Search

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid ( 1 ) and dinophysistoxin-2 ( 2 ) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24- O -β-d-glucosides of 1 and 2 ( 4 and 5 , respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1 , 2 , dinophysistoxin-1 ( 3 ), and a synthetic specimen of 7-deoxy- 1 ( 7 ), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1 - 5 , the 24- O -β-d-glucoside of dinophysistoxin-1 ( 6 ), 7 , 7-deoxy- 2 ( 8 ), and 7-deoxy- 3 ( 9 ) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum   lima cultures was also examined by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and was found to contain 1 , 3 , 7 , and 9 . However, although 4 - 6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin-2 (DTX2) from shellfish ( M. edulis ), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima , that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL -1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L -1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.

Considerable efforts are being made worldwide to replace in vivo assays with instrumental methods of analysis for the monitoring of marine biotoxins in shellfish. Analysis of these compounds by the preferred technique of liquid chromatography tandem mass spectrometry (LC-MS/MS) is challenged by matrix effects associated with the shellfish tissues. In methods validation, assessment of matrix interferences is imperative to ensure the validity and accuracy of results being produced. Matrix interferences for the analysis of okadaic acid (OA) and azaspiracid 1 (AZA1) were assessed using acidic methods on electrospray triple stage quadrupole (TSQ) and hybrid quadrupole time of flight (QToF) instruments by the use of matrix matched standards for different tissue types. Using an acidic method no matrix interference and suppression was observed on the TSQ for OA and AZA1 respectively, whilst the opposite was observed on the QToF; matrix enhancement for OA and no matrix interference for AZA1. The suppression of AZAs on the TSQ was found to be due to interfering compounds being carried over from previous injections. The degree of suppression is very much dependant on the tissue type ranging from 15 to 70%. Several strategies were evaluated to eliminate these interferences, including the partitioning of the extract with hexane, optimisation of the chromatographic method and the use of on-line SPE. Hexane clean up did not have any impact on matrix effects. The use of an alkaline method and a modified acidic method eliminated matrix suppression for AZA1 on the TSQ instrument while an on-line SPE method proved to be effective for matrix enhancement of OA on the QToF., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Using high-performance liquid chromatography with mass spectrometry, the influence of conventional steaming and another heat treatment on the level of okadaic acid and dinophysistoxin-2 in mussels (Mytilus edulis) was investigated. Concentration increases correlated with water loss during steaming, and increased distribution of okadaic acid and dinophysistoxin-2 from the digestive glands to the remainder tissues was observed as a result of the processes examined. This suggests that the analysis of whole flesh tissues, as opposed to dissected digestive glands, is more appropriate for regulatory purposes, particularly if cooked samples are being analysed. A systematic heat treatment experiment showed that while okadaic acid group toxins are stable during cooking processes, degradation does occur in mussel tissues after prolonged exposure to high temperatures. The findings of the studies reported here have importance in terms of the methodology applied in regulatory phycotoxin monitoring programmes. Therefore, options for sample pretreatment are discussed.