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.

Microscopic Characteristic and Chemical Composition Analysis of Three Medicinal Plants and Surface Frosts.

The accumulation of chemical constituents of some medicinal plants, such as Paeonia ostii T. Hong et J. X. Zhang, Houpoëa officinalis (Rehder and E. H. Wilson) N. H. Xia and C. Y. Wu. and Atractylodes lancea (Thunb.) DC, can precipitate on the surface and form frosts after natural or artificial intervention. The characteristics of these three medicinal plants and their frosts were analyzed by light microscope, polarizing microscope, stereomicroscope, and metalloscope. The results of ordinary Raman of P. ostii and H. officinalis showed that the frosts of P. ostii matched paeonol, while that of H. officinalis matched magnolol and honokiol. In P. ostii and its frost, 19 peaks were identified by UPLC-Q/TOF-MS, and the main component was paeonol. Eleven components were identified in H. officinalis and its frosts, and the main components were magnolol and honokiol. A. lancea and its frosts were analyzed by gas chromatography-mass spectrometry (GC-MS), 21 were identified, and its main components were hinesol and ß-eudesmol. These three medicinal plants accumulate compounds and precipitate frosts on the surface. The results show that the components of the frosts provide a basis for quality evaluation and research on similar medicinal plants, and reveals the scientific connotation of "taking the medicinal materials' precipitated frosts as the best" of P. ostii, H. officinalis, and A. lancea, to some extent.

An LC-ESI-MS/MS method for the simultaneous determination of pronuciferine and roemerine in some Papaver species.

Papaver species, well known for their alkaloids, have been used for the treatment of several diseases, such as inflammation, diarrhea, depression, and sleep disorders in certain parts of Anatolia. In this study, four Papaver species (P. lacerum, P. syriacum, P. glaucum and P. rhoeas) were collected from different localities of Turkey. Methanolic extracts were prepared from the aerial parts of the plants. A rapid analytical method was developed for the simultaneously quantitative analysis of two alkaloids, pronuciferine and roemerine, using liquid chromatography tandem mass spectrometry. Multiple reaction monitoring in the positive ionization mode was used for detection. Pronuciferine and roemerine were analyzed on a C18 column (2.1 × 50 mm, 3 µm) with the mobile phase run in the gradient mode with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min. The transitions 312.1→283.1 m/z and 280.0→249.0 m/z were used to monitor pronuciferine and roemerine, respectively. The assay was linear in the concentration range of 0.01 µg/mL to 1 µg/mL (r = 0.996 for roemerine, r = 0.998 for pronuciferine). The validation studies revealed that the method was linear, sensitive, accurate, precise, selective, repeatable, robust, and rugged. Finally, the developed method was applied to quantify pronuciferine and roemerine in the selected species. The amounts of pronuciferine and roemerine were respectively found as 8.5 to 48 µg/g and 4.4 to 43,000 µg/g.

Toxicological effects of soil contaminated with spirotetramat to the earthworm Eisenia fetida.

The aim of this study was to evaluate the potential toxicity of spirotetramat to the earthworm Eisenia fetida in a natural soil environment. Many biochemical markers, viz., superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione S-transferase (GST), cellulase, and malondialdehyde (MDA) contents were measured after exposure to 0.25, 1.25, and 2.5mgkg(-1) for 2, 7, 14, 21, and 28days. In addition, the comet assay was performed on earthworm coelomocytes to assess the level of genetic damage. The results demonstrate that the SOD activity and MDA content were significantly stimulated by the highest dose (2.5mgkg(-1)) of spirotetramat for the entire period of exposure. The activities of CAT and POD increased significantly by 2d and 21d, respectively, but the activities of both were significantly inhibited after prolonged exposure (28d). After an initial increase on the 2nd day, the cellulase activity in the high-dose treatment group was significantly inhibited for the entire remaining exposure period. The comet assay results demonstrate that spirotetramat (⩽2.5mgkg(-1)) can induce low and intermediate degrees of DNA damage in earthworm coelomocytes. The results indicate that spirotetramat may pose potential biochemical and genetic toxicity to earthworms (E. fetida), and this information is helpful for understanding the ecological toxicity of spirotetramat on soil invertebrate organisms.

Analysis of the neurotoxin anisatin in star anise by LC-MS/MS.

The aim of this work was to develop an analytical method capable of determining the presence of anisatin in star anise. This neurotoxin may induce severe side effects such as epileptic convulsions. It is therefore of prime importance to have rapid and accurate analytical methods able to detect and quantify anisatin in samples that are purportedly edible star anise. The sample preparation combined an automated accelerated solvent extraction with a solid-supported liquid-liquid purification step on EXtrelut®. Samples were analysed on a porous graphitic carbon HPLC column and quantified by tandem mass spectrometry operating in the negative ionisation mode. The quantification range of anisatin was between 0.2 and 8 mg kg⁻¹. The applicability of this validated method was demonstrated by the analysis of several Illicium species and star anise samples purchased on the Swiss market. High levels of anisatin were measured in Illicium lanceolatum, I. majus and I. anisatum, which may cause health concerns if they are misidentified or mixed with edible Illicium verum.

Rapid control of Chinese star anise fruits and teas for neurotoxic anisatin by Direct Analysis in Real Time high resolution mass spectrometry.

After ingestion, products containing Chinese star anise (Illicium verum) contaminated or adulterated with Japanese star anise (Illicium anisatum) or other Illicium species, can cause epilepsy, hallucinations, and nausea due to the rare neurotoxic sesquiterpene dilactone anisatin that is present in Japanese star anise. Thus a rapid, simple and unambiguous method for distinguishing between the morphologically similar Chinese star anise and toxic Japanese star anise is important for food safety issues. Direct Analysis in Real Time (DART) ambient ionisation coupled with orbitrap high resolution mass spectrometry allowed the recording of mass spectra of anisatin in solid star anise fruits in seconds without any prior sample pretreatment. Spectra could be obtained in both positive ([M+NH(4)](+) at m/z 346.1496, C(15)H(24)NO(8)) and negative mode ([M-H](-) at m/z 327.1074, C(15)H(19)O(8)) and gave the same outcome provided a mass resolution of at least 27,000 is available. The anisatin signal was typically >1000 times larger in Japanese star anise than in Chinese star anise thus allowing an unequivocal qualitative determination. Herbal teas containing star anise fragments too small to be visually recognised, could be analysed by preparing a tea in 6 min and subsequently sampling ∼2 µL of tea on a glass rod. None of the 8 investigated retail teas contained significant quantities of anisatin. Spiking a complex herbal tea containing Chinese star anise with an equally concentrated tea prepared from Japanese star anise provided a linear calibration curve (R(2) ≥ 0.995) after normalising on a native constituent of Chinese star anise (standard addition method). This showed that adulteration down to 1% (w/w) is still measurable. Compared with existing PCR, TLC, GC-MS and HPLC-ESI-MS/MS procedures, the proposed DART-HRMS procedure is faster and simpler and moreover measures the actual biotoxin.

[Simultaneous determination of atractylone, hinesol, beta-eudesmol, atrctylodin in Atractylodes lancea and hierarchical cluster analysis].

OBJECTIVE: To develop a GC method for simultaneous determination of 4 compounds (atractylone, hinesol, beta-eudesmol and atractylodin) in Atractylodes lancea. METHOD: A HP-1 capillary column (0.25 mm x 30 m, 0.25 microm) was used. The detector was FID:Inlet temperature was 250 degrees C. The detector temperature was 250 degrees C. The column temperature was set at 145 degrees C and held for 25 min after injection, then programmed at 10 degrees C x min(-1) to 250 degrees C and held for 10 min at the temperature. The carrying gas was nitrogen, split ratio was 40:1. Injection volume was 2 microL, Cluster analysis was performed by SPSS13.0 software. RESULT: The linear ranges for atractylone, hinesol, beta-eudesmol and atractylodin were 0.0122. 32 (r = .9998), 0.008-1.68 (r = 0.9998), 0.009-1.76 (r = 0.9999), 0.016-3.20 g x L(-1) (r = 0.9997), respectively. The average recoveries (n = 3) of atractylone, hinesol, beta-eudesmol and atractylodin were 98.0%-99.0%, 97.7%-99.4%, 98.4%-99.2%, 97.8%-99.7%, respectively. The samples analyzed were divided into two classes. CONCLUSION: This method is simple, specific, repeatable and stable. It can be applied for the simultaneous determination of 4 compounds (atractylone, hinesol, beta-eudesmol and atractylodin) in A. lancea, which will provide the basis for the quality control of A. lancea. The contents of 4 active compounds were significantly different between geo-authentic and non-authentic producing areas.

[Fingerprint of volatile oil of Atractylodes lancea by GC-MS].

To study the fingerprint of the volatile oil of Atractylodes lancea (Thunb.) DC., and to offer the characteristic data for the quality evaluation, GC-MS analysis was performed for 17 samples of different areas used as Atractylodes lancea (Thunb.) DC. Nine kinds of same components were selected. TIC profiles were evaluated by "Computer Aided Similarity Calculation". The characteristic peaks in chromatograms were identified by comparing mass data with literatures. Hierarchical clustering analysis was performed by SPSS based on the relative peak area (RPA) of identified peak to atractydin in 17 samples. The mutual mode fingerprint plots of genuine Atractylodes lancea (Thunb.) DC. have been established, the matching of active components was characteristic that atractylon, hinesol, beta-eudesmol, atractydin as (0.89 - 1.12): (0.11 - 0.15) : (0.48 - 0.61) : 1. The difference of resemblance of not genuine samples with genuine samples was remarkable. Two categorizations were clustered. Atractylodes lancea (Thunb.) DC. from genuine and Tangshan and Nanshan, Jiangsu Prov. were in a group, while those from Anhui and Hubei Prov. were in another group. The characteristic fingerprint of genuine Atractylodes lancea (Thunb.) DC. combined with the study of the matching of active components for the quality control and the resemblance calculation of fingerprints and SPSS hierarchical clustering analysis provided a new analytic method for the quality evaluation and discrimination of Atractylodes lancea (Thunb.) DC.

Dissipation kinetics of spiromesifen on tea (Camellia sinensis) under tropical conditions.

Spiromesifen (Oberon) is a new insecticide and miticide of chemical class ketoenol active against white flies (Bemisia spp., Trialeuroides spp.) and spider mites (Tetranychus and Panonychus spp.). Due to its potential significance in insect resistance management, it is important to establish its behaviour on crop and environment. In the present study, the degradation/dissipation of spiromesifen on tea crop under tropical environmental conditions was studied and its DT(50) (t(1/2)), and DT(90) (time to reduce to 90% of the initial value) were estimated. Spiromesifen was sprayed on tea crop after first rain flush at four different locations @ 96 and 192ga.i.ha(-1). Samples of tea leaves were drawn at 0, 1, 3, 5, 7, 10, 15, 21 and 30 days after treatment and that of soil at 10 days after treatment and at harvest from 0 to 15 and 15 to 30cm layers. After crude extraction of tea leaves for spiromesifen residues with acetone:water, the contents were partitioned with cyclohexane:ethyl acetate and cleaned up on Florosil column. Soil residues were also extracted similarly. Quantification of residues was done on GC-MS in Selected Ion Monitoring (SIM) mode in mass range 271-274m/z. The LOQ of this method was found to be 0.05microgg(-1) while LOD being 0.015microgg(-1). The DT(50) of spiromesifen when applied at recommended doses in tea leaves was found to be 5.0-8.5 days. Ninety-nine percent degradation was found to occur within 33-57 days after application. In soil, no residues of spiromesifen were detectable 10 days after treatment.

Liquid chromatography/tandem mass spectrometry of unusual phenols from Yucca schidigera bark: comparison with other analytical techniques.

Qualitative and quantitative analyses of phenolic compounds are of interest for both medicinal and food plants. In the present work, the phenolic fraction from Yucca schidigera, a plant bearing the GRAS (Generally Recognized as Safe) label approved by the US Food and Drug Administration, was studied. Crude extracts of Y. schidigera bark were investigated by liquid chromatography/UV spectrophotometry with diode-array detection, liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), in order to develop and optimize simple and rapid techniques to determine both stilbenes and yuccaols for the purposes of quality control of collected material. With optimal LC and MS conditions, stilbenes and yuccaols were quantified with all the proposed methods and the results were compared. Sensitivity was evaluated and the results indicated that MS/MS detection in the multiple reaction monitoring mode is easily applicable to this plant and allows the rapid and direct identification and quantification of these peculiar compounds in crude plant extracts.

[Studies on chemical constituents in essential oil from wild Atractylodes lancea in dabie mountains].

The chemical constituents in essential oil of wild Atractylodes lancea (Thumb.) DC. from Dabie Mountains were studied. The essential oil was extracted by simultaneous distillation and extraction equipment and analyzed by gas chromatography-mass spectrometry method. 49 compounds were identified and they represent 92.49% of the total peak aeras. The extraction method and solvent selection of the essential oil were studied. The oil yields and major constituents from Atractylodes lancea (Thumb.) DC. from Dabie Mountains were contrasted to that had been reported. The results showed that Atractylodes lancea (Thumb.) DC. from Dabie Mountains had more essential oil (10.14%) and there were more constituents in it. The main constituents in the essential oil were hinesol, beta-eudesmol, 1H-cyclopropa (a) naphthalene, 1a,2,3,5,6,7,7a,7b-octaphdro-1,1,7,7a-tetramethyl-, [1aR-(1a. alpha, 7. alpha, 7a. alpha,7b. alpha-)] -and gamma-eudesmol. Both the major constituents and contents were different from that had been reported.

[The naphtha composing characteristics of geoherbs of Atractylodes lancea].

OBJECTIVE: To find the chemical diversity and characteristics of A. lancea on two levels--individuals and populations, and to discover the chemical essentials for forming geoherbs. METHOD: 47 rhizomes of A. lancea were collected in 7 populations, and 6 naphtha components (1. elemol, 2. hinesol, 3. beta-eudesmol, 4. atractylone, 5. atractylodin, 6. atractylenolid I) in the rhizomes were determined by GC-MS combination. Principal Component Analysis and Cluster Analysis were carried out by SPSS. RESULT: Cluster Analysis of the 6 main components indicated that the chemical components of geoherbs were different from those of the non-geonerbs of A. lancea. Other analysis showed as follows: 1. The general oil of geoberbs were lower than that of non-geoherbs(P < 0.01), but components yielding more than 1% (% of the total oil) were more than non-geoherbs(P < 0.01); 2. Hinesol mixing beta-eudesmol was more in non-geoherbs, which atractylodin mixing atractylone was more in geoherbs(P < 0.001); 3. Principal Component Analysis implied that atractylone was the most important component to discriminate geoherbs and non-geoherbs of A. Lancea. CONCLUSION: The naphtha composing characteristics of geoherbs was the special proportionment sale, viz. atractylone: hinesol: beta-eudesmol: atractylodin being(0.70~2.00):(0.04~0.35):(0.09~0.40):1.

[TLC-FT-SERS study on ingredients of Isrhynchophylline].

A new method for analysing the ingredients of Isrhynchophylline in Uncaria Rhynchophylla Jacks by thin layer chromatography (TLC) and the surface-enhanced Raman spectroscopy (SERS) is reported in this paper. The results show that the characteristic spectra bands of Isrhynchophylline situated at the thin layer with the amount of sample about 2.5 micrograms were obtained. The difference between SERS and solid spectra was found. Great enhancement of the 1,615 cm-1 spectral band was abstained. Molecule was absorbed in surface silver sol by pi electrons in phenyl and by pair of electrons in N together. An absorption model of Isrhynchophylline and silver sol was proposed. This method can be used to analyse the chemical ingredients with high sensitivity.

Chlorinated alkaloids in Menispermum dauricum DC: root culture.

Feeding experiments using (36)Cl showed that Menispermum dauricum root culture produces four alkaloids containing chlorine. They included the novel alkaloids dauricumine and dauricumidine as well as the known alkaloids acutumine and acutumidine. The structures of novel alkaloids were established by spectroscopic, crystallographic, and chemical methods. These four alkaloids were labeled with (36)Cl, isolated, and fed independently to root cultures. Mutual conversion between acutumine and acutumidine, and between dauricumine and dauricumidine by N-methylation and N-demethylation, was demonstrated. Moreover, dauricumine was converted to acutumine and acutumidine. Epimerization of acutumidine to dauricumidine or vice versa was not observed. These results suggest that dauricumine is the first chlorinated alkaloid formed in cultured M. dauricum roots. Skewed distribution of radioactivity derived from labeled dauricumine is proof that epimerization at C-1 proceeds at a lower rate than N-demethylation.

Random amplified polymorphic DNA analysis and variation of essential oil components of Atractylodes plants.

Total DNAs were prepared from the leaves of Atractylodes lancea DE CANDOLLE, A. chinensis KOIDZUMI, A. lancea var. simplicifolia KITAMURA, A. japonica KOIDZUMI ex KITAMURA and A. ovata DE CANDOLLE. The DNAs were subjected to random amplified polymorphic DNA (RAPD) analysis. Some primers showed the definitive polymorphic DNA patterns in A. lancea, A. japonica and A. ovata. The RAPD of A. lancea var. simplicifolia and one of A. chinensis gave similar patterns to those of A. lancea, but one of the other A. chinensis gave a similar pattern to A. japonica. Furthermore, quantitative analysis of atractylon, hinesol, beta-eudesmol and atractylodin in the rhizomes was done using gas chromatography. Though atractylon was detected not only in A. japonica and A. ovata but also in some of A. lancea, their RAPD profiles revealed the presence of intraspecific variation with A. lancea.