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.

A sensitive LC-MS/MS assay for brevisulcenal and brevisulcatic acid toxins produced by the dinoflagellate Karenia brevisulcata.

A toxic dinoflagellate, Karenia brevisulcata, devastated almost all marine life in Wellington Harbour, New Zealand during the late summer of 1998. Brevisulcatic acids (BSXs) and brevisulcenals (KBTs), both polycyclic ether toxins, have been identified as the causative agents. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the sensitive and specific determination of BSXs and KBTs in culture medium, seawater and shellfish. Acidified algal culture, or seawater, was extracted using reverse phase solid phase extraction cartridges. Shellfish tissue homogenate was blended with methanol-water (9:1) and partitioned with hexane to remove non-polar lipids. This extraction protocol is similar to that used for analysis of lipophilic shellfish toxins. LC-MS/MS (triple quadrupole) was used for quantitative analysis with gradient elution (acidic buffer), positive electrospray ionization and multiple-reaction monitoring. Purified toxins were available for 4 KBTs (KBT-F, -G, -H and -I) and 4 BSXs (-1, -2, -4, and -5), and were used to calibrate the instrument responses. Relative response factors were used for semi-quantitative analysis of BSX-3 and BSX-6, using BSX-1 and BSX-4 respectively. Calibration curves for all toxins monitored were linear over the concentration range tested (5-200 ng mL(-1)) with r(2) values >0.99. The method limit of quantitation was determined to be 2 ng mL(-1) for BSXs and KBTs, except KBT-I, which was 5 ng mL(-1). Validation data was generated for culture medium and shellfish. Toxin recoveries were typically >70% with relative standard deviations <20% across all of the matrices tested. In addition, toxins specific to K. brevisulcata were able to be detected in seawater at a cell concentration of 10,000 cells L(-1), which represents the suggested trigger level for this harmful algal species. This method shows suitable performance characteristics to be regarded a useful tool to monitor toxin levels in a variety of sample matrices during future bloom events.

A deep cavitand with a fluorescent wall functions as an ion sensor.

The synthesis and characterization of a deep cavitand bearing a fluorescent benzoquinoxaline wall is reported. Noncovalent host-guest recognition events are exploited to sense small charged molecules including acetylcholine. The cavitand also exhibits an anion dependent change in fluorescence that is used to differentiate halide ions in solution.

Feasibility of using a surface plasmon resonance-based biosensor to detect and quantify yessotoxin.

Yessotoxin (YTX) is a disulfated polyether toxin produced by marine dinoflagellates. Although there is no clear evidence that YTX is toxic to humans, it is a major cause of false positives in DSP toxin detection by mouse bioassay. We developed a new detection and quantification method for yessotoxin using a BiaCore X Surface plasmon resonance (SPR)-based biosensor. The assay is based in the interaction of YTX with phosphodiesterase enzymes (PDE), one of its cellular targets. The injection of several YTX concentrations (3-12 microM) over immobilized PDE I, showed a dose dependent binding signal, which K(obs) (observed rate constant) allowed us to obtain a calibration curve with a linear fit. The detection of yessotoxin using SPR-based biosensor allows the quantification of the toxin with an automated and repetitive method at concentrations in the range of the 1 mg kg(-1) European regulatory limit.

Extraction and cleaning methods to detect yessotoxins in contaminated mussels.

Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from mussels that does not interfere with this detection method. YTX concentrations were measured in spiked mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally contaminated mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.

Feasibility of gamma irradiation as a stabilisation technique in the preparation of tissue reference materials for a range of shellfish toxins.

The effect of gamma-irradiation on concentrations of hydrophilic and lipophilic phycotoxins has been investigated by use of HPLC-UV and LC-MS. Pure toxins in organic solvents and toxins in mussel (Mytilus edulis) tissues were irradiated at three different doses. In solution all toxin concentrations were reduced to some extent. Most severe decreases were observed for domoic acid and yessotoxin, for which the smallest dose of irradiation led to almost complete destruction. For pectenotoxin-2 the decrease in concentration was less severe but still continuous with increasing dose. Azaspiracid-1 and okadaic acid were the least affected in solution. In shellfish tissue the decrease in toxin concentrations was much reduced compared with the effect in solution. After irradiation at the highest dose reductions in concentrations were between ca. 5 and 20% for the lipophilic toxins and there was no statistical difference between control and irradiated samples for azaspiracids in tissue. Irradiation of shellfish tissues contaminated with domoic acid led to a more continuous decrease in the amount of the toxin with increasing dose. The effect of irradiation on the viability of microbial activity in shellfish tissues was assessed by using total viable counting techniques. Microbial activity depended on the type of shellfish and on the pretreatment of the shellfish tissues (with or without heat treatment). As far as we are aware this is the first investigation of the effectiveness of irradiation as a technique for stabilising tissue reference materials for determination of phycotoxins. Our results suggest that this technique is not effective for materials containing domoic acid. It does, however, merit further investigation as a stabilisation procedure for preparation of shellfish tissue materials for some lipophilic toxins, in particular azaspiracids. Chemical structures of the toxins investigated in the study.

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.

Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure.

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3',5'-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein-dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.

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).

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.

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.

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.

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.

Nano liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry for the determination of yessotoxin in marine phytoplankton.

Studies of yessotoxin involving confirmation of fragmentation processes using a high-resolution orthogonal hybrid quadrupole time-of-flight (QqTOF) mass spectrometer and nanoLC hybrid quadrupole TOF MS have been undertaken. The fragmentation of YTX was studied in negative mode using nano electrospray (nanoESI) QqTOF mass spectrometry. Three major molecule-related ions were observed, [M - 2Na + H]-, [M - Na]- and [M - 2Na]2-, and fragmentation of the latter was studied in detail. This showed that product ions were formed as a consequence of charge-remote fragmentation processes that included a strong directional cleavage of the polyether rings of YTX. NanoLC coupled with QqTOF MS was used to determine YTX in small samples of the phytoplankton, Protoceratium reticulatum, by monitoring the [M - 2Na]2- ion at m/z 570. A PepMap C18 nanoLC column (75 microm x 10 cm, 100 A, 3 microm, LC Packings) was used and the solvent was acetonitrile/water (90:10 (v/v)) containing 1 mM ammonium acetate, at a flow rate of 400 nl/min, for 30 min. Calibrations obtained with YTX standard solutions were linear over four orders of magnitude, 0.75-250 ng/ml; r2 = 0.9947-0.9998. Phytoplankton cells (ca. 100-300) were picked, extracted with methanol/water (40:60), and the YTX concentration was determined over the range 0.011-0.020 ng/cell. The detection limit (3 x S/N) of this method was ca. 0.5 pg YTX on-column.

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.

Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves.

A simple and sensitive in situ method for monitoring the occurrence of toxic algal blooms and shellfish contamination events has been developed. The technique involves the passive adsorption of biotoxins onto porous synthetic resin filled sachets (SPATT bags) and their subsequent extraction and analysis. The success of the method is founded on the observation that during algal blooms significant amounts of toxin, including the low polarity lipophilic compounds such as the pectenotoxins and the okadaic acid complex toxins, are dissolved in the seawater. The results of field trials during Dinophysis acuminata and Protoceratium reticulatum blooms are presented. These data prove the concept and demonstrate that the technique provides a means of forecasting shellfish contamination events and predicting the net accumulation of polyether toxins by mussels. As an early warning method it has many advantages over current monitoring techniques such as shellfish-flesh testing and phytoplankton monitoring. In contrast to the circumstantial evidence provided by genetic probe technologies and conventional phytoplankton monitoring methods, it directly targets the toxic compounds of interest. The extracts that are obtained for analysis lack many of the extraneous lipophilic materials in crude shellfish extracts so that many of the matrix problems associated with chemical and biological analysis of these extracts are eliminated. Analyses can confidently target parent compounds only, because analytical and toxicological uncertainties associated with the multiplicity of toxin analogues produced by in vivo biotransformation in shellfish tissues are reduced. Time integrated sampling provides a good simulation of biotoxin accumulation in filter feeders and the high sensitivity provides lengthy early warning and conservative estimates of contamination potential. The technique may reduce monitoring costs and provide improved spatial and temporal sampling opportunities. When coupled with appropriate analytical techniques (e.g. LC-MS/MS multi-toxin screens, ELISA assays, receptor binding assays), the technique has the potential to offer a universal early warning method for marine and freshwater micro-algae toxins.