Liquid chromatography-electrospray ionization mass spectrometry of the diarrhetic shellfish-poisoning toxins okadaic acid, dinophysistoxin-1 and pectenotoxin-6 in bivalves

Determination of diarrhetic shellfish-poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-6 (PTX6) was carried out by liquid chromatography (LC) followed by on-line atmospheric pressure electrospray ionization-mass spectrometric (ESI-MS) detection with a heated capillary interface. Mass spectra of authentic OA, DTXI and PTX6 standards exhibited abundant [M-H] at m/z 803, 817 and 887, respectively. Linearity of peak area obtained by selected-ion monitoring (SIM) for [M-H]- of each toxin was confirmed over a wide range of concentrations from 10 pg to 30 ng. LC-ESI-MS analysis of OA, DTX1 and PTX6 in scallops and mussels, collected at the same site (Mutsu Bay, Japan), was carried out. Scallops and mussels collected at the same site showed different toxin profiles. Although PTX6 was detected from scallops, it was not detected from mussels.     

Detailed profiles of 7-O-acyl esters in plankton and shellfish from the Portuguese coast

In bivalve mollusks from the Portuguese coast contaminated by diarrhetic shellfish poisoning (DSP), most of the parent toxins, okadaic acid (OA) or dinophysistoxin-2 (DTX2), are found esterified, and toxicity assessment is only performed after an alkaline hydrolysis step to recover the parent molecules in their free form. The presence of 7-O-acyl esters with fatty acids (FAs) has already been confirmed previously in Mytilus galloprovincialis and Donax trunculus samples. This paper reports the presence of acyl esters in a wider range of estuarine and offshore bivalve species found by direct analysis in LC-MS. The total of acyl esters found in each species represented the percentages commonly found by hydrolysis in those species in previous years, justifying the majority of the esters commonly found in shellfish. This implies that any diol esters remaining after digestion of toxic microalgae would represent only a minor contribution to the ester's contents. Esters with C14:0, C16:0, C16:1, C20:5 and C22:6 FAs were the most abundant, followed by esters with C18:0, C18:1, C18:2, C18:3 and C18:4. This is the first report of OA and DTX2 esters with odd FAs: C15:0, C17:0, C17:1, and probably a branched FA: iso-C16:0. Esters with iso-C16:0 where found in high percentages particularly in two species of estuarine clams, where they represented 13-34% of total esters found. Esters were also found in plankton, predominantly with C16:0. Total esters in plankton were not higher than 10%, not enough to justify per se the high levels found in bivalves.     

Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry

Quantitative determination by liquid chromatography (LC) coupled with mass spectrometry (MS) was achieved for the following 10 toxins found in association with diarrhetic shellfish poisoning: okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoylokadaic acid (palOA), 7-O-palmitoyldinophysistoxin-1 (pa1DTX1), pectenotoxin-1 (PTX1), pectenotoxin-2 (PTX2), pectenotoxin-2 seco acid (PTX2SA), pectenotoxin-6 (PTX6), yessotoxin (YTX), and 45-hydroxyyessotoxin (YTXOH). Toxins in 2 g of the adductor muscle or the digestive glands of scallops, Patinopecten yessoensis, were extracted with 18 ml of methanol-water (9:1, v/v), freed of polar contaminants by partition between chloroform and water, and treated by solid-phase extraction on a silica cartridge column. Samples containing YTXOH were purified separately on a buffered reversed-phase column. Chromatographic separation was achieved by the following combinations of columns and mobile phases: a Symmetry C18 column with acetonitrile-0.05% acetic acid (7:3, v/v) for OA, DTX1, PTX6 and PTX2SA; a Develosil ODS column with the same mobile phase for PTX1 and PTX2; a Capcellpak column with methanol-2.5% acetic acid (98:2, v/v) for palOA and palDTX1; and an Inertsil ODS column with methanol-0.2 M ammonium acetate (8:2, v/v) for YTX and YTXOH. Carboxylic acid toxins were selectively monitored on [M-H]- ions, sulfated toxins on [M-Na]-ions, and neutral toxins on [M+NH4]+ ions. Average recoveries of the toxins spiked to tissue homogenates ranged from 70 to 134%. Detection limits in the muscle ranged from 5 to 40 ng/g and those in the digestive glands from 10 to 80 ng/g.     

Ionspray mass spectrometry of marine toxins. IV. Determination of diarrhetic shellfish poisoning toxins in mussel tissue by liquid chromatography/mass spectrometry.

An improved liquid chromatographic/mass spectrometric (LC/MS) method utilizing gradient elution and ion-spray ionization is described for the sensitive determination of okadaic acid and dinophysistoxin-1, the principal toxins implicated in cases of diarrhetic shellfish poisoning. The method was used to confirm the presence of both toxins, together with a recently identified isomer of okadaic acid, dinophysistoxin-2, in various samples of cultivated blue mussels (Mytilus edulis) from Canadian and European waters. The method provided a mass detection limit of 0.4 ng for each toxin, thus allowing detection of 40 ng per g of whole mussel tissue (or approximately 10 ng/g if only the digestive glands were used in the assay). Quantitative results obtained by LC/MS were in good agreement with those obtained by derivatization and high-performance liquid chromatography with fluorescence detection.     

Enzymatic hydrolysis of esterified diarrhetic shellfish poisoning toxins and pectenotoxins

Okadaic acid (OA) and dinophysistoxins-1 and -2 (DTX1, DTX2), the toxins responsible for incidents of diarrhetic shellfish poisoning (DSP), can occur as complex mixtures of ester derivatives in both plankton and shellfish. Alkaline hydrolysis is usually employed to release parent OA/DTX toxins, and analyses are conducted before and after hydrolysis to determine the concentrations of nonesterified and esterified toxins. Recent research has shown that other toxins, including pectenotoxins and spirolides, can also exist as esters in shellfish, but these toxins cannot survive alkaline hydrolysis. A promising alternative approach is enzymatic hydrolysis. In this study, two enzymatic methods were developed for the hydrolysis of 7-O-acyl esters, “DTX3,” and the carboxylate esters of OA, “diol-esters.” Porcine pancreatic lipase induced complete conversion of DTX3 to OA and DTXs within one hour for reference solutions. The presence of mussel tissue matrix reduced the rate of hydrolysis, but an optimized lipase concentration resulted in greater than 95% conversion within four hours. OA-diol-ester was hydrolyzed by porcine liver esterase and was completely converted to OA in less than 30 min, even in the presence of mussel tissue matrix. Esters and OA/DTX toxins were all monitored by LC–MS. Further experiments with pectenotoxin esters indicated that enzymatic hydrolysis could also be applied to esters of other toxins. Enzymatic hydrolysis has excellent potential as an alternative to the conventional alkaline hydrolysis procedure used in the preparation of shellfish samples for the analysis of toxins.     

Sensitive HPLC-fluorometric and HPLC-MS determination of diarrhetic shellfish poisoning (SP)-toxins as 4-bromomethyl-7-methoxycoumarin esters

 Extracts containing the diarrhetic shellfish poisoning (DSP) toxins okadaic acid (OA), dinophysistoxin-2 (DTX₂), and dinophysistoxin-1 (DTX₁) were purified on a silica gel cartridge and derivatized with 4-bromomethyl-7 methoxycoumarin (BrMmc). After pre-column derivatization the BrMmc derivatives of the DSP toxins were directly injected into an HPLC system, isocratically eluted, and quantified by fluorescence detection. The signals of the esters showed good linearity in the fluorescence detector within the examined contamination range of 0.03 mg DSP/kg to 2.5 mg DSP/kg. The detection limits for the DSP toxins as 7-Mmc esters were 0.04 ng (corresponding to 0.05 mg DSP/kg). The chromatographic conditions allow to couple the HPLC device with mass spectrometry. The method was tested with various mussel tissue samples. Received: 14 December 1995/Revised: 26 January 1996/Accepted: 30 January 1996     

Sensitive HPLC-fluorometric and HPLC-MS determination of diarrhetic shellfish poisoning (SP)-toxins as 4-bromomethyl-7-methoxycoumarin esters

 Extracts containing the diarrhetic shellfish poisoning (DSP) toxins okadaic acid (OA), dinophysistoxin-2 (DTX₂), and dinophysistoxin-1 (DTX₁) were purified on a silica gel cartridge and derivatized with 4-bromomethyl-7 methoxycoumarin (BrMmc). After pre-column derivatization the BrMmc derivatives of the DSP toxins were directly injected into an HPLC system, isocratically eluted, and quantified by fluorescence detection. The signals of the esters showed good linearity in the fluorescence detector within the examined contamination range of 0.03 mg DSP/kg to 2.5 mg DSP/kg. The detection limits for the DSP toxins as 7-Mmc esters were 0.04 ng (corresponding to 0.05 mg DSP/kg). The chromatographic conditions allow to couple the HPLC device with mass spectrometry. The method was tested with various mussel tissue samples. Received: 14 December 1995/Revised: 26 January 1996/Accepted: 30 January 1996     

Confirmation of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish as their anthrylmethyl derivatives using UV radiation

A rapid and simple method for confirmation of the diarrhetic shellfish poisons (DSP): okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) using fluorescence detection following derivatization with 9-chloromethylanthracene, has been established as an alternate to LC/MS. Exposure of the anthrylmethyl derivatives of OA, DTX-1 and DTX-2 to near UV light (300-400 nm) resulted in the loss of these compounds to below detection limits within 30 min, with a concurrent appearance of two additional compounds. Based on the mass spectral evidence, we propose that these newly formed compounds are the decarboxylation products of the derivatized diarrhetic shellfish poisons. UV radiation is, therefore, proposed as a rapid and simple confirmation technique for these DSP in mussel samples.     

液相色谱-串联质谱法同时测定贝类中大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素

建立了同时测定贝类中大田软海绵酸(okadaic acid, OA)及其衍生物鳍藻毒素(dinophysistoxin-1, DTX-1)、蛤毒素(pectenotoxin-2, PTX-2)和虾夷扇贝毒素(yessotoxin, YTX)的液相色谱-串联质谱分析方法。样品经甲醇提取,固相萃取柱净化,C18色谱柱分离,经含甲酸和甲酸铵的乙腈-水溶液为流动相梯度洗脱,选择反应监测(SRM)模式检测,正、负离子切换扫描,基质标准校正,外标法定量。结果表明,OA、DTX-1和YTX的线性范围为2.0～200.0 μg/L,定量限(以信噪比(S/N)≥10计)为1.0 μg/kg; PTX-2的线性范围为1.0～100.0 μg/L,定量限为0.5 μg/kg;几种化合物的添加平均回收率为83.1%～105.7%,相对标准偏差(RSD)为3.16%～9.29%。成功应用本法对黄海灵山湾海域采集的贝类样品进行了分析,发现部分样品中含有大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素。     

Results of a European interlaboratory method validation study for the quantitative determination of lipophilic marine biotoxins in raw and cooked shellfish based on high-performance liquid chromatography–tandem mass spectrometry. Part I: collaborative study

A European interlaboratory collaborative study was conducted to validate a method for the quantitative determination of lipophilic marine biotoxins based on high-performance liquid chromatography–tandem mass spectrometry. During this study, the diarrhetic shellfish poisoning toxins okadaic acid, dinophysis toxin1 and 2 including their esters, the azaspiracids 1-3, pectenotoxin2, and the yessotoxins were investigated at concentration levels near the limit of quantification and near the legal limit. Naturally contaminated blue mussels, both raw and cooked and spiked extracts of clams and oysters were studied and results were obtained for 16 test samples from 16 laboratories representing eight different countries. This article summarizes the study outcome concerning validation key parameters like specificity, linearity, limit of detection, accuracy/recovery, and precision. Further, influences of cooking of mussels before homogenization or hydrolysis on method robustness have been evaluated.     

Discovery of fatty acid ester metabolites of spirolide toxins in mussels from Norway using liquid chromatography/tandem mass spectrometry

Cultured mussels sampled in the spring of 2002 and 2003 from Skjer, a location in the Sognefjord, Norway, tested positive in the mouse bioassay for lipophilic toxins. In a previous report, it was established that a number of spirolides, cyclic imine toxins produced by the phytoplankton Alexandrium ostenfeldii, were present in the mussels and were responsible for the observed toxicity. The main toxin proved to be a new compound named 20-methyl spirolide G. In subsequent studies, a delayed onset of spirolide-like symptoms in the mouse bioassay exceeding the usual time limit of 20 min was observed in some samples, with symptoms and death appearing as long as 45-50 min after injection. It is well known that shellfish can extensively metabolize other toxins, such as okadaic acid and the dinophysistoxins, to fatty acid acyl esters and it is also known that a delayed onset of toxic symptoms with such metabolites can occur. Analyses performed with liquid chromatography/tandem mass spectrometry (LC/MS/MS) have revealed a complex mixture of esters of 20-methyl spirolide G in the contaminated mussels. Precursor ion scanning has delineated the range of fatty acid esters involved, while product ion scanning has provided information on structure. Identity was also supported through reaction of 20-methyl spirolide G with palmitic anhydride, which produced a derivative with a retention time and spectrum identical with one putative metabolite, 17-O-palmitoyl-20-methyl spirolide G.     

Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins

A liquid chromatography (LC)-based method with mass spectrometric (MS) detection was developed for simultaneous determination of various algal and cyanobacterial toxins extracted from phytoplankton occurring world-wide in marine waters and lakes. The method enables quantification of saxitoxin, anatoxin-A, domoic acid, nodularin, microcystins, okadaic acid and dinophysistoxin-1 with a single chromatographic run. In addition, the applied chromatographic conditions allow isolation and identification of substances suspected to be "new" microcystins (cyclic peptides) by fraction collection, hydrolysis, derivatisation of resulting free amino acids with the modified chiral Marfey's reagent N-alpha-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) and enantioselective analysis of the amino acid derivatives by LC-ESI-MS.     

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).     

Dynamic adsorption of diarrhetic shellfish poisoning (DSP) toxins in passive sampling relates to pore size distribution of aromatic adsorbent

Solid-phase adsorption toxin tracking (SPATT) technology was developed as an effective passive sampling method for dissolved diarrhetic shellfish poisoning (DSP) toxins in seawater. HP20 and SP700 resins have been reported as preferred adsorption substrates for lipophilic algal toxins and are recommended for use in SPATT testing. However, information on the mechanism of passive adsorption by these polymeric resins is still limited. Described herein is a study on the adsorption of OA and DTX1 toxins extracted from Prorocentrum lima algae by HP20 and SP700 resins. The pore size distribution of the adsorbents was characterized by a nitrogen adsorption method to determine the relationship between adsorption and resin porosity. The Freundlich equation constant showed that the difference in adsorption capacity for OA and DTX1 toxins was not determined by specific surface area, but by the pore size distribution in particular, with micropores playing an especially important role. Additionally, it was found that differences in affinity between OA and DTX1 for aromatic resins were as a result of polarity discrepancies due to DTX1 having an additional methyl moiety.     

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.     

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.     

Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish.

Two acidic analogues of the polyether marine toxin, pectenotoxin-2 (PTX-2), responsible for diarrhetic shellfish poisoning (DSP), have been isolated from the toxic marine phytoplankton (Dinophysis acuta), collected in Irish waters. Liquid chromatography with fluorimetric detection (LC-FLD) analyses of the extracts of bulk phytoplankton samples, following derivatisation with 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP), showed a complex toxin profile with peaks corresponding to okadaic acid (OA) and its isomers, dinophysistoxin-2 (DTX-2) and DTX-2C, as well as other unidentified lipophilic acids. LC-UV analysis showed the presence of a diene moiety in these new compounds and two acids have been isolated. LC coupled with mass spectrometry (MS) and tandem mass spectrometry (LC-MS-MS) were used to gain structural information. Through flow injection analysis (FIA)-MS, both in positive and negative ion modes, the molecular weight of 876 for both compounds was determined. Collision Induced Dissociation (CID) from each parent ion, as performed both in positive and negative ion mode, produced mass spectra which were very similar to those obtained for authentic PTX-2 (mw 858). These new compounds have been confirmed to be pectenotoxin-2 seco acids (PTX-2SAs) and they are closely related to PTX-2 except that they contain an open chain carboxylic acid rather than a lactone ring. Toxic mussels also contained these pectenotoxin-2 analogues.     

Structure and stereochemistry of a new cytotoxic polychlorinated sulfolipid from Adriatic shellfish

A detailed analysis of the causative toxins contained in the hepatopancreas of toxic mussels from the northern Adriatic sea has been carried out. Along with some DSP (diarrhetic shellfish poisoning) type toxins, such as okadaic acid, yessotoxin, and their derivatives, which are involved in a number of human intoxications throughout the world, we have now isolated a new cytotoxin, a polychlorinated sulfolipid 1, whose gross structure has been elucidated by spectral analysis, including various 2D NMR techniques. The relative stereochemistry of 1 was elucidated by successful application of the J-based configuration analysis developed for acyclic compounds using carbon-proton spin-coupling constants ((2,3)J(C,H)) and proton-proton spin-coupling constants ((3)J(H,H)); its absolute stereochemistry was established by the Mosher method. Compound 1 possesses in vitro cytotoxicity against WEHI 164 and RAW 264.7 cells.     

Phycotoxins in seafood--toxicological and chromatographic aspects.

Two typical clinical types of algae-related seafood poisoning have attracted medical and scientific attention: paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP). Therefore, it became necessary to establish methods for the evaluation of possible hazards caused by contamination of seafood with these phycotoxins. Bioassays with mice or rats are the common methods for the determination of the toxin content of seafood. However, biological tests are not completely satisfactory because of a lack of sensitivity and pronounced variations. Additionally, there is growing opposition against animal testing. Therefore, many efforts have been undertaken to determine phycotoxins by chromatographic methods. PSP determination is mainly based on high-performance liquid chromatographic (HPLC) separation by ion-pair chromatography followed by postcolumn oxidation of the underivatized toxins in alkaline solution and fluorescence detection. HPLC methods for the determination of the DSP toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are characterized by precolumn derivatization with 9-anthryldiazomethane (ADAM) and/or 4-bromomethyl-7-methoxycoumarin (Br-Mmc), followed by chromatographic separation of the DSP esters formed and fluorescence detection. The chromatographic methods discussed in this review allow the rapid, sensitive and non-ambiguous determination of individual species of the two most important phycotoxins in seafood, PSP and DSP.     

Characterization of sodiated glycerol phosphatidylcholine phospholipids by mass spectrometry

Fast atom bombardment mass spectrometry in the positive mode was used for the characterization of sodiated glycerol phosphatidylcholines. The relative abundance (RA) of the protonated species is similar to the RA of the sodiated molecular species. The sodiated fragment ion, [M + Na - 59](+), corresponding to the loss of trimethylamine, and other sodiated fragment ions, were also observed. The decomposition of the sodiated molecule is very similar for all the studied glycerol phosphatidylcholines, in which the most abundant ion corresponds to a neutral loss of 59 Da. Upon collision-induced dissociation (CID) of the [M + Na](+) ion informative ions are formed by the losses of the fatty acids in the sn-1 and sn-2 positions. Other major fragment ions of the sodiated molecule result from loss of non-sodiated and sodiated choline phosphate, [M + Na - 183](+), [M + Na - 184](+.) and [M + Na - 205](+), respectively. The main CID fragmentation pathway of the [M + Na - 59](+) ion yields the [M + Na - 183](+) ion, also observed in the CID spectra of the [M + Na](+) molecular ion. Other major fragment ions are [M + Na - 205](+) and the fragment ion at m/z 147. Collisional activation of [M + Na - 205](+) results in charge site remote fragmentation of both fatty acid alkyl chains. The terminal ions of these series of charge remote fragmentations result from loss of part of the R(1) or R(2) alkyl chain. Other major informative ions correspond to acylium ions.