DEVELOPMENT OF ANALYTICAL METHODS AND REFERENCE MATERIALS FOR CYANOBACTERIAL TOXINS
MetadataShow full item record
Cyanobacterial toxins present a real and growing threat to humans and animals due to the projected increases in algal blooms resulting from increasing global temperature and pollution. Wild animals, livestock, pet animals and humans can be poisoned from contaminated drinking water. With the discovery of cyanobacterial toxins present in nutritional supplements, a new concern looms over consumers with threats of neurotoxin and hepatotoxin related damage from exposure to these products. To this end, work on the development of a freeze dried algal reference material was pursued for future use in environmental and nutritional supplement analysis. The first stage of the project was to prepare needed calibration standards, starting with homoanatoxin a, a homologue of the highly neurotoxic anatoxin-a compound. The resulting reference material (RM-hATX) had a homoanatoxin-a concentration of 20.2 ± 0.7 ?M, and proved to be stable while stored at temperatures of 80°C. Reference samples for dihydro and epoxy analogues of anatoxin-a and homoanatoxin-a were then prepared by semi-synthesis. The second stage of the project was the development of new analytical methods for the anatoxins. A derivatization reaction in which dansyl chloride was coupled with a novel cleanup step produced anatoxin derivatives suitable for liquid chromatography (LC) with mass spectrometry (MS) or fluorescence detection (FLD). Limits of quantitation were 60 ng L-1 and 1.6 ?g L-1 for the developed LC-MS/MS and LC-FLD methods, respectively, with the limit of quantitation significantly better than that of a previously developed method for the underivatized toxins based on HILIC MS/MS. Quantitative results for anatoxins in various algal samples using all three methods of analysis of were compared and it was found that there were no significant differences between the three methods. Unfortunately, experiments showed that the various toxin analogues did not elicit equimolar responses in either LC-MS/MS or LC FLD, thus indicating the importance of having individual calibration standards for quantitative analysis. The LC-MS/MS and LC-FLD methods were paired with a previously developed method for the analysis of hepatotoxic microcystins to screen a small number of nutritional supplement samples for cyanobacterial toxins. Microcystins were detected in all five Aphanizomenon flos-aquae samples examined. This method involved a fifteen-fold pre-concentration using a solid phase extraction cartridge, which gave a 98% recovery of microcystins. The third phase of the project was the preparation and testing of a preliminary algal matrix reference material as a feasibility study for the eventual production of a CRM. After selecting various algal cultures and samples that could be blended together, a freeze dried algal reference material was prepared and packaged. This material (RM-BGA) was then characterized using several methods including the two new dansylation-based procedures.