Document Type

Theses, Ph.D


This item is available under a Creative Commons License for non-commercial use only



Publication Details

Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Dublin Institute of Technology, 2015.


The two main groups of biotoxins which affect the Irish shellfish industry are azaspiracids (AZAs) and the okadaic acid (OA) group (OA, DTX2, DTX1 and their esters) toxins. Since AZAs were first identified in 1998, well over 30 analogues have been reported. Structural and toxicological data have been described for AZA1–5 (isolated from shellfish). LC-MS/MS is the EU reference method for detection of the AZAs (AZA1, -2 and -3) and the OA group toxins in raw shellfish with the regulatory limit set at 160 μg/kg for each toxin group. Limited supplies of purified toxins for certified reference materials (CRMs) were available for AZA1−3. Little knowledge was also available on the relevance of the additional AZA analogues that had been reported, in terms of human health protection. The analysis of marine biotoxins by LC-MS/MS can be severely affected by matrix interferences. Here, a study was performed on two instruments; a quadrapole time of flight (QToF) and a triple stage quadrupole (TSQ) to assess matrix interferences for AZA1 and OA using a number of tissue types. Enhancement was observed for OA on the QToF while matrix suppression was observed for AZA1 on TSQ. The enhancement on the QToF was overcome by use of an on-line SPE method and matrix matched calibrants, while the suppression on the TSQ was found to be due to late eluting compounds from previous injections and was overcome by employing either a column flush method or an alkaline mobile phase. The isolation of 11 AZA analogues (AZA1−10 and 37-epi-AZA1) from shellfish using an improved procedure (7 steps) is described. Recoveries increased ~2-fold (~ 52%) from previously described isolation procedures. The preparative isolation procedure developed for shellfish was optimised for Azadinium spinosum bulk culture extracts such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of ~70% was achieved, and isolation from 1,200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 (purities >95%).