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).

Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection.

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC-FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2-84.9% at concentrations of 10 micrograms/l. The R.S.D. values were 1.7-3.9% (n = 8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.

Anatoxin-a in Irish freshwater and cyanobacteria, determined using a new fluorimetric liquid chromatographic method.

A new sensitive high-performance liquid chromatographic (HPLC) method was used to determine anatoxin-a in freshwater, following blooms of cyanobacteria (blue-green algae). Anatoxin-a was converted into a highly fluorescent derivative using 4-fluoro-7-nitro-2,1,3-benzoxadiazole and HPLC analysis gave good linear calibrations even at low concentration ranges (1-10 micrograms/liter, r = 0.997). The detection limit for anatoxin-a was 0.02 ng/ml, and this new HPLC method should prove useful for the routine analysis of potable waters. Anatoxin-a was discovered in three major lakes in Ireland using this method and identification was confirmed using gas chromatraphy-mass spectrometry (GC-MS), following acetylation. Anatoxin-a was found in Anabaena, a planktonic cyanobacterium, as well as in a benthic Oscillatoria species. This is the first identification of anatoxin-a in Irish freshwater and this toxin was also implicated as the causative agent in incidents of fatal canine neurotoxicosis.

First identification of microcystins in Irish lakes aided by a new derivatisation procedure for electrospray mass spectrometric analysis.

Recent animal and bird deaths at several lakes in Ireland were indicative of possible cyanobacterial poisoning. Using protein phosphatase inhibition assays, microcystins (MCs) were identified in extracts of cyanobacteria from several lakes at concentrations ranging from 1.6 to 168 micrograms/g. This is the first report of MCs in Irish freshwaters. The protein phosphatase inhibition assay was used to screen fractions during HPLC purification of the MCs in cyanobacteria (Anabaena and Oscillatoria) and water samples from Corbally and Caragh Lakes. MC-LR, MC-HtyR, MC-FR, and MC-YR and 3 unidentified MCs of m/z values 1028.5, 981, 1042.7 were isolated from the Corbally sample; while the Caragh Lake sample contained largely MC-LR and MC-YR. A new microanalytical technique was developed for the confirmation of MCs which involved the derivatisation of the methyldehydroalanine group of MCs with 2-aminoethanethiol. Electrospray mass spectrometry of these products showed characteristic double-charged ions, and this novel technique was useful for differentiating MCs from co-eluting impurities in HPLC fractions of cyanobacterial extracts.

Determination of the cyanobacterial neurotoxin, anatoxin-a, by derivatisation using 7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F) and HPLC analysis with fluorimetric detection.

A sensitive HPLC analysis has been developed for the determination of anatoxin-a in cyanobacterial bloom material and in raw and treated waters. Efficient extraction of this toxin from its matrix can be achieved using solid-phase extraction with a weak cation exchanger. HPLC analysis with fluorimetric detection (lambda ex 470 nm, lambda em 530 nm) was possible after derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). This new analytical protocol permits the detection of anatoxin-a at low concentrations (0.1 micrograms/L) and gave a good linear calibration (1-20 micrograms/L, r = 0.997).