Macrocyclic-, polycyclic-, and nitro musks in cosmetics, household commodities and indoor dusts collected from Japan: implications for their human exposure.

This paper reported the occurrence and concentrations of macrocyclic-, polycyclic- and nitro musks in cosmetics and household commodities collected from Japan. The high concentrations and detection frequencies of Musk T, habanolide, and exaltolides were found in commercial products, suggesting their large amounts of production and usage in Japan. Polycyclic musks, HHCB and OTNE, also showed high concentrations in cosmetics and products. The estimated dairy intakes of Musk T and HHCB by the dermal exposure to commercial products were 7.8 and 7.9 µg/kg/day in human, respectively, and perfume and body lotion are dominant exposure sources. We also analyzed synthetic musks in house dusts. Polycyclic musks, HHCB and OTNE, showed high concentrations in samples, but macrocyclic musks were detected only in a few samples, although these types of musks were highly detected in commercial products. This is probably due to easy-degradation of macrocyclic musks in indoor environment. The dairy intakes of HHCB by dust ingestions were 0.22 ng/kg/day in human, which were approximately five orders of magnitudes lower than those of dermal absorption from commercial household commodities.

Potent sex attractant of the gypsy moth: its isolation, identification, and synthesis.

The sex attractant emitted by the female gypsy moth has been identified as cis-7,8-epoxy-2-methyloctadecane. The structure was verified by spectral, gas chromatographic, and biological comparisons with the synthesized compound. Nine closely related isomers were considerably less effective.

Advanced studies for the application of high-performance capillary electrophoresis for the analysis of yessotoxin and 45-hydroxyyessotoxin.

Yessotoxins (YTXs) are a group of polyether toxins which have been previously reported as responsible for seafood contamination in several places worldwide. Despite their toxicity, which is not yet fully discussed, YTXs have been reported as an interference in the success of mouse bioassay for the determination of diarrhetic shellfish poisoning (DSP) toxins, and therefore, efficient and reliable analytical methodologies are required to evaluate their presence, avoiding false positives for DSP. High-performance capillary electrophoresis (HPCE) is presented in this work as an alternative to HPLC technique widely used for the analysis of YTXs. Improvements in the applicability of HPCE have been carried out through the development of different CE modes as well as different detection modes. With this aim, micellar electrokinetic chromatography (MEKC) has been considered for an increased selectivity while an increased sensitivity was achieved by using sample stacking. Moreover, the coupling of CE with mass spectrometry allowed the confirmation of YTXs present in the contaminated samples evaluated in this work. The results obtained showed the potential of CE as an alternative to HPLC for the analysis of YTXs present in naturally contaminated samples.

Detection and identification of glycoyessotoxin A in a culture of the dinoflagellate Protoceratium reticulatum.

The toxin composition of a culture of the dinoflagellate Protoceratium reticulatum was investigated using LC-FLD, after derivatization with DMEQ-TAD (4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalimylethyl)-1,2,4-triazoline-3,5-dione)). Besides yessotoxin (YTX), the new YTX analogue, glycoyessotoxin A (G-YTXA) was detected in culture medium as well as in cells. The conditions for extraction were optimized and the production profile established. Retention time of the resulting fluorescent G-YTXA adduct was identified by comparison of the appropriate standard. Additionally, both G-YTXA and the DMEQ-TAD-G-YTXA adduct were confirmed by LC-MS showing ion peaks at m/z 1273 [M-2Na+H](-) and m/z 1618 [M-2Na+H](-), respectively. The LC-MS(n) displayed a fragmentation pattern similar to that of the YTX series.

Study of the effect of temperature, irradiance and salinity on growth and yessotoxin production by the dinoflagellate Protoceratium reticulatum in culture by using a kinetic and factorial approach.

A complete first order orthogonal plan was used to optimize the growth and the production of yessotoxin (YTX) by the dinoflagellate Protoceratium reticulatum in culture by controlling salinity, temperature and irradiance. Initially, an approach to the kinetic data of cellular density and YTX production for each one of the experimental design conditions was performed. The P. reticulatum growth and YTX production were fitted to logistical equations and to a first-order kinetic model, respectively. The parameters obtained from this adjustment were used as dependent variables for the formulation of the empirical equations of the factorial design tested. The results showed that in practically all the cases for both, P. reticulatum growth and YTX production, irradiance is the primary independent variable and has a positive effect in the range 50-90 micromol photons m(-2) s(-1). Additionally, in certain specific cases, temperature reveals significant positive effects when maintained between 15 and 23 degrees C and salinity in the range of 20-34 displays negative effects. Despite the narrow ranges used in the work, results showed the suitability of factorial analysis to evaluate the optimal conditions for growth and yessotoxin production by the dinoflagellate P. reticulatum.

[Lipophilic toxin profiles associated with diarrhetic shellfish poisoning in scallops, Patinopecten yessoensis, collected in Hokkaido and comparison of the quantitative results between LC/MS and mouse bioassay].

Lipophilic toxins associated with diarrhetic shellfish poisoning (DSP) in scallops, Patinopecten yessoensis, collected in Hokkaido, Japan were quantified by liquid chromatography-mass spectrometry (LC/MS). Pectenotoxin-6 (PTX6) and yessotoxin (YTX) were the dominant toxins in the scallops, although the percentages of these toxins were different depending on the production area or the sampling period. The quantitative results obtained for the scallops in LC/MS and in mouse bioassay (MBA) were compared. Fifty of the 55 samples found to be exceeding the local quarantine level (0.025 MU/g whole meat) in Hokkaido by LC/MS were quantified by MBA as being below the quarantine level. It is suggested that this discrepancy is due to poor detection of YTX by MBA. These results indicate that LC/MS is a better method than MBA in terms of sensitivity and accuracy to quantify known lipophilic toxins, including YTX.

Systematic assignment of the configuration of flexible natural products by spectroscopic and computational methods: the bistramide C analysis.

[reaction: see text] The combination of NMR NOE, chemical shift, and J-coupling measurements with molar rotation and circular dichroism (CD) determinations, including RI-DFT BP86/aug-cc-pVDZ calculations, reduced a candidate pool of 1024 possible stereoisomers of (+)-bistramide C to a single absolute configuration assignment for the 10 stereogenic carbons of the marine natural product.

Comparison of ELISA and LC-MS analyses for yessotoxins in blue mussels (Mytilus edulis).

Blue mussels (Mytilus edulis) collected from Flødevigen Bay, Norway, in 2001 and 2002 were analysed for yessotoxins (YTXs) by ELISA and yessotoxin (YTX), 45-hydroxyYTX, and carboxyYTX by LC-MS. Results from the two methods were compared to evaluate the ELISA. The response in the ELISA was 3-13 times higher than LC-MS, probably due to the antibodies binding to other YTX analogues not included in the LC-MS analysis. Nevertheless, the correlation between ELISA and LC-MS was good, with r2 values> or =0.8. The results indicate that the ELISA is a reliable method for estimating the total level of YTXs in mussels, and are consistent with extensive metabolism of algal YTXs in mussels. YTX was a minor component in the blue mussels at all times compared to 45-hydroxyYTX and especially carboxyYTX, except when the P. reticulatum bloom occurred. The results also indicate the presence of significant amounts of YTX analogues in addition to those measured by LC-MS. All samples below 4 mg/kg by ELISA were below the current EU regulatory limit of 1 mg/kg by LC-MS. Therefore, we propose using ELISA as a screening tool with a cut-off limit at 4 mg/kg for negative samples, whereas samples above this limit would be reanalyzed by LC-MS.

Yessotoxins in Norwegian blue mussels (Mytilus edulis): uptake from Protoceratium reticulatum, metabolism and depuration.

The Protoceratium reticulatum cell density at Flodevigen reached a maximum of 2200 cells/L on 16 May 2001. The levels of yessotoxins (YTXs) in blue mussels (Mytilus edulis) at the same site increased sharply by 14 May and peaked on 28 May, after which they steadily declined. No other algal species present showed a similar pattern of correspondence. Together with the recent finding that Norwegian strains of P. reticulatum produce YTXs, these results indicate that P. reticulatum causes yessotoxin (YTX) contamination of shellfish in Norway, and that only relatively low cell densities are necessary for this to occur. The mussels from Flodevigen were analyzed by LC-MS for YTX, 45-hydroxyYTX, carboxyYTX, and a new yessotoxin believed to be 45-hydroxycarboxyYTX, and by ELISA for YTXs. The seasonal variations in toxin content versus time measured by the two methods were qualitatively very similar, although the response in the ELISA was 3-9 times higher due to the antibodies detecting other YTXs that were not detected by the LC-MS method. Changes in the LC-MS profile for YTXs, and in the ratio of YTXs by LC-MS to YTXs by ELISA with time, were consistent with extensive metabolism of YTX in the mussels. Kinetic analysis of the LC-MS data showed an initial half-life of 20 days for YTX, and for YTX+45-hydroxyYTX, in the mussels. Similar analysis of the ELISA data gave a half-life of 24 days for YTXs. The depuration rate remained consistent over a 3-month period during which the temperature remained at 13-16 degrees C.

Persistence of yessotoxin under light and dark conditions.

The change in concentration of the disulfated polyether yessotoxin (YTX) produced by a culture of the marine dinoflagellate Protoceratium reticulatum was measured in laboratory experiments under light and dark conditions. Experimental cultures were inoculated and grew at a growth rate of 0.14 d(-1) until stationary phase was reached, after approximately 21 days. Cultures were maintained in the stationary phase until 31 days after inoculation. Cells of P. reticulatum contained a concentration of approximately 10-15 pg YTX cell(-1) during stationary phase but this was considerably lower (<5 pg cell-1) during the growth phase. Low amounts of 45-hydroxy-YTX were also detected. At day 32, P. reticulatum was killed by cooling to 1 degrees C (confirmed microscopically) and YTX concentrations were measured periodically under light and dark conditions. YTX concentrations decreased rapidly to approximately 10% of the initial concentration within the first 3 days and depleted to near zero within a week in the light treatment. In the dark environment, YTX persisted longer with approximately 10% of the initial YTX concentration still remaining after 18 days.

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins.

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study.

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).

Quantitation of radio-labelled vitamin K1 and vitamin K1 epoxide in plasma by high pressure liquid chromatography.

A convenient, accurate and reproducible high pressure liquid chromatographic method for the quantitation of radio-labelled vitamin K1 and vitamin K1 epoxide in plasma is described. The method involves the determination of total ether extractable radioactivity, and a chromatographic separation to determin the relative quantities of radio-labelled vitamin K1 and vitamin K1 epoxide. The method is useful over a wide range of ratios of the two compounds, and has a coeffcient of variation of approximately 5%.

Seasonal, geographic and individual variation of okadaic acid content in cultivated mussels in Sweden.

In Western Europe the dinoflagellate toxin, okadaic acid (OA) has been the main cause of diarrheic shellfish poisoning (DSP). Chemical determination of OA in mussels by homogenization of the hepatopancreas, extraction, purification, reaction with 9-anthryldiazomethane (ADAM), HPLC-separation, and fluorometric quantification has been used for weekly monitoring of mussel growing farms and to control harvested mussels. Within a week, substantial rises (from 0.41 to 5.4 micrograms OA/g hepatopancreas) as well as great reductions (from 7.2 to 1.8 micrograms/g hepatopancreas) were recorded. The rapid rise implies that weekly sampling is not sufficient to ensure that mussels are free from toxic levels of OA. The rapid decrease reveals that efficient toxin clearance mechanisms exist in the mussels. Substantial OA clearance occurs also at low temperatures (1.4-3 degrees C). Within a mussel growing site the OA concentrations could differ considerably between adjacent mussels (0.63 and 4.2 micrograms OA/g hepatop.) and even more between mussels grown at different depths along the same rope (0.63 and 10 micrograms OA/g hepatop.). These data emphasize the importance of sampling in studies on DST in mussels. Great differences between the different mussel growing sites were also observed. These data have been discussed with respect to the spread of the toxin by the sea, and the possibilities of reducing the exposure of the mussels to the toxic algae.

Preparation of mouse monoclonal antibodies to okadaic acid and their binding activity in organic solvents.

Seven of 20 mouse monoclonal antibodies to OA, OA8-2, OA10-8, OA22-22, OA227-11, OA296-1, OA423-3, and OA958-2, were studied as to their binding to OA in organic solvents. OA423-3 (IgG1-kappa) and OA958-2 (IgG1-kappa) in 90-100% methanol retained their binding activities with both immobilized and free antibodies. Whereas OA8-2 (IgG2a-kappa), OA10-8 (IgG1-kappa), OA22-22 (IgG2a-kappa), OA227-11 (IgG1-kappa), and OA296-1 (IgM-kappa) did not bind to OA in over 50-60% methanol. The results of a non-competitive inhibition assay for OA indicated that in a methanolic or ethanolic solution, the binding ability of immobilized OA423-3 decreased as the concentration of each alcohol increased. The concentration of OA at the midpoint between the upper and lower plateaus of the inhibition curve was 0.18 ng/ml in 0% methanol and 570 ng/ml in 100%, respectively. In 0-50% of each of acetone, diethyl ether, and benzene in methanol, the binding ability of OA423-3 remained at the level in 100% methanol. OA958-2 showed similar binding properties to OA423-3. No relationship between the subclass of the immunoglobulin and the binding activity of the antibody in organic solvents was observed. These results indicate that the OA423-3 and OA958-2 antibodies are useful for the development of a new ELISA method for OA in organic solvents.

Adrenoceptor-induced changes of intracellular K+ and Ca2+ in astrocytes and neurons in rat cortical primary cultures.

The calcium- and potassium sensitive fluorescent dyes fura-2 and K+-binding benzofuran isophtalate (PBFI) were used to detect changes in [Ca2+]i and [K+]i in type 1 astrocytes and neurons in mixed astroglial/neuronal rat cortical primary cultures after adrenoceptor stimulation. Noradrenalin (NA), phenylephrine (phe; alpha1-agonist), clonidine (clon; alpha2-agonist) and isoproterenol (iso; beta-agonist) were used. All agonists were able to increase [Ca2+]i and decrease [K+]i in the astrocytes with the exception of clon, which could not induce potassium responses. In the neurons, NA and phe evoked calcium transients while clon and iso did not. NA and clon were able to elicit reductions in [K+]i but no responses were seen after phe or iso stimulation. In neurons, the NA-evoked reductions in [K+]i always appeared immediately and gradually (after 30-50 s) returned to baseline even in the presence of the agonists. On the other hand, in the astrocytes, the NA-induced reductions in [K+]i appeared with some latency and always persisted at the lower level in the presence of the agonists. In addition, external tetraethylammonium (TEA) could severely reduce the NA-induced K+ responses in the astrocytes. The results indicate a clear heterogeneity regarding both adrenoceptor expression and response characteristics between astroglial cells and neurons.

Influence of the extraction procedure on recovery of okadaic acid from experimentally contaminated mussels.

Hepatopancreas samples from mussels (Mytilus galloprovincialis) experimentally contaminated with okadaic acid were analysed with Yasumoto's mouse bioassay and HPLC. A likely effect of some components of the hepatopancreas on the results (matrix effect) was evaluated, and a possible loss of toxin during the extraction phase was quantified. Experiments were conducted by comparing two different extraction procedures. Under our experimental conditions, the results obtained from mouse bioassay showed no matrix effect with either procedure. A certain quantity of the actual amount of okadaic acid contained in the sample was found to be lost after the extraction, i.e. 10.2-17.0% in samples extracted with acetone alone and 9.8-18.5% in samples extracted with acetone and ether.

Detection of the marine toxins okadaic acid and domoic acid in shellfish and phytoplankton in the Gulf of Mexico.

Liquid chromatographic analyses of extracts from shellfish and phytoplankton from the Gulf of Mexico indicated the presence of the marine toxins okadaic acid (0.162 microgram/g shellfish) and domoic acid (2.1 pg/cell phytoplankter). These toxins are causative agents of diarrhetic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. The presence of DSP and ASP toxins in a region with no previous record of outbreaks may indicate a potential for human poisoning under conditions appropriate for accumulation of these toxins in shellfish.

Quantification of diarrhetic shellfish toxins and identification of novel protein phosphatase inhibitors in marine phytoplankton and mussels.

Liquid chromatography (LC)-linked protein phosphatase 1/2A (PP-1/PP2A) bioassay was used to quantitatively identify diarrhetic shellfish toxins in marine phytoplankton (cultured and natural assemblages) and commercially available mussels. Using this approach, multiple protein phosphatase inhibitor profiles of varying composition were found in diarrhetic mussels from Holland and Canada. Based on LC elution positions and relative activity versus PP-1 and PP-2A, at least six inhibitors distinct from known diarrhetic shellfish toxins were identified and termed mussel phosphatase inhibitor (MPI) 19,22,23,25,33 and 42. The levels of these inhibitors, in okadaic acid equivalent units, varied from 100 pg to 3350 ng per g shellfish tissue. The combined levels of PP-1/2A inhibitors in all instances superseded that of okadaic acid/dinophysistoxin-1 and may contribute to the diarrhetic shellfish toxin profile of the contaminated mussels. The efficacy of LC-protein phosphatase bioassay was established for cultured phytoplankton where picogram levels of okadaic acid could be detected from microgram extracts of Prorocentrum lima. Analyses of plankton net tows from estuarine mussel culture sites in Eastern Canada revealed a heterogeneous population of protein phosphatase inhibitors, with dinophysistoxin-1 being most prevalent. This toxin was predominant for at least 2 months in mussel populations in the immediate vicinity of plankton sampling sites. The results are consistent with a hypothetical model in which marine bacteria, cyanobacteria and dinoflagellates combine to produce a variety of protein phosphatase inhibitors effective against signal transduction pathways in higher eukaryotes.

Studies of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland using multiple tandem mass spectrometry.

Diarretic shellfish poisoning (DSP) is a toxic syndrome associated with the consumption of bivalve molluscs. The DSP toxins are polyether compounds, which include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs) and pectenotoxin seco acids (PTX2SAs). These toxins originate in marine dinoflagellates, including Dinophysis spp. Phytoplankton samples were collected from the southwest coast of Ireland and D. acuta was the predominant species. Monocultures of D. acuta cells were prepared by hand picking from microscope slides in order to confirm their toxin profiles. There was a remarkable consistency in the toxin profiles in all of the phytoplankton samples collected during the summer months, irrespective of location, depth or mesh size. Analysis using liquid chromatography-multiple tandem mass spectrometry (LC-MS/MS) revealed that DTX2 and OA were the predominant toxins at a consistent ratio. The average toxin composition was: DTX2 (53+/-5%), OA (26.5+/-2.3%) and total pectenotoxins (20.8+/-4.7%). Toxin profiles in D. acuta from Europe were distinctly different from those found in New Zealand, where PTX2 was the predominant toxin and DTX2 was absent.