Discovery of gymnodimine fatty acid ester metabolites in shellfish using liquid chromatography/mass spectrometry.

RATIONALE: Gymnodimines (GYMs) are fast-acting toxins that belong to the cyclic imine group, a subclass of lipophilic marine toxins. GYMs are considered to be emerging toxins but have not yet been linked to incidents of human poisoning, Limited knowledge on the metabolism of GYMs means that a proper risk assessment has not been possible and caution must be taken when establishing the relevance of GYMs in terms of food safety of marine products. METHODS: A series of mass spectrometric experiments involving precursor and product ion scans, selected reaction monitoring (SRM), and high-resolution mass spectrometry (MS) were used to detect and confirm 10-O-acyl esters of gymnodimine-A (1). RESULTS: We have detected for the first time the presence of a range of acyl ester derivatives of GYMs in shellfish samples from the Gulf of Gabes, Tunisia. The MS fragmentation pathways of 1 and its esters were also elucidated. Partial synthesis of a palmitic acid ester of 1 facilitated confirmation of identity and calibration of SRM analyses. Evidence of acyl ester metabolites of gymnodimine-B and -C was also obtained. CONCLUSIONS: A semi-quantitative analysis indicated that the majority of GYMs present in the sample were in the acylated form (>90%), suggesting that these compounds must not be neglected when trying to understand the risks associated with GYMs. There is a clear need for toxicology studies on these esters and assessment of bio-availability to humans.

Spatial and temporal variations of a saxitoxin analogue (LWTX-1) in Lyngbya wollei (Cyanobacteria) mats in the St. Lawrence River (Québec, Canada).

The concentration of the saxitoxin analogue LWTX-1 was quantified in samples of the benthic filamentous cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck collected in two fluvial lakes of the St. Lawrence River (Canada) over the 2006-2013 period. The study was aimed at documenting the spatial (between fluvial lakes, between sites within each lake) and temporal (inter-annual, monthly) variations of toxin concentration in relation with hydrological (water level), physical (water temperature, conductivity, transparency), chemical (nutrients in overlying water) and biological (L. wollei biomass and mat condition) characteristics. Toxin concentration was hypothesized to vary seasonally with biomass accumulation and environmental conditions. Toxin concentrations measured in Lake Saint-Louis (51±40µg LWTX-1g-1 DM, N=29 days in 2007, 2009-2011) were double those in Lake Saint-Pierre (25±31µg LWTX-1g-1 DM, N=26 days in 2006-2008, 2012-2013); however, August 2007 measurements taken from both lakes did not differ significantly. Ten of the twelve highest values (>100µg LWTX-1g-1 DM) were obtained from Lake Saint-Louis, between April and October in 2007, 2010 or 2011. Under ice samples showed intermediate concentrations of LWTX-1 (42±9µg LWTX-1g-1 DM, N=2). Concentrations of LWTX-1 were positively correlated with Secchi depth (r=0.59, p<0.001), L. wollei biomass (Spearman r=0.31, p<0.01) and %N in filaments (r=0.48, p<0.001), suggesting toxin production was linked to mat growth and metabolism rather than water quality. Although LWTX-1 has been reported to have a low toxicity, monitoring of L. wollei abundance is required to assess the environmental and human health risks posed by this taxon in the St. Lawrence - Great Lakes system.

Synthesis, characterization, and Ca2+ antagonistic activity of diltiazem metabolites.

Diltiazem is a calcium antagonist widely used in the treatment of angina and hypertension. The contributions of metabolites of diltiazem to the vasorelaxant effects of diltiazem were investigated. The synthesis and spectroscopic characterization of eight major cis-diltiazem metabolites are described. Three of the compounds--N, O-didemethylated metabolite (21), O-demethylated metabolite (22), and diltiazem N-oxide (27)--have been recently reported and have not previously been synthesized. The identities of all eight synthetic metabolites have been verified with samples obtained from human urine using combined LC-MS/MS. The Ca2+ antagonistic activities of diltiazem and its metabolites (except 27) were studied on hamster aorta preparations depolarized with KCl. The order of potencies (IC50 +/- SE, microM) is as follows: diltiazem (0.98 +/- 0.47) greater than 17 (2.46 +/- 0.38) greater than or equal to 23 (3.27 +/- 1.02) greater than 26 (20.2 +/- 10.5) greater than 22 (40.4 +/- 15.4) greater than or equal to 25 (45.5 +/- 18.1) greater than 21 (112.2 +/- 33.2) greater than or equal to 24 (126.7 +/- 24.2). Structure-activity relationships are also discussed.

Characterization of flame-generated C10 to C 160 polycyclic aromatic hydrocarbons by atmospheric-pressure chemical ionization mass spectrometry with liquid introduction via heated nebulizer interface.

Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) generated from fuel-rich combustion of ethylene-naphthalene mixtures in a jet-stirred-plug-flow reactor were chemically characterized by combined mass spectrometric techniques to yield product composition data that cover the molecular mass region from simple PAHs (naphthalene, 128 u) to large molecules comparable in molecular size (1792 u) to nanoparticles of soot. Two techniques based on atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) were investigated: (1) APCI-MS combined with high-performance liquid chromatography through a heated nebulizer interface was found suitable for PAHs up to C36 (448 u). (2) For the characterization of larger PAHs beyond C36, direct liquid introduction (DLI) of sample into an atmospheric-pressure chemical ionization mass spectrometer through a heated nebulizer gave protonated molecular ions for PAHs over the m/z 400-2000 range. Although unequivocal elemental composition information is unattainable from the unit-resolution DLI/APCI-MS data, by starting with structural data from identified C16 to C32 PAHs, and applying PAH molecular growth principles, it was possible to generate PAH molecular maps from the DLI/APCI-MS data from which values for the elemental composition could be derived for all major peaks.

First report of the cyanobacterial toxin cylindrospermopsin in New Zealand.

Cylindrospermopsin, a hepatotoxin produced by cyanobacteria, has been unambiguously detected in cyanobacteria collected from a recreational lake in the Wellington region of New Zealand. To our knowledge this is the first report of cylindrospermopsin in New Zealand. Cylindrospermopsin and several microcystins were identified by reversed phase high-performance liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) using an atmospheric-pressure ionization source and an ionspray interface. The presence of cylindrospermopsin in New Zealand highlights the risk of direct cyanobacterial toxin exposure in recreational waters.

An idiotypic-anti-idiotypic competitive immunoassay for quantitation of okadaic acid.

A competitive indirect enzyme-linked immunosorbent assay for the measurement of okadaic acid, a marine toxin, was developed. The assay uses a murine monoclonal anti-idiotypic antibody bearing an internal image of okadiac acid epitope to capture an anti-okadaic acid monoclonal antibody in the presence of free okadaic acid. Bound anti-okadaic acid antibody is detected with peroxidase-conjugated anti-mouse immunoglobulin antiserum. If present, free toxin will lessen the amount of anti-okadaic acid antibody binding to its corresponding anti-idiotypic antibody in a dose-dependent manner that can be quantified from the standard curve. The assay permits reliable measurement of okadaic acid in the 9-81 ng/ml range. The intra- and interassay coefficients of variation in the measurement of OA in the toxin spiked mussel samples averaged 9% and 12%, respectively. The assay is rapid, accurate, reproducible and relatively simple to perform. It may be of potential use to laboratories involved in monitoring the toxin levels in plankton, seafood or sponges.

Detection of new 7-O-acyl derivatives of diarrhetic shellfish poisoning toxins by liquid chromatography-mass spectrometry.

A novel method for the detection of acylated diarrhetic shellfish poisoning toxins is reported. Direct determination of these compounds is possible using high performance liquid chromatography coupled with ion-spray mass spectrometry. An extract, purified from the digestive glands of toxic mussels (Mytilus edulis) contaminated with okadaic acid, dinophysistoxin-1, and a recently reported analog, dinophysistoxin-2, was also shown to contain small amounts of dinophysistoxin-3, a mixture of 7-O-acyl ester derivatives of dinophysistoxin-1. In addition, acyl ester derivatives of okadaic acid and dinophysistoxin-2 were also detected by direct LC-MS analysis and confirmed by analysis of their hydrolysis products. This is the first report of the detection of other naturally occurring 7-O-acyl esters similar to dinophysistoxin-3.

Comparative toxicity of the diarrhetic shellfish poisons, okadaic acid, okadaic acid diol-ester and dinophysistoxin-4, to the diatom Thalassiosira weissflogii.

Quantitative structure-activity relationships were determined for the diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), OA diol-ester and dinophysistoxin-4 (DTX-4), using a sensitive bioassay procedure with the diatom Thalassiosira weissflogii. OA diol-ester was found to be nearly as toxic as OA. This result contradicted the accepted idea that only the free acid toxins, such as DTX-1 and OA, are potent phosphatase inhibitors. Postassay analyses using liquid chromatography-mass spectrometry (LC-MS) of cultures incubated with OA diol-ester showed that the ester had partially decomposed to OA, which explained some but not all of the observed toxicity. The formation of OA during the bioassay raised the possibility that cells exposed to inactive DSP toxin esters could metabolically activate them. This was examined in an additional experiment which showed that the hydrolysis of both DTX-4 and OA diol-ester was spontaneous and apparently not mediated by the presence of T. weissflogii cells. However, cells of T. weissflogii challenged with OA diol-ester rapidly metabolized most of the toxin to a more water-soluble product. From interpretation of mass spectral data obtained using ion-spray LC-MS, the metabolite was identified as an oxygenated diol-ester of OA, implying that it was the product of a monooxygenase-detoxification pathway. It is postulated that OA diol-ester, as a lipid-soluble, uncharged molecule with a propensity to hydrolyse to OA, may facilitate the transfer of OA across cell walls and membranes.

Improved method for preparation and use of 9-anthryldiazomethane for derivatization of hydroxycarboxylic acids. Application to diarrhetic shellfish poisoning toxins.

Application of a method for the "in situ" generation of 9-anthryldiazomethane (ADAM) to the derivatization of the carboxyl function in diarrhetic shellfish poisoning (DSP) toxins revealed the formation of artifact products. Using liquid chromatography-mass spectrometry, it was determined that these artifacts were due to base-catalyzed reactions between the solvent, ethyl acetate, and the hydroxyl groups of the analyte to produce O-acetylated ADAM derivatives. Using a new formulation, with tetrahydrofuran as solvent, it was possible to eliminate these artifact reactions. Various reaction parameters have also been re-optimized to ensure quantitative derivatizations. An assessment method was developed that was useful not only for optimizing reaction parameters, but also for evaluating the reagent potency before use on important samples. Finally, application of the method to the determination of DSP toxins in plankton and mussel tissue was demonstrated.

Mass spectra of sterically crowded trialkylsilyl ether derivatives of steroids.

The electron-impact mass spectra of the title compounds have some important features which give these derivatives certain advantages over the widely studied trimethylsilyl analogues. There is significantly less extensive fragmentation, and abundant ions at (M - t.Bu)+ or (M - i.Pr)+ serve as indicators of molecular weight and should be useful for selected ion monitoring. From various precursors, the ease of elimination of HX2SiOH, via a proposed multi-centre transition state, appears to depend upon conformational and stereochemical factors, as well as the position of the parent silyloxy group, RX2SiO, on the steroid skeleton. This particular fragmentation appears to be a powerful diagnostic method for distinguishing between stereoisomers, being especially useful for differentiation between epimers. In addition, the presence of a 17-silyloxy function promotes a characteristic cleavage of ring B in a skeletally saturated steroid. Elimination of silanol, RX2SiOH, at various stages in the fragmentations of bis-silylated steroids is also an important process, but other familiar features of the spectra of steroid trimethylsilyl ethers, though usually present, are very much suppressed.

Sterically crowded trialkylsilyl ether derivatives for the analysis of steroid metabolites.

The applications of sterically crowed trialkyksilyl ether derivatives to the analysis and characterization by thin-layer chromatography, gas chromatography and mass spectrometry, of metabolites of 2alpha, 3alpha-cyclopropano-5alpha-androstan-17beta-ol in the rabbit are described. These derivatives are complementary to the familiar trimethysilyl ether derivatives, but have greater hydrolytic stability (an advantage for TLC), generally give better GC separations, and have characteristic mass spectra. Isomer differentiation by GC and MS is also more readily achieved than via the TMSi ether derivatives. These properties should make SCTASi ethers useful derivatives for studies of steroid metabolism.

Metabolism of 1,8-dinitropyrene by Salmonella typhimurium.

Earlier work has shown that many nitroaromatic and nitroheterocyclic compounds are directly 'activated' to their ultimate mutagenic forms through the action of bacterial nitroreductase enzymes. However, in the case of 1,8-dinitropyrene (DNP) and certain other nitroarenes the pathway of activation is more complex and neither the identity of the ultimate mutagens nor the nature of the DNA adducts formed are known. We now show that Salmonella typhimurium strains TA98 and TA1538, which are sensitive to DNP and have wild type nitroreductase complements, do metabolize DNP to 1-amino-8-nitropyrene (ANP) and 1,8- diaminopyrene (DAP) but that these compounds are much weaker mutagens than DNP. These two strains (TA98 and TA1538) contain two separable components of nitroreductase activity as determined using nitrofurazone as the substrate. The major component, at least, is capable of reducing both 1-nitropyrene (NP) and DNP although the rates are much lower than with nitrofurazone. TA98NR , a mutant of TA98 that is resistant to nitrofurazone and NP but not to DNP, lacked the major nitroreductase but retained two minor components. In contrast, a mutant ( DNP6 ) which is resistant to DNP (but not to NP) contained a full complement of nitroreductases. When the metabolism of [3H]DNP by crude extracts of TA98 was re-examined, previously undetected metabolites were found. These were more polar than DAP and ANP and were also seen when TA98NR was used as the source of enzyme. These metabolites were not formed when enzymes from TA98DNP6 or TA98NR / DNP6 were used. This work supports the notion that some enzymic activity other than (or in addition to) nitroreductase is required for the activation of DNP and that the new polar metabolites may be related to this process.

Dinitropyrene-resistant Salmonella typhimurium are deficient in an acetyl-CoA acetyltransferase.

Salmonella typhimurium strain TA98NR which is sensitive to 1,8-dinitropyrene mutagenesis possesses acetyl-CoA dependent acetyltransferase activity, while a strain selected for resistance to 1,8-dinitropyrene (TA98/1, 8-DNP6) is deficient in this activity. Acetyltransferase competent strains can acetylate 1,8-diaminopyrene, forming 1-N-acetylamino-8-aminopyrene and 1,8-N,N'-diacetyldiaminopyrene. The coincidence of dinitropyrene resistance and acetyltransferase deficiency implicates acetylation as an important process in the metabolic activation of dinitropyrene to a mutagenic intermediate.

Amaranth suppresses the mutagenicity of 2-acetylaminofluorene by lowering the concentration of NADPH in top agar.

Addition of 1 mg amaranth (FD&C Red No. 2) to the top agar of Salmonella/S9 assay plates decreased the yield of revertants induced by 20 micrograms 2-acetylaminofluorene (AAF) by over 50% and additional amaranth completely eliminated the mutagenic response. Similar suppression of AAF mutagenicity was seen with sulfonazo III, another azo dye. The suppressive effect of amaranth was greatest at low S9 concentrations and decreased as the amount of S9 was increased. When N-hydroxyacetylaminofluorene (N-OH-AAF) was used as mutagen, amaranth had little or no effect on either the number of revertants obtained or the S9 optimum. Similarly, 1-naphthylamine-4-sulfonic acid (a reduction product of amaranth) did not significantly affect the mutagenicity of AAF. The rate of metabolism of [14C]AAF by the S9 preparations was shown to be markedly decreased by amaranth, as were the levels of both the phenolic metabolites and of N-OH-AAF. Thus, it appeared that amaranth acts by blocking the conversion of AAF to N-OH-AAF and that this effect is caused by the amaranth itself and not by its constituent amines. Further experiments indicated that amaranth greatly decreased the levels of NADPH formed in reaction mixtures comparable to S0 mix in top agar and that such reaction mixtures also metabolized amaranth to colourless compounds. It appears likely that in top agar, NADPH reacts with amaranth at a fast enough rate to limit severely the level of the reduced co-factor (which must be formed from NADPH+ by the action of endogenous glucose-6-phosphate dehydrogenase) and thus decreases the rate of activation of mutagens by other NADPH-dependent processes.

Further studies on the analysis of DSP toxin profiles in galician mussels.

Further studies on mussel samples from Galicia, Spain, have revealed the presence of okadaic acid (OA), dinophysistoxin 2 (DTX2), and the fatty acid acyl esters of both of these toxins as the "DTX3" complex. Measurements were performed with an improved in situ method for the formation of 9-anthryldiazomethane (ADAM) derivatives followed by liquid chromatography with fluorescence detection. Base hydrolysis of DTX3 toxins gave free OA and DTX2, which were determined following ADAM derivatization. Results were confirmed by liquid chromatography/mass spectrometry analyses, and in most of the samples, free DTX2 was the most abundant toxin. However, the OA/DTX2 ratio in the DTX3 conjugated form was different, with OA being the most abundant in all cases. This difference could be due to different rates of metabolism of OA and DTX2 to the acyl esters or due to contamination of the shellfish by the two toxins at different points in time, resulting in less acyl ester formation for one toxin versus the other. The second possibility would be reasonable if two different source organisms were producing the toxins.

Analysis of diarrhetic shellfish poisoning toxins in shellfish tissue by liquid chromatography with fluorometric and mass spectrometric detection.

Diarrhetic shellfish poisoning (DSP) is a severe gastrointestinal illness caused by consumption of shellfish contaminated with toxigenic dinoflagellates. The main toxins responsible for DSP are okadaic acid (OA), DTX-1, DTX-2, and DTX-3, the latter being a complex mixture of 7-O-acyl derivatives of the first 3. In this study, existing methods based on liquid chromatography (LC) combined with mass spectrometry (LC-MS) and LC with fluorometric detection (LC-FLD) of anthryldiazomethane (ADAM) derivatives were improved upon to achieve a high degree of accuracy and precision for the determination of DSP toxins in a new mussel tissue reference material (MUS-2). All experimental parameters were examined comprehensively, and a new internal standard and a new solid-phase extraction cleanup method were introduced. Quantitative extraction of DSP toxins from shellfish tissue was achieved by exhaustive extraction with aqueous 80% methanol. Cleanup was accomplished by partitioning the crude aqueous methanol extract with hexane to remove lipids and then with chloroform to isolate the toxins. A further cleanup based on an aminopropylsilica column was useful for LC-MS and looks promising for the ADAM/LC-FLD method. The internal standard, 7-O-acetylokadaic acid, synthesized by partial acetylation of OA, improved accuracy and precision by correcting for incomplete recoveries in extraction, cleanup, and derivatization steps and for volumetric errors and instrumental drift. An improved silica cleanup after ADAM derivatization also was developed by controlling the activities of both sorbent and solvents. The methods were tested with various mussel tissue samples. The resulting improved methods will be useful to analysts involved in routine monitoring of DSP toxins.

Phycotoxins.

The 1997-1998 period brought many new developments to the phycotoxin field. There were several reviews on phycotoxins in general, on their toxicological evaluation, and on their analysis. The ecophysiology, biosynthesis, and metabolism of polyether toxins and paralytic shellfish poisoning (PSP) toxins were also reviewed. The proceedings of the Eighth International Conference on Harmful Algae (Vigo, Spain, June 25-29, 1997) have been published and provide an excellent source of information on phycotoxins and toxic plankton bloom research. In addition, the much anticipated proceedings of the IX International IUPAC Symposium on Mycotoxins and Phycotoxins (Rome, Italy, May 27-31, 1996) have been published. Further evidence was provided to support the theory that Prorocentrum lima is the source organism for diarrhetic shellfish poisoning (DSP) toxins in Nova Scotian shellfish. In another study, different Prorocentrum species and isolates were analyzed for DSP toxins. In addition to detecting some new compounds, such as a DTX1 isomer, it was found that toxins were produced by both axenic and nonaxenic batch cultures, indicating that bacteria are probably not involved in the biosynthesis. The source organism for the spirolides, a family of fast-acting toxins reported from Nova Scotia, Canada, was determined to be Alexandrium ostenfeldii, a species that is found worldwide. The biogenetic origin of yessotoxin was reported to be Protoceratium reticulatum, another widely occurring organism. A great deal of attention and research funding has been directed at the serious problems associated with Pfiesteria piscicida. Analysts are eagerly awaiting publication of toxin structures, which will then allow the development of analytical methods. An incident of the mass mortality of California sea lions was reported in the Monterey area in May 1998. Analyses of tissue and urine samples revealed the presence of domoic acid. High levels of domoic acid were also found in anchovies and sardines, a common food source of sea lions. This is reminiscent of an incident of mass bird mortality in 1992 in the same region. Toxicological studies of domoic acid continue with one investigation on the effect of pH on toxicity in the mouse assay and others examining toxic effects in rats and cynomolgus monkeys. A study on the uptake and depuration of domoic acid in the Dungeness crab was reported. On October 20, 1997, EU (European Union) directive CE97/61 established a regulatory limit of 20 ppm for domoic acid in European shellfish, the same level as in North America. A detailed study on the oral toxicity of DSP toxins in mice was reported. Recent work by several researchers has revealed the genotoxic potential of okadaic acid and other DSP toxins. Previous work had clearly demonstrated the tumor-promoting potential of DSP toxins, but this recent evidence, which shows mutations in the progeny of okadaic acid-treated cells and the formation of DNA-adducts, increases concerns over the hazards associated with DSP-contaminated shellfish. The toxicology of yessotoxin was evaluated by Ogino et al. The toxin showed weak cytotoxicity, but was not orally lethal to mice at 10 mg/kg, and did not cause intestinal fluid accumulation, inhibition of protein phosphatase 2A (PP2A), or hemolytic effects. Similarly, Tubaro et al. saw no evidence for diarrheogenicity of homoyessotoxin isolated from mussels and from the proposed planktonic producer, Lingulodinium polyedrum. All this provides further evidence that yessotoxin should not be classed as a DSP toxin. A number of new toxins have been detected and identified. Two analogues of yessotoxin, homoyessotoxin, and 45-hydroxyhomoyessotoxin were isolated from mussels of the Adriatic Sea and identified by Satake et al. A recent DSP event in Ireland associated with cultured mussels led to the identification of azaspiracid, a unique marine toxin with spiro ring assemblies. (ABSTRACT TRUNCATED)

Screening for okadaic acid by immunoassay.

Increasing incidences of phytoplankton blooms with the potential danger of toxin release into the food chain have necessitated the search for new diagnostic methods that can detect toxins quickly and reliably. A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantitate okadaic acid in shellfish and phytoplankton extracts. To determine the specificity of the assay, a number of toxins, such as calyculin A, brevetoxin-1, and dinophysistoxins-1, -2, and -3 were analyzed. Both dinophysistoxins-2 and -1 could be detected by the assay but in concentration ranges 10- and 20-fold higher than that for okadaic acid, respectively. Dinophysistoxin-3, calyculin A, or brevetoxin-1 could not be detected with this assay. To validate the accuracy of the method, 18 mussel and 7 phytoplankton extracts were analyzed in parallel for okadaic acid content by ELISA and liquid chromatography combined with either fluorescence or mass spectrometric detection. Very high correlation between the results was found.

Determination and confirmation of the amnesic shellfish poisoning toxin, domoic acid, in shellfish from Scotland by liquid chromatography and mass spectrometry.

During 1998 and early 1999, shellfish samples from sites in Scotland were found to contain the amnesic shellfish poisoning toxin, domoic acid (DA). Two different techniques, liquid chromatography (LC) with UV diode-array detection and LC with mass spectrometric (MS) detection, were used to detect and confirm DA in shellfish extracts. The LC/UV method was validated for routine monitoring by recovery experiments on spiked mussel and scallop tissues with a certified mussel tissue used as reference material. Crude extracts of selected samples as well as extracts cleaned with strong anion exchange (SAX) were analyzed by both LC/UV and LC/MS. Good correlation (linear regression r2 = 0.996, slope = 0.93) between the 2 methods was found for cleaned extracts. Analyses of crude extracts by LC/UV produced false-positive results in 2 crab samples, whereas LC/MS analyses gave accurate results. It was concluded that LC/UV is a valid approach for routine monitoring of DA in shellfish when cleanup is performed with a SAX cartridge to prevent false positives. A variety of shellfish species were surveyed for DA content, including Pecten maximus (king scallops), Chlamys opercularis (queen scallop), Mytilus edulis (blue mussels), Cancer pugaris (crab), and Ensis ensis (razor fish). The highest concentration of DA was 105 microg/g in Pecten maximus.

Determination of polycyclic aromatic compounds by high-performance liquid chromatography with simultaneous mass spectrometry and ultraviolet diode array detection.

A major problem in the determination of polycyclic aromatic compounds (PACs) in environmental samples is the extreme complexity of the extracts, even after extensive fractionation. The combination of high-performance liquid chromatography (HPLC) with simultaneous mass spectrometry (MS) and ultraviolet diode array detection (DAD) is a powerful tool for the identification and quantitation of such species with a high degree of confidence. HPLC allows the selective separation of a wide variety of PACs, including thermally labile and high molecular weight compounds. Electron ionization MS with the moving belt interface provides high sensitivity and selectivity, as well as structural information such as molecular weight, functional groups, and elemental composition. The diode array detector helps to differentiate isomeric structures and confirm compound identity.