida) was based on recently developed qPCR assays, whose performance was successfully validated and tested with specificity tests and spike experiments. The multi-method approach of on-board live microscopy, qPCR assays and chemical AZA-analysis revealed the presence of Amphidomataceae in the North Atlantic including the three targeted toxigenic species and their respective AZA analogues (AZA-1, -2, -33, -38, -39). Azadinium spinosum was detected at the majority of Irish stations with a peak density of 8.3 x 104 cells L-1 and AZA (AZA-1, -2, -33) abundances up to 1,274 pg L-1. Amphidoma languida was also present at most Irish stations but appeared in highest abundance in a bloom at a central North Sea station with a density of 1.2 x 105 cells L-1 and an AZA (AZA-38, -39) abundances of 618 pg L-1. Azadinium poporum was detected sporadically at the Irish south coast and North Sea and was rather low in abundance during this study. The results confirmed the wide distribution and frequent occurrence of the target species in the North Atlantic area and revealed, for the first time, bloom abundances of toxigenic Amphidomataceae in this area. This emphasizes the importance of future studies and monitoring of amphidomatacean species and their respective AZA analogues in the North Atlantic.

A novel hydrophilic interaction liquid chromatography method for the analysis of arterolane; Isolation and structural elucidation of its major degradation product by LC-HRMS and NMR.

Adequate retention of arterolane (ART) has not been published in the literature till date; the hydrophilic interaction liquid chromatographic (HILIC) method with improved retention and separation of arterolane from its degradation products are reporting here for the first time. The present study discusses the comparative retention of the drug-using Reverse Phase C18 and HILIC columns, indicating the effective method (Syncronis HILIC-150 x 4.6 mm, 5µ). It was observed that the buffer concentration (ammonium acetate) in the mobile phase plays a crucial role in the retention of ART. Forced degradation studies of ART were conducted as per ICH Q1 (R2) prescribed conditions. The drug is not stable in hydrolytic (acid, base and neutral), oxidative and photolytic conditions and observed four unique degradation products (DPs). The optimized method for the analysis of degraded samples comprises of Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) technique having Agilent HILIC plus (100 × 4.6 mm, 3.5µ) column and acetonitrile (ACN) and 10 mM ammonium acetate 75:25 (% v/v) mobile phase with 0.4 mL/minute flow. Initially, the structure of all four DPs was proposed on the basis of accurate mass and their fragmentation pattern. The major degradation product (DP4) was isolated, and its structure was confirmed by HRMS and 1H NMR, 13C NMR, HMBC, DEPT 135 and Deuteriated NMR spectroscopy. The formation of DPs might be due to the breakdown of (1 r,3r,5 r,7r)-2-methoxyadamantan-2-ol from ART (DP4), and subsequent diol formation at 1,2,4-trioxolane moiety (DP3).

Magnetic nanostructures for marine and freshwater toxins removal.

Marine and freshwater toxins contaminate water resources, shellfish and aquaculture products, causing a broad range of toxic effects in humans and animals. Different core-shell nanoparticles were tested as a new sorbent for removing marine and freshwater toxins from liquid media. Water solutions were contaminated with 20 µg/L of marine toxins and up to 50 µg/L of freshwater toxins and subsequently treated with 250 or 125 mg/L of nanoparticles. Under these conditions, carbon nanoparticles removed around 70% of saxitoxins, spirolides, and azaspiracids, and up to 38% of diarrheic shellfish poisoning toxins. In the case of freshwater toxins, the 85% of microcystin LR was eliminated; other cyclic peptide toxins were also removed in a high percentage. Marine toxins were adsorbed in the first 5 min of contact, while for freshwater toxins it was necessary 60 min to reach the maximum adsorption. Toxins were recovered by extraction from nanoparticles with different solvents. Gymnodinium catenatum, Prorocentrum lima, and Microcystis aeruginosa cultures were employed to test the ability of nanoparticles to adsorb toxins in a real environment, and the same efficacy to remove toxins was observed in these conditions. These results suggest the possibility of using the nanotechnology in the treatment of contaminated water or in chemical analysis applications.

Residues, dissipation and risk evaluation of spiroxamine in open-field-grown strawberries using liquid chromatography tandem mass spectrometry.

The dissipation dynamic and residues of spiroxamine in open-field-grown strawberries were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spiroxamine application was performed according to Egyptian good agricultural practices recommendation. A QuEChERS-based extraction method along with direct analysis with an LC-MS/MS analytical method were optimized and validated, and the specificity of the techniques used was considered satisfactory. Good linearity (R2 > 0.999) was obtained for spiroxamine within the range of 0.001-0.1 µg/ml. The mean recoveries varied between 97.1 and 108.2%, with inter- and intra-day precision (RSD) <4.9%. The limit of quantitation for spiroxamine was 0.001 mg/kg. The results indicated that spiroxamine degradation in strawberry followed first-order kinetics (R2 > 0.9929) with an estimated half-life value of 4.71 days. Considering the Australian maximum residue limit (0.05 mg/kg) in strawberry and based on the results from residue trials with a preharvest interval of 14 days for strawberry, compliance can be expected. The present results could provide guidance to fully evaluate the risks of spiroxamine residues, preventing any potential health risk to consumers.