Luca Lucentini

First report of pectenotoxin-2 (PTX-2) in algae (Dinophysis fortii) related to seafood poisoning in Europe

Pectenotoxin-2 (PTX-2), a polyether-lactone included in the neutral class of diarrhoetic shellfish poisoning (DSP) toxins, has been unambiguously detected in Dinophysis fortii collected in the northern Adriatic Sea (Emilia Romagna coasts). This is the first report of such a toxin in Europe. This lipid soluble toxin was identified both in crude methanolic phytoplankton extract and in the neutral fraction obtained by extract chromatography on a basic alumina column. The techniques used were reversed phase high-performance liquid chromatography followed either by UV diode-array detection (LC-UV-DAD) or by mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS-MS) using an atmospheric-pressure ionization source and an ionspray interface. Okadaic acid (OA) was also found in the D. fortii specimens and quantified as 15 pg/cell. Although quantitation of PTX-2 was not possible due to the lack of pure toxin, the high PTX-2:OA ratio suggested PTX-2 was significant in the D. fortii specimens. The presence of PTX-2 in a region with no previous report of DSP neutral toxic compounds may indicate a risk of human poisoning. Serious efforts should therefore be made to develop suitable routine methods capable of detecting the presence of PTXs in biological materials of marine origin, in order to assure the wholesomeness of seafood products.

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry.

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC-APCI-MS-MS) for the quantitation of anabolic hormone residues (17beta-19-nortestosterone, 17beta-testosterone and progesterone) and their major metabolites (17alpha-19-nortestosterone and 17alpha-testosterone) in bovine serum and urine is reported. [2H2]17Beta-testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid-liquid extraction and purified by C18 solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]+, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC-MS-MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from -5.90 to -3.18% and from -6.40 to -2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.

New approach to the direct detection of known and new diarrhoeic shellfish toxins in mussels and phytoplankton by liquid chromatography-mass spectrometry.

A new approach using combined liquid chromatography-mass spectrometry (LC-MS) with ionspray ionization is proposed for the direct detection of known and new toxins in mussels and phytoplankton. A first stage reversed-phase, negative ion mode, selected ion monitoring (SIM) LC-MS analysis was performed in order to detect DSP toxins in the same chromatographic run with a total run time of 20 min. The toxins analysed included yessotoxin (YTX), okadaic acid (OA) and four of its analogues, dinophysistoxins (i.e. DTX-1, DTX-2, DTX-2B, DTX-2C), and pectenotoxins (PTXs), involving PTX-2, two PTX-2 secoacids (PTX-2SAs), PTX-2SA, 7-epi-PTX-2SA, and AC1, the three isomeric toxins structurally related to PTX-2 recently identified in Irish phytoplankton. Positive samples can, therefore, be analyzed through reversed-phase, positive ion mode SIM LC-MS, in order to perform complete chromatographic separations of the structurally related toxins within the OA and PTX groups. Detailed toxin profiles of a number of toxic phytoplankton and shellfish, from different marine areas, were easily obtained through the new approach. PTX-2SAs and AC1 were found in phytoplankton and shellfish from Ireland as well as in Italian shellfish. Moreover, for the first time there was evidence of the presence of PTX-2 in Irish phytoplankton. YTX was present in Italian shellfish. Four isomeric OA toxins were detected in samples from Ireland with OA, DTX-2 and DTX-2B present in shellfish, and OA, DTX-2 and DTX-2C in phytoplankton. In contrast, OA was the only toxin from this group to be detected in Italian mussels.

Improved ion chromatography-integrated pulsed amperometric detection method for the evaluation of biogenic amines in food of vegetable or animal origin and in fermented foods

An improved method for the simultaneous determination of underivatized biogenic amines, cadaverine, putrescine, spermidine, histamine, tyramine and some amino acids precursors, histidine and tyrosine, in food products, based on ion-exchange chromatography (IC) with integrated pulsed amperometric detection (IPAD) has been developed. The method was successfully used for the analysis of biogenic amines and amino acids in food both of vegetable (kiwi, Actinidia chinensis) and animal origin, (fish, pilchard), as well as in fermented foods, such as cheese (Emmenthal) and dry sausages (salami). The method was also successfully used to study the changes in biogenic amines during the ripening of dry fermented sausages (salami). The analytes were extracted from foods with perchloric acid and the extracts were purified by liquid-liquid partition using n-hexane. Determination of biogenic amines was performed through cation-exchange chromatography with isocratic elution and IPAD. The detection limits for the analytes under investigation were found to range from 1.25 to 2.50 ng, at a signal-to-noise ratio of 3:1. Average recoveries ranged from 85.5 to 97.4% and R.S.D. values ranged from 3.4 to 8.8. The proposed method offers a number of advantages over our previous IPAD method, such as the application to a larger number of analytes and matrices, a simpler extraction procedure and clean-up, isocratic elution using low acid and base concentrations, an improved chromatographic separation and a lower detection limit.

Liquid chromatography with fluorimetric, mass spectrometric and tandem mass spectrometric detection for the investigation of the seafood-toxin-producing phytoplankton, Dinophysis acuta

Abstract The diarrhoeic shellfish poisoning (DSP) toxins, okadaic acid (OA) and its isomer, dinophysistoxin-2 (DTX-2), were determined in the marine phytoplankton, Dinophysis acuta , harvested in Ireland. Unialgal samples (22–100 cells) were extracted and derivatised using 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP) and analysed by liquid chromatography (LC). Isocratic elution on a C 18 reversed-phase column, with fluorimetric detection, was used to determine OA (58±7 pg/cell) and DTX-2 (78±14 pg/cell). The detection limit was 0.1 ng OA/20 μl injection using ADAM. Gradient LC, using a polymeric bonded phase, successfully separated mixtures containing both the ADAM and BAP derivatised toxins. Identification of DSP toxins was confirmed using isocratic micro LC with tandem mass spectrometric (μLC–MS–MS) analysis of the free toxins and μLC–MS of the BAP-derivatised toxins with an ionspray (IS) interface, coupled to an atmospheric pressure ionisation (API) source. Collision induced dissociation (CID) ion mass spectra of the protonated molecule, [M+H] + , at m / z 805 for OA and DTX-2, identified three diagnostic fragment ions for each analyte which were used for selected reaction monitoring (SRM) LC–MS–MS analysis. The detection limit for OA and DTX-2 was 0.025 ng/0.2 μl injected. These studies showed that D. acuta was the progenitor of DTX-2 in shellfish.

Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Confirmatory analysis of 17β-boldenone, 17α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine by liquid chromatography–tandem mass spectrometry

Abstract A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for confirmatory analysis of 17β-boldenone (17β-BOL), 17α-boldenone (17α-BOL) and androsta-1,4-diene-3,17-dione (ADD) in bovine urine was developed. [ 2 H 2 ]17β-Testosterone (17β-T-d 2 ) was used as the internal standard. Sample preparation involved enzymatic hydrolysis and purification on a C 18 solid-phase extraction column. Chromatographic separation of the analytes was obtained using an RP-C 18 HPLC column. LC–MS–MS detection was carried out with an atmospheric pressure chemical ionisation (APCI) source equipped with a heated nebulizer (HN) interface operating in the positive ion mode. For unambiguous hormone confirmation, three analyte precursor–product ion combinations were monitored during multiple-reaction monitoring (MRM) LC–MS–MS analysis. Overall recovery (%), repeatability (relative standard deviations, RSD, %) and within-laboratory reproducibility (RSD, %) ranged from 92.2 to 97.7%, from 6.50 to 2.94% and from 13.50 to 5.04%, respectively, for all analytes. The limit of quantification in bovine urine was 0.20 ng ml −1 for 17β-BOL and ADD and 0.50 ng ml −1 for 17α-BOL. The validated method was successfully applied for determination of 17β-BOL, 17α-BOL and ADD in a large number of bovine urine samples collected within the national Official Residue Control Program.

Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography-mass spectrometry

Direct detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and some of their related compounds in toxic mussels (Mytilus galloprovincialis) is reported using ionspray liquid chromatography-mass spectrometry (LC-ISP-MS). This was employed to analyse diarrhoetic shellfish poisoning (DSP) toxins in mussels collected from coastal areas of the northern and southern Adriatic Sea. DTX-1 was found in some samples from both the northern and southern Adriatic and this is the first report of the unambiguous identification of this toxin in Italian mussels. The low levels found indicate that this toxin did not play a significant role in toxicity in these samples. Okadaic acid was found in all the mussels examined, although its concentration was not always sufficient to account for DSP toxicity. Furthermore, two related compounds of OA were detected in all the samples and one related DTX-1 compound was observed in some samples from the northern Adriatic. All three compounds are still to be identified, but it is possible that these substances are involved in mussel DSP toxicity in the Adriatic Sea.

Efficient isolation of the rare diarrhoeic shellfish toxin, dinophysistoxin-2, from marine phytoplankton

The rare diarrhoeic shellfish poisoning (DSP) toxin, dinophysistoxin-2 (DTX-2), which is an okadaic acid (OA) isomer, has been isolated from a marine phytoplankton biomass that consisted mainly of Dinophysis acuta. Using a large double plankton net (length 5.9 m), bulk phytoplankton samples were collected off the south-west coast of Ireland and extracted with methanol and chloroform. Liquid chromatography coupled with ionspray mass spectrometry and tandem mass spectrometry (LC-MS, LC-MS-MS) showed the sample contained DTX-2 and OA, at a concentration of 80 pg/cell and 60 pg/cell, respectively. Flash chromatography using silica, sephadex LH20 and C18-silica, followed by preparative reversed-phase LC, separated DTX-2 from OA. The efficiency of the separation procedures was substantially improved by the use of a bioscreen to detect DSP toxins in eluate fractions and the application of a new derivatisation procedure for the chromatographic elucidation of toxin profiles with fluorimetric detection (LC-FLD). Thus, 1/1000th aliquots of eluate fractions were assayed using protein phosphatase-2A for the presence of inhibitory compounds. Positive fractions were further analysed for DSP toxins by LC-FLD following derivatisation using the hydrazine reagent, luminarine-3. The identity and purity of the free isolated DTX-2 was confirmed using flow injection analysis (FIA) and liquid chromatography (FIA-MS, LC-MS and LC-MS-MS).

Comparison of mouse bioassay, HPLC and enzyme immunoassay methods for determining diarrhetic shellfish poisoning toxins in mussels

Mussel specimens (Mytilus galloprovincialis) collected from two different areas of the Adriatic Sea were analysed for diarrhoetic shellfish poisoning (DSP) toxin by three methods: mouse bioassay, the DSP Check enzyme immunoassay kit, and high-performance liquid chromatography (HPLC). The results obtained confirm that Yasumotos mouse bioassay, capable of detecting all the components of the DSP group, is still necessary to determine the wholesomeness of the product. The ELISA method has not always given quantitatively reliable results. The HPLC method is advantageous in terms of sensitivity, accuracy, specificity and rapidity. However, its application is limited so far to the determination of okadaic acid in mussels.