Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish

We performed a neuroblastoma cell (Neuro2a) culture assay modified slightly from a method reported previously to provide a simple and sensitive evaluation of paralytic shellfish poisoning (PSP) toxicity in shellfish. The cell bioassay was just as sensitive for C-toxins as for gonyautoxins. The sensitivity of our cell bioassay was 4 times that of the current standard mouse bioassay. Using the cell bioassay, we evaluated PSP toxicity in 361 shellfish samples collected from Mikawa Bay and Ise Bay, Aichi Prefecture, Japan, from April 1999-March 2002. The results were compared with those obtained in a postcolumn derivatization liquid chromatographic analysis. PSP toxins were detected in 236/361 samples by both assays, and there was a fairly good correlation (r = 0.9001, n = 236, p < 0.001) between the results from the two assays. We applied this cell bioassay when short-necked clams in the bay turned poisonous in 2001. The chronological changes in PSP toxicity in the short-necked clams were analyzed and compared with those of the cell density of poisonous plankton (Alexandrium tamarense) occurring in the bay. The PSP toxicity in shellfish peaked 2 weeks after the cell density reached a maximum. We recommend using the cell bioassay for routine monitoring of PSP toxicity in shellfish living in natural marine environments.     

Liquid chromatographic determination of paralytic shellfish poisons in shellfish after prechromatographic oxidation.

A liquid chromatographic method for quantitating paralytic shellfish poison toxins in shellfish has been developed in which the toxins are converted to fluorescent purines by prechromatographic oxidation under mildly basic conditions with hydrogen peroxide or periodate. The addition of ammonium formate to the periodate oxidation reaction greatly improved the yield of fluorescent derivatives for neosaxitoxin, gonyautoxin-1, B-2, and C-3 compared to the same reaction without ammonium formate. As little as 3-6 ng of each of the nonhydroxylated toxins and 7-12 ng of the hydroxylated compounds per gram of shellfish could be detected. Reversed-phase chromatography using ammonium formate in the mobile phase improved the chromatography of neosaxitoxin and B-2 compared to results obtained earlier. Because the oxidation products of neosaxitoxin and B-2 could not be separated, parent compounds were separated before oxidation by using an SPE-COOH ion exchange cartridge. The repeatability coefficient of variation for the oxidation reactions ranged from 3 to 8% for the peroxide reaction, and from 4 to 11% for the periodate reaction, depending upon the individual toxin determined and its concentration in the extract (0.04-0.55 micrograms/g). The method was compared to the mouse bioassay and the postcolumn oxidation method. In most cases, results were comparable.     

A study of ten toxins associated with paralytic shellfish poison using prechromatographic oxidation and liquid chromatography with fluorescence detection.

Ten paralytic shellfish toxins [saxitoxin, neosaxitoxin, B-1, B-2, gonyautoxin 1, 2, and 3 (i.e., GTX-1, GTX-2, and GTX-3), C-1, C-2, and C-3] were oxidized at room temperature under mildly basic conditions with hydrogen peroxide or periodic acid. The products were then analyzed by liquid chromatography (LC). The N-1-hydroxylated toxins (neosaxitoxin, B-2, GTX-1, and C-3) formed fluorescent products after periodate oxidation at ca pH 8.7, but did not form fluorescent derivatives with peroxide oxidation. The non-N-1-hydroxylated toxins (saxitoxin, B-1, GTX-2, GTX-3, C-1, and C-2) formed highly fluorescent derivatives with both peroxide and periodate oxidations. Individual toxins produced mainly single fluorescent peaks by reverse-phase LC. However, all GTX toxins eluted with the same retention time. Also, C-1 and C-2 eluted together, as did neosaxitoxin and B-2. The non-N-1-hydroxylated toxins could be detected in quantities as low as 20-50 pg/injection, while the N-1-hydroxy analogues could be detected at levels as low as 100-500 pg/injection. UV absorption and fluorescence emission spectra were similar for the oxidation products of all toxins examined (max. 333 +/- 2 nm absorption, 389 +/- 4 nm fluorescence emission).     

Improved solid-phase extraction procedure in the analysis of paralytic shellfish poisoning toxins by liquid chromatography with fluorescence detection

The analysis of shellfish extracts for the determination of paralytic shellfish poisoning (PSP) toxins by liquid chromatography with fluorescence detection repeatedly showed the presence of a compound suspected to interfere with gonyautoxin 4. The first aim of this study was to confirm by liquid chromatography coupled to tandem mass spectrometry that this compound was not gonyautoxin 4. The second part of this work was to improve a nonvolumetric C(18) solid-phase extraction (SPE) procedure to evaluate the removal of the interference associated with the recovery of PSP toxins. The cleanup procedure was modified into a volumetric SPE procedure and proved to efficiently and totally remove the interference while recovering from 78 to 85% of the PSP toxins available as commercial standards (saxitoxin, neosaxitoxin, gonyautoxins 1-4) and considered as major PSP toxins in human intoxication, with 85% recovery for gonyautoxin 4. The efficiency of this cleanup procedure was checked on shellfish extracts containing this interference and originating from France and Turkey.     

High pressure liquid chromatographic determination of toxins associated with paralytic shellfish poisoning

A high pressure liquid chromatographic procedure is described for assay of toxins associated with paralytic shellfish poisoning (PSP). The method is applicable to saxitoxin, neosaxitoxin, gonyautoxins I through IV, and their sulfocarbamoyl derivatives. Toxins are separated on a bonded phase cyano column and detected by fluorescence following alkaline oxidation (NH+4 and periodic acid). The utility of the HPLC procedure for research and monitoring is discussed.     

Removal of lipids and diarrhetic shellfish poisoning toxins from blue mussels (Mytilus edulis) during acid and alkaline isolation of proteins

Diarrhetic shellfish poisoning (DSP) toxins pose a serious health risk for consumers of bivalves and other shellfish, as well as a huge economic burden for the bivalve-producing farmers. In this work, the aim was to utilize a solubilization-based protein-isolation method to produce a low-DSP toxin protein isolate from toxic blue mussels that are unsuitable for the whole shellfish market. A homogenate of whole mussel meat was solubilized at low pH (2.8) or high pH (11.1), followed by centrifugation and reprecipitation of the solubilized mussel proteins at the isoelectric pH. In a second centrifugation, precipitated proteins were collected. These processes resulted in 81 (acid solubilization) and 72% (alkaline solubilization) reduction in the initial DTX-1 toxin content of the mussel meat. No other DSP toxins were found in the protein isolates. Acid processing of mussel meat resulted in 50% reduction in the total lipid content, while alkaline treatment did not significantly affect the lipid content. The effect of citric acid and calcium chloride addition to the mussel meat-water homogenate on lipid and toxin content was also investigated. A poor correlation factor was surprisingly obtained between reductions in DTX-1 toxin and lipids in protein isolates from processed toxic mussels. Results from an analytical mass balance of the DTX-1 toxin during acid processing showed that 61% of this toxin ended up in the aqueous supernatant after the second centrifugation. The present study presents a promising alternative way of utilizing mussels for food production in periods when they are toxic.     

Rapid determination of methyl mercury in fish and shellfish: method development

The AOAC official first action method for methyl mercury in fish and shellfish was modified to provide more rapid determination. Methyl mercury is isolated from homogenized, acetone-washed tissue by addition of HCl and extraction by toluene of the methyl mercuric chloride produced. The extract is analyzed by electron capture gas chromatography (GC) on 5% DEGS-PS treated with mercuric chloride solution. The quantitation limit of the method is 0.25 micrograms Hg/g. Swordfish, shark, tuna, shrimp, clams, oysters, and NBS Research Material-50 (tuna) were analyzed for methyl mercury by the AOAC official first action method. All products also were analyzed by the modified method and the AOAC official method for total Hg. In addition, selected extracts obtained with the modified method were analyzed by GC with Hg-selective, microwave-induced helium plasma detection. There was no significant difference between the results for the various methods. Essentially all the Hg present (determined as total Hg) was in the organic form. Coefficients of variation from analyses by the modified method ranged from 1 to 7% for fish and shellfish containing methyl mercury at levels of 0.50-2.30 micrograms Hg/g. The overall average recovery was 100.5%.     

Evaluation of laboratory performance of the AOAC method for PSP toxin in shellfish

Laboratory performance of the official AOAC method for paralytic shellfish poison (PSP) toxin in shellfish was evaluated. Two series of naturally toxic shellfish split samples were distributed (15 in 1979 and 19 in 1982) to state shellfish-monitoring laboratories which participate in the National Shellfish Sanitation Program. The laboratories performed bioassays on duplicate 100 g portions of each 220 g split sample. Bioassays were consistent among the laboratories and compared favorably with those of previous studies.     

Liquid chromatographic determination and stability of the Fusarium mycotoxin moniliformin in cereal grains

Moniliformin is a mycotoxin produced by Fusarium subglutinans and other Fusarium species. A rapid, liquid chromatographic method for its determination in corn and wheat is described. Samples are extracted in acetonitrile-water (95 + 5); following defatting with n-hexane, an aliquot of the extract is evaporated and cleaned up on small C18 and neutral alumina columns successively. Reverse-phase liquid chromatography (LC) is conducted on a C18 column with 10 or 15% methanol or acetonitrile in aqueous ion-pair reagent as mobile phase, with detection by ultraviolet absorption at 229 and 254 nm. Average recoveries of moniliformin (potassium salt) added to ground corn and wheat at levels of 0.05-1.0 micrograms/g were 80% (n = 20) and 85% (n = 12), respectively, and the limit of detection was ca 0.01-0.18 micrograms/g, depending on LC conditions. Analysis of 24 samples of wheat, 4 samples of rye, and 12 samples of corn showed moniliformin in only 2 corn samples (0.06 and 0.2 micrograms/g). Moniliformin was also detected in a sample of artificially damaged (slashed) corn (0.2 micrograms/g) and selected kernels of corn that were field-inoculated with F. subglutinans and F. moniliforme (50 micrograms/g and 0.5 micrograms/g, respectively). In stability studies, moniliformin (potassium salt, 1 microgram/g) in ground corn and ground wheat heated at 50, 100, and 150 degrees C for 0.5-2 h decomposed moderately, e.g., 55% remained in corn after 0.5 h at 100 degrees C.     

Polybrominated diphenyl ethers in retail fish and shellfish samples purchased from Canadian markets

Fish and shellfish retail samples (n = 122) were purchased from three Canadian cities in the winter of 2002 and analyzed for a total of 18 polybrominated diphenyl ether (PBDE) congeners. The samples (salmon, trout, tilapia, Arctic char, mussels, oysters, shrimp, and crab) represented the range of fish and shellfish commercially available to Canadian consumers at the time of purchase. Trout and salmon (geometric mean SigmaPBDE = 1600 and 1500 pg/g, wet weight, respectively) were found to contain significantly higher amounts of PBDEs than the mussel, tilapia, and shrimp groups (geometric mean SigmaPBDE = 260, 180, and 48 pg/g, wet weight, respectively). These differences in SigmaPBDE concentrations among fish and shellfish products were partly driven by differences in lipid content among the samples. Mean SigmaPBDE concentrations in domestic samples were also significantly greater than in imported samples, possibly reflecting global environmental distribution of PBDEs. These concentration differences will contribute to variations in dietary exposure to PBDEs when assorted fish and shellfish items from various origins are consumed.     

Lipophilic toxins analyzed by liquid chromatography-mass spectrometry and comparison with mouse bioassay in fresh, frozen, and processed molluscs

The search for alternative methods to the mouse bioassay (MBA) has intensified over recent years. The present work analyzes seven different species of shellfish (clams, small scallops, small clams, mussels, oysters, cockles, and edible whelks) in fresh, frozen boiled, and canned presentations using liquid chromatography-mass spectrometry (LC-MS/MS), and the results are compared with the same samples analyzed through MBA. The toxins studied were OA, DTX1, DTX2, YTX, PTX2, and AZA1, which are legislated in the EU, and SPX1, which is not regulated yet. Consistent results between LC-MS/MS and MBA were found in 69% of the samples, whereas 26% of MBA showed "false-positive" results with respect to the toxins analyzed. No "false negatives" were observed. The possibility of LC-MS/MS as an alternative or complementary technique to MBA is discussed.     

Liquid chromatographic determination of chloramphenicol residues in meat: interlaboratory study

A liquid chromatographic (LC) method for the determination of chloramphenicol (CAP) residues in meat at the 10 microgram/kg level was tested in an interlaboratory study. The method used, based on aqueous extraction and sample cleanup with a cartridge containing Extrelut, was published earlier. A prestudy to familiarize collaborators with the method was performed before the actual interlaboratory precision study. The meat samples used in the precision study were prepared by diluting dosed chicken and pig muscle tissues with blank tissues from other species. Fourteen laboratories received 20 meat samples; 13 laboratories actually participated in the study. Two blank samples and 2 positive samples each of pig, calf, chicken, lamb, and cow meat were tested. The chloramphenicol concentrations in the positive samples ranged from 6.5 to 21 micrograms/kg. The overall mean reproducibility coefficient of variation was 17.9% after the results per laboratory were corrected for the mean recovery obtained within each sample series. The overall mean recovery was 55.1% with a coefficient of variation of 18.0% at the 10 micrograms/kg level. The limit of detection, based on chromatograms of blank samples, was estimated to be 1.5 micrograms/kg of chloramphenicol. No false positives or false negatives were observed in the concentration range tested; only 2 false positive results above the detection limit (1.7 and 6 micrograms/kg) on a total number of 60 blank analyses (3.3%) were observed.     

Liquid chromatographic determination of polycyclic aromatic hydrocarbons in fish and shellfish

A simple and accurate analytical method for determination of polycyclic aromatic hydrocarbons (PAHs) in fish and shellfish is presented, which is considered to be useful for routine analyses and for screening purposes. The procedure involves alkaline digestion, extraction with n-hexane, silica gel column chromatography, and liquid chromatographic (LC) determination with fluorometric detection. During development of the analytical method for determination of PAHs, it was found that benzo[a]pyrene, a representative PAH, was decomposed easily by the analytical procedure, and this tendency was investigated for the experimental conditions used. Benzo[a]pyrene was decomposed by the coexistence of alkaline conditions, light, and oxygen; by peroxides in aged ethyl ether; and by oxygen when absorbed on silica gel. Thus, to obtain good recoveries and precise analytical results, these decomposition conditions must be avoided. The following precautions are recommended: protection from light through all analytical steps; addition of Na2S to alkaline digestion mixture as an antioxidant; complete removal of peroxides from ethyl ether just before use; quick column chromatography on silica gel; and prevention of air from contact with adsorbent. When this simple method was applied to fish and shellfish samples, very good recoveries of PAHs from fortified fish samples were obtained, and no serious interferences were observed in fish and shellfish extracts.     

Basis for a new procedure to eliminate diarrheic shellfish toxins from a contaminated matrix.

The natural contamination of shellfish with diarrheic shellfish toxins (DSP) has important public health implications. To avoid the economic effects of toxic episodes on shellfish farmers and the related industry, research on artificial methods alternative to the natural detoxification of shellfish is needed. Because the usual thermal processes are not efficient, alternative technologies have to be studied. Here preliminary results are presented about the lability of the DSP toxin okadaic acid in a supercritical atmosphere of carbon dioxide with acetic acid. Most of the toxin is eliminated (up to 90%), and the biological activity against its target enzyme is also severely affected (up to 70% reduction). Detoxification of contaminated shellfish requires a partial dehydration, and the detoxification yield is lower than that obtained with free toxin. Mass spectrometry experiments suggest that acetylation of the toxin molecule is not the basis of the inactivating mechanism, but a conformational change is suggested. This is the first report of the use of supercritical fluids to inactivate toxins.     

Liquid chromatographic determination of the processing aid 4-hexylresorcinol in shrimp.

A rapid, sensitive, liquid chromatographic (LC) method has been developed for determination of residuals of the processing aid, 4-hexylresorcinol, on shrimp meat. An aqueous homogenate of shrimp meat is extracted with ethyl acetate followed by precolumn preparation on a silica Sep-Pak cartridge. LC determination is preformed with a Nova-Pak C18 column, with UV detection at 214 nm. Sensitivity was 0.006 micrograms, and recovery from shrimp meat samples of known 4-hexylresorcinol addition was 94%. Shrimp treated with 4-hexylresorcinol under the recommended dip protocol had mean residuals of 1.18 ppm, with a standard deviation of 0.13 ppm.     

Rapid, semimicro method for determination of polycyclic aromatic hydrocarbons in shellfish by automated gel permeation/liquid chromatography

A simple, rapid, easily automated method is described for the determination of polycyclic aromatic hydrocarbons (PAHs) in shellfish such as American lobster (Homarus americanus) and blue mussel (Mytilus edulis). PAHs are extracted from small amounts (1-8 g) of tissue by saponification in 1N ethanolic potassium hydroxide followed by partitioning into 2,2,4-trimethylpentane. This solution is evaporated just to dryness by rotary evaporation and the residue is dissolved in cyclohexane-dichloromethane (1 + 1) for gel permeation chromatography (GPC) on Bio-Beads SX-3. The GPC procedure is ideal as a screening method in the range 25-18 000 ng PAHs/g tissue. If individual PAH measurements are required, the appropriate GPC fraction is collected and PAHs are separated by reverse phase liquid chromatography (LC) with fluorometric detection. Individual PAHs at concentrations as low as 0.25-10 ng/g can be determined. Recoveries of added fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[ghi]perylene, and indeno[1,2,3-cd]pyrene were quantitative, with relative standard deviations ranging from 0.0 to 16.9%.     

Use of solid-phase extraction systems to improve the sensitivity of Artemia bioassays for trichothecene mycotoxins

Solid-phase extraction was used to preconcentrate trichothecene mycotoxins from rivers and streams in order to develop and improve a rapid and sensitive bioassay using the brine shrimp Artemia salina. For T-2 toxin, HT-2 toxin, and 4,15-diacetoxyscirpenol, LC50 values obtained were 172, 600, and 700 micrograms/L, respectively. The LC50 for 4-deoxynivalenol was 21 mg/L. A more than 5-fold increase in sensitivity was observed when solid-phase extraction (SPE) was used in conjunction with the Artemia bioassay. For T-2 toxin, HT-2 toxin, and 4,15-diacetoxyscirpenol, LC50/SPE values after solid-phase extraction were 21, 83, and 130 micrograms/L. The use of river and stream waters and chlorinated water did not seem to interfere with the bioassay.     

Comparison of liquid chromatographic and bioassay procedures for determining depletion of intramuscularly injected tylosin

Crossbred pigs weighing 80-110 kg were injected intramuscularly in the ham with 8.8 mg/kg tylosin. Animals were slaughtered in groups of 3 at intervals of 4 h, and 1, 2, 4, and 8 days after injection, and samples of blood, injected muscle, uninjected muscle, liver, and kidney were analyzed by liquid chromatography (LC) and by bioassay using Sarcina lutea as the test organism. The LC method was far more sensitive with a detection limit of less than 0.1 ppm, while the detection limit by bioassay was about 0.5 ppm in tissue. Results by bioassay and LC sometimes differed considerably for tissue samples. Residues in all tissues were below the tolerance limit of 0.2 ppm at 24 h, except in the injected muscle in one animal. Residues were not detected in any tissue of any animal at 48 h after treatment.     

Human exposure to perfluorinated compounds in Catalonia, Spain: contribution of drinking water and fish and shellfish

In this study, the concentrations of 15 perfluorinated compounds (PFCs) were analyzed in 30 water samples collected in Catalonia (Spain) at three stages of the drinking water treatment process in several water purification plants. In addition, the concentrations of 13 PFCs were determined in samples of fish and shellfish collected from coastal areas of Catalonia. The intake of PFCs through both pathways, drinking water intake and fish and shellfish consumption, was also estimated. In water samples, the highest mean concentrations corresponded to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) (1.81 and 2.40 ng/L, respectively), whereas perfluorodecanosulfonate (PFDS) and perfluorotetradecanoic acid (PFTDA) were under their respective limits of detection in all analyzed samples. The results show that although the current treatment processes caused slight reductions in PFC concentrations, these processes did not mean significant changes in the amounts of PFCs already contained in the raw water. Among the analyzed PFCs in fish and shellfish, only seven compounds could be detected in at least one composite sample. PFOS showed the highest mean concentration (2.70 ng/g fw), being detected in all species with the exception of mussels. With regard to PFOA (mean, 0.074 ng/g fw), the highest concentrations were detected in prawn and hake (0.098 and 0.091 ng/g fw, respectively). The current exposure to PFCs through consumption of fish and shellfish indicates that it should not be of concern for the consumers. The amounts ingested are well below the recommended tolerable daily intakes, at least for those PFCs for which information is available.     

Simultaneous detection of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry

A sensitive method has been developed for the simultaneous determination of residues of 25 β?-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry (HPLC-LIT-MS). This method is based on a new procedure of hydrolysis and extraction by 5% trichloracetic acid, and then cleaned up by mixed strong cation exchange (MCX) cartridges coupled with a novelty cleanup step by methanol. Methanol and 0.1% formic acid were used as mobile phases for gradient elution, while a Supelco Ascentis Express Rp-Amide column was used for LC separation. ESI positive ion scan mode was used with consecutive reaction monitoring (CRM, MS3). Nine β?-agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 48 analytes were from 5 to 200 μg/L; the coefficient of correlation was not less than 0.995. Blank pork muscle, blank liver, and blank kidney were selected as representative matrix for spiked standard recovery test. The recoveries of each compound were in the range of 46.6-118.9%, and the relative standard deviations were in the range of 1.9-28.2%. Decision limits (CCα, α = 0.01) of 48 analytes in muscles, liver, and kidney samples ranged from 0.05 to 0.49 μg/kg, and the detection capability (CCβ, β = 0.05) ranged from 0.13 to 1.64 μg/kg. This method was successfully applied to 110 real animal origin food samples including meat, liver, and kidney of pig and chicken samples.